# EMERGING CHALLENGES OF CARDIOVASCULAR AND METABOLIC DYSFUNCTIONS IN CARDIO-ONCOLOGY: FROM BENCH TO BEDSIDE

EDITED BY : Canan G. Nebigil, Tienush Rassaf and Michael W.Y. Chan PUBLISHED IN : Frontiers in Cardiovascular Medicine

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ISSN 1664-8714 ISBN 978-2-88966-095-7 DOI 10.3389/978-2-88966-095-7

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# EMERGING CHALLENGES OF CARDIOVASCULAR AND METABOLIC DYSFUNCTIONS IN CARDIO-ONCOLOGY: FROM BENCH TO BEDSIDE

Topic Editors:

Canan G. Nebigil, École supérieure de biotechnologie Strasbourg (ESBS), France Tienush Rassaf, University Hospital Essen, Germany Michael W.Y. Chan, National Chung Cheng University, Taiwan

Citation: Nebigil, C. G., Rassaf, T., Chan, M. W. Y., eds. (2020). Emerging Challenges of Cardiovascular and Metabolic Dysfunctions in Cardio-oncology: From Bench to Bedside. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-095-7

# Table of Contents


Katie Livingston, Rachel A. Schlaak, Lindsay L. Puckett and Carmen Bergom

*30 Emerging Challenges of Radiation-Associated Cardiovascular Dysfunction (RACVD) in Modern Radiation Oncology: Clinical Practice, Bench Investigation, and Multidisciplinary Care*

Moon-Sing Lee, Dai-Wei Liu, Shih-Kai Hung, Chih-Chia Yu, Chen-Lin Chi, Wen-Yen Chiou, Liang-Cheng Chen, Ru-Inn Lin, Li-Wen Huang, Chia-Hui Chew, Feng-Chun Hsu, Michael W. Y. Chan and Hon-Yi Lin


Himani Kumari, Wan-Hong Huang and Michael W. Y. Chan

# Editorial: Emerging Challenges of Cardiovascular and Metabolic Dysfunctions in Cardio-Oncology: From Bench to Bedside

Canan G. Nebigil <sup>1</sup> \*, Michael W. Y. Chan<sup>2</sup> and Tienush Rassaf <sup>3</sup>

*<sup>1</sup> Regenerative Nanomedicine, UMR 1260, INSERM, University of Strasbourg, Strasbourg, France, <sup>2</sup> Department of Biomedical Sciences, National Chung Cheng University, Chiayi City, Taiwan, <sup>3</sup> Department of Cardiology and Vascular Medicine, West German Heart- and Vascular Center, University Hospital Essen, Essen, Germany*

Keywords: cardiooncology, cardiotoxicity, antracyclines, biomarkers, GPCR (G protein coupled receptors), epigenetics (methylation/demethylation), HPSC-cardiomyocytes, HDLP

**Editorial on the Research Topic**

**Emerging Challenges of Cardiovascular and Metabolic Dysfunctions in Cardio-Oncology: From Bench to Bedside**

## INTRODUCTION

#### Edited by:

*Jun-ichi Abe, University of Texas MD Anderson Cancer Center, United States*

#### Reviewed by:

*Claudia Penna, University of Turin, Italy Neal Lee Weintraub, Augusta University, United States*

> \*Correspondence: *Canan G. Nebigil nebigil@unistra.fr*

#### Specialty section:

*This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine*

> Received: *29 May 2020* Accepted: *14 July 2020* Published: *26 August 2020*

#### Citation:

*Nebigil CG, Chan MWY and Rassaf T (2020) Editorial: Emerging Challenges of Cardiovascular and Metabolic Dysfunctions in Cardio-Oncology: From Bench to Bedside. Front. Cardiovasc. Med. 7:148. doi: 10.3389/fcvm.2020.00148*

The number of cancer survivors is increasing and up to 30 new cancer therapies are approved each year with only incompletely characterized side effects (1). Many anti-cancer drugs including traditional, new targeted kinase inhibitors and immunotherapies are associated with cardiovascular and metabolic adverse effects and may have dramatic impact on morbidity and mortality (2). The exact mechanisms how chemotherapeutics induce metabolic disturbances are mostly unclear. Chemotherapeutics-induced oxidative stress and mitochondrial dysfunction may promote metabolic disturbance (3).

Cardio-oncology is a relatively new discipline, aiming at finding an optimal balance between the efficacy of anticancer treatments and the management of their adverse cardiovascular and metabolic effects. It includes the prevention, diagnosis, and treatment of these complications in cancer patients. Identifying markers/predictors of disease risk, ensuring safety of novel cancer therapeutics, developing cardioprotective drugs are the emerging challenges in cardio-oncology (4).

Cardiovascular disease and cancer not only share common genetic, cellular, and signaling mechanisms such as chronic inflammation but also exhibit common risk factors such as obesity and diabetes (5). Dyslipidemia, hypertriglyceridemia, altered levels of low-density lipoprotein cholesterol (LDL-C), and low high-density lipoprotein cholesterol (HDL-C) have been observed in cancer survivals (6). In this regard, about half of the cancer survivors have obesity issue (7). Hypertension, Insulin resistance, hyperinsulinemia, and impaired glucose control that directly affect insulin sensitivity have been observed in cancer survivals (8). Chemotherapies that adversely affect metabolism may amplify cardiac and vascular toxicity, and patient management represents a major economical and clinical burden.

In this edition, we covered the expert reviews on biomarkers and signaling pathways of cardiovascular toxicity and metabolic alteration, and characterization of possible target molecules to prevent or treat cardiovascular damages induced by the cancer therapy.

Kumari et al., focused on epigenetic modifications by doxorubicin (DOX) that can either be used as molecular markers for cancer prognosis or represent molecular targets to attenuate DOX-induced cardiotoxicity in cancer patients.

Kluck et al., outlined emerging preclinical evidence that high density lipoprotein and its precursor protein apolipoprotein A1 may also protect against doxorubicin-induced cardiotoxicity.

Schwach et al., described that human pluripotent stem cell derived cardiomyocytes can be used as a screening platform to test cardioprotective agents against anti-cancer mediated oxidative stress generation and mitochondrial dysfunction, disruption of calcium homeostasis, and changes in transcriptome and proteome, triggering apoptotic cell death.

Mrotzek et al., underlined new studies on the mechanisms and severity of radiation-induced cardiovascular side effects and clinical management and treatment options.

Cardinale et al., discussed in particular troponins as a biomarker of subclinical cardiotoxicity and angiotensinconverting enzyme inhibitors (mainly enalapril) to prevent LVEF reduction in case of early detection of cardiotoxicity and prompt heart failure treatment.

Parichatikanond et al., discussed the molecular mechanisms of TGF-β in the pathogenesis of cardiac fibrosis and cancer and provide in vitro and in vivo evidences regarding antifibrotic and anticancer actions of TGF-β inhibitors.

Schlaak et al. outlined how inherited genetic variants promote differences in mitochondrial gene expression that may contribute to susceptibility of cancer patients to mediated cardiotoxicity.

Lee's et al. group recommended combined managements with control of comorbidities (such as hypertension, hypercholesterolemia, and diabetes, smoking cessation), and close monitoring and discussed use of statins and angiotensinconverting enzyme inhibitors (ACEIs) in the treatment of cardiovascular disorders induced by anti-cancer drugs.

Livingston et al., presented the evidence that understanding of mitochondria-dependent mechanisms of radiation-induced heart dysfunction can help to develop potential therapeutic targets to assist in prevention and treatment of radiation-induced heart damage.

Audebrand et al. emphasized newly identified cardioprotective agents targeting G protein coupled receptors (GPCRs) of adrenalin, adenosine, melatonin, ghrelin, galanin, apelin, prokineticin, and cannabidiol, provoking further drug development studies for the treatment of human heart failure induced by anticancer drugs.

# CONCLUSION AND FUTURE CHALLENGES

The adverse effects of anticancer treatments including cardiovascular toxicity and metabolic syndrome and their

# REFERENCES

1. Rassaf T, Totzeck M, Backs J, Bokemeyer C, Hallek M, Hilfiker-Kleiner D, et al. Onco-cardiology: consensus paper of the german cardiac society, the german society for pediatric cardiology and congenital heart defects and the german society for hematology and medical oncology. Clin Res Cardiol. (2020). doi: 10.1007/s00392-020-01636-7

relations with the genetic and environmental factors are still needs to be discovered. Research in cardio-oncology should aim at elucidating the mechanisms involved in cardiovascular toxicity as well as metabolic disturbances. A better understanding of the mechanisms of these adverse effects of anti-cancer therapies may lead to the identification of novel targets for drug development. Currently, evaluation of anticancer therapyinduced cardiovascular toxicity and metabolic disturbance have limitations. Therefore, identification of new early biomarkers of subclinical cardiovascular dysfunctions and metabolic disorders is a key challenge. Research in cardio-oncology should also aim at elucidating the efficacity and toxicity of the new cancer treatments. Thus, the coordinated efforts of oncologists, endocrinologists, and cardiologist are required to overcome these life-threatening problems especially in cancer survivals.

We believe that the topic of "Emerging challenges of cardiovascular and metabolic dysfunctions in cardiooncology" provides new challenges and potential future directions to the readers from basic scientists, cardiologist, endocrinologists, and oncologist in developing field of cardio-oncology.

# AUTHOR CONTRIBUTIONS

CN wrote the editorial information. MC and TR have been corrected and edited. All authors contributed to the article and approved the submitted version.

# FUNDING

This study was supported by grants from European ERA-NET, ERA-CVD-JC2016, French government managed by Agence Nationale de la Recherche (ANR-16-ECVD-0005- 01) (CN); the Ministry of Science and Technology, Taiwan (MOST 106-2923-B-194-001-MY3), the Center for Innovative Research on Aging Society (CIRAS) from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by Ministry of Education (MOE) in Taiwan (MC), and IFORES research grant from the Medical Faculty, University Duisburg-Essen, Germany (TR).

# ACKNOWLEDGMENTS

We thank to all authors, reviewers, and editors.


insulin resistance, and adiposity as a predictors of metabolic syndrome in child survivors of lymphoma and acute lymphoblastic leukemia of a developing country. BMC Cancer. (2017) 17:125. doi: 10.1186/s12885-017-3097-8

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Nebigil, Chan and Rassaf. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Targeting GPCRs Against Cardiotoxicity Induced by Anticancer Treatments

#### Anais Audebrand, Laurent Désaubry and Canan G. Nebigil\*

Laboratory of CardioOncology and Therapeutic Innovation, CNRS, Illkirch, France

Novel anticancer medicines, including targeted therapies and immune checkpoint inhibitors, have greatly improved the management of cancers. However, both conventional and new anticancer treatments induce cardiac adverse effects, which remain a critical issue in clinic. Cardiotoxicity induced by anti-cancer treatments compromise vasospastic and thromboembolic ischemia, dysrhythmia, hypertension, myocarditis, and cardiac dysfunction that can result in heart failure. Importantly, none of the strategies to prevent cardiotoxicity from anticancer therapies is completely safe and satisfactory. Certain clinically used cardioprotective drugs can even contribute to cancer induction. Since G protein coupled receptors (GPCRs) are target of forty percent of clinically used drugs, here we discuss the newly identified cardioprotective agents that bind GPCRs of adrenalin, adenosine, melatonin, ghrelin, galanin, apelin, prokineticin and cannabidiol. We hope to provoke further drug development studies considering these GPCRs as potential targets to be translated to treatment of human heart failure induced by anticancer drugs.

#### Edited by:

Carlo Gabriele Tocchetti, University of Naples Federico II, Italy

#### Reviewed by:

Claudia Penna, University of Turin, Italy Alessandra Cuomo, Federico II University Hospital, Italy

> \*Correspondence: Canan G. Nebigil nebigil@unistra.fr

#### Specialty section:

This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine

> Received: 13 November 2019 Accepted: 23 December 2019 Published: 24 January 2020

#### Citation:

Audebrand A, Désaubry L and Nebigil CG (2020) Targeting GPCRs Against Cardiotoxicity Induced by Anticancer Treatments. Front. Cardiovasc. Med. 6:194. doi: 10.3389/fcvm.2019.00194 Keywords: GPCRs, cardiotoxicity, melatonin, ghrelin, galanin, apelin, prokineticin, cannabidiol

# INTRODUCTION

New anticancer treatments have improved overall mortality (1). However, most of the anticancer drugs display a wide array of cardiovascular toxicities, leading to interruption of cancer therapies and maladaptive remodeling in hearts, affecting the short- and long-term quality of life (2–4). Oxidative stress and inflammation are inter-reliant processes involved in cardiovascular diseases and cancers (5, 6), along with apoptosis (7, 8) and necrosis (9). Tissue resident and circulating inflammatory cells (such as macrophages, mast cells, neutrophils, and monocytes) can also release both reactive oxygen species (ROS) and reactive nitrogen species (RNS) to induce an oxidative stress (6). Due to negligible detoxification capacity, the heart is particularly susceptible to ROS and RNS injury (10). Thus, high levels of ROS and RNS can debilitate cardiac cellular signaling pathways and can augment the gene expression of proinflammatory (11) and antioxidant defenses as the major cause for necrosis and apoptosis.

Classic chemotherapeutics particularly anthracyclines are the prototype of drugs causing cardiotoxicity (12). They can induce acute cardiotoxicity, including reversible hypotension, pericarditis and transient electrocardiographic abnormalities (changes in the ST-T waves, QT prolongation), and vasodilatation (13). However, after completion of cumulative dose regimens, anthracyclines promote irreversible cardiomyopathy (classified as type (1) cardiotoxicity), leading to heart failure (HF) (13, 14). Doxorubicin (DOX), the most frequently used anthracyclines can

**7**

cause irreversible type 1 cardiotoxicity via accumulation of ROS and RNS (15, 16). They also target Topoisomerase IIβ (Top IIβ) in cardiomyocytes to induce DNA damage and apoptosis. Recently, the anthracycline mediated cardiotoxicity has been reviewed by Nebigil (17).

Targeted therapies also provoke some degree of cardiotoxicity. Targeting key tyrosine kinases (TKs) with TK antibodies and inhibitors has a remarkable achievement in cancer management. However, they also induce cardiotoxicity, because they block pathways that also regulate myocardial function (18). This cardiotoxicity is often reversible, and thus classified as type 2 cardiotoxicity (19, 20). It results in ultrastructural changes in cardiomyocytes, with reversible cardiac dysfunctions such as elevated blood pressure, thromboembolism, pericardial thickening, and arrhythmia (21). Type 1 and 2 forms of cardiotoxicity can overlap, when the classic and targeted therapeutics used together or subsequently. For example, in patient treated with anthracyclines earlier, trastuzumab, a monoclonal antibody anti-HER-2 can cause irreversible cardiac damage and left ventricular (LV) dysfunction (18, 22, 23). On the other hand, 27%, of patients who received both anthracycline and trastuzumab encountered cardiac dysfunction, while this rate was of 2-16% for patients treated with anthracyclines alone (24).

Recent studies have demonstrated that patients treated with immune checkpoint inhibitors (25) also develop myocarditis due to immune-related adverse events (6, 26). The therapeutic mechanisms of inhibitors mostly rely on blocking either the cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) or programmed cell death protein-1 (PD-1) pathways, while activating the host's immune system against cancer (27). CTLA-4 and PD-1 act as immune response inhibitors (6, 28). They suppress the T-cell response in order to prevent autoimmunity and maintain T-cell tolerance. Cardiac immune-related adverse events appear more frequently in patients treated with CTLA-4 antagonists compared with PD-1 inhibitors (29) and the myocarditis risk increases with combination therapy, leading to discontinuation in approximately 50% of patients (30, 31) probably due to targeting PD-1 and CTLA-4 in cardiomyocytes as well.

# CLINICALLY USED CARDIOPROTECTIVE AGENTS AGAINST CARDIOTOXICITY

There are several cardioprotective therapeutics that have been used against anticancer-mediated cardiotoxicity. Their properties are summarized in **Table 1**.

# Antioxidants

Beneficial effects of antioxidants on LV remodeling and amelioration of contractility have been demonstrated in many experimental models of HF. For example, vitamin C effectively mitigates DOX-induced oxidative stress and apoptosis in rats (35). Resveratrol, a polyphenolic compound has also both prophylactic and therapeutic benefits in reversing DOX induced apoptosis and fibrosis in rat myocardium (36). Baicalein, a bioflavonoid can alleviate cardiotoxicity in mice (37). However, elimination of ROS and RNS by antioxidant drugs may be detrimental and even impair physiological cellular functions (58). There is also a risk of loss of oncological efficacy, because of the overlapping mechanisms with cardioprotective effects. Nevertheless, in clinic these approaches did not significantly improve survival rate and they may even increase mortality if they do not have other pharmacological properties (32, 59).

# Dexrazoxane

Dexrazoxane is an iron chelator and detoxifying agent that can prevent anthracycline-associated cardiotoxicity. It also acts on Topoisomerase IIβ to promote cardioprotective effects. Dexrazoxane is the only Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved cardioprotective drug to against chemotherapeutics-mediated HF (38, 60). However, its use in children and adolescent were forbidden by EMA in 2011, because it increases risk of infection, myelosuppression and second primary malignancies. These restrictions by EMA have been partially altered based on the new findings in 2018 (39). Only use of dexrazoxane was allowed in patients who have received a cumulative DOX at the dose of 300 mg/m (2) and are continuing with this medicine. Although dexrazoxane is a valuable option to prevent cardiotoxicity, it induces a severe leukopenia in 78% of cancer patients (40). Use of dexrazoxane is not recommended with non-anthracycline chemotherapy regimens.

# Statin

Statins are used to lower low-density lipoprotein (LDL) and cholesterol amount in the blood on patients suffering to arterosclerosis (61). The mechanism involved in this action is due to inhibition of HMG-CoA reductase, which is involved the biosynthesis of cholesterol. Statins also display significant vasodilatation, platelet inhibition, anti-inflammatory, and antioxidant effects due to their pleiotropic effects (62, 63). Statin (atorvastatin) could be effective in maintenance of LV ejection fraction (LVEF) in patients treated with anthracycline (42). Moreover, it could limit oxidative stress and vascular inflammation (64) and activate autophagy (43) to promote cardioprotective effects against dasatinib. Statins also inhibits Top IIβ mediated DNA damage via Rac1 inhibition. Recent

**Abbreviations:** GPCR, G protein-coupled receptor; DOX, Doxorubicin; HF, Heart failure; ROS, Reactive oxygen species; RNS, Reactive nitrogen species; Top Iiβ, Topoisomerase Iiβ; LVEF, LV ejection fraction; HER2, Hergulin2; CTLA-4, Tlymphocyte associated antigen-4; PD-1, Programmed cell death protein-1; LDL, low-density lipoprotein; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; PI3K, Phosphoinositide 3-kinases; MAPK, Mitogen-activated protein kinases; β-ARs, β-adrenergic receptors; ? -ARs???-adrenergic receptors; CaMKII, Calmodulin-dependent protein kinase II; Ang-II, Angiotensin II; AT-1R and AT-2R, Angiotensin receptors; RAS, Renin-angiotensin system; ARB, Angiotensin-II receptor blockers; IP3, Inositol trisphosphate; DAG, Diacylglycerol; PKD1, Anchored protein kinase D1; ATP, Adenosine-triphosphate; A1R, A2AR, A2BR and A3R, Adenosine receptors; MT1 and MT2, Melatonin receptors; mPTP, Mitochondrial permeability transition pore; GHS-R, Ghrelin receptor, growth hormone secretagogue receptor; VEGF, vascular endothelial growth factor; GalR1, GalR2 and GalR3, Galanin receptors; APJ, Apelin receptor; ACE2, Angiotensinconverting enzyme 2; PARP, Poly(ADP-ribose) polymerase; PROK1 and PROK2, Prokineticins 1 and 2; PKR1 and PKR2, Prokineticin receptors; hiPSC-CMs, Inducible pluripotent stem cell derived cardiomyocytes.



meta-analyses suggest that statins are at least equally potent as dexrazoxane in the prevention of anthracycline-induced cardiotoxicity (65). Calvillo-Argüelles and colleagues have found that in HER2<sup>+</sup> breast cancer patients treated with trastuzumab with or without anthracycline, the concomitant statin use was associated with a lower risk of cardiotoxicity (44). Although, several studies on the influence of statin therapy on development of cancer risk resulted in conflicting results, the recent metaanalyses suggested that statin can reduce cancer-mediated mortality (46). However, there are some studies show that statin induces myopathies that may be due to decreased synthesis of mevalonic acid, leading to decreased energy generation and muscle injury. Another side effect associated with statin usage is new-onset diabetes (47). Many of the beneficial effects of a statin is due to inhibition of heterotrimeric G proteins, including Ras and Rho or Rac1 signaling (45). Thus, the specific Rho and Rac inhibitors may be more preferable targets for future chemo-preventive strategies.

## GPCRs

As seven transmembrane (7TM) domain proteins, G proteincoupled receptors (GPCRs) represent the largest family of cell surface proteins (66). GPCRs regulate many physiological processes in every tissue, making the GPCR superfamily a major target for therapeutic intervention (67). The binding of agonists to GPCRs not only initiates the "classical," signaling cascades through heterotrimeric G proteins (composed of the three subunits, Gα, Gβ, and Gγ). It can also activate Gprotein-independent pathways involving β-arrestin (68, 69). Indeed, β-arrestins are identified as scaffolding proteins for MAP kinases and serine/threonine kinases cascades (70). The discovery that some GPCRs prefer to activate G-protein- or arrestin-mediated pathways has given rise to efforts to produce signal biased drugs (71). The drug discovery efforts aim to produce "biased" and/or allosteric ligands with less adverse effects without compromising their efficacy (72). In cardiovascular system, GPCRs can lead to hypertrophy, apoptosis, contraction, and cardiomyocytes survival. Some of the GPCR targeted therapeutics are used in clinic for treatment of heart failure and cardiotoxicity (**Table 1**).

# Preventive and Prophylactic Strategies Targeting GPCRs Against Anticancer-Induced Cardiotoxicity β-Blockers

β-adrenergic receptors (β-ARs) play a crucial role in cardiovascular regulation. It exists 3 types of β-ARs: β1, β<sup>2</sup> and β3. Cardiac adrenergic receptor corresponding to β1-ARs whereas β2-ARs are localized on blood vessels. β1-ARs, are coupled to the Gα<sup>s</sup> and activate adenylyl cyclase to exert a positive inotropic, chronotropic and dromotropic effects in the heart. Indeed, β1-ARs increase heart rate, cardiac contractility and myocardial oxygen demand, thus promoting myocardial ischemia in patients with coronary heart disease. More importantly, persistent β1-ARs induce myocyte apoptosis and hypertrophy by activating CaMKII. On the opposite, persistent β2-ARs activation protects myocardium through a Gαi-mediated pathway, and activating PI3K, and Akt kinase probably via small G proteins (73). Administration of β2-AR agonist and β1-AR antagonist seems to be better than β2-AR antagonist in HF prevention. Interestingly, β3-AR is activated by catecholamines at higher concentration than those required to activate β1-AR and β2-AR (73). Thus, β3-AR plays an important protective role in the cardiovascular system during sympathetic over-stimulation.

It exists three mains β-AR blockers. The first generation of β-blockers, such as propranolol, inhibits both β<sup>1</sup> and β2- ARs. The second generation of β-blockers (metoprolol) are cardioselective (β1-ARs).

The third generation of β-blockers (carvedilol and nebivolol) are vasodilators that not only inhibit β1 and α1-adrenoreceptors, but they also activate β3-adrenergic receptors (74). Carvedilol also reduces ROS generation and apoptosis in cardiomyocyte (49). Nebivolol has a vasodilatory effect mediated by nitric oxide release and avoid vasoconstriction to decrease blood pressure in hypertensive patients (50). Two clinical studies showed that carvedilol prevent cardiotoxicity in female patients diagnosed with breast cancer (75, 76). This cardioprotective effects has been attributed to its antioxidant and anti-apoptotic properties rather than its β-AR blocking activity, because carvedilol inhibits mitochondrial complex-I that promotes cardiotoxicity (77). This cardioprotective effect of carvedilol is superior than metoprolol and atenolol for preventing cardiomyocytes against DOXinduced apoptosis (78). In contrast, Avila and his colleague showed that carvedilol has no impact on the LVEF reduction induced by anthracycline in breast cancer patients (53). The recent meta-analyses on cancer patients have demonstrated that the use of β-blockers is not associated with cancer prognosis (51). Indeed, several studies on the influence of β-blockers on cancerspecific survival rate resulted in conflicting results (51, 52). The beneficial effects of non-selective β1 and β2 blockers could be due to their antioxidant effects (28).

### Angiotensin Converting Enzyme Inhibitors (ACEI) and Angiotensin (AngII) Receptor Blockers (ARB)

Renin-angiotensin-aldosterone (RAAS) system regulates the cardiac and renal functions. Ang-II interacts with two GPCRs: AT-1R and AT-2R that are associated with opposite functions (79). However, most of the effects of renin-angiotensin system (RAS) are mediated by AT-1R, which promotes vasoconstriction, inflammation, fibrosis, hypertrophy, and releasing of catecholamine and aldosterone. AT-2 is implicated to vasodilatations, inhibition on cell growth, apoptosis, and bradykinin releasing. Increasing of Ang-II also stimulates sympathetic system and the production of aldosterone, leading to LV hypertrophy (80). Reduction of excessive Ang-II and aldosterone decrease cardiovascular morbidity and mortality. Indeed, AT-1R blockers ACE inhibitors are of paramount importance in treatment of cardiovascular diseases, including hypertension (54).

Several clinical trials indicate that Angiotensin-II receptor blockers (ARB) alleviate anthracycline cardiotoxicity (55), however, prospective trials are still needed for further validation. The expression of AngII and AT-1R have been found in many cell types of the tumor microenvironment (56). Thus, the RAS may alter remodeling of the tumor microenvironment and the immuno-suppressive milieu, thereby affecting tumor growth. In contrast, meta-analysis derived from the results of a group of trials demonstrated that ARB may promote the occurrences of new tumors (especially lung cancer) (57). These findings warrant further investigation.

The cardioprotective effects of combined ACEIs/ARBs and βblockers have been evaluated during anthracycline, trastuzumab, or sequential chemotherapy. The combination of carvedilol and enalapril has been shown to preserve the LV function in adult patients treated with anthracyclines (81). However, other trials with combination of enalapril with metoprolol (82) or candesartan with metoprolol (83), ended up with disappointing results. Indeed, Guglin and his colleague recently demonstrated that both lisinopril and carvedilol do not prevent the cardiotoxicity of trastuzumab monotherapy in breast cancer patients (48). However, both drugs significantly alleviated the cardiotoxicity of anthracycline and trastuzumab sequential therapy. Although, ARBs, ACEIs, and β-blockers are necessary for treatment of HF, long-term studies are essential to validate whether ARBs have cardioprotective effects against the chronic or late-onset types of cardiotoxicities induced by cancer treatments.

# Newly Discovered GPCR Agonist Against Anticancer-Mediated Cardiotoxicity

We discus here newly identified GPCR agonists that exhibit cardioprotective effects against anti-cancer drugs in in vitro and in vivo preclinical models (**Figure 1** and **Table 2**).

# Alpha Adrenergic Receptor (Dabuzalgron)

Both the adrenergic receptors alpha 1 (α-AR1) and alpha 2 (α-AR2) bind catecholamines (epinephrine and norepinephrine). The α-AR1 couples to Gαq type, resulting in activation of phospholipase C, increasing Inositol trisphosphate (IP3) and diacylglycerol (DAG), and ultimately increasing the intracellular Ca2<sup>+</sup> levels, leading to smooth muscle contraction and glycogenolysis (104). Cardiac α1-ARs activate phospholipase C and MAPK to promote ischemic preconditioning (105), cardiac hypertrophy (106)and cardiac cell survival (107). The knockout of α1A/α1B-adrenoceptor in mice develops small hearts (108) and aggravates the pressure overload–induced HF. In support of this study a large-scale clinical trial showed that doxazosin, an inhibitor of α-AR1 signaling, increases HF in hypertension patients (109). The α2-AR acts via Gαi/o to an inhibit adenylyl cyclase, decreasing the available cAMP (110). It also decreases neurotransmitter release and central vasodilation.

Dabuzalgron is a selective α1AR agonist that has been clinically examined against urinary incontinence (111). Recent study in mice showed that dabuzalgron displayed a strong cardioprotection against DOX-induced cardiotoxicity (84). It reduces ROS production and fibrosis, enhances contractile function, and preserves myocardial ATP content via regulating mitochondrial function, in DOX-treated mice. Cardioprotective signaling pathways of α1-AR is not limited to activation of MAPK1/2 pathways (84), it also activates pro-survival pathways such as A kinase anchoring protein-Lbc (AKAP-Lbc) and its anchored protein kinase D1 (PKD1) in cardiotoxicity mice models (112). Future studies should determine whether dabuzalgron can be used to treat chemotherapeutics-mediated HF in cancer patients.

#### Adenosine Receptor Agonists

Adenosine is a naturally occurring nucleoside formed by the degradation of ATP. Extracellular adenosine concentrations rise in response to hypoxia and other stress (113). However, chronic adenosine elevation can increase inflammation, cytokine release, and induces brain dopamine depletion, fibrosis and kidney damage (114). The adenosine receptors A1R, A2AR, A2BR, and A3R can sense an imbalance of demand and supply of oxygen and nutrients (115). Adenosine exerts a significant cardioprotective effect during cardiac ischemia by activation of the A1R and A3R (86, 116). However, full A1R agonists have promote several cardiovascular adverse effects due to its off-target activation as well as desensitization of A1R, leading to tachyphylaxis (117). In contrast, a selective partial agonist for A1AR improves cardiac function without promoting atrioventricular blocks, bradycardia, or unfavorable effect on blood pressure (118, 119).

A selective A3R agonist (Cl-IB-ME) mitigates bradycardia, elevated serum creatine kinase levels and cardiac histopathological changes in DOX-treated mice. Cardioprotective effect of Cl-IB-ME involves the inhibition of ROS production and inflammation induced by DOX in vivo (85). A3AR activation also prevents perioperative myocardial ischemic injury (120), protects ischemic cardiomyocytes by preconditioning (121), and induces ischemic tolerance that is dependent on KATP channels (122). This cardioprotective effects A3R agonists were absence in A3AR deficient mouse cardiomyocytes, showing an A3AR-mediated effect. On the opposite to A1AR, A3AR is expressed at very low levels in adult ventricular cardiomyocytes. The efficacy of two A3AR agonists is currently examined in multiple clinical trials (123).

#### Melatonin Receptor Agonists

Melatonin is a pineal gland hormone synthesized from the amino acid tryptophan and is secreted into both the bloodstream and cerebrospinal fluid. It regulates circadian, seasonal, and transgenerational time cycles. Melatonin acts through 2 GPCRs, MT1, and MT2 that are linked to Gαi/Gα<sup>o</sup> or Gαq/Gα<sup>11</sup> to induce TABLE 2 | Newly discovered cardioprotective agents targeting GPCRs.


anti-adrenergic effects (124). These melatonin receptors are ubiquitously present in central and peripheral organs, including the cardiovascular system. Melatonin regulates blood pressure and heart rate either normalizing the circadian rhythm of blood pressure and ameliorating nocturnal hypertension, or directly acting on heart and blood vessels (125). They also regulate the renin-angiotensin system (126) and mitochondrial function (127).

Melatonin inhibits necrosis and apoptosis, and improves DOX-mediated cardiac dysfunction without compromising the antitumor effect of DOX in mice (87) and rats (88). The mechanism involved in cardioprotective

**12**

effect against DOX-cardiotoxicity has been attributed to its antioxidant effect (89) and suppression of lipid peroxidation (90). Recent studies showed that melatonin activates AMPK, PGC1α (91), and sirtuins (92) to attenuate acute DOX-cardiotoxicity via alleviating mitochondrial oxidative damage and apoptosis. Indeed, high doses of melatonin are essential to reach adequate subcellular concentrations to exert these cardioprotective effects (128).

Ramelteon, is a dual MT1 and MT2 melatonin receptor agonist used for insomnia that displays a strong cardioprotective effect in the models of ischemic HF induced by the coronary artery ligation (129), chronic intermittent hypoxia-induced HF (130), and isoproterenol-induced myocardial infarction (131, 132). Unfortunately, the effect of ramelteon in anticancermediated cardiotoxicity has not been studied yet. Melatonin can also enhance antitumor effects of anthracycline in animal model (93). Thus, the combined treatment of anthracyclines and melatonin needs to be further explored in cancer patients.

#### Ghrelin Receptor Agonists

Ghrelin is a growth hormone-releasing and orexigenic peptide that acts through growth hormone secretagogue receptor (GHS-R) in the brain. However, expression of GHS-R in cardiovascular system is controversial. Ghrelin regulates energy balance, body weight maintenance, and metabolism (133). Roles of ghrelin in protecting heart function and reducing mortality after myocardial infarction are partly due to its role on the cardiac vagal afferent nerve terminals (inhibition of cardiac sympathetic and activation of cardiac parasympathetic nerve activity) (134). Ghrelin significantly decreased blood pressure and heart rate in healthy human (135) and prevents the arrhythmia in the mice model of myocardial infarction (136).

Ghrelin significantly improves LV functions and attenuates fibrosis (137) and development of cachexia (138) in rat HF model. Ghrelin inhibits the DOX -induced cardiotoxicity in mice hearts and cardiomyocytes by blocking AMPK activity and activating the p38-MAPK pathway, which suppresses excessive autophagy (94). A ghrelin-containing salmon extract given per os was found to alleviate the cardiotoxicity of DOX in mice, mimicking cardioprotective effect of synthetic ghrelin (95). Cardioprotective effect of ghrelin can also be due to its angiogenic properties in ischemic tissue (139–141). Ghrelin via GHS-R ameliorates impaired angiogenesis by increasing VEGF levels in the ischemic hearts of diabetic rats (140) and in a rat myocardial infarction model (142). Despite the potent synthetic agonist of GHS-R, RM-131 plays an anticatabolic effect in chronic HF models of rat (143), its role in anti-cancer drug mediated cardiotoxicity has not been studied yet.

#### Galanin Receptor Agonists

Galanin is a neuropeptide present in the nervous system and some organs (144) that uses 3 kinds of GPCRs called GalR1, GalR2 and GalR3 that are all expressed in the cardiovascular system (145). The elevated sympathetic activity during cardiac failure stimulates the release of galanin. This neuropeptide is a one of the sympathetic co-transmitters together with ATP and neuropeptide Y (NPY), in addition to norepinephrine. Galanin released by sympathetic nerves may diminish vagal neurotransmission (146). Indeed, galanin via GalR1 inhibits vagal bradycardia (147). In accord with this study, GalR1 inhibitor, M40 improves cardiac function and attenuate remodeling after myocardial infarction in rats (148). In contrast, an peptide agonist of galanin receptors and the full-length galanin reduce infarct size and the cardiac damage markers in ischemia and reperfusion rat model (96). Indeed, the natural N fragments of Galanin that have more affinity to GalR2 than GalR1 and GalR3 (145) limit acute myocardial infarction in rats in vivo (149). Moreover, natural galanin and GalR2 agonist have shown to increase cell viability by suppressing caspase-3 and 9 activity against hypoxic insults in other cells (97).

The GalR1-3 agonist [RAla14, His15]-galanin (2-15) exhibits cardioprotective properties against DOX-mediated cardiac injury in rats. Coadministration of this agonist with DOX has prevented the increase in plasma CK-MB activity and improved the parameters of cardiac function and caused weight gain. The obtained results demonstrate the ability of a novel agonist of galanin receptors GalR1-3 to attenuate DOX-induced cardiotoxicity (150). To conclude, galanin peptides via GalR1- 3 alleviate the cardiac dysfunctions induced by DOX. The role of GalR1-3 agonist on anti-tumor effect of DOX in cancer mice model needs to be studied.

#### Apelin Receptor Agonists

Apelin is an endogenous peptide that acts trough the APJ receptor that is 54% identical with AngII receptor. However, angiotensin II does not bind to APJ (151). Mature apelin, apelin-36, and its shorter forms (apelin-17, -12, and -13) result from the cleavage of pre-pro-apelin. Apelin itself can also be cleaved in vitro by the angiotensin-converting enzyme 2 (ACE2) (152). Apelin has a positive inotropic effect in vitro (153) and is involved in lowering arterial blood pressure (154), inducing arterial vasodilation (155), and improving cardiac output (156). It protects the heart against ischemia/reperfusion-mediated injury and promotes angiogenesis (157).

Moreover, in APJ knockout mice exhibited more severe heart injury, including impaired contractility functions and survival rate after DOX treatments as compare to wild type mice receiving DOX (98). On the other hand, apelin protects H9c2 cardiomyocytes overexpressing APJ against DOX-mediated cell death. These findings all together have suggested that the suppression of APJ expression can worsen DOX-induced cardiotoxicity. Impairment of the endogenous apelin-APJ system may partially depress the protective signaling in DOX-treated hearts (98). Apelin-13 pretreatment attenuates cisplatin-induced cardiotoxicity by inhibiting apoptosis in cardiomyocytes via activation of MAPKs and PI3K/Akt signaling in vitro and in vivo in mice heart (99). The mechanism of cardioprotection in vivo involves an attenuation of the ROS and superoxide anion accumulation, inhibition of DNA damage, and suppression of PARP and caspases as well as an improvement in angiogenesis.

Importantly, high levels of apelin and APJ have been found in several cancer types that may be connected with obesity. For example, increase levels of Apelin-12 in colon cancer patients with obesity (158), or elevated levels of apelin-36 in endometrial and breast cancer patients with obesity (159–161) have been found. The role of AJP agonist on anti-tumor effect of anticancer agents in cancer mice model needs to be studied. Thus, promoting APJ signaling in heart may represent an interesting strategy to alleviate the cardiotoxicity of anticancer treatments.

#### Prokineticin Receptor Agonists

Prokineticins are peptides found in milk and macrophages (162). These peptides are called prokineticin because of their first identified biological activity was a prokinetic effect on smooth muscle cells of the gastrointestinal tract (163). Prokineticins exist as two isoforms, PROK1 and PROK2 that are expressed in all mammalian tissues (164). They are angiogenic factors (165) and induce mitogenic and survival pathway in lymphocytes and hematopoietic stem cells (166), neuronal cells (167, 168), cardiomyocytes (169), and endothelial cells (170). PROK1 and PROK2 exert their biological activity on prokineticin receptors 1 and 2 (PKR1 and PKR2) (171).

We have showed that PROK2/PKR1 can induce angiogenesis, while PROK2/PKR2 signaling promotes endothelial cell fenestration and disorganization (170). In cardiomyocytes PKR1 signaling activates Gα11/Akt pathway to reduce cardiomyocyte death (169), while PKR2 signaling induces hypertrophic cardiomyopathy (172). Indeed, PKR1 gene therapy promotes resistance to ischemia, protects heart against myocardial infarction, and ameliorates heart structure and function (169). Overexpression of PKR1 in transgenic mice hearts promotes neovascularization, suggesting a novel myocardial-epicardial interaction that is involved in differentiation of epicardial progenitor cells (EPDCs) in to vasculogenic cells type by a paracrine PROK2/PKR1 signaling (173).

PKR1 signaling controls epithelial mesenchymal transformation (EMT) during heart (174) and kidney development (175). PKR1 controls fate of tcf21<sup>+</sup> fibroblast (176) and Wt1<sup>+</sup> epicardial cells (174). PKR1 epigenetically controls stemness and differentiation of these cells, unraveling a new neovasculogenic pathway vs. adipogenesis (177). PKR1 inhibits adipogenesis and reduce adipocyte accumulation under high fat diet regime of mice (178, 179). PKR1 controls

**14**

trans-endothelial insulin uptake, preadipocyte proliferation and adipogenesis (180). Lack of PKR1 in mice induces developmental defect in heart and kidney and in adult stage insulin resistance and obesity (181, 182).

In 2015, Gasser et al. discovered the first PKR1 agonists called IS20 (183). This agonist prevents the formation of cardiac lesions and ameliorates the cardiac function and survival after myocardial infarction in mice. IS20 inhibits DOX-mediated cardiotoxicity in cultured cardiac cells including cardiomyocytes, endothelial and progenitor cell as well as in mice models of acute and chronic cardiotoxicity. Importantly, these small molecules did not alter cytotoxic effect of DOX in cancer cells and in vivo cancer cell line- derived xenograft mice model (100). This study also described how classic chemotherapeutics, anthracyclines affect cardiac cells in dose-and time-dependent manner and how they impair NFR2 defense mechanism. These results indicate that PKR1 is a target for development of cardioprotective drugs.

#### Cannabidiol

Cannabidiol is the most abundant non-psychoactive, derived cannabinoid (184). In the low nanomolar range, cannabidiol act as an antagonist of cannabinoid 1 receptor (CB1R) and cannabinoid 2 receptor (CB2R), while it has agonist/inverse agonist actions at micromolar concentrations (185, 186). Cannabidiol activate TRPV1 channel and several GPCRs, including the orphan receptor GPR55, the putative Abn-CBD receptor, α1-adrenoreceptors, 5HT1A receptors and µ opioid receptors (187). Several studies showed cardioprotective effects of cannabidiol in animal models of myocardial ischemic reperfusion injury (188), and myocardial infarction (189). It also ameliorates cardiac functions in diabetic cardiomyopathy (186).

Cannabidiol protects hearts against DOX-induced cardiac injury, in rats (101) and in mice (102). It improves cardiac dysfunction by (i) attenuating ROS /RNS accumulation, (ii) preserving mitochondrial function and biogenesis, (iii) promoting cell survival, and (v) decreasing myocardial inflammation. The involvement of CB<sup>1</sup> and CB<sup>2</sup> signaling were not clarified in these studies. Recent data has shown that CB1R and CB2R receptors have opposite effects. Indeed CB1R antagonists and CB2R agonists both protect the heart against clozapine-toxicity (190). Thus, CB1R antagonist reduces DOX-induced cardiotoxicity and decreased cortical cerebral infarction (191). By contrast, two CB2R agonists JWH-133, AM 1241 alleviate quetiapine cardiotoxicity (192). Moreover, cannabidiol by itself display cytotoxicity in many cancer cell lines, and anti-tumor effects in cancer mice models (103), suggesting that cannabidiol may have a synergistic effect with antineoplastic drugs in the use of cardioprotective agents. In fact, the cannabinoid HU-331 has been shown to be more potent and less cardiotoxic than DOX (193). Indeed, Insys Therapeutics has obtained FDA orphan drug designation for Cannabidiol for the treatment of multiform glioblastoma and childhood epilepsy.

# CONCLUSION

Cardiotoxicity induced by anti-cancer therapy may occur when the anticancer agent targets a common signaling pathway that are essential to maintain the functions of both cardiac and cancer cells. It can also involve off-target effects due to non-selective actions of anti-cancer agents. The choice of the cardioprotective therapeutic approach relies on the delicate balance between the efficiency of anti-neoplastic drugs and the management of cardiovascular complication.

Cardioprotective utility of GPCR ligands will require validation of preferentially expression of these GPCRs in both cancer and cardiac cells, and identification of their signaling (e.g., G-protein- or arrestin-mediated pathways) and functional roles (**Figure 2A**). Whether these cardioprotective ligands interfere with the anti-tumor effect of the chemotherapeutics should be studied as well. The human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), iPSC-CM-derived 3D cultures and organoids provide human-based model systems to explore the molecular mechanisms of cardiotoxicity and cardioprotection (194). They may also serve as a platform for personalized medicine. Thus, GPCR ligand efficacy can be optimized and their side-effects can be examined in hiPSC-CMs and organoids.

In addition, most of the data regarding the efficacy of cardioprotective GPCR-ligands against cancer therapy mediatedcardiotoxicity have been obtained from small animal models of cardiotoxicity and cancer cell-derived xenograft mice models. Therefore, further studies in bigger animals are necessary to examine their efficacy and adverse effects before these findings can be translated to a human study.

Interestingly, certain cancer cell types may retain a GPCR expression pattern via serving novel biomarkers and/or as valuable therapeutic targets. For example, GPR161 is functionally expressed in breast cancer (195) and GPRC5A in pancreatic cancer (196) and GPR68 in the tumor microenvironment (197). However, both CD97 and GPR56 are highly express in multiple cancer types and in normal tissues (198). Moreover, many mutated GPCRs such as GPR110, GPR112, GPR125, GPR126, GPR98, and GPR110 have been found in certain cancers (199). These findings suggest that different types of cancers may be characterized by a specific onco-GPCR-ome (67). It could be interesting to examine if there is a "GPCR signature" in heart as well. In precision medicine, selectively targeting GPCRs in specific cancers can lead to a novel class of anti-cancer drugs with less adverse cardiac effects, after defining their expression and their role in heart (**Figure 2B**).

# AUTHOR CONTRIBUTIONS

AA, LD, and CN participated in writing and drawings of the manuscript.

### FUNDING

The publication was made possible by grants from European ERA-NET, ERA-CVD-JC2016, French government managed by Agence Nationale de la Recherche (ANR-16-ECVD-0005-01) Centre National de la Recherche Scientifique (CNRS).

# REFERENCES


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Audebrand, Désaubry and Nebigil. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Role of Mitochondrial Dysfunction in Radiation-Induced Heart Disease: From Bench to Bedside

#### Katie Livingston1,2, Rachel A. Schlaak 2,3, Lindsay L. Puckett 1,2 and Carmen Bergom1,2,4 \*

<sup>1</sup> Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>2</sup> Cancer Center, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>3</sup> Department of Pharmacology & Toxicology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>4</sup> Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI, United States

Radiation is a key modality in the treatment of many cancers; however, it can also affect normal tissues adjacent to the tumor, leading to toxic effects. Radiation to the thoracic region, such as that received as part of treatment for breast and lung cancer, can result in incidental dose to the heart, leading to cardiac dysfunction, such as pericarditis, coronary artery disease, ischemic heart disease, conduction defects, and valvular dysfunction. The underlying mechanisms for these morbidities are currently being studied but are not entirely understood. There has been increasing focus on the role of radiation-induced mitochondrial dysfunction and the ensuing impact on various cardiac functions in both preclinical models and in humans. Cardiomyocyte mitochondria are critical to cardiac function, and mitochondria make up a substantial part of a cardiomyocyte's volume. Mitochondrial dysfunction can also alter other cell types in the heart. This review summarizes several factors related to radiation-induced mitochondrial dysfunction in cardiomyocytes and endothelial cells. These factors include mitochondrial DNA mutations, oxidative stress, alterations in various mitochondrial function-related transcription factors, and apoptosis. Through improved understanding of mitochondria-dependent mechanisms of radiation-induced heart dysfunction, potential therapeutic targets can be developed to assist in prevention and treatment of radiation-induced heart damage.

Keywords: mitochondria, radiation-adverse effects, radiation-induced cardiovascular toxicity, oxidative stress, radiation, cardiomyocyte, endothelial cell, apoptosis

# RADIATION-INDUCED CARDIAC DISEASE

It has long been recognized that high-dose radiation exposure to the heart can cause cardiac dysfunction, manifesting months to decades following treatment. In 1924, radiation-induced histologic changes to the heart were first reported following radiation treatment of a patient for Hodgkin's lymphoma (1). Since that time, it has been established that therapeutic radiation to the thoracic region, for treatment of lymphomas, breast and lung cancers and pediatric malignancies can cause cardiac injuries (2). Even low doses of radiation can lead to radiation-induced heart dysfunction (RIHD), as demonstrated in epidemiologic cohorts of atomic bombing survivors and occupational exposures (1, 3, 4). Radiation can cause various structural changes to cardiac

#### Edited by:

Canan G. Nebigil, École supérieure de biotechnologie Strasbourg (ESBS), France

#### Reviewed by:

Guido Iaccarino, University of Naples Federico II, Italy Ariane Vieira Scarlatelli Macedo, Santa Casa of Sâo Paulo, Brazil

> \*Correspondence: Carmen Bergom cbergom@mcw.edu

#### Specialty section:

This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine

> Received: 15 December 2019 Accepted: 05 February 2020 Published: 21 February 2020

#### Citation:

Livingston K, Schlaak RA, Puckett LL and Bergom C (2020) The Role of Mitochondrial Dysfunction in Radiation-Induced Heart Disease: From Bench to Bedside. Front. Cardiovasc. Med. 7:20. doi: 10.3389/fcvm.2020.00020 tissue, including the cardiac vasculature, leading to complications, such as pericarditis, coronary artery disease, ischemic heart disease, congestive heart failure, conduction defects and valvular dysfunction (5, 6).

Darby et al. completed a population-based case-control study of women who underwent radiotherapy for breast cancer. In this study, for every gray (Gy) of mean dose to the heart (the average mean dose was 4.9 Gy), the rate of major coronary events (myocardial infarction, coronary revascularization, or death from ischemic cardiac disease) increased by 7.4% with no upper limit. Cardiac events occurred within the first 5 years and continued several decades post-radiotherapy (7). Other studies have also examined the association between mean heart dose and cardiac events/disease, finding an ∼4–16% increased risk per Gy of mean heart dose (7–9). Studies have also suggested RIHD can occur in non-small cell lung cancer patients within 2 years post-radiation exposure (8–11). In a number of lung cancer studies, mortality rates were cardiac dose-dependent, either based on mean heart dose (12) or with the percent of heart receiving 5 Gy (13), 30 Gy (13), or 50 Gy (10). In pediatric and young adult cancer patients who received cardiac radiation, there is over a 6-fold relative risk of RIHD, defined as congestive heart failure, myocardial infarction, pericardial disease, and/or valvular abnormalities (14). Cardiac events were also found to be dose-dependent, with the highest risk of events found when the mean heart dose was >30 Gy (15).

As demonstrated from these studies, cardiac exposure should be minimized when possible for radiation therapy to the thoracic region. There have been many advances in reducing cardiac exposure by improving both imaging and radiotherapy techniques (16–21). However, heart radiation exposure often remains unavoidable. There are currently no widely used methods to reverse RIHD, thus the primary way to reduce cardiotoxicity is through improved treatment planning. There is a need for preclinical studies to understand radiationinduced changes on a cellular and molecular level, with the hope of discovering new targetable pathways. Currently, there are several hypotheses on the predominant causes of RIHD, with most identified using animal models. One cause is the formation of fibrosis, distinguished by collagen deposition in and surrounding cardiomyocytes (1, 6, 22). An additional cause is macrovascular and microvascular injury, developed in a multifactorial manner by endothelial cell damage and adhesion, and activation of inflammatory and atherosclerotic responses (1, 6, 23–28). Signaling pathways, including apoptosis and mitochondrial dysfunction, have also been linked to RIHD (29, 30). This review will focus on the role of radiationinduced mitochondrial dysfunction in RIHD. The biologic pathways described in this review are illustrated in **Figure 1**, and the discussed clinical and preclinical studies are summarized in **Table 1**.

# MITOCHONDRIA AND OXIDATIVE STRESS

The role of cardiomyocyte mitochondria is critical to cardiac function, with each cardiomyocyte having abundant mitochondria that make up ∼30% of cell volume (43, 44). Mitochondria have a role in stress responses, cell death, and metabolic processes. They are essential for energy production, which is created by products of glycolysis and fatty acid metabolism via oxidative phosphorylation in the respiratory chain, yielding reactive oxygen species (ROS) biproducts, such as superoxide, peroxide and hydroxyl radicals. Wang et al. reviewed the normal mitochondrial mechanisms, as well as manners in which equilibrium can be interrupted in the heart after radiation. In homeostasis, ROS facilitate cellular functions, including immune responses, signal transduction and apoptosis. ROS can be neutralized by antioxidants when their concentrations are in excess (29). If this highly regulated process is disrupted, increased production or decreased removal of ROS can lead to cellular and DNA damage (29).

Stress-induced mitochondrial damage can cause a loss of mitochondrial membrane potential, leading to the mitochondria undergoing either fission or fusion. Fission helps mitigate stress by fusing parts of damaged mitochondria with normal mitochondria and is regulated by proteins including Opa1 and Mitofusin-1 and−2 (Mfn-1 and Mfn-2). Fission is needed to help create new mitochondria, but also serves as quality control through facilitating apoptosis during high levels of cellular stress. Fission is mediated by proteins including Drp1 and Mft (45, 46). If damaged mitochondria need to be eliminated, kinase PINK1 yields as a sensor of mitochondrial damage and signals to induce mitophagy (46). These complex dynamic mitochondrial processes are imperative in cardiomyocytes, as these cells obtain more than 90% of their energy from mitochondrial respiration (35, 40). This makes mitochondria within these energy-demanding cells an ideal study model to characterize mitochondrial changes that occur after radiation exposure.

It was discovered in the late 1960s that radiation can drastically alter the structural appearance of mitochondria, both short- and long-term. Seven years after high dose radiation (52 Gy) to the mediastinal region in a human patient, electron microscopy revealed cardiomyocyte mitochondria that were variably swollen with decreased number and disorganization of cristae, often with fused outer double membranes (31). Changes in mitochondrial structural integrity occurred as early as 48 h following exposure in rabbit myocardial cells which had received a single dose of either 10 or 13 Gy (32). These findings have led to additional studies on the role of mitochondrial function in RIHD.

On a molecular level, ionizing radiation directly modifies DNA, including single- and double-stranded breaks, base damage, and cross-links, all of which can lead to cell death if not repaired properly. Indirectly, radiation can lead to ROS formation, which can cause cellular stress and death (1). Mitochondrial DNA (mtDNA) is a major radiation target because it lacks the protective effects of histones (47). In addition, it is generally repaired less efficiently than nuclear DNA (48) and has a mutation rate 10–1,000 times higher than nuclear DNA, making it an ideal model to study the mutational effects of radiation (33, 48, 49). This has been most notably reflected with the mutation called common deletion a 4,977 base pair deletion within mtDNA that has become a marker for oxidative damage. Increased levels of the common

deletion have been noted in human cardiac cells undergoing oxidative stress secondary to atrial fibrillation (50). Other studies have noted radiation-induced development of common deletion within human cell lines, both in low (0.1 Gy) and therapeutic doses (>1 Gy) (33, 48, 51), although no studies to date have specifically analyzed radiation-induced common deletion in cardiomyocytes. All mtDNA genes are essential for the biogenesis and function of mitochondria, so mutations leading to altered overall gene expression would be expected to cause a deficiency in energy metabolism and enhanced production of ROS, leading to oxidative stress (40).

Alterations in proteins that affect ROS generation and oxidative stress may also enhance RIHD. Manganese superoxide dismutase (MnSOD), a mitochondrial matrix enzyme that protects against oxidative stress by converting superoxide to H2O2, can decrease cardiac injury severity. Mice deficient in MnSOD died within the first 10 days of life and exhibited dilated cardiomyopathy, among other abnormalities. A study by Nojiri et al. generated cardiac-specific MnSOD-deficient mice, and these mice developed congestive heart failure with severe cardiac muscle degeneration and significantly reduced ATP production, demonstrating that alterations in enzymes important for maintenance of ROS levels can lead to oxidative stressdependent heart disease (52). Other studies have shown that MnSOD can play an important role in ischemia-reperfusion cardiac injury as well (52).

G protein-coupled receptor kinase (GRK) is another protein reported to regulate ROS in response to stress. Studies have shown data that GRK may act both a protector against and a promotor for death following ischemic injury (45, 53). Removal of cardiac-specific GRK2 has been linked to embryonic cardiovascular development, adult cardiac dilatation, early atherosclerosis and inhibited angiogenesis in mice (42, 54–57). Franco et al. evaluated cell cultures with knockdown or overexpressed GRK2 3–8 h after exposure of a single dose of 4 Gy. Knockdown of GRK caused morphologic mitochondrial changes, reduced membrane potential and reduced mitochondrial function (42). The overexpression of GRK2 protected mitochondria from radiation damage (42). Furthermore, it was demonstrated that in the presence of heat shock proteins, GRK2 interacts with mitofusins (MFN-1 and MFN-2), key regulators of mitochondrial fission and fusion (42, 58). Additional studies are required to understand the chronic response of GRK to radiation, but GRK-related pathways may be important targets to drive mitochondrial protection from radiation-induced damage.

Several other studies have noted radiation-induced mitochondrial changes in terms of oxidative stress and respiratory capacity in mice (34). These changes have been noted h to months following radiation exposure. Five and 24 h after 3 Gy total body irradiation, C57BL/6 mice had immediate changes in cardiac structure and function. On murine cardiac tissue proteomic analysis, mitochondrial proteins represented the protein class most sensitive to radiation, with increased levels of proteins involved in oxidative phosphorylation, including ATP synthase, NADH dehydrogenase and cytochrome c oxidase (34).

Chronic low dose exposure of ionizing radiation can cause heart disease, as noted in atomic bomb survivors and nuclear power industry workers (3, 4, 59). As previously mentioned,

#### TABLE 1 | Summary of studies investigating the role of mitochondria in RIHD.


RT, Radiation therapy; mito, mitochondria; AT, ataxia telangiectasia; KSS, Kearns Sayre Syndrome; MnSOD, manganese superoxide dismutase; TBI, total body irradiation; WT, wildtype; CO, cardiac output; SV, stroke volume; EF, ejection fraction; FFA, free fatty acids, MPT, membrane permeability transition; RT, radiation therapy.

doses as low as 0.1 Gy caused accumulation of the common deletion in human cell lines (51). However, other studies have shown minimal effect with these low doses. Barjaktarovic et al. studied C57BL/6N mice that received either 0.2 Gy, 2 Gy to the heart or sham radiation. Four weeks postexposure, cardiac mitochondria were examined for proteomic and functional alterations. After 2 Gy, both functional and proteomic alterations were observed. Proteomic analysis revealed a total of 25 downregulated proteins, in three biological areas: oxidative phosphorylation, pyruvate metabolism and cytoskeletal structures. Functional impairment was reflected as partial deactivation of mitochondrial Complex I and III, decreased succinate-driven respiratory capacity, increased ROS levels and enhanced oxidation of mitochondrial proteins. At the lower dose (0.2 Gy), only proteomic changes were identified, suggesting a dose-dependence of mitochondrial dysfunction after cardiac radiation (35). This group then investigated the late cardiac effects at 40 weeks post-exposure, at which time respiratory capacity of the mitochondria was still reduced after 2 Gy. This suggests that radiation can cause non-transient alterations of oxidative stress in mitochondria (36).

# GLUTATHIONE S-TRANSFERASE ALPHA 4 (GSTA4-4)/Nrf2 PATHWAY

Another method of measuring oxidative stress is by quantifying downstream transcription factors. During the process of lipid peroxidation, 4-hydroxynonenal (4-HNE) concentrations increase, which directly activates nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2, gene name NFE2L2), a transcription factor that targets a number of antioxidant proteins. Nrf2 is a redox-sensitive factor that controls oxidative responses within cells and has a role in endothelial function and cardiac protection (22, 60). The repressor protein Keap1 binds and sequesters Nrf2, promoting Nrf2 ubiquitin-mediated degradation. The protein phosphoglycerate mutase family member-5 (PGAM5) is attached to the mitochondrial membrane and can form a complex with Keap1 and Nrf2 (37, 61, 62). Under oxidative conditions, Nrf2 is released from the complex, allowing nuclear accumulation of Nrf2 (60, 62). The role of Nrf2 was indirectly verified by the impact of glutathione S-transferase alpha 4 (GSTA4-4), which is an enzyme that removes 4-HNE. A study with Gsta4-null mice noted enhanced resistance to the cardiotoxic effects from doxorubicin, suggesting a compensatory mechanism may have caused cardiac protection (37). Boerma et al. conducted a similar study with the Gsta4-null mice with local heart irradiation to a total dose of 18 Gy. Six months post-radiation exposure, the wildtype mice had reduced cardiac output, stroke volume and ejection fraction, with associated increased levels of cardiac troponin-I levels when compared to the Gsta4-null mice. Additionally, the Gsta4-null mice had increased mRNA levels of PGAM5 and Nrf2. Nrf2 was also significantly elevated in the shamirradiated Gsta4-null mice when compared to wild-type mice. When comparing the levels of 14 different Nrf2 target genes, none were significantly elevated in wild-type irradiated mice; seven genes were significantly elevated in irradiated Gsta4-null mice compared to non-irradiated Gsta4-null mice, suggesting a stronger activation of the Nrf2 pathway in the irradiated Gsta4 null mice (37).

Anotherstudy found that genes on rat chromosome 3 can alter RIHD by using consomic rat strains to identify genetic variants that cause differences in cardiac radiosensitivity. One week after 24 Gy of localized cardiac radiation, changes in expression of numerous gene pathways, including mitochondrial function, were seen between the sensitive and resistant rat hearts. Nrf2 was found to be an upstream regulator of many of the enriched pathways (61). Other preclinical investigations have studied the protective role of the Nrf2 pathway on radiation injury to cardiomyocytes and other cell lines, including embryonic fibroblasts and breast and lung epithelial cells (37). These results taken together suggest Nrf2 and GSTA-4 pathways may be promising targets for reducing mitochondrial dysfunction from cardiac radiation exposure.

# PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-α (PPAR-α)

Cardiac muscle preferentially relies on fatty acid energy production via oxidative phosphorylation over glucose metabolism. Peroxisome proliferator-activated receptor-α (PPAR-α) is a highly expressed transcription factor in tissues with elevated lipid metabolic turnover, including cardiac tissue. When PPAR-α is downregulated, lipid metabolism is impaired, as noted in PPAR-α-null mice (38), suggesting PPAR-α regulates energy equilibrium. When C57BL/6 mice were exposed to 8 or 16 Gy of cardiac radiation, PPAR-α was phosphorylated by ERK-MAPK causing decreased transcriptional activity, leading to increased free fatty acid levels and reduced levels of mitochondrial complexes I, III and V (38). The finding that radiation-induced PPAR-α alterations cause decreased expression of energy metabolism and mitochondrial respirationrelated genes has been corroborated in human subjects with chronic radiation exposure. In the 1940s, the Mayak Production Association built a nuclear facility in Russia, where workers were chronically exposed to incidental radiation during their occupational duties. Epidemiologic cohorts were analyzed to identify workers who died from heart disease and individual dosimetric monitors were used to determine radiation exposure. Studies in this cohort of individuals found significant increases in heart disease associated with total external gamma-ray doses, even after adjusting for confounding factors, such as smoking exposure (63–65). In a separate study, the protein expression from post-mortem heart samples were examined from a subset of workers exposed only to external gamma rays who had a diagnosis of ischemic heart disease and a primary cause of death of ischemic heart disease. Total doses of external exposure in this cohort ranged from 100 mcGy to more than 5 Gy. Proteomic analysis from 29 individuals identified a dose-dependent increase in phosphorylation of PPAR-α with a corresponding dose-dependent decrease in mitochondrial proteins, such as complexes I, III, and Nrf2 (4). PPAR-α has already shown an effect in other cardiovascular risk factors in preclinical and clinical studies (for dyslipidemia and diabetes mellitus) and is now an encouraging targetable agent for potential mitigation of RIHD (6, 66).

# MITOCHONDRIA AND APOPTOSIS

Mitochondria are critical for some methods of programmed cell death, or apoptosis. For the intrinsic pathway, the inner and outer mitochondrial membranes must be permeabilized to release apoptotic factors, such as cytochrome c. The Bcl-2/Bax family of proteins help regulate and stabilize the membranes and govern the predilection for mitochondrial membrane permeabilization (29, 67). Once Bax is activated, it translocates from the cytoplasm to the mitochondrial membrane, where it can induce membrane permeability transition (MPT). MPT is characterized by mitochondrial swelling, depolarization of the membrane and uncoupled oxidative phosphorylation. It can also be induced by calcium influx and ROS. Radiation may also cause mitochondrial-mediated apoptosis due to the close association between the endoplasmic reticulum and mitochondria. When cardiomyocytes are irradiated, the endoplasmic reticulum releases a flux of excess calcium ions (Ca2+) that facilitate permeabilization of mitochondria (29). Animal studies have shown increased levels of the Bax/Bcl2 ratio expression following irradiation. In one analysis, increased expression levels of Bax/Bcl2 and increased apoptotic nuclei were seen in Wistar rats 5 months after cardiac radiation (20 Gy), with an associated increase of fibrotic tissue and cardiomyocyte hypertrophy (39). Sridharan et al. investigated the time course of radiation-induced changes to mitochondria in rats sacrificed 2 h to 9 months following a single dose of radiation, ranging from 3 to 21 Gy. Levels of Bax and Bcl2 were significantly increased by 6 h post-exposure. The Bax/Bcl2 ratio was elevated from 6 h to 6 months after irradiation, but not significantly elevated at 9 months. These findings were associated with apoptotic nuclei at 6 and 24 h and 2 weeks following radiation (40). One study identified cleaved caspase 3, an apoptosis activator, as late as 13 months post local heart radiation (15 Gy) to Wistar rats (40, 41). Additionally, the study completed by Sridharan et al. noted increased radiation-induced susceptibility to MPT, measured by increased calcium-stimulated mitochondrial swelling. At time points ranging from 6 h to 9 months post-radiation, in a dosedependent manner, irradiated cardiac mitochondria were more susceptible to calcium-induced swelling. Previously, studies have noted only a transient depolarization of the mitochondrial membrane potential and MPT after radiation. This suggests that radiation may cause an enhancement in the susceptibility of MPT and pore opening in mitochondria to subsequent stressors (40).

# ENDOTHELIAL CELL MITOCHONDRIA

Radiation exposure can induce endothelial cell activation, shifting endothelial cells into a pro-inflammatory state. When exposure is repeated or prolonged, the endothelium can alter its protective physiology, which can lead to exhaustion and a decrease in vascular function. This endothelial dysfunction leads to decreased vascular tone, inflammation and atherosclerosis, all of which may contribute to cardiovascular disease (28). Concentrations of mitochondria are relatively low in endothelial cells compared to cardiomyocytes and mitochondria produce a lower portion of total endothelial cell energy. However, endothelial cell mitochondria have been found to play important roles in cellular signaling (28). Radiation-induced endothelial cell mitochondrial dysfunction may contribute to RIHD, though data on this topic is currently limited. Endothelial cell functions that can be altered by radiation include Ca2<sup>+</sup> regulation, apoptosis and oxidative stress signaling. Radiationinduced release of Ca2<sup>+</sup> from the endoplasmic reticulum leads to increased mitochondrial Ca2<sup>+</sup> uptake, yielding membrane swelling and release of apoptotic factors (29). However, Ca2<sup>+</sup> plays numerous roles in signaling pathways and intracellular functions that theoretically may be affected by radiation (e.g., inner membrane calcium uniporter, mitochondrial Ca2<sup>+</sup> activation of dehydrogenase enzymes and ATP synthase and TNF-α-induced inflammation). These concepts have just begun to be addressed preclinically in the setting of radiation exposure (28).

Baselet et al. illustrated that dysregulation of the Bcl2 pathway (intrinsic apoptosis pathway) yields endothelial inflammation, apoptosis and senescence, all of which are coupled with atherosclerotic development (28). Along similar lines, cells can undergo senescence, the irreversible arrest of endothelial cell renewal, after extensive cell division or exposure to stressors, including radiation. Previous in vitro and in vivo studies have noted evidence of endothelial cell senescence following local radiation exposure. In human umbilical vein endothelial cells (HUVECs), mitochondrial membrane potential was altered 2 days after irradiation with 1.5, 4, and 10 Gy. The membrane potential returned to baseline levels at days 5 and 6 with 1.5 and 4 Gy, respectively; however, mitochondrial activity remained reduced in cells irradiated with 10 Gy (28). The underlying mechanisms of radiation-induced senescence are not fully established, though mechanisms may involving the p53-p21 and the IGF1-PI3K-Akt/mTOR pathways that may be attributable to the downregulation of Silent Information Regulator-1 (SIRT1) (68). SIRT1 is a NAD-dependent deacetylase that regulates many proteins involved in mitigating oxidative stress, and although its relationship to RIHD has not been explored, SIRT1-deficiency increased the sensitivity of thymocytes to apoptosis (69). Similar to cardiomyocytes, studies of in vitro endothelial cells noted increased production of ROS 24– 72 h post-radiation exposure (5–20 Gy) (28). In addition, Nrf2 upregulation has also been implicated in oxidative stress-induced endothelial dysfunction (1, 28). Furthermore, proteomic data on C57BL/6 mice receiving 8 or 16 Gy of local heart irradiation revealed expression of proteins associated with mitochondrial dysfunction within endothelial cells (70).

# CONCLUSION

Radiation exposure to the thoracic region can cause a variety of cardiac injuries. Numerous preclinical animal and cell models have studied the mechanisms behind RIHD, though these are not yet fully elucidated. Here we have reviewed several factors related to radiation-induced cardiomyocyte and endothelial cell mitochondrial dysfunction, including mtDNA mutations, oxidative stress, alterations in various transcription factors and apoptosis. These factors ultimately play a role in the complex mitochondrial dynamics that can change the fate of cardiac cells. Through further understanding of mitochondriadependent mechanisms of RIHD, potential therapeutic targets can be developed to prevent and/or treat radiation-induced heart damage.

# AUTHOR CONTRIBUTIONS

All authors contributed to conception and design of the review, wrote sections of the manuscript, contributed to manuscript revision, read, and approved the submitted version.

# FUNDING

This work was supported by the Medical College of Wisconsin (MCW) Cancer Center and the Dr. Nancy Sobczak Fund for Breast Cancer (CB). Support was also received from NIH/NHLBI 1R01HL147884 (CB), the Mary Kay Foundation Grant No. 017- 29 (CB), a Susan G. Komen Grant #CCR17483233 (CB), and the Michael H. Keelan, Jr., MD, Research Foundation Grant and the Cardiovascular Center at the Medical College of Wisconsin (CB).

# ACKNOWLEDGMENTS

We apologize to the authors whose work was not cited due to space limitations.

# REFERENCES


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Livingston, Schlaak, Puckett and Bergom. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Emerging Challenges of Radiation-Associated Cardiovascular Dysfunction (RACVD) in Modern Radiation Oncology: Clinical Practice, Bench Investigation, and Multidisciplinary Care

Moon-Sing Lee1,2†, Dai-Wei Liu2,3†, Shih-Kai Hung1,2,4, Chih-Chia Yu1,5, Chen-Lin Chi 2,6 , Wen-Yen Chiou1,2,4, Liang-Cheng Chen1,2,4, Ru-Inn Lin1,5, Li-Wen Huang1,2,4 , Chia-Hui Chew1,2,4, Feng-Chun Hsu<sup>1</sup> , Michael W. Y. Chan<sup>5</sup> \* and Hon-Yi Lin1,2,4,5 \*

#### Edited by:

*Carlo Gabriele Tocchetti, University of Naples Federico II, Italy*

#### Reviewed by:

*Alessandra Cuomo, Federico II University Hospital, Italy Sherry-Ann Brown, Mayo Clinic, United States*

\*Correspondence:

*Michael W. Y. Chan biowyc@ccu.edu.tw Hon-Yi Lin doc16021@gmail.com*

*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine*

> Received: *15 December 2019* Accepted: *31 January 2020* Published: *21 February 2020*

#### Citation:

*Lee M-S, Liu D-W, Hung S-K, Yu C-C, Chi C-L, Chiou W-Y, Chen L-C, Lin R-I, Huang L-W, Chew C-H, Hsu F-C, Chan MWY and Lin H-Y (2020) Emerging Challenges of Radiation-Associated Cardiovascular Dysfunction (RACVD) in Modern Radiation Oncology: Clinical Practice, Bench Investigation, and Multidisciplinary Care. Front. Cardiovasc. Med. 7:16. doi: 10.3389/fcvm.2020.00016* *<sup>1</sup> Department of Radiation Oncology, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Dalin, Taiwan, <sup>2</sup> School of Medicine, Tzu Chi University, Hualien, Taiwan, <sup>3</sup> Department of Radiation Oncology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, <sup>4</sup> Cancer Centre, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Dalin, Taiwan, <sup>5</sup> Department of Biomedical Sciences, National Chung Cheng University, Chia-Yi, Taiwan, <sup>6</sup> Department of Anatomic Pathology, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Dalin, Taiwan*

Radiotherapy (RT) is a crucial treatment modality in managing cancer patients. However, irradiation dose sprinkling to tumor-adjacent normal tissues is unavoidable, generating treatment toxicities, such as radiation-associated cardiovascular dysfunction (RACVD), particularly for those patients with combined therapies or pre-existing adverse features/comorbidities. Radiation oncologists implement several efforts to decrease heart dose for reducing the risk of RACVD. Even applying the deep-inspiration breath-hold (DIBH) technique, the risk of RACVD is though reduced but still substantial. Besides, available clinical methods are limited for early detecting and managing RACVD. The present study reviewed emerging challenges of RACVD in modern radiation oncology, in terms of clinical practice, bench investigation, and multidisciplinary care. Several molecules are potential for serving as biomarkers and therapeutic targets. Of these, miRNAs, endogenous small non-coding RNAs that function in regulating gene expression, are of particular interest because low-dose irradiation, i.e., 200 mGy (one-tenth of conventional RT daily dose) induces early changes of pro-RACVD miRNA expression. Moreover, several miRNAs, e.g., miR-15b and miR21, involve in the development of RACVD, further demonstrating the potential bio-application in RACVD. Remarkably, many RACVDs are late RT sequelae, characterizing highly irreversible and progressively worse. Thus, multidisciplinary care from oncologists and cardiologists is crucial. Combined managements with commodities control (such as hypertension, hypercholesterolemia, and diabetes), smoking cessation, and close monitoring are recommended. Some agents show abilities for preventing and managing RACVD, such as statins and angiotensin-converting enzyme inhibitors (ACEIs); however, their real roles should be confirmed by further prospective trials.

Keywords: radiation, cardiovascular dysfunction, miRNA, late sequelae, toxicity

# INTRODUCTION

Radiotherapy (RT) is an essential treatment modality in managing cancer patients (1, 2). Biologically, RT delivers ionizing radiation (IR) to eradicate cancer cells mainly through reacting with H2O to generate reactive oxygen species (ROS) to target multiple intra-cellular organelles, such as nucleus (mainly DNA), mitochondria, and cell membrane (3–5). Many IR-associated normal tissue damages are acute toxicities, characterizing potentially reversible and self-limited; however, some types of damages develop late sequelae, which are highly irreversible and progressively worse (4–6). For example, though the incidence is rare, irradiated cancer patients who had IR dose sprinkling to the cardiovascular system may encounter radiation-associated cardiovascular dysfunctions (RACVDs) (7–9), including blood pressure reduction (10), carotid stenosis (11), pericardial disease (12), myocardial infarction (13), pericardial/myocardial fibrosis (14, 15), valvular heart disease (16), arrhythmia (17), and subsequent heart failure (18–20). On clinical presentation, many RACVDs are late RT sequelae, developing a few years later after RT (21). Notably, as time elapsed, the risk of RACVD is larger in the third decade than that of the first two decades after IR exposure (22).

RACVD is a well-known treatment-related toxicity in the field of cardio-oncology (23–25). Other anti-cancer therapies, such as chemotherapy (26–29), targeted therapy (30–33), and immunotherapy (34–36), may also induce cardiovascular dysfunctions (37–39). As a result, when these therapies are prescribed concurrently or sequentially with RT, the risk of RACVD is increased substantially, especially in vulnerable pediatric (40, 41) or elderly cancer patients (42, 43). Besides, other RT-associated adverse events may occur with RACVD, such as ischemic stroke (44, 45) and lung fibrosis (46, 47), which may further impair patients' survival and life quality.

Several cardiovascular pathophysiological dysfunctions are associated with RT, such as late fibrosis/stenosis in the irradiated cardiovascular structures, mainly the endothelium (including endothelial cells and its stroma) and smooth muscle cells (2, 4, 5, 48). Epigenetic dysregulation, e.g., DNA methylation regulating gene expression without changes of sequence, demonstrates profound effects on the development of RACVD. For instance, differentially methylated enhancer of diacylglycerol kinase alpha (DGKA) reduces pro-fibrotic fibroblast activation, involving in radiation-associated tissue fibrosis and vascular stenosis (49). Similarly, microRNAs (miRNAs) also have been found to regulate the innate endothelium response to IR (50).

Clinically, moderate- to high-dose IR to the cardiovascular system increases the risk of RACVD (2, 4, 5). More notably, lowdose IR with a single 200 mGy (i.e., one-tenth of conventional RT daily dose of 200 cGy) has been observed to induce early damage of RACVD, demonstrating expression changes of miRNAs, e.g., miR-21 and miR-146b, and their regulated proteins in primary human coronary artery endothelial cells (51). This finding suggests that miRNAs as potential biomarkers for early detecting RACVD. Furthermore, some miRNAs have been reported as potential targets in managing RACVD, e.g., miR-15b (52), miR-21 (51–54), and miR-126-5p (55).

Hence, the present study aimed to review clinical challenges, potential biomarkers, and therapeutic targets of RACVD, with a focus on the role of miRNA. Emerging challenges of multidisciplinary care and example agents for prevention are also reviewed.

# CLINICAL CHALLENGES AND EMERGING ISSUES FOR DETECTING, MANAGING, AND PREVENTING RACVD

# Clinical Challenges in Improving Detection, Management, and Prevention of RACVD

Several factors affect the risk of RACVD (**Table 1**). As a result, current treatment guidelines recommend several methods to reduce the risk of RACVD (1, 2, 56). For example, in patients at high risk, radiation oncologists always consider alternative treatment choice of deferred RT, adopt rigorous dose constraints on the cardiovascular system, or implement advanced irradiation techniques. However, even implementing advanced techniques, the occurrence of RACVD cannot be avoided totally. Several issues are still challenging in clinical practice.

### Clinical Challenges of Decreasing the Risk of RACVD in Modern Radiation Oncology

Clinically, the overall incidence of RACVDs is rare but substantially encountered in irradiated patients with mediastinum lymphoma (8, 44, 66), head and neck (10, 45), esophagus (63), lung (13, 61, 62), and breast (12, 21, 56, 64) cancers. High-risk features of RACVD development are as follows: left-side breast irradiation (21, 65), combination with anthracycline-based chemotherapy (65), and vulnerable patient populations [e.g., pre-existing cardiac risk factors/heart disease (21, 57) and BRCA1/2 mutation carriers (60)]. For example, for a typical 50-year-old woman with pre-existing cardiac risk factors, an estimated 20-year risk of death from ischemic heart disease after breast RT is up to 1.6%, which is higher than that of those patients with no RT (i.e., 0.9%) (21, 56, 75). Moreover, in irradiated left breast cancer patients, each additional Gray (Gy) of the mean heart dose (MHD) increases the relative risk of major cardiac events by 7.4% (21).

Radiation oncologists implement several methods to decrease IR dose to the heart for minimizing the risk of RACVD (76), such as prone positioning (77), heart block with electronic compensation (57), heart-sparing three-dimensional printing technique (78), continuous positive airway pressure (CPAP) (79), real-time position management (RPM) inspiration gating (80, 81), proton-beam irradiation (82–85), and deep-inspiration breath-hold technique (DIBH) (86–90). However, even with the highly recommended visual-guided DIBH technique, residual variations of the heart position are still noticeable (91). As a result, the actual heart dose may be underestimated, burdening a higher risk of cardiac toxicities than that of estimation from the RT treatment planning.

For reducing the risk of cardiac toxicities, modern irradiation techniques aim to decrease irradiation dose to the heart. Diminishing the mean heart dose (MHD) is the main goal

#### TABLE 1 | Factors affect the risk of RACVD.


\**The dose to OARs is different according to the irradiated sites and cancer disease extension. Radiation oncologists always judge the pros and cons of RT to achieve better tumor control and fewer toxicities, i.e., judging for maximum tolerated dose (MTD) or as low as reasonably achievable (ALARA) (67, 74).*

\*\**Multimodality treatment is the cornerstone in managing cancer patients. However, combined treatments irreversibly enhance the risk of RTCVD.*

\*\*\**Statin used in irradiated cancer patients with hypercholesterolemia may demonstrate double benefits of decreasing the blood level of cholesterol and the risk of RACVD.*

\*\*\*\**ACEIs and angiotensin II receptor antagonists used in irradiated patients with hypertension may have double benefits of controlling blood pressure and decreasing the risk of RACVD. "V5" represents the percent volume of organ at risk (i.e., the heart) that is irradiated with an IR dose of* ≥*5 Gy. V25, V30, V40, and V45 are similar representations.*

*ACEI, angiotensin-converting enzyme inhibitor; ALARA, as low as reasonably achievable; BMI, body mass index; COPD, chronic obstructive pulmonary disease; Dmax, maximal dose; Gy, gray; MHD, mean heart dose; MTD, maximum tolerated dose; OAR, organ at risk; RACVD, radiation-associated cardiovascular dysfunction; RT, radiotherapy; SABR, stereotactic ablative body radiotherapy; VEGF, vascular endothelial growth factor.*

based on data estimated from conventional tangential technique (21, 92–94). Nevertheless, attenuating IR doses to coronary artery (95–97) and other cardiac substructures, such as left anterior descending artery (LAD) and left ventricle (LV), are more reasonable and suitable in modern precise RT departments (2, 66, 95, 98). However, long-term results investigated dose effects on these cardiac substructures are pending.

Another emerging challenge in clinical radiation oncology is the concept-shifting on treatment consideration. Previously, radiation oncologists always apply IR dose to organs at risk (OARs) according to the principle of "as low as reasonably achievable (ALARA) (99)." However, in some patient populations that required very aggressive managements, the treatment concept frequently shifts to maximum tolerated dose (MTD) for gaining the ultimate tumor control (67, 74). Undoubtedly, adopting MTD increases the heart dose and then burdens a higher risk of RTCVD than that of ALARA.

### Challenges of Clinical Detection for RACVD

Early detection of RACVD is challenging. Some clinical predictors have been reported for stratifying patients at risk, such as dosimetric parameters of RT (61), cardiac risk index (100), and coronary calcium score (101). Moreover, biomarkers are clinically helpful for detecting RACVD (102), such as cardiac troponins (e.g., troponin I or T) and natriuretic peptides (e.g., B-type natriuretic peptide (BNP) or pro-BNP) (103). On imaging, echocardiography is the pivotal method to detect cardiac anatomic and functional changes of RACVD (104–106). Profound RACVD may show a reduction of LV ejection fraction, and subclinical disease may reveal early signs of decreased global longitudinal strain (107–109). Recently, other advanced imaging modalities are attractive for detecting RACVD (110), such as cardiac computed tomography (111–113) and cardiovascular magnetic resonance (CMR) (114–116).

In recent precision cardio-oncology, it is a promise direction that applies combined omic-data and metabolic-function nuclear images (117), such as single-photon emission computed tomography (SPECT) (118) and positron emission tomography (PET) (119–122). Of these, PET that demonstrated metabolic changes of the heart is the most expecting image marker for detecting RACVD. However, identifying suitable isotopes of PET for early detecting RACVD is still challenging.

### Challenge of Clinical Managements for RACVD

Unfortunately, there is still no effective method to restore RT-associated late sequelae, including RACVD, because their disease courses are generally irreversible (2, 4, 6, 56). However, several pre-clinic studies have suggested potential targets for therapeutic interventions, such as HMGB1 (123) and miR-212 (124). Moreover, selective irradiation to the heart induces early overexpression of pro-hypertrophic miR-212, leading the miR-212 intervention as a reasonable approach for RACVD (124).

### Clinical Prevention for RACVD and Future Challenges

Some clinical agents may be used to prevent the occurrence of RACVD. For instance, statins, HMG-CoA reductase inhibitors prescribed for managing hypercholesterolemia, significantly reduces the risk of stroke [hazard ratio (HR) = 0.68; 95% confidence interval (CI), 0.48–0.98; P = 0.0368] and demonstrates a trend to decrease the risk of RACVD (HR = 0.85; 95% CI, 0.69–1.04; P = 0.0811) in irradiated cancer patients (71).

The detailed mechanism of statin in protecting the cardiovascular system is unclear. Some potential mechanisms are proposed. Firstly, statin inhibits RhoA GTPase (125), which is essential to mediate the irradiation inhibition of endothelial cell migration (126–128). Secondly, statin decreases cardiac endothelial cell permeability via activating ERK5 (129). Thirdly, statin enhances the release of Nitric Oxide (NO), which is crucial for improving endothelial function via regulating miR-221/222 (130). Fourthly, statin diminishes IR-induced responses of cardiac Connexin-43 and miR-21 (53) that involves in the process of cardiac fibrosis (52).

Clinical strategies, such as close monitoring, smoking cessation (58, 131), prescribing angiotensin-converting enzyme inhibitors (ACEIs), and β-blockers, are useful to prevent anthracycline-associated cardiac toxicities (132, 133). In the literature, ACEIs also showed a potential for preventing RACVD. For example, Captopril, one of ACEIs prescribed for hypertension or heart failure, has been found to decrease pulmonary endothelial dysfunction in irradiated rats (72). Similarly, Candesartan, an Angiotensin II Receptor Antagonist, has been reported to reduce the risk of RACVD in left breast irradiated patients (73). Thus, a potential mechanism of ACEI for cardioprotection may be demarcated reasonably by inhibiting angiotensin II to decrease the expression of TGF-β, a well-known pro-fibrogenic factor of post-IR late fibrosis (134, 135). However, these methods required further data support to demarcate their real roles in preventing the development of RACVD.

### Future Challenge: Mixed-Agent-Associated Cardiotoxicity in Combined Treatments

The major clinical problem is that many cancer patients were managed with multimodality treatments. As a result, the incidence of multi-treatment-associated CVDs, such as combined anthracycline-based chemotherapy and RT (136), is much higher than that of isolated RACVD. This phenomenon increases the difficulty of prevention and management, mostly requiring combined care from multidisciplinary team members, including radiation oncologists, medical oncologists, and cardiologists.

# Emerging Challenge of Bench Studies to Improve Early Detection, Management, and Prevention of RACVD, Focusing on the Role of miRNA in Acting as a Biomarker and Therapeutic Target

As mentioned above, in addition to currently clinical use biomarkers, such as cardiac troponins (e.g., troponin I) and natriuretic peptides (e.g., BNP) (103), several pre-clinical studies have been investigated to explore underlying mechanisms of RACVD, such as TGF-β and PPAR-α signaling pathways (137, 138), damage-associated molecular patterns (DAMPs) (139), and miRNA modulations (138). Of these, endogenous small noncoding miRNAs that function in regulating gene expression (140) grasp more interest in terms of biomarkers (141–143) and therapeutic targets (144–146) (**Table 2**).

### Emerging Challenges for Investigating Biological Mechanisms of RACVD

Detail mechanisms of RACVD are not well-recognized. Some potential mechanisms and pathways have been proposed. For example, IR may impair corin function and inhibit natriuretic peptides to accelerate senescence of cardiac and endothelial cells, contributing to the development of RACVD (151). Besides, several pathways have been identified with involvement into the process of RACVD, such as the 5 lipoxygenase (5-LO)/leukotriene pathway (152), the miRNA-34a/sirtuin-1 signaling pathway (149), the Reactive Oxygen Species (ROS)-mediated p16 pathway (153), and the TGF-βassociated signaling (154).

TABLE 2 | Examples of miRNAs involved in the process of RACVD that are potential for severing as biomarkers or therapeutic targets.


*HRW, hydrogen-risk water (H*<sup>2</sup> *water); IR, ionizing radiation; mGy, micro-Gray; MIF, macrophage migration inhibitory factor; NO, Nitric Oxide; RACVD, radiation-associated cardiovascular dysfunction.*

Moreover, some molecules may play roles in the process of RACVD, such as peroxisome proliferator-activated receptor gamma (PPAR-γ) (155), Growth differentiation factor 15 (GDF15) (153), and RhoA GTPase (125) that is essential to mediate the irradiation inhibition of endothelial cell migration. More recently, by using RNA-seq, differential gene-expression profiles have been identified in mice models, such as Nrf2, PDK1, and sirtuins (156). However, despite these lines of evidence, the whole picture of RACVD development is still not well-demarcated.

Another emerging challenge of investigating bio-mechanisms of RACVD comes from the difference of biological effects among different irradiation sources, e.g., proton vs. photon beams. Although proton and photon beams activate similar canonical radiation response pathways, distinct vascular genomic responses have been observed in the murine aorta (157). That is, models established according to photon radiation may not accurately predict the risk of RACVD associated with proton radiation.

#### Emerging Challenge of Bench Studies for Early Detecting and Managing RACVD, Focusing on the Example Role of miRNA

In the literature, many clinical studies assessed circulating miRNA levels in peripheral blood for diagnosing, predicting, and monitoring human diseases (158–163), including cardiac and vascular disease (CVD) (164–169). For example, the combination of miR-34a-5p and fibrinogen levels have been reported as a useful tool in differentiating pre-thrombotic status in patients with stable coronary artery disease (165). Moreover, the plasma expression level of miR-423-5p has been reported to serve as a promising biomarker for stratifying patients with coronary artery disease (168).

Similarly, several miRNAs have been found to involve in the process of RACVD (147, 170, 171). For example, via exosomesbased delivery to vascular smooth muscle cells, miR-30, miR-210, and miR-155 play roles in developing vascular calcification, which is one of the end events of RACVD that induces coronary artery stenosis and ischemic heart disease (148). Remarkably, IR-induced miRNAs expression behaves in a dose- and timedepended manner (150, 172). For instance, at 2 h after 2-Gy IR, the expression of miRNA-221 and miRNA-222 are significantly increased, but the level of miRNA-155 is decreased. On the other hand, at 24 h after 2-Gy IR, miRNA-146a and miRNA-155 are significantly overexpressed, but miRNA-222 is downregulated (150). These patterns of miRNA expression changes require attention in further prospective studies that intend to demarcate the role of miRNAs in association with RACVD.

Although it requires further efforts to bridge miRNAs from bench to bedside, some miRNAs are attractive in early detecting and managing RACVD (124). For instance, applying miR-126-5p therapy potentially improves endothelial recovery and prevents post-irradiation vascular re-stenosis (55). Besides, inhibiting miR34a by macrophage migration inhibitory factor (MIF) has been reported to reduce radiation-induced cardiomyocyte senescence via targeting SIRT1, implicating a novel strategy for managing RACVD (149). Moreover, molecular hydrogen, i.e., hydrogen-rich water (HRW; H<sup>2</sup> water), shows protective effects on IR-induced heart damage via regulating miRNA-1, -15b, and -21 (52).

In conjunction with miRNAs, circular RNAs (circRNAs) have been identified to involve in the regulatory network of the cardiovascular system. In biological function, circular RNAs may interact with RNA-binding proteins and act as miRNA sponges that inhibit the function of correspondingly matched miRNAs (173), demonstrating an ability for serving as novel biomarkers to early detect cardiovascular disease (174).

Applying circulating miRNA levels of peripheral blood is an immediately translatable mean for screening/monitoring RACVD. When researchers selected their miRNA targets by a literature review (such as targets that listed in the present study), miRNA database search, or miRNA-specific sequencing, they can subsequently conduct prospective clinical studies to validate their targets of interest under the pre-defined purpose of detecting, screening, or monitoring RACVD by using blood samples. However, testing details of circulating miRNAs (such as measuring methods, timing, and cut-off point values) are still required to be validated by prospectively clinical trials.

In the ClinicalTrials.gov (175), two actively recruiting trials integrate circulating miRNA as predicting biomarkers to detect RACVD in irradiated breast cancer patients, entitled: (1), Pre- or Postoperative Accelerated Radiotherapy (POP-ART; Identifier: NCT03783364) and (2), Breast Cancer and Cardiotoxicity Induced by Radiotherapy: the BACCARAT Study (Identifier: NCT02605512). Of these, the BACCARAT study investigates the role of several types of circulating biomarkers in detecting RACVD, including B-type natriuretic peptide, TGF-β1, and several miRNAs (e.g., miR-1, miR-34, miR-126, and miR-155). The results of the two trials are highly anticipated.

One potential limitation of applying miRNA in clinical practice is that the expression level of specific miRNAs would be varied in different tissues and testing time points. Therefore, the studies proceeding on the ClinicalTrials.gov may be very informative. Before the information of these clinical trials is available, in the authors' consensus opinion, integrating miRNAs as a component of circulating biomarkers for detecting RACVD may be critically considered in future clinical trials and practice that apply RT. Several measuring time points that similar to the protocol of the BACCARAT study are suggested as follows: before RT, the middle term of the RT course, and five time points after RT (i.e., 1 day, 6 months, 2, 5, and 10 years).

Why the time points of 2, 5, and 10 years should be considered testing and measuring? The main reason is that RACVD is a wellknown RT toxicity; it characterizes not only acute cardiovascular damage but also late sequelae of cardiovascular dysfunction that may be encountered a few years or decades after RT (21, 56, 75). Thus, long-term series measuring (i.e., 2-, 5-, and 10-years after RT) of target miRNA levels is useful for early detecting and monitoring the occurrence and severity altering of RACVD.

### Emerging Challenge: Novel Agents and Managements for Treating RACVD

As mentioned above, TGF-β-associated signaling gains a substantial interest in investigating the process of RACVD. For example, reducing irradiation-induced TGF-β1 production through blocking the NF-kB signaling pathway has been reported to provide a new insight in inhibiting irradiation-induced myocardial fibrosis (154). Besides, Protein Kinase C (PKC) has been reported to play a role in the process of RACVD (48). Remarkably, inhibiting PKC, such as applying RNA-interference techniques (176), could be a reasonable approach for managing IR-induced vascular dysfunction (48).

Some radioprotection agents, such as L-arginine, show protection effects on blood vessels of urinary bladder wall in patients treated with pelvic RT (177). Furthermore, IRdamaged vascular dysfunction has been observed to be restored by quercetin-filled phosphatidylcholine liposomes and mesenchymal stem cell injection (48). However, the real clinical roles of these agents and interventions on the cardiovascular system require further evidence to define.

# Emerging Challenge: Further Multidisciplinary Cooperation Among Radiation Oncologists, Cardiologists, and Molecular Biologists

Multidisciplinary care is required for preventing, detecting, and managing RACVD in irradiated cancer patients (178). In conjunction with the improvement of detection methods, increasing awareness and integrating works between oncologists and cardiologists are essential (179). Managing comorbidities adequately [e.g., hypertension, hypercholesterolemia, and diabetes control (180)], exercise therapy (181), and smoking cessation (58, 131) are all useful to decrease the risk of anticancer-treatment-related CVD (182), including RACVD. For multidisciplinary management, standard recommendation and structure/infra-structure requirements for patient care are ongoing established (183–188). For example, establishing consensus guidance to train RT staffs to delineate cardiac substructures decreases inter-observer variation and increases the accuracy of dose estimation, helping in implementing further randomized clinical trials and then daily clinical practice (189, 190).

Remarkably, several radiation-associated toxicities, including RACVDs, are diagnosed by a ruling-out—not ruling-in—way (2, 6). That is, diagnosing RACVD requires excluding other heart diseases, such as infectious disease or prior-existing subclinical cardiovascular dysfunctions. This work requires tight cooperation and interaction among multidisciplinary team members, such as radiation oncologists, medical oncologists, and cardiologists. Further consensus and recommendations are encouraged to establish in a multidisciplinary manner.

# CONCLUSION

Overall, the incidence of RACVD is rare in irradiated cancer patients. When it happened, however, RACVD may significantly impair patients' survival and life quality, particularly in vulnerable patient populations. Radiation oncologists implement many clinical efforts to reduce the risk; the incidence of RACVD is decreased but still substantial.

# REFERENCES


Further efforts from bench studies are emergently required to improve early detection, management, and prevention. For example, miRNAs play active roles in serving as biomarkers and therapeutic targets. Remarkably, integrating cooperation among multidisciplinary team members, such as oncologists and cardiologists, is encouraged and ongoing.

In the ClinicalTrials.gov (175), more than 20 clinical trials are actively or not yet recruiting for investigating challenging issues of RACVD, mainly focusing on early detection (e.g., circulating and imaging biomarkers) and aggressively avoidance/prevention (e.g., DIBH and proton therapy). Results from these ongoing trials are hopeful for resolving clinical obstacles of RACVD in the future.

# AUTHOR CONTRIBUTIONS

All authors contributed to the brainstorming of the ideas generation. M-SL and D-WL also contributed to first draft writing. S-KH, C-CY, C-LC, W-YC, L-CC, R-IL, L-WH, C-HC, and F-CH also contributed to literature review and interpretation. H-YL and MC also co-corresponded to overall manuscript communication and final approval.

# FUNDING

The present study was supported by the Ministry of Science and Technology, Taiwan (Grant No. 106-2923-B-194-001- MY3), and the Buddhist Tzu Chi Medical Foundation (Grant Nos. TCMMP105-09-02, TCMMP106-02-02, and DTCRD1062-E-18).

neck tumors. Am J Hypertens. (2018) 31:235–39. doi: 10.1093/ajh/ hpx158


contemporary radiotherapy for breast cancer. Circulation. (2017) 135:1388– 96. doi: 10.1161/CIRCULATIONAHA.116.025434


mastectomy for patients with left breast cancer. Breast J. (2019) 25:682– 6. doi: 10.1111/tbj.13304


calcification after radiation therapy. J Cardiovasc Imaging. (2019) 27:268– 79. doi: 10.4250/jcvi.2019.27.e38


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Lee, Liu, Hung, Yu, Chi, Chiou, Chen, Lin, Huang, Chew, Hsu, Chan and Lin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Differences in Expression of Mitochondrial Complexes Due to Genetic Variants May Alter Sensitivity to Radiation-Induced Cardiac Dysfunction

Rachel A. Schlaak <sup>1</sup> , Anne Frei <sup>2</sup> , Gopika SenthilKumar 3,4, Shirng-Wern Tsaih<sup>5</sup> , Clive Wells <sup>6</sup> , Jyotsna Mishra<sup>7</sup> , Michael J. Flister <sup>8</sup> , Amadou K. S. Camara<sup>7</sup> and Carmen Bergom1,2,9,10 \*

<sup>1</sup> Department of Pharmacology & Toxicology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>2</sup> Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>3</sup> Medical Scientist Training Program, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>4</sup> Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>5</sup> Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>6</sup> Electron Microscope Facility, Department of Microbiology & Immunology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>7</sup> Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>8</sup> Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>9</sup> Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI, United States, <sup>10</sup> Cancer Center, Medical College of Wisconsin, Milwaukee, WI, United States

#### Edited by:

Canan G. Nebigil, École supérieure de biotechnologie Strasbourg (ESBS), France

#### Reviewed by:

Nilufer Turan Dural, Brown University, United States Abdelrahman Ibrahim Abushouk, Harvard Medical School, United States

> \*Correspondence: Carmen Bergom cbergom@mcw.edu

#### Specialty section:

This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine

> Received: 08 January 2020 Accepted: 11 February 2020 Published: 05 March 2020

#### Citation:

Schlaak RA, Frei A, SenthilKumar G, Tsaih S-W, Wells C, Mishra J, Flister MJ, Camara AKS and Bergom C (2020) Differences in Expression of Mitochondrial Complexes Due to Genetic Variants May Alter Sensitivity to Radiation-Induced Cardiac Dysfunction. Front. Cardiovasc. Med. 7:23. doi: 10.3389/fcvm.2020.00023 Radiation therapy is received by over half of all cancer patients. However, radiation doses may be constricted due to normal tissue side effects. In thoracic cancers, including breast and lung cancers, cardiac radiation is a major concern in treatment planning. There are currently no biomarkers of radiation-induced cardiotoxicity. Complex genetic modifiers can contribute to the risk of radiation-induced cardiotoxicities, yet these modifiers are largely unknown and poorly understood. We have previously reported the SS (Dahl salt-sensitive/Mcwi) rat strain is a highly sensitized model of radiation-induced cardiotoxicity compared to the more resistant Brown Norway (BN) rat strain. When rat chromosome 3 from the resistant BN rat strain is substituted into the SS background (SS.BN3 consomic), it significantly attenuates radiation-induced cardiotoxicity, demonstrating inherited genetic variants on rat chromosome 3 modify radiation sensitivity. Genes involved with mitochondrial function were differentially expressed in the hearts of SS and SS.BN3 rats 1 week after radiation. Here we further assessed differences in mitochondria-related genes between the sensitive SS and resistant SS.BN3 rats. We found mitochondrial-related gene expression differed in untreated hearts, while no differences in mitochondrial morphology were seen 1 week after localized heart radiation. At 12 weeks after localized cardiac radiation, differences in mitochondrial complex protein expression in the left ventricles were seen between the SS and SS.BN3 rats. These studies suggest that differences in mitochondrial gene expression caused by inherited genetic variants may contribute to differences in sensitivity to cardiac radiation.

Keywords: radiation, radiation-induced heart damage, mitochondria, consomic rats, oxidative phosphorylation, echocardiogram, cardiotoxicity

# INTRODUCTION

Radiation therapy (RT) is used in over half of all cancer patients to treat malignancies and improve patient survival (1). RT can be administered to the thoracic region in treating chest tumors including Hodgkin lymphoma and breast and lung cancers. Despite advances in planning and delivering techniques (2–5), these techniques are not universally available and/or utilized by all providers (6), and RT to the thoracic region even with these techniques can still result in some exposure of the heart that can lead to cardiotoxicity (7, 8). Irradiation to the heart and surrounding vasculature may lead to toxicities including pericarditis, ischemic heart disease, myocardial fibrosis, cardiomyopathy, arrhythmias, and/or valvular abnormalities, collectively referred to as radiation-induced heart dysfunction (RIHD) (9–11). These normal tissue side effects may arise months to decades after RT, potentially leading to increased morbidity and mortality (12–14).

Cardiomyocytes are the most abundant cell type in the heart occupying roughly 70–85% total volume, and ∼30% of the heart volume consists of cardiomyocyte mitochondria (15–17). The heart demands very high levels of adenosine triphosphate (ATP) for healthy function (18), and therefore mitochondrial function is crucial in maintaining heart health by coupling respiration with oxidative phosphorylation to generate ATP (7, 19, 20). Mitochondria are known to have roles in metabolism, cell death, and stress responses including combating reactive oxygen species (ROS). In addition to causing direct effects to DNA that may lead to cell death, radiation also causes indirect effects including the production of ROS. The mitochondria function to protect against ROS-induced cellular damage, and therefore, mitochondria play a role in protecting the normal heart tissue against radiation induced toxicity (21).

We previously reported that the inbred Dahl saltsensitive/Mcwi (SS) rat strain was more sensitive to localized image-guided cardiac radiation than the Brown Norway (BN) strain, and that substitution of chromosome 3 from the BN strain into the SS background (SS.BN3 consomic rats) confers dramatic resistance to radiation-induced cardiac dysfunction when compared to the SS strain (22). Consomic chromosome substitution studies can be used to map complex genetic modifiers of pathophysiologic phenotypes (23–26). In our previous consomic rat study with the SS strain that was relatively sensitive to localized cardiac radiation when compared to the SS.BN3 consomic strain, the top genetic pathways differentially expressed between SS and SS.BN3 consomic rat ventricles 1 week after radiation included mitochondrial-related genes (22). However, expression of mitochondrial genes was not measured in unirradiated SS and SS.BN3 rat hearts, and protein expression of mitochondrial complexes was not examined. There is a need to better understand the mechanisms of mitochondrial dysfunction that may lead to RIHD. Here we examined changes in gene expression of all mitochondria-encoded genes and nuclear-encoded mitochondria oxidative phosphorylation complex genes between the sensitive SS and comparatively resistant SS.BN3 rat hearts that were not treated with radiation (sham treated). We also examined mitochondrial morphology using transmission electron microscopy, as well as the protein levels of mitochondrial oxidative phosphorylation complexes in isolated mitochondria from the left ventricles of SS and SS.BN3 rats after localized cardiac radiation. These results suggest that genetic changes can lead to altered expression of mitochondrial oxidative phosphorylation complexes that may contribute to differences in responses to localized cardiac irradiation. Better understanding of the role of mitochondrial dysfunction in RIHD may lead to targeted therapeutics to protect and/or mitigate RIHD while maintaining therapeutic effects of radiation therapy.

# MATERIALS AND METHODS

# Rats and Irradiation Procedure

The rat cardiac irradiation procedure has been reported elsewhere (22). In brief, female SS and SS.BN3 rats [Medical College of Wisconsin (23)] aged 10–12 weeks were randomized into different treatment groups. Animals were anesthetized with 3% isoflurane and given localized heart irradiation using a the high-precision image-guided X-RAD SmART irradiator (Precision X-Ray, North Branford, CT). A 24 Gy × 1 fraction was given to the isocenter of the heart, with equally weighted anteriorposterior and 2 lateral beams (1:1:1, 225 kVp, 13 mA, 0.32 mm Cu, 2.69 Gy/min) using a 1.5 cm collimator. Pilot V1.8 Imaging Software (University Health Network, Toronto, Canada) was used to create two-dimensional projections over 360◦ to provide CT scans in sagittal, coronal, and axial views, with each projection on the heart centered to fit into the collimator. Monte Carlobased treatment planning was utilized to calculate radiation dose (MAASTRO Radiotherapy Clinic, Netherlands). Age-matched sham-irradiated animals were included in the study. Animals were irradiated and housed in pathogen-free conditions with a 12:12 light:dark cycle and access to a standard diet (0.4% salt) and water. All procedures were performed according to the American Guidelines for the Ethical Care of Animals and approved by our Institutional Animal Care and Use Committee.

# Echocardiography

The echocardiogram procedure for rats has been reported elsewhere (22). In brief, echocardiography with M-mode was used to assess cardiac function on irradiated and sham treated rats at baseline, 3- and 5- months post-RT. An echocardiograph Vivid 7 with an 11-MHz M12L linear-array transducer and EchoPac software (General Electric, Wauwatosa, WI) was used to perform the examinations. Imaging was conducted in the shortaxis view at mid-level of the left ventricle, by a sonographer with three consecutive heartbeats measured where the average was utilized for analyses (27, 28). For strain analysis, images were processed with EchoPac Q analysis software (General Electric, Wauwatosa, WI). A cardiac cycle was defined from peak one R wave to the peak of the following wave. The endocardial border was traced during an end-systolic frame in the short-axis view at mid-ventricle to calculate radial and circumferential strain. The computer produced a profile of radial (myocardial deformation toward the center) and circumferential (myocardial deformation along the curvature) strain percentage over time.

# RNA-Sequencing

The RNA-sequencing protocol was previously reported (22, 23). Briefly, total RNA was extracted by TRIzol (Thermo Fisher Scientific, Waltham, MA) from the left ventricle tissue of 11–13 weeks old female mock-treated SS and SS.BN3 rats (N = 4–5/group) from a group of rats matched to 1 week post-radiation rats (not reported here, but previously reported). For RNA-seq, a library preparation was made for each sample, indexed for multiplexing, and sequenced using an Illumina HiSeq2500 (Illumina, San Diego, CA). The Trim Galore program (v0.4.1) was used to trim bases with a Phred quality score <20 [https://www.bioinformatics.babraham.ac.uk/ projects/trim\_galore/]. The RSEM program function "rsemprepare-reference" (v1.3.0) was used to extract the transcript sequences from the Rat genome (Rnor6.0, Ensembl release 98) (30) and to generate Bowtie2 indices (Bowtie2 v2.2.8) (31), followed by read alignment and expression quantification using the "rsem-calculate-expression" function. Differential expression (DE) analysis was performed using the Bioconductor package DESeq2 version 1.12.4 (29) to compute log2 fold changes and FDR-adjusted p-values. Statistical significance was determined at an FDR threshold of 0.05. Data were analyzed for molecular and functional pathway enrichment using the IPA tool (Qiagen). All raw sequencing data can be accessed from the Sequence Read Archive, BioProject ID PRJNA525087 (https://www.ncbi. nlm.nih.gov/bioproject/PRJNA525087).

# Transmission Electron Microscopy (TEM)

Rat left ventricle was harvested 1 week after 1 × 24 Gy cardiac RT or sham from adult female SS and SS.BN3 rats (N = 2– 5/group) and fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer pH 7.2. The samples were then post-fixed in 1% OsO<sup>4</sup> on ice for 1 h, followed by dehydration in a graded methanol series, and an embedding in EPON 812 (EMS, Hatfield, PA). Ultra-thin sections (60 nm) were cut, stained with uranyl acetate and Reynolds lead citrate, and examined with a Hitachi H600 Transmission Electron Microscope (TEM) (Hitachi High Technologies America Inc., Pleasanton, CA). Representative images to assess cardiac mitochondrial morphology were captured at 20,000X magnification.

# Mitochondrial Isolation and Western Blot Analyses

Rat hearts were harvested 12 weeks after either 1 × 24 Gy localized cardiac radiation or sham treatment (22). Heart mitochondria isolation has previously been reported (32, 33). In brief, fresh heart tissue was minced in ice cold isolation buffer [200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM 3- (N-morpholino) propanesulfonic acid, and 1 mM EGTA, with 0.1% bovine serum albumin, pH 7.15]. The minced tissue was homogenized in the presence of 5 U/ml protease (P5459, Sigma Life Science, St. Louis, MO) followed by differential centrifugation at 4◦C. The final pellet was resuspended in isolation buffer and protein concentration was determined by the Bradford method. For Western blot analysis, isolated mitochondria were lysed using a RIPA buffer containing protease and phosphatase inhibitors, centrifugated, and the supernatant was collected. Total protein was assessed using a BCA Protein Assay Kit (23225, Thermo Scientific, Rockford, IL). Mitochondrial protein lysates were loaded and separated using SDS-PAGE and then transferred onto a PVDF membrane. The following antibodies were used in the present study using mitochondrial lysates: total OXPHOS rodent WB antibody cocktail (1:2500; ab110413; Abcam) and anti-COX IV antibody Mitochondrial Loading Control (1:5000; ab16056; Abcam).

# Statistical Analysis

Analyses of the western blotting were evaluated by a Student's t-test. Blots were imaged on ImageQuant LAS 4000 (GE Healthcare Life Sciences, Marlborough, MA), and analyzed using ImageQuant TL software (version 8.1.0.0). All western blotting results reported are representative of 3 technical replicates. The criterion for significance was P < 0.05. Data are reported as means ± SE. For our RNA-sequencing studies (22), power analysis was determined using a combination of simulated and experimental data approach previously described (34). We performed 100 simulations based on a RNAseq count data from our previous study (35). This analysis suggested that 4 replicates per group in a 2-group comparison would provide more than 90% power to detect genes differentially expressed at FDR 0.05 level. All power calculations and animal numbers for our studies were also performed by a non-biased statistician (S.-W.T.).

# RESULTS AND DISCUSSION

Previously, we have demonstrated that the SS rat strain is more sensitive to localized image-guided cardiac radiation that the SS.BN3 consomic rat strain, which differs only in substitution of chromosome 3 from the BN strain, as measured by pleural effusions, echocardiogram indices of left-sided heart failure and strain, as well as mortality. We also demonstrated that the SS and SS.BN3 strains had differentially expressed mitochondria-related genes in the left ventricle 1 week after radiation, as measured with RNA-sequencing (22). In this study, we examined the differential expression of oxidative phosphorylation genes from both the mitochondrial and nuclear genomes between SS and SS.BN3 left ventricles in rats 1 week after sham radiation treatment, at 11–13 weeks of age (N = 4/condition), which is the same time period reported previously after radiation (22). Of the mitochondrialencoded genes coding for oxidative phosphorylation complexes, 13 of 13 genes are differentially expressed between SS and SS.BN3 rats at FDR < 0.05 (**Figure 1A**, **Supplemental Table 1B**). Expression of these genes was significantly higher in the protected SS.BN3 rats in comparison to the more sensitive SS rats. In addition, of the 80 nuclear encoded rat genes involved in encoding mitochondrial complexes I–V, 74 of 80 genes were differentially expressed between SS and SS.BN3 rats at FDR < 0.05 (**Figure 1B**, **Supplemental Table 1A**). Interestingly, these genes had higher expression in SS rats compared to the SS.BN3 rats. We subsequently examined whether there were changes in mitochondrial morphology between the SS and SS.BN3 rat left ventricles. TEM was performed on mitochondria isolated from rat hearts at 1 week post-radiation or sham treatment. Longitudinal views of tissue were examined for each condition,

mitochondrial complexes that drive oxidative phosphorylation.

where total mitochondria and irregular shaped mitochondria were counted. This revealed no morphological differences between SS and SS.BN3, with representative images shown in **Figure 2**.

Our data in **Figure 1**, along with previously published data (22), demonstrate that changes in gene expression of oxidative phosphorylation complex genes are differentially expressed in the left ventricles of both the non-irradiated rats and rats irradiated with a single dose of 24 Gy to the whole heart. However, the functional consequences of these changes at later time points had not been examined. We isolated mitochondria from SS and SS.BN3 rats (N of 3–4 per group) 12 weeks post-treatment with 24 Gy of localized heart radiation or sham (no radiation). We then performed Western blotting on the isolated mitochondria to examine protein expression of mitochondrial complexes I– V. This revealed no significantly significant changes between complex I–V in the unirradiated SS vs. SS.BN3 heart, but significant increases were seen in complexes I, III, and V in the SS.BN3 vs. SS hearts (**Figures 3A–F**, **Supplemental Figure 1**). **Figure 3** shows representative results from 3 technical replicates of each Western blot. Protein expression levels were assess using NADH: Ubiquinone Oxidoreductase Subunit B8 (NDUFB8; complex I) (**Figure 3A**), Succinate dehydrogenase ubiquinone iron-sulfur subunit (SDHB; complex II) (**Figure 3B**), Ubiquinol Cytochrome C Reductase Core Protein 2 (UQCRC2; complex III) (**Figure 3C**), Mitochondrial Cytochrome C Oxidase I (MT-CO1; complex IV) (**Figure 3D**), and Mitochondrial ATP Synthase 5A (ATP5A; complex V) (**Figure 3E**). Representative Western blots of the mitochondrial lysates are shown in **Figure 3F**, N = 3– 4/group. complexes I, III, and V showed increased expressions in the SS.BN3 vs. SS with RT lysates (complex I: P = 0.004, 2/3 blots significant; complex III: P = 0.02, all 3 blots were significant; CV: P = 0.004, all 3 blots were significant). There was also a trend in complex IV with increased expression in SS.BN3 vs. SS with RT (1/3 blots significant). Although the OXPHOS antibody cocktail consists of a mixture of five antibodies to detect the five different complex subunits, different subunits were quantified at different exposure times to be in the linear

detection range, as shown in **Figure 3F**. The full blots at different exposure times are shown in **Supplemental Figure 1**. These results indicate that genetic changes in rat chromosome 3 can lead to significant changes in mitochondrial complex expression several weeks after high-dose cardiac radiation exposure, at a time when echocardiogram changes are seen demonstrating differences in left ventricular heart function between the SS and SS.BN3 rats (22). M-mode echocardiogram imaging, performed previously (22) demonstrated cardiac dysfunction in SS rats compared to SS.BN3 rats displaying hyperdynamic systolic function (**Figure 4A**). Analysis of both radial and circumferential strain at 3 and 5 months post-RT revealed the SS rat hearts had significantly decreased myocardium deformation, consistent with decreased systolic dysfunction (**Figures 4B,C**).

A number of studies have implicated mitochondrial changes in the development of cardiac dysfunction following radiation, both in pre-clinical models and in human studies (7, 36–39). In C57BL/6N mice that received sham, 0.2 Gy, or 2 Gy of heart radiation, functional and proteomic alterations were seen 4 weeks following irradiation. This included changes in proteins related to oxidative phosphorylation (36, 40–42). Functionally, partial deactivation of complexes I and III were observed in mice receiving 2 Gy of cardiac radiation. In a separate publication, this group also examined the long-term effects of cardiac radiation, finding that respiratory capacity was still reduced 40 weeks after 2 Gy of cardiac radiation (38). In separate studies, C57BL/6 mice treated with 8 or 16 Gy of cardiac radiation demonstrated increased free fatty acids and reduced levels of complexes I, III, and V (39). Studies of mitochondrial-related proteins in the left ventricles of decreased nuclear workers exposed to varying levels of radiation (external exposure ranges from 100 mcGy to >5 Gy) revealed dose-dependent reductions in complexes I, III, and V, and changes in complexes II and IV in those with the highest radiation exposures (42).

There are limitations from this study that should be acknowledged. We examined the effects of RIHD with the treatment dose of 1 × 24 Gy cardiac RT. We have previously reported similar cardiac trends by using a fractionated regimen of 9 Gy × 5 (22). The dosing regimen was determined based on previous studies of studying RIHD from cardiac RT in rats (7, 43–47). To better mimic cancer patient thoracic RT, future studies are needed with both partial heart irradiation and increased fractions of smaller daily radiation dose to more closely resemble the radiation exposure observed. In addition to cancer patients receiving thoracic RT, recent studies report the using 25 Gy cardiac RT in a single fraction to treat ventricular tachycardia (48, 49). Our rat model of cardiac RT is very relevant to this clinical model of treatment and could be further used to study side effects and biologic changes that occur from this high dose cardiac RT. Additional considerations include how

these findings can be translated into future applications. Other than the 13 mitochondrial encoded genes, many genes involved in mitochondrial dysfunction, sirtuin signaling and cardiac hypertrophy were also found to be differentially expressed between SS and SS.BN3 rats (22). Candidates involved in these pathways as well as mitochondrial gene transcription, translation, and regulation could be further tested to investigate their roles in radiation-induced cardiotoxicity. The use of pharmacologic modulators of these pathways and transgenic models could also be pursued to further elucidate mechanisms of RIHD to prevent and/or mitigate effects observed in patients receiving radiation therapy.

In this current study, we demonstrate changes in the levels of oxidative phosphorylation complexes between genetically similar rats, differing only in the single nucleotide polymorphisms on chromosome 3, that demonstrate dramatic differences in the development of radiation-induced cardiotoxicity after localized radiation exposure to the heart (22). These results demonstrate that there are differences in gene expression of both mitochondrial-encoded and nuclear-encoded genes for the oxidative phosphorylation complexes in the left ventricles of unirradiated SS and SS.BN3 rats (**Figure 1**), as well as the left ventricles of SS and SS.BN3 rats 1 week after 24 Gy of localized cardiac irradiation (22). It is unclear why there are differences in the direction of differential expression of oxidative phosphorylation complex genes encoded by mitochondrial vs. nuclear genomes. In general, the mitochondrial genome is more likely to experience DNA damage than nuclear DNA following radiation due to the lack of protective effect from histones (50), as well as less efficient DNA repair (51, 52). However, as our results here demonstrate, differences in mitochondrial-encoded genes are seen between SS and SS.BN3 left ventricles even without radiation treatment **(Figure 1**). Although large numbers of mitochondrial genes are differentially expressed in SS vs. SS.BN3 rats in unirradiated and irradiated left ventricles, no gross changes in mitochondrial morphology were seen in the left ventricles 1 week after radiation or sham treatments (**Figure 2**). However, at a later timepoint of 12 weeks following 24 Gy of localized cardiac radiation, differences in expression of complex I, III, and V proteins were seen in isolated mitochondrial in the SS vs. SS.BN3 samples. Taken together, these results indicate that inherited genetic variants can lead to differences in oxidative phosphorylation gene expression that may contribute to differences in radiation-induced cardiac dysfunction.

# DATA AVAILABILITY STATEMENT

The datasets generated for this study can be found in https:// www.ncbi.nlm.nih.gov/sra, NCBI Accession No. PRJNA525087.

# ETHICS STATEMENT

The animal study was reviewed and approved by Medical College of Wisconsin Institutional Animal Care and Use Committee.

# AUTHOR CONTRIBUTIONS

RS, MF, AC, JM, and CB conceived and designed the research. RS, AF, GS, and CW performed the experiments. RS, AF, CW, S-WT, and CB analyzed data and interpreted results. RS, AF, and S-WT prepared figures. RS and CB drafted the manuscript. RS, GS, JM, AC, and CB edited and revised manuscript. All authors approved the final version of manuscript.

# FUNDING

This work was supported by NIH 1R01HL147884 (CB). Additional support was provided by the Mary Kay Foundation Award Grant No. 017-29 (CB), Susan G. Komen **<sup>R</sup>** Grant CCR17483233 (CB), the Nancy Laning Sobczak, Ph.D., Breast

# REFERENCES


Cancer Research Award (CB), the Medical College of Wisconsin Cancer Center (CB), the Michael H. Keelan, Jr., MD, Research Foundation Grant (CB), and the Cardiovascular Center at the Medical College of Wisconsin (CB).

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2020.00023/full#supplementary-material

Supplemental Figure 1 | Full immunoblots against OXPHOS antibody cocktail in heart mitochondrial lysates from SS and SS.BN3 rats with either 24 Gy RT or sham treatment at different exposure times of (A) 4 s for C-V, (B) 8 s for C-III, (C) 30 s for C-IV, and (D) 5 min for C-I and C-II.

Supplemental Table 1A | Expression of Nuclear-Encoded Mitochondrial Complex Genes in SS.BN3 vs. SS Left Ventricles 1 Week After 24 Gy of Localized Heart Radiation.

Supplemental Table 1B | Expression of Mitochondrial-Encoded Genes in SS.BN3 vs. SS Left Ventricles 1 Week After 24 Gy of Localized Heart Radiation and Differentially Expressed Mitochondrial Genes in SS versus SS-BN3 Left Ventricles Mitochondrial-Encoded Genes.


dynamic near-infrared fluorescence imaging. Biomed Optics Express. (2018) 9:543–56. doi: 10.1364/BOE.9.000543


linked to radiation-induced ischemic heart disease in mayak nuclear workers. Oncotarget. (2016) 8:9067–78. doi: 10.18632/oncotarget.10424


**Conflict of Interest:** MF currently is a Principal Research Scientist at Abbvie, but at the time of his contributions to the manuscript, he was employed at the Medical College of Wisconsin. CB receives research funding from Innovation Pathways, Palo Alto, CA.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Schlaak, Frei, SenthilKumar, Tsaih, Wells, Mishra, Flister, Camara and Bergom. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Therapeutic Targets for the Treatment of Cardiac Fibrosis and Cancer: Focusing on TGF-β Signaling

Warisara Parichatikanond<sup>1</sup> , Theerut Luangmonkong<sup>1</sup> , Supachoke Mangmool <sup>2</sup> and Hitoshi Kurose<sup>3</sup> \*

*<sup>1</sup> Department of Pharmacology, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand, <sup>2</sup> Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand, <sup>3</sup> Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan*

Transforming growth factor-β (TGF-β) is a common mediator of cancer progression and fibrosis. Fibrosis can be a significant pathology in multiple organs, including the heart. In this review, we explain how inhibitors of TGF-β signaling can work as antifibrotic therapy. After cardiac injury, profibrotic mediators such as TGF-β, angiotensin II, and endothelin-1 simultaneously activate cardiac fibroblasts, resulting in fibroblast proliferation and migration, deposition of extracellular matrix proteins, and myofibroblast differentiation, which ultimately lead to the development of cardiac fibrosis. The consequences of fibrosis include a wide range of cardiac disorders, including contractile dysfunction, distortion of the cardiac structure, cardiac remodeling, and heart failure. Among various molecular contributors, TGF-β and its signaling pathways which play a major role in carcinogenesis are considered master fibrotic mediators. In fact, recently the inhibition of TGF-β signaling pathways using small molecule inhibitors, antibodies, and gene deletion has shown that the progression of several cancer types was suppressed. Therefore, inhibitors of TGF-β signaling are promising targets for the treatment of tissue fibrosis and cancers. In this review, we discuss the molecular mechanisms of TGF-β in the pathogenesis of cardiac fibrosis and cancer. We will review recent *in vitro* and *in vivo* evidence regarding antifibrotic and anticancer actions of TGF-β inhibitors. In addition, we also present available clinical data on therapy based on inhibiting TGF-β signaling for the treatment of cancers and cardiac fibrosis.

## Edited by:

*Tienush Rassaf, University Hospital Essen, Germany*

#### Reviewed by:

*Alessandra Cuomo, Federico II University Hospital, Italy Scott Levick, University of Sydney, Australia*

> \*Correspondence: *Hitoshi Kurose hikurose@phar.kyushu-u.ac.jp*

#### Specialty section:

*This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine*

> Received: *30 November 2019* Accepted: *24 February 2020* Published: *10 March 2020*

#### Citation:

*Parichatikanond W, Luangmonkong T, Mangmool S and Kurose H (2020) Therapeutic Targets for the Treatment of Cardiac Fibrosis and Cancer: Focusing on TGF-*β *Signaling. Front. Cardiovasc. Med. 7:34. doi: 10.3389/fcvm.2020.00034* Keywords: anticancer, antifibrotic, cancer, cardiac fibrosis, inhibitors of TGF-β signaling, transforming growth factor-β (TGF-β)

# INTRODUCTION

Transforming growth factor-β (TGF-β) is a crucial member of the TGF-β superfamily and its sophisticated signaling pathways have pleiotropic effects that regulate several systems throughout the body such as cell growth, cell differentiation, apoptosis, motility and invasion, tissue remodeling, angiogenesis, and the immune response (1–6). TGF-β signaling dysfunctions are frequently found in tumors and these dysfunctions play critical roles in tumor progression (e.g., development and metastasis) (7–9). In addition, TGF-β is a major profibrotic mediator that plays an important role in the development of fibrosis (10). Due to the significant implication of TGF-β signaling in cancer as well as in fibrosis (**Figure 1**), drug research into treatments for cancer and

fibrosis has aimed to develop various approaches to inhibit TGFβ signaling. Thus, the number of lead compounds used either in animal models or in clinical studies related to cancer and fibrosis is currently growing. Targeting TGF-β signaling pathways could be a novel therapeutic strategy to treat a variety of fibrotic disorders and cancers.

The synthesis and secretion of TGF-β, including its activity, is markedly increased in experimental models of fibrosis and in patients with tissue fibrosis (e.g., liver, lung, kidney, and heart). Fibrosis is an important pathophysiological phenomenon in many tissues. It is characterized by fibroblast activation and accumulation, an imbalance of extracellular matrix (ECM) production and degradation, and myofibroblast differentiation, which results in the accumulation of fibrotic scar and tissue stiffness, leading to distortions of organ architecture and function [Reviewed in (11, 12)].

Among fibrotic conditions in various organs, cardiac fibrosis is a major pathologic disorder associated with a great number of cardiovascular diseases resulting from an excessive ECM protein deposition in the heart [Reviewed in (11, 12)]. The etiologies of cardiac fibrosis and myocardial stiffness are multifactorially developed in response to multiple risk factors (13, 14) include myocardial infarction (MI), hypertension (15), diabetes (16, 17), aging (16), and excessive alcohol consumptions (18, 19) leading to the excessive deposition of ECM. After cardiac injury, alterations in ECM homeostasis, the upregulation and release of growth factors and cytokines, and differentiation of fibroblasts into myofibroblasts dynamically modulate cardiac fibroblast characteristics and functions, leading to myocardial fibrosis. Myocardial fibrosis is associated with fibrotic scar formation, myocardial stiffness, and the progression of heart failure (HF) (20–23). Treatment of HF and cardiac fibrosis still has limited efficacy and currently there is no drug approved for the treatment of cardiac fibrosis. The main reason is that the underlying mechanism of fibrosis is still unclear. However, cardiovascular diseases remain the leading global cause of death (22, 23) and understanding the pathogenesis of fibrotic myocardial remodeling is crucial to identifying innovative treatment strategies for patients with cardiac fibrosis.

In the heart, activation of cardiac fibroblasts mainly by TGFβ leads to alterations in cardiac ECM and cardiac remodeling that play a major role in the development and progression of heart diseases (10, 22). A significant number of preclinical and clinical studies have reported that inhibition of TGFβ signaling pathways by various strategies exhibited potential effectiveness for the treatment of cardiac fibrosis. Cancers and fibrotic diseases share the most common pathologies associated with the activity of TGF-β (1, 2). Here, we review the molecular mechanisms and signaling pathways of TGF-β and their effect on cancer and cardiac fibrosis, and we also summarize the role of inhibition of TGF-β for anticancer and antifibrotic therapies.

# Introduction of Cancer

Cancer is defined as a collection of diseases relating to atypical cell growth. In physiological process, new cells can grow, divide, and replace senescent or damaged cells. However, this systemically process fails when cancer develops as aged or injured cells remain survive, together with a proliferation of unneeded new cells. These unnecessary cells can divide, spread, and invade nearby tissues without stopping. Also, the harm cells can possibly travel through the blood or lymph system to invade remote tissues. This atypical cell growth and spreading is known as carcinogenesis (24). Widespread and recognized theory of carcinogenesis is the DNA mutations that disrupt the normal balance between proliferation and cell death. Variants of inherited genes and environmental factors might play a pivotal role in DNA mutations. In addition, viruses containing oncogenes are recently known as a trigger of cancer cell growth (24).

#### Therapeutic Targets for Treatment of Cancers

Treatment of cancers can be achieved using several strategies such as surgery, radiation, and especially drugs. Chemotherapy is a conventional treatment by using toxic drugs to kill cancer cells. Beyond fast-growing cancer cells, traditional anticancer drugs using for chemotherapy damage healthy cells that rapidly grow and divide, leading to multiple adverse effects (25). Newer drugs for the treatment of cancers were subsequently developed for a preferable safety issue and prevailing therapeutic efficacy (25). Hormonal therapy is another strategy to cease the growth of cancer which required certain hormones. Due to the blockade, undesired effects of anti-hormone drugs can be seen depending on types of interfered hormone (26, 27). Targeted therapy is a type of cancer treatment using drugs targeting particular molecules required for the pathogenesis of individual cancer. Nevertheless, treated cancer cells can gradually resist to targeted therapy, and conventional chemotherapy might be needed to be co-administered in the regimen for a better outcome (28). Immunotherapy is a novel treatment method by enhancing immune system for eradicating cancer cells. Despite solely activated self-immune cells, overactive immunity against cancer also influences healthy cells and tissues resulting in various adverse effects (29). Described anticancer drug classes and representative drugs among each class are demonstrated in **Table 1**. However, in-depth review regarding mechanism of drug action, clinical effectiveness, and safety profile of these anticancer drugs are beyond our scope. Furthermore, it should be noted that although anticancer drugs appears to be diverse and abundant, we still need distinct agents to deal with innumerable types of advanced cancers in clinical practice, especially multi-drug resistant cancers (30). Therefore, in this review, we focus on the role of TGF-β and its signaling on the treatment of cancer.

### Introduction of Cardiac Fibrosis

Cardiac fibrosis is a pathological remodeling process following cardiac injury, MI, and other heart diseases. Cardiac fibrosis disrupts the communication and function of myocytes and nonmyocyte cells in the heart, leading to contractile dysfunction and arrhythmia. Fibrosis also accelerates the remodeling processes that exhibit detrimental effects on the heart (23, 31).

The imbalance between production and degradation of interstitial ECM proteins leads to progressively increased cardiac stiffness and diastolic dysfunction (23). Lines of existed evidence demonstrates that the pathogenesis of diastolic dysfunction caused by cardiac fibrosis (32, 33). In the fibrotic heart, collagens mainly from activated myofibroblasts undergoes crosslinking process contributing to the progression of diastolic dysfunction and the restricted cardiac chamber compliance (34, 35). In addition, ECM overproduction and deposition between the layers of cardiac myocytes results in the disruption of myocardial electrophysiological functions, which leads to contractile dysfunction and an increased risk of cardiac arrhythmia (36, 37). In fact, TGF-β induced cardiac fibrosis is seriously involved in the pathogenesis of arrhythmia by disturbing electrical signal conduction, leading to the generation of re-entry circuits (10).



*GnRH, gonadotropin releasing hormone; IFN, interferon; PRR, pattern recognition receptor; SERMs, selective estrogen receptor modulators.*

### Myofibroblasts

In the heart, cardiac fibroblasts can be transdifferentiated into myofibroblasts with contractile, migratory, and secretory properties (**Figure 2**). Myofibroblast is a key regulator that accelerates the fibrotic response in many conditions associated with HF. Regardless of the etiology of cardiac fibrosis, myofibroblast transdifferentiation is a hallmark of the fibrotic response in the heart [Reviewed in (20, 23)].

Myofibroblasts are the activated form of fibroblasts. They overexpress α-smooth muscle actin (α-SMA) and contain contractile bundles of actin filaments resembling the myofibrils of smooth muscle cells and associated proteins organized into prominent stress fibers (38). The incorporation of α-SMA into contractile bundles is a major characteristic of differentiated myofibroblasts and significantly increases contractile function. Thus, α-SMA has been suggested to be the most significant marker of myofibroblasts (39). Although α-SMA is found in human myocardial scars, the other structural ECM proteins such as collagens, vimentin, and desmin are also present in fibrotic scars (40). Fibroblast differentiation into myofibroblast is controlled by a variety of growth factors and cytokines. Among them, TGF-β is

a strong inducer that stimulates myofibroblast formation (**Figure 2**).

Fibroblasts are abundant in normal hearts and can differentiate into myofibroblasts via profibrotic mediators such as TGF-β (41, 42). This process suggests that the activation of resident fibroblasts represents a major source of myofibroblasts in hearts with fibrosis. In addition, proliferating myofibroblasts are commonly found in high numbers in the infracted area of the heart (41, 42).

Following cardiac fibroblast activation, inflammatory cells (e.g., macrophages, monocytes, and mast cells) infiltrate the site of remodeling myocardium and secrete various types of profibrotic mediators, including growth factors and cytokines [Reviewed in (43)]. These mediators have been found to promote myofibroblast formation, but the most significant and common inducer is TGF-β (44). TGFβ accelerates the differentiation of resident fibroblasts, epithelial cells, and endothelial cells into myofibroblasts (44). Thus, agents that inhibit myofibroblast differentiation might provide a tool to prevent the maladaptive myocardial remodeling that occurs in response to profibrotic stimuli and for fibrosis prevention.

## Overproduction of ECM Proteins

Alterations in ECM homeostasis, especially in terms of ECM overproduction, lead to cardiac dysfunction. Several mediators, including angiotensin II (Ang II), and TGF-β, regulate ECM production by cardiac fibroblasts (45). In response to cardiac injury, myocardial fibrosis results from an imbalance of both ECM synthesis and degradation, leading to an accumulation of collagen type I and III in the heart (20, 23). Deposition of ECM proteins is significantly increased in the hearts of patients with cardiac diseases (46). In addition, the levels of cardiac fibrosis are associated with cardiac dysfunction (46). Moreover, ECM deposition and fibroblast activation contribute to the impairment of ventricular compliance and filling due to increased ventricular stiffness (20, 23). Furthermore, overproduction of ECM interrupts the electrophysiological functions in the heart, leading to arrhythmias (10).

## Therapeutic Targets for Treatment of Cardiac Fibrosis

According to cardiac fibrosis is associated with cardiac remodeling and is involved in the pathogenesis of HF, the prevention and reversal of cardiac fibrosis is an important therapeutic target for the treatment of HF. Numerous signaling pathways, through a variety of profibrotic mediators (e.g., Ang II, endothelin-1 [ET-1], and TGF-β), have been implicated in the activation of cardiac fibroblasts and the development of cardiac fibrosis. Modulation of these signaling pathways using inhibitors is of great interest for the treatment and prevention of cardiac fibrosis. Below, we summarize the update and important roles of several agents that act against cardiac fibrosis (**Table 2**). Although, both angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) have already demonstrated significant efficacy in reducing cardiac fibrosis in human and animal models of HF, neither ACEIs nor ARBs have been approved for the treatment of cardiac fibrosis. Further studies are required to establish the molecular mechanisms of ACEIs and ARBs not only for treatment but also for reversal of fibrotic remodeling in HF.

# TGF-β SIGNAL TRANSDUCTION

TGF-β is a member of the TGF-β superfamily, which is comprised of TGF-β, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activin and inhibin (65). Members of this diversify superfamily are the pleiotropic multifunctional polypeptides that play a role in a wide range of physiological cellular activities such as growth, proliferation, differentiation, and apoptosis (65). Among these polypeptides, TGF-β has been proven to be one of the major factors driving the fibrotic response in most organs (2). In mammals, there are 3 isoforms of TGF-β: TGF-β1, TGF-β2, and TGF-β3. These highly homologous polypeptides, encoded by various genes, are synthesized, processed and regulated in a similar fashion. However, these 3 isoforms are secreted by various types of cells and signals through the same receptors, but they exhibit distinct patterns of distribution in different tissues (3, 66). Even though any isoform can be found in fibrotic tissues, the progression of organ fibrosis, in particular cardiac fibrosis, is predominantly attributed to TGF-β1 (67). To date, information on isoformspecific activities of various isoforms of TGF-β in a specific

#### TABLE 2 | Therapeutic targets/strategies for treatment of cardiac fibrosis.


pathology is lacking and needs further investigation. Next, the signaling of TGF-β, excluding conclusions regarding specific isoforms, is discussed in detail.

The synthesis, release, and activation of TGF-β is a complex process (**Figure 3**). Following intracellular biosynthesis, a dimer of TGF-β is secreted as an inactive protein complex (latent TGF-β), which is retained in the ECM. Active TGF-β1 can be liberated from ECM by multiple activators such as reactive oxygen species (ROS), plasmin, thrombospondin-1, and αvβ6 integrin (68). Once active TGF-β is released from ECM, it binds to transmembrane TGF-β receptor type II (TβRII) of a target cell. This receptor-ligand interaction induces serine/threonine kinase activity of TβRII for autophosphorylation (69). The canonical pathway of TGF-β signaling is initiated after phosphorylated TβRII forms a stable heteromeric complex with TGF-β receptor type I (TβRI), also known as activin receptor-like kinase 5 (ALK5), for the transphosphorylation of residual phosphate to TβRI (70). This receptor binding complex, which is a heterotetrameric combination between two molecules of TβRII and another two of TβRI, recruits and phosphorylates the downstream signaling proteins Smad2 or Smad3, which are called receptor-activated Smads. After phosphorylation, Smad2 or Smad3 is released and forms an intracellular complex with Smad4, the mediator Smad. This intracellular complex between Smad2/4 or Smad3/4 moves from the cytoplasm into the nucleus, where it binds to promoter regions of the genes involved in physiological process of induction of specific gene expression (71). For an example of fibrogenesis, gene encoding α-SMA, collagens, and fibronectin are significantly upregulated via the Smad3-dependent pathway (72). The expression of these fibrosis-related genes plays a pivotal role in the cellular transdifferentiation that generates myofibroblasts and the production/deposition of ECM by myofibroblasts in fibrotic tissue (72). In addition to fibrogenesis, the Smadmediated signaling pathway is also a significant intracellular process activated by TGF-β that increases genes associated with carcinogenesis (73). Furthermore, the activation of TGF-β signaling results in the expression of Smad7, an inhibitory SMAD, which acts as a negative regulator by interacting with Smad2 or Smad3, thereby mitigating signaling through receptor-activated Smads and further decreasing TGF-β actions (74).

Beyond canonical pathways or Smad-mediated signaling, TGF-β might mediate signaling directly by activating kinase enzymes via non-Smad signaling pathways, which are also known as non-canonical pathways (**Figure 4**). The non-Smad signaling pathways are initially propagated by either or both phosphorylated TβRI and TβRII for modulating downstream cellular responses. It has been reported that crosstalk between canonical and non-canonical pathways appeared to occur in most TGF-β-mediated effects (75). Epithelial-to-mesenchymal transition (EMT) plays a significant role in the pathogenesis of cancer. In part, this process requires an activation of ERK by TGF-β to upregulate the genes involving in remodeling of cell-matrix adhesion, thereby promoting the motility of the transformed cells (76). Also, EMT might be induced by TGF-β via both TβRI and TβRII through the activation

of TNF receptor-associated factor 6 (TRAF6). TRAF6 is capable of recruiting TGF-β-activated kinase 1 (TAK1) to subsequently allow the activation of c-Jun amino terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38- MAPK) (77). In addition, the TRAF6-TAK1-JNK/p38 pathway is believed to be an essential pathway for TGF-β-induced apoptosis (78). Similar to the ERK and JNK/p38-MAPK pathway, the Ras homolog gene family member A (RhoA) is also a signaling mediator of EMT. TGF-β-induce RhoA degradation by phosphorylating partitioning-defective 6 (Par6), which subsequently recruits Smad-specific E3 ubiquitin protein ligase (Smurf1) to loosen tight junctions and rearrange the actin cytoskeleton, a prerequisite step for EMT (79). Another non-Smad signaling pathway contributing to TGF-βpromoted EMT is the phosphoinositide 3-kinase (PI3K)/Akt (protein kinase B) pathway, which subsequently activates the mammalian target of rapamycin (mTOR) and phosphorylation of S6 kinase (S6K) (80, 81). In addition, TGF-β1 signaling can be regulated at the post-transcriptional level via the expression of microRNAs (miRNAs), and the expression of miRNAs might play a role in TGF-β1-mediated EMT also (82).

# TGF-β Signaling in the Development of Cancers

For the ultimate outcome of TGF-β-mediated responses in any pathological condition, it is apparent that a combination of canonical and non-canonical pathways are coordinated (1). Cancers and fibrotic diseases are the most common pathologies associated with the activity of TGF-β. Currently, most putative drugs affecting TGF-β for the treatment of cardiac fibrosis were initially developed for the management of cancer; therefore, we next discuss the signaling of TGF-β in carcinogenesis.

In the pathogenesis of cancer, TGF-β acts as a tumor suppressor in early stages of the disease. However, in later stages, TGF-β turns into a tumor promoter. This paradoxical role of TGF-β is due to a bypass of the cytostatic effect of TGF-β in tumor cells (4). The tumor suppressive effect of TGF-β is derived from various cellular effects. TGF-β stabilizes the cell cycle of epithelial cells by upregulating multiple cyclin-dependent kinases: p15, p21, and p27, via the canonical pathway (83). Also, via the Smad-dependent pathway, TGF-β downregulates genes associated with cell proliferation, such as c-Myc (84). In addition, the canonical pathway contributes to the tumor suppressive effects of TGF-β by inducing gene encoding Bcell lymphoma 2 (BCL2) and subsequently activating BIM for apoptotic processes in human B cells (85). Conversely, noncanonical pathways might mediate the apoptotic effect of TGF-β by inducing caspase-8 expression and activating BID in human gastric carcinoma cells (86). The difference in signaling of TGF-βmediated apoptosis indicates that the cellular context is essential for controlling the main pathway in the tumor suppressive effects of TGF-β. The tumor promoting effects of TGF-β such as EMT, invasion, metastasis, and angiogenesis emerge when cancer progresses to a later stage (5, 87). The upregulation of miR-106b-25 cluster targets Smad7 to ameliorate the TGF-β signaling that is not generally found in normal tissues is an excellent example of this phenomenon. In human breast cancer, increased miR-106b-25 leads to the inhibition of tumor suppressive protein p21 and BIM, thereby allowing tumor cells to grow via the

activation of TGF-β (88). Interestingly, TGF-β also regulates the functions of various immune cells, including the modulation of cytokines released from these cells. Impairment of TGF-β signaling pathways leads to immune dysregulation, fibrosis, and cancer [Reviewed in (7)]. TGF-β is produced as a complex with latency associated peptide (LAP). This complex associates with ECM by binding to latent TGF-β binding protein (LTBP) or glycoprotein A repetitions predominant (GARP) expressed on T cells, especially on Tregs, or platelets. Integrins bind to the complex and stimulate the release of TGF-β from the complex. The release of active TGF-β promotes oncogenesis and immune tolerance in breast cancer (89). Inhibition of αvβ8 integrins potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers. Cancer cells can evade host immunity by mobilizing active TGF-β1 through αvβ8 integrins (90). Thus, TGF-β acts as a significant suppressor of immune responses during tumor progression.

In general, tissue fibrosis is considered a main step in triggering cancer development. An apparent example is hepatocellular carcinoma, the most common form of liver cancer. Cirrhosis, which is known as the end-stage of liver fibrosis, occurs in most patients who ultimately develop hepatocellular carcinoma (91). Interestingly, the progression of fibrosis to cancer in the heart is rare. The low incidence of cardiac cancer might be due to the fact that cardiac cells, in particular cardiomyocytes, are fully differentiated cells. Moreover, the regenerative capacity of cardiomyocytes is considered to be negligibly low. Thus, cardiomyocytes appear to resist further transformation and proliferation processes such as EMT in the development of cancer (92). Accordingly, signaling of TGFβ in fibrogenesis of the heart might not be identical to that occurring in other organs where progressive fibrosis ultimately develops cancers.

# TGF-β Signaling in the Development of Cardiac Fibrosis

During tissue injury, TGF-β expression is increased to play a role in the tissue repair process and scar formation. In the heart tissue following MI, TGF-β signaling plays an important role in reparative, angiogenetic, and fibrotic responses by modulating inflammation (93). Studies on mice and dogs have revealed that TGF-β1 and TGF-β2 were upregulated in the early phase after MI, and then TGF-β3 was increased in a later stage post-infarction myocardium (94). Among various cells that release TGF-β, a significant amount of TGF-β might be released from infiltrated macrophages that migrate to the injured area to engulf the damaged cardiomyocytes, as shown in a mouse model (95). On the other hand, a study using a porcine model of chronic coronary constriction revealed that cardiomyocytes were a significant source of TGF-β (96). Another study suggested that TGF-β was found in the extracellular fluid of ischemic canine myocardium tissue (97). Multiple pathways involving integrins and thrombospondin-1 were found to be associated with the release of TGF-β from the cardiac ECM-bound TGF-β (98, 99). Following the release of active TGF-β, TGF-β binds to the receptors, as described earlier, to activate intracellular responses in the infarcted tissue. The TGF-β-mediated effects can be classified into 4 actions in the following order: cardiomyocyte survival, immune cell-related action, formation of myofibroblasts, and production/deposition of ECM, all of which modulate the effects on myocardial endothelial cells.

TGF-β-mediated effects on cardiomyocyte survival in MI appear to be dependent on the time period after MI. In the early phase, exogenous TGF-β administered before or immediately after ischemic injury to an isolated perfused heart showed cardioprotective effects by reducing the amount of superoxide anions, maintaining coronary relaxation, and reducing injurious responses of exogenous TNF-α (100). Similarly, a study has shown that the infarct size of intact rat hearts receiving TGF-β during early reperfusion was reduced, and this reduction was due to activation of MAPK (101). However, the mechanism underlying cardioprotection remains poorly understood. Conversely, a proapoptotic effect of TGFβ via interplay with Ang II was demonstrated in a study using rat cardiomyocytes (102). The findings showed that the actions of exogenous TGF-β are likely dependent on the timing of administration.

Immune cells play a pivotal role in fibrogenesis, and TGFβ regulates both the phenotype and function of the immune cells. It is worth noting that TGF-β can be either a pro- or anti-inflammatory mediator of the immune response in in vitro studies [Reviewed in (93)]. Factors that determine the effects of TGF-β include the types of cytokines and the origin of the tissue (103). In an in vivo study, TGF-β suppressed T cell-mediated inflammation in genetically modified mice with T cell-specific loss of TβRII. Thus, the results from this in vivo study implicate an immunosuppressive effect of TGF-β (104). Nevertheless, the specific TGF-β-mediated effects on the phenotype of immune cells, together with its signaling and significance in the regulation of fibrosis, in the infarcted tissue remain unknown in the infarcted tissue.

TGF-β-mediated effects on the formation of myofibroblasts and on the induction of transformed myofibroblasts to further produce/deposit ECM are currently recognized central to the role of TGF-β in the pathogenesis of fibrosis. In cardiac fibrosis, Smad3-deficient mice that underwent reperfused MI showed significantly less fibroblast proliferation and ECM when compared to those of wild-type mice (105, 106). Even though the origin of the cells that underwent transformation has been debated (107), a recent study using fibroblast-specific, TGF-β signaling pathway knockout mice demonstrated that myofibroblasts in cardiac fibrosis are derived from resident fibroblasts, which activated via the TGF-β-Smad2/3 signaling pathway (72). These results suggest that the canonical pathway of TGF-β is principally involved in the pathogenesis of cardiac fibrosis. Interestingly, it was found that the Smad3-dependent pathway is essential for the upregulation of connective tissue growth factor (CTGF), which in turn acts as a mediator to stimulate fibroblast differentiation and collagen synthesis (108). Beyond the formation of myofibroblasts, genes encoding collagen type I and III were upregulated in cardiac fibroblasts isolated from rabbit hearts following treatment with TGF-β (109). The TAK1/p38-MAPK pathway in the cardiomyocytes of non-infarcted myocardium was found to be activated in rats after acute MI, suggesting a role for this non-canonical pathway in ventricular hypertrophy and remodeling (110). Nevertheless, the significance of Smad-independent pathways in the transformation of cardiac fibroblasts appears to be less proven than that of renal and pulmonary fibrosis (111, 112). Finally, a study on TGF-β-overexpressed mice showed increase expression of tissue inhibitors of matrix metalloproteinases (TIMPs), which regulate the remodeling of ECM in the cardiac tissue. However, the signaling of TGF-β was not evaluated in this study (113).

In addition to cardiomyocytes, immune cells, and transformed myofibroblasts, vascular endothelial cells might also play an important role in cardiac fibrosis. It has been found that endothelial cells served as a source of chemokines and played a role in recruiting neutrophils and monocytes to the heart after MI (114). Interestingly, although TGF-β plays a role in angiogenesis in cancers (8), information on the effects of TGFβ on angiogenesis in infarcted myocardium is limited at present. Moreover, although most cardiac myofibroblasts originate from resident fibroblasts, a study has shown that endothelial cells might be activated by the TGF-β via Smad3-dependent pathway and transform into myofibroblasts, thereby inducing cardiac fibrosis (115).

# TGF-β INHIBITORS FOR THE TREATMENT OF CANCERS AND CARDIAC FIBROSIS

# Inhibitors of TGF-β Signaling for the Treatment of Cancers

TGF-β suppresses cell proliferation leading to apoptosis in the early phase of tumor development, whereas it aggravates tumor invasion and metastasis via boosting immune escape, angiogenesis, and EMT of tumors at an advanced stage (116). The paradoxical impact of TGF-β signaling in various tumors raises concerns that anti-TGF-β signaling might lead to a poor prognosis due to its tumor suppressor role. This concern has delayed progression in the development of TGF-β inhibitors as therapeutic agents. In addition, some experimental models have revealed that TβRI inhibitors aggravated the potential for cardiotoxicity (117).

However, several potential approaches to interfering with TGF-β signaling to prevent TGF-β production and block its signaling pathway have emerged. Next, we summarize the results of TGF-β inhibitors that have been studied in preclinical or clinical trials on carcinogenesis. The studies can be mainly categorized into 3 levels: (1) The ligand level: Direct blockage of TGF-β ligand synthesis by antisense molecules; (2) The ligandreceptor level: Inhibition of TGF-β ligand-receptor interaction using monoclonal antibodies or soluble TGF-β decoy receptors (traps); and (3) The intracellular level: Suppression of the TGF-β signaling pathway by tyrosine kinase inhibitors that disturb the downstream signaling of TGF-β related proteins (9, 118). The examples of current therapeutic agents in preclinical and clinical development in oncology are summarized in **Tables 3**, **4**.

#### Trabedersen (AP12009)

#### **Preclinical data**

Trabedersen (AP12009, Antisense Pharma) is a synthetic, 18 oligomer phosphorothioate antisense oligonucleotide (ASO). It was developed as an ASO specifically targeting human TGFβ2 mRNA, which leads to a reduction in TGF-β2 expression, cellular proliferation, and cellular migration in various types of tumors in vitro and in vivo, including gliomas (119), melanoma (120), pancreatic carcinomas (121, 122), and colorectal cancer (123). Trabedersen has been shown to reduce cell proliferation, tumor growth, cell migration or metastasis, and vascularization TABLE 3 | Preclinical studies of TGF-β inhibitors for cancer treatment.


in human pancreatic cancer cells and in mouse model of human metastatic pancreatic cancer (122).

#### **Clinical data**

After several preclinical studies provided evidence of potential clinical efficacy, trabedersen was moved to phase I/II trials in patients with recurrent high-grade gliomas (119, 140, 149). Trabedersen was initially assessed for its safety and efficacy in phase I/II dose escalation studies in patients with highgrade gliomas and found a significant increase of median survival time after recurrence, exceeding that of standard chemotherapy (149). Similarly, prolonged survival and high response rates after treatment with trabedersen were observed in phase I/II studies in patients with recurrent or refractory malignant glioma, WHO grade III or IV (119). However, trabedersen was further compared with standard chemotherapy (temozolomide or procarbazine/lomustine/vincristine) in patients with recurrent or refractory malignant glioma (WHO grade III or IV) in a phase IIb trial. The results revealed that trabedersen did not control tumor growth, but delayed responses were observed after discontinuation of treatment (140).

#### Belagenpumatucel-L Vaccine

The principle of anti-TGF-β cancer vaccines is to deliver antisense molecules of TGF-β into cancer cells and overturn the effects of immunosuppression in host cells, as well as to enhance antitumor immunity (9). Belagenpneumatucel–L (Lucanix, NovaRx) is a TGF-β2, antisense, gene-modified non-viral based allogenic tumor cell vaccine. It was developed from non-small cell lung cancer (NSCLC) and modified to express ASO, which leads to suppression of the immunosuppressive activity implicit in TGF-β2 overexpressing cancer cells (141).

#### **Clinical data**

Currently, an anti-TGF-β cancer vaccine, belagenpumatucel-L, has entered a phase III study to determine whether it improves overall survival (OS) and might be useful for stimulating immune reactions. A dose-related survival difference was achieved in patients who received belagenpumatucel-L at least 2.5 × 10<sup>7</sup> cells/injection in a phase II trial involving patients with stages II, III, and IV NSCLC. Moreover, immune function measurements revealed an increase in cytokine production, including IFN-γ, IL-6, and IL-4, among clinical responders, who also displayed an elevated antibody-mediated response to the vaccine human leukocyte antigens (HLAs) (141). Likewise, a further study to evaluate its safety and response at the previously defined optimal dose found the median survival of patients with fewer than 2 circulating tumor cells (CTCs) at baseline was longer than patients with 2 or more CTCs. Thus, plasma levels of CTCs are associated with the OS of patients with stage IV NSCLC (142). Nevertheless, in a phase III trial with 532 patients with stage III/IV NSCLC who did not progress after platinumbased induction chemotherapy with or without irradiation, TABLE 4 | Clinical studies of TGF-β inhibitors for cancer treatment.


belagenpumatucel-L did not increase survival compared with placebo (143).

### Galunisertib (LY2157299)

#### **Preclinical Data**

#### Fresolimumab (GC1008)

#### **Clinical Data**

Fresolimumab (GC1008, Genzyme/Sanofi) is a fully human monoclonal antibody blocking pan-TGF-β (TGF-β1, TGF-β2, and TGF-β3) [Reviewed in (150)]. Fresolimumab demonstrated acceptable safety and preliminary evidence of antitumor activity in a phase I trial on patients with previously treated malignant melanoma or renal cell carcinoma (151). In a phase II trial on 13 patients with malignant pleural mesothelioma, 3 patients showed stable disease for at least 3 months, and those who produced antitumor antibodies had an increased median OS. However, treatment with fresolimumab had no effect on the expression of NK, CD4+, or CD8<sup>+</sup> T cell activating and inhibitory markers, other than a decrease in the expression of CD244 (also known as 2B4) and CD266 (best known as DNAM1) on NK cells (144). A phase II trial on 23 patients with metastatic breast cancer undergoing radiotherapy has reported that fresolimumab in combination with focal radiotherapy significantly increased OS and was well-tolerated in a dose-dependent manner. Higher doses of fresolimumab correlated with an improved CD8<sup>+</sup> pool, leading to a favorable systemic immune response and longer median OS (145).

Galunisertib monohydrate (LY2157299, Eli Lilly) is a smallmolecule inhibitor of TβRI that robustly downregulate the phosphorylation of Smad2 in pancreatic, lung, colorectal (129), and ovarian cancer (130). Galunisertib effectively demonstrated potent inhibition of both canonical and noncanonical pathways in a variety of in vitro hepatocellular carcinoma cells regardless of TGF-β pathway protein expression (131, 132). Nevertheless, the antiproliferative activity of TGF-β pathway inhibitors is quite limited. It has been reported that TGF-β inhibited cell proliferation while inducing apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7). However, cancer cells were sensitive to TGF-β-dependent growth inhibition and displayed limited sensitivity to galunisertib in another group of cell lines expressing high quantities of TGF-β and Smad7 and showing significantly reduced Smad3 signaling (SK-HEP1, SK-Suni, SK-Sora, JHH6, HLE, HLF, and FLC-4) (132, 133). Despite limited antiproliferative activity in vitro, galunisertib exhibited antiproliferative effects in ex vivo models, indicating that inhibition of TGF-β can exert anticancer properties (131, 133). Nevertheless, from the reports on several preclinical studies, treatment with TGF-β inhibitors as monotherapy might display limited efficacy. However, the immunological effects of galunisertib are strongly augmented in combination with other checkpoint inhibitors (152, 153).

#### **Clinical data**

Among small molecule inhibitors, galunisertib is one of the most advanced. It has shown promising results in clinical trials due to its safety profile, with no cardiac potential toxicity in humans, which was a primary concern with first-generation TGF-β inhibitors (154). A phase I study on 28 patients with Grade IV glioma showed galunisertib was well-tolerated. The dose limiting toxicities included pulmonary embolism and thrombocytopenia, but no cardiotoxicities were observed (155). In addition, the safety of galunisertib was confirmed by a first-in-human dose study with 79 cancer patients with glioma and solid tumors treated with galunisertib as monotherapy or in combination with lomustine. No medically relevant cardiac toxicity or signs of cardiovascular injury were found, including increased blood pressure, troponin I, BNP, or hs-CRP or reductions in cystatin C levels (156). Likewise, no safety concerns or dose limiting toxicities was observed after treatment with galunisertib in patients with glioblastoma based on a pharmacokinetic/pharmacodynamic (PK/PD) model (157). Galunisertib as monotherapy and as second-line therapy after sorafenib failure in a subset of 109 patients with hepatocellular carcinoma yielded a median OS of 8.3 months in a phase II trial (147). Interestingly, patients who had decreased expression levels of specified blood biomarkers [e.g., alpha-fetoprotein (AFP), TGF-β1, and CDH1] had improved clinical outcomes, indicating that the effects of galunisertib might be more pronounced in patients with a poor prognosis due to elevated AFP at baseline (147). Similarly, galunisertib in combination with gemcitabine improved OS with minimal added toxicity in a phase II study on patients with locally advanced or metastatic pancreatic adenocarcinoma who were considered candidates for first-line chemotherapy with gemcitabine (146).

### Vactosertib (EW-7197) and EW-7195

#### **Preclinical Data**

Vactosertib (EW-7197 or TEW-7197), a novel small molecule inhibitor of ALK5, has been recently developed as a more potent and specific antitumoral compound than galunisertib. Vactosertib and EW-7195 expressed potent antimetastatic activity in vivo via an inhibition of TGF-β1-induced Smad/TGFβ signaling, cell migration, invasion, EMT, and breast tumor metastasis to the lung in xenografted nude mice and transgenic MMTV/cNeu mice (134, 135). In addition, vactosertib expressed the potential to boost cytotoxic T lymphocyte function in 4T1 orthotopic-grafted mice and prolonged the lifespan of 4T1 breast tumor-bearing mice (134).

#### **Clinical data**

Vactosertib is currently being tested in phase I/II clinical trials for several cancer types in combination with chemotherapy or antibodies against immune checkpoints. A phase I study is evaluating the safety and tolerability of the drug in combination with paclitaxel in 12 metastatic gastric cancer patients (NCT03698825). The phase Ib/IIa trials include a study of vactosertib in combination with durvalumab in patients with advanced NSCLC who progressed following platinumbased chemotherapy (N = 63) (NCT03732274). A combination with pembrolizumab is being employed for metastatic or locally advanced colorectal or gastric/gastroesophageal junction adenocarcinoma (N = 67) (NCT03724851), and a combination with imatinib is being employed for patients with advanced desmoid tumors (N = 24) (NCT03802084). The latest phase II trial aims to determine whether administration of vactosertib with durvalumab will provide meaningful increases in the overall response rate in patients with urothelial cancers that fail to achieve a CR with anti-PD-1/PD-L1 based regimens (N = 48) (NCT04064190).

Remarkably, given TGF-β signaling plays a crucial role in fibrotic states, vactosertib has recently been investigated as an antifibrotic agent to delay the development of fibrosis in primary organs including the liver, kidney, and lung. Vactosertib was found to suppress fibrosis-induced accumulation of ROS and ECM proteins (collagen, α-SMA, fibronectin, and integrins) in the liver, lungs, and kidneys of mice due to its antifibrotic mechanism via inhibition of both TGF-β1/Smad2/3 and ROS signaling (158). A study on a rat model of Peyronie's disease showed that vactosertib suppressed phospho-Smad2 expression and recruitment of inflammatory cells, leading to a decline in fibrotic plaques (159). Thus, vactosertib and EW-7195 could be a promising antifibrotic compound for the treatment of fibrotic diseases.

#### Tasisulam (LY573636)

#### **Clinical Data**

Tasisulam has completed many trials in various oncologic diseases, including phase I studies on patients with essential thrombocythemia and acute myeloid leukemia (NCT00718159) and solid tumors (NCT01214668) and phase II trials on patients with ovarian cancer (NCT00428610), metastatic breast cancer (NCT00992225), NSCL cancer (NCT00363766), and malignant melanoma (NCT00383292). A phase II study on tasisulam as second- or third-line treatment for 101 patients with unresectable or metastatic soft tissue sarcoma reported that tasisulam demonstrated modest activity with a median OS of 8.71 months (148). Consequently, the synergistic and additive effects of tasisulam combined with other anticancer agents are currently of interest. Currently there is an ongoing phase I trial of tasisulam in combination with sunitinib, a multiple tyrosine kinase, in renal cancer patients (NCT01258348), and with pemetrexed, an inhibitor of purine synthesis, in patients with solid tumors (NCT01215916).

#### **M7824 (MSB0011359C)**

Interestingly, recent preclinical study has been reported that M7824 (MSB0011359C) which is a dual inhibitor of programmed death ligand 1 (PD-L1) and TGF-β inhibited tumor growth and metastasis more effectively than treatment with TGF-β inhibitor alone. Thus, M7824 (an inhibitor of PD-L1 and TGF-β) exhibits potent and superior antitumor effects compared to that of TGFβ inhibitor monotherapy and is likely to help minimize potential side effects (160).

# Inhibitors of TGF-β Signaling for the Treatment of Cardiac Fibrosis

The renin-angiotensin system (RAS) inhibitors are currently used as standard therapy for HF and have been shown to inhibit activation of fibroblast and differentiation into myofibroblast. However, cardiac fibrosis persists in patients with HF even when treated with these conventional RAS inhibitors, indicating a need to develop novel and effective antifibrotic therapies for heart disease (161). Currently, due to its established role in cardiac fibrosis, there is great interest in inhibiting the TGF-β signaling pathway (6, 161). TGF-β is considered a mediator of cancer and fibrosis. Thus, blockades of TGF-β signaling activity using receptor antagonists, inhibition via antibody or antisense oligonucleotide, or even using gene deletion of TGF-β signaling molecules are potential therapeutic strategies.

Anti-TGF-β1 neutralizing antibodies have also been under investigation as potential antifibrotic agents by interfering with TGF-β signaling. Administration with anti-TGF-β1 antibody attenuated cardiac fibrosis and diastolic abnormalities in a rat model of pressure overload (47) (**Table 2**). Although these antibodies attenuated fibroblast activation and collagen synthesis, no improvements in overall cardiac functions were found in pressure-overloaded rats (47). Furthermore, anti-TGF-β neutralizing antibody inhibited ECM proteins synthesis and reduced cardiac fibrosis in a rat model induced by a chronic blockade of nitric oxide synthesis (162). However, in a mouse model of MI, a neutralizing anti-TGF antibody administered before or after coronary artery ligation resulted in increased mortality rates and left ventricular (LV) dilation after MI (163).

Alternative approaches have included inhibition of the expression of TGF-β using antisense oligonucleotides (164), and the use of a soluble TβRII, which either acts by adsorbing TGFβ or acting as a dominant negative receptor (165). Inhibitors of ALK5 (TβRI) are under investigation for antifibrotic effects in the heart. Inhibitor of ALK5 which decrease TGF-β activity can rescue cardiac dysfunction and ameliorate cardiac remodeling in post-MI hearts (50). Moreover, ALK5 inhibitors can also suppress the collagen synthesis and attenuate the progression of fibrosis in animal model of pressure overload induced by transverse aortic constriction, and inhibit TGF-β-mediated collagen synthesis in cardiac fibroblasts (51) (**Table 2**).

In addition to the canonical Smad-mediated signaling pathway, TGF-β also stimulates the non-canonical MAPK signaling pathways such as JNK-dependent and p38-MAPKdependent pathways (166–168). These MAPK signaling pathways are involved in TGF-β-mediated activation of TAK1 which is thought to play a role in cardiac fibrosis and remodeling. Cardiac specific overexpression of the active form of TAK1 induced myocardial hypertrophy and HF (166–168), suggesting that TAK1 is a major effector of TGF-β signaling. Blockade of TAK1 activity attenuated TGF-β-mediated ECM protein overproduction in cardiac fibroblasts (48) (**Table 2**). In addition to inhibition of TAK1, inhibition of p38-MAPK is being investigated for its efficacy in the treatment of cardiac fibrosis. Inhibitors of p38-MAPK suppress myofibroblast activation and expression of ECM proteins and α-SMA induced by TGF-β, while overexpression of p38-MAPK induces myofibroblast differentiation in cardiac fibroblasts (49).

Two promising antifibrotic agents include tranilast and pirfenidone, which inhibit the actions of TGF-β as well as other pathogenic growth factors by unclear mechanisms (169). Current agents and therapeutic targets in preclinical and clinical development for the treatment of cardiac fibrosis and heartrelated diseases are summarized in **Tables 5, 6**.

#### GW788388

#### **Preclinical data**

GW788388 is a potent inhibitor of both ALK5 and TGβRII with an improved pharmacokinetic profile (184) and minimal toxic effects (185). Several studies have been demonstrated that GW788388 pre-clinically reduces cardiac fibrosis in various models. GW788388 inhibited the development of cardiac fibrosis by suppression of collagen I and fibronectin synthesis, increased survival, and improved cardiac function in an experimental murine model of Chagas heart disease (170). Deletion of SCN5A, a gene encoding the main cardiac sodium channel NaV1.5, has been associated with inherited progressive cardiac conduction disease. GW788388 chronically inhibited TGF-β receptors and prevented fibrosis in a Scn5a heterozygous knockout (Scn5a+/−) mouse model of progressive cardiac conduction disease (171). Furthermore, treatment with GW788388 attenuated systolic dysfunction and delayed LV remodeling by reducing the phosphorylated Smad2, α-SMA, and collagen I in a rat model of HF following MI (50). Taken together, GW788388 appears to be a promising antifibrotic agent, although further studies are warranted.

### Pirfenidone

#### **Preclinical data**

Pirfenidone is an oral antifibrotic drug initially approved for the treatment of idiopathic pulmonary fibrosis (186). Pirfenidone inhibited TGF-β expression and also inhibited the profibrotic effects of TGF-β signaling (187). Thus, pirfenidone might be a promising agent for the treatment of cardiac fibrosis. A reduction in ventricular hypertrophy without lowering systolic blood pressure has been detected in the deoxycorticosterone acetate (DOCA)-salt hypertensive rats after pirfenidone treatment (172). Moreover, pirfenidone decreased total and non-scar myocardial fibrosis, which has been associated with decreased infarct scarring, improved LV function, and decreased ventricular tachycardia in rat MI model (173). Administration of pirfenidone reversed cardiac fibrosis, including renal fibrosis, and attenuated myocardial stiffness in streptozotocin (STZ)-diabetic rats (176).

Given pirfenidone has significant antifibrotic and antiinflammatory properties, the anti-inflammatory effects of pirfenidone have been investigated. Pirfenidone inhibited NLRP3 expression and formation, contributing to a reduction in IL-1β synthesis, and attenuation of IL-1β-induced inflammatory and profibrotic responses in a mouse model with transverse aortic constriction (TAC)-induced LV remodeling (174). Similar effects were observed in murine pressure-overload injury; pirfenidone increased survival and attenuated fibrosis through suppression


TABLE 6 | Clinical studies of TGF-β inhibitors for treatment of cardiac fibrosis.


of myocardial fibrosis and vascular permeability in pressureoverloaded hearts (175). Therefore, pirfenidone might be a potential treatment for cardiac fibrosis.

#### **Clinical data**

Although pirfenidone has shown efficacy in the treatment of idiopathic pulmonary fibrosis in humans (186), clinical trials for the treatment of cardiac fibrosis are ongoing and the results have not yet been published. A phase II study of pirfenidone in patients with hypertrophic cardiomyopathy associated with LV diastolic function aims to examine the effectiveness of pirfenidone in improving heart function and reducing of myocardial fibrosis. The study was completed with unpublished data (NCT00011076). Another phase II trial is ongoing and will finish in Jan 2020. This trial is exploring the antifibrotic effects of pirfenidone on patients with chronic heart failure with preserved ejection fraction (HFpEF) and cardiac fibrosis by determining changes in myocardial ECM volume and investigating the relationship between myocardial fibrosis and myocardial energetics (PIROUETTE study, NCT02932566) (188).

#### Tranilast

#### **Preclinical data**

Tranilast has been used to treat allergic disorders (e.g., allergic rhinitis, asthma, and atopic dermatitis); however, tranilast might also be useful for other medical conditions due to its ability to suppress TGF-β expression and activity. The molecular mechanisms underlying its antifibrotic actions are not completely understood, but tranilast might inhibit several profibrotic growth factors such as TGF-β and platelet-derived growth factor (PDGF) (22). The effects of tranilast on inhibition of cardiac fibrosis have also been supported by multiple animal models of cardiomyopathy. In STZ-induced (mRen-2)27 diabetic rats, tranilast treatment attenuated cardiac matrix deposition in association with reductions in phospho-Smad2 of the heart (177). In a similar model, administration of tranilast attenuated cardiac dysfunction and structural abnormalities in diabetic cardiomyopathy with improved LV systolic and diastolic function, while tranilast did not affect Smad phosphorylation but it significantly attenuated TGF-β-induced p44/42 MAPK phosphorylation (178).

The underlying mechanisms of the antifibrotic effects of tranilast have been attributed to its regulation of TGF-β signaling and to suppression of the infiltration of inflammatory cells, including monocytes and macrophages. The mRNA levels of TGF-β1, plasminogen activator inhibitor 1 (PAI-1), monocyte chemotactic protein-1 (MCP-1), IL-6, procollagens were attenuated, and myocardial fibrosis and collagen accumulation were suppressed in DOCA/salt hypertensive rats receiving tranilast (179). Similar findings were observed in other animal models of renovascular hypertensive rats (180) and hypertensive (mRen-2)27 rats (182). Interestingly, tranilast-mediated inhibition of cardiac fibrosis is independent of changes in blood pressure in these studies, suggesting that tranilast directly targeted cardiac fibrosis and might be beneficial for HF treatment in addition to current therapeutic strategies (181).

#### **Clinical data**

Restenosis after percutaneous coronary intervention (PCI) is a major adverse outcome following stent placement. In limited trials, administration of tranilast reduced the frequency of angiographic restenosis after PCI (189). Accordingly, the Prevention of Restenosis With Tranilast and Its Outcomes (PRESTO) trial was designed as a phase III trial with a large group of patients after PCI to investigate major adverse cardiovascular events of tranilast. It was found that tranilast did not improve restenosis or its clinical sequelae in patients receiving successful PCI (183). However, the number of events of MI was significantly reduced with tranilast treatment. The most commonly reported adverse events were laboratory test abnormalities consisting of hyperbilirubinemia, elevations in hepatic enzymes, and increased serum creatinine (183).

# CONCLUSION

TGF-β is a multifunctional cytokine regulator acting through transmembrane serine/threonine kinase receptors and intracellular Smad transcriptional regulators. Once TGF-β is activated, it regulates ECM remodeling and promotes a fibroblast to myofibroblast transition, which is essential for fibrotic processes. Given TGF-β plays a major role in various stages of cancer progression and in the development of cardiac fibrosis, TGF-β and its signaling pathway offer opportunities for novel treatment strategies in patients with cancer and

# REFERENCES


cardiac fibrosis. Research on the underlying mechanisms and the therapeutic targets of TGF-β inhibitors for cancer and cardiac fibrosis has advanced significantly in recent decades. The inhibitors of TGF-β signaling for cancer and fibrosis have been extensively studied in animal models and clinical studies; however, translation of these findings into human pathologic conditions has been limited due to the broad range of responses to TGF-β and its role in tissue homeostasis. Currently, various types of TGF-β inhibitors are challenged and tested their efficacies in patients with cancers. A few of TGF-β inhibitors are subjected into the clinical studies for treatment of cardiac fibrosis. The development of more specific agents targeting TGF-β signaling pathways such as M7824, a bifunctional fusion protein composed of TGF-β trap, and a monoclonal antibody against programmed death ligand 1 (PD-L1) are likely to help minimize potential side effects and enhances efficacy for treatment of cancers. Furthermore, the combination of anti-TGF-β therapies with various mechanisms of action might have greater efficacy against cancer and cardiac fibrosis.

# AUTHOR CONTRIBUTIONS

WP, TL, and SM wrote the manuscript. HK reviewed and edited. All authors agree to submit the manuscript.

# ACKNOWLEDGMENTS

This work was supported in part by grants from JSPS KAKENHI Grant Number JP17H01525 and the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (2017K1A1A2004511) (to HK), and Thailand Research Fund [Grant RSA6080061] (to SM).


first one to five hours after reperfusion. Circulation. (1997) 95:684–92. doi: 10.1161/01.CIR.95.3.684


of monocyte/macrophage infiltration in DOCA/salt hypertensive rats. J Hypertens. (2004) 22:1007–15. doi: 10.1097/00004872-200405000-00024


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Parichatikanond, Luangmonkong, Mangmool and Kurose. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cardiotoxicity of Anthracyclines

Daniela Cardinale<sup>1</sup> \*, Fabiani Iacopo<sup>1</sup> and Carlo Maria Cipolla<sup>2</sup>

<sup>1</sup> Cardioncology Unit, European Institute of Oncology, IRCCS, Milan, Italy, <sup>2</sup> Cardiology Division, European Institute of Oncology, IRCCS, Milan, Italy

Cardiotoxicity is a feared side effect that may limit the clinical use of anthracyclines. It may indeed affect the quality of life and survival of patients with cancer, regardless of oncological prognosis. This paper provides an overview of anthracycline-induced cardiotoxicity in terms of definition, classification, incidence, risk factors, possible mechanisms, diagnosis, and treatment. We also report effective strategies for preventing cardiotoxicity. In addition, we discuss limiting current approaches, the need for a new classification, and early cardiotoxicity detection and treatment. Probably, anthracycline-induced cardiotoxicity is a continuous phenomenon that starts from myocardial cell injury; it is followed by left ventricular ejection fraction (LVEF) and, if not diagnosed and cured early, progressively leads to symptomatic heart failure. Anthracycline-induced cardiotoxicity can be detected at a preclinical phase. The role of biomarkers, in particular troponins, in identifying subclinical cardiotoxicity and its therapy with angiotensin-converting enzyme inhibitors (mainly enalapril) to prevent LVEF reduction is a recognized and effective strategy. If cardiac dysfunction has already occurred, partial or complete LVEF recovery may still be obtained in case of early detection of cardiotoxicity and prompt heart failure treatment.

#### Edited by:

Canan G. Nebigil, École supérieure de biotechnologie Strasbourg (ESBS), France

#### Reviewed by:

Sherry-Ann Brown, Mayo Clinic, United States Giselle Melendez, Wake Forest University, United States

#### \*Correspondence:

Daniela Cardinale daniela.cardinale@ieo.it

#### Specialty section:

This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine

> Received: 14 November 2019 Accepted: 18 February 2020 Published: 18 March 2020

#### Citation:

Cardinale D, Iacopo F and Cipolla CM (2020) Cardiotoxicity of Anthracyclines. Front. Cardiovasc. Med. 7:26. doi: 10.3389/fcvm.2020.00026 Keywords: cardiotoxicity, anthracyclines, early detection, troponin, prevention, reversibility, ACE-inhibitors, beta-blockers

# INTRODUCTION

Anthracyclines are cytostatic antibiotics (1), introduced into the clinical field in the 1960s. As of 2012, anthracyclines were among the most diffused chemotherapeutic agents, and they still represent the base of treatment in many solid cancers and hematological malignancies (1, 2).

Unfortunately, anthracyclines are considered the principal culprit drugs behind chemotherapy-induced cardiotoxicity (1–5). The pathognomonic manifestation of anthracycline-induced cardiotoxicity is a hypokinetic cardiomyopathy progressively leading to heart failure, first described in 1967 (6). The onset of anthracycline-cardiomyopathy, also at the pre-clinical stage, may negatively affect the cardiovascular outcome of patients as also limit the chemotherapeutic strategies (4, 5).

# INCIDENCE AND RISK FACTORS

The risk of anthracycline-induced heart failure increases as the cumulative dose administered increases: 3–5% with 400 mg/m<sup>2</sup> and as high as 18–48% at 700 mg/m<sup>2</sup> (4). However, there is a different level of risk for each patient scheduled for anthracycline therapy: patients less than 5 years old or more than 65 years old, with prior or concurrent chest irradiation, pre-existing heart disease, or already known cardiovascular risk factors, have an increased risk for cardiotoxicity



AF, atrial fibrillation; CABG, coronary artery bypass graft; CAD, coronary artery disease; CV, cardiovascular; LV, left ventricular; LVEF, left ventricular ejection fraction; LVH, left ventricular hypertrophy; PCI, percutaneous coronary intervention; VHD, valvular heart disease.

(**Table 1**) (4, 7). Moreover, anthracycline-induced cardiotoxicity risk increases with the use of other agents that may increase its incidence. In particular, trastuzumab, while very effective in treating breast cancer, interferes with myocyte survival pathways, crucial in countering the toxic effects of anthracyclines (5, 7, 8).

# MECHANISMS

The specific mechanisms of anthracycline cardiotoxicity still remain unclear. A potential mechanism is the generation of reactive oxygen species (ROS), changes in iron metabolism, and Ca2þ signaling. In 2014, topoisomerase (Top) 2β was indicated as the critical mediator of anthracycline's cardiac toxic effect (9). Top2 can uncoil deoxyribonucleic acid (DNA) filaments during DNA replication, transcription, or recombination. The anthracycline inhibition of Top2β causes mitochondrial dysfunction and leads to activation of cell death pathways and ROS deposit (2, 3, 10).

The cardiomyocyte has always been considered the main cellular target of anthracycline toxic effect in the heart, as their destruction results in the progressive development of cardiac dysfunction. More recently, however, other cell types—such as cardiac progenitor cells, cardiac fibroblasts, and endothelial cells—have been identified as potential additional targets, creating a more complex and intriguing scenario in the pathogenesis of anthracycline-induced cardiomyopathy (**Figure 1**) (11). So far, the principal mechanisms, with potential differential impact and grade of involvement in different cell types, are oxidative stress, DNA damage, senescence, and cell death.

# CLASSIFICATION

A previous and more dated classification identified three distinct types of anthracycline-induced cardiotoxicity (**Table 2**): acute, occurring after a single dose, or a single course, with the onset of symptoms within 14 days from the end of treatment, which is usually reversible; early-onset chronic, occurring within 1 year, the principal form of cardiotoxicity, from a clinical and epidemiological stand-point, presenting as a dilated-hypokinetic cardiomyopathy, with progressive evolution toward heart failure; and late-onset chronic, developing years, possibly decades, after the end of anthracycline therapy. The two chronic forms are considered irreversible, with a poor prognosis and a limited to heart failure therapy. This classification stems back to early 1980s, and it is mainly based on small retrospective studies reporting the occurrence of heart failure symptoms in childhood cancer survivors (12–14). In particular, in a milestone study, Steinherz et al. reported cases of heart failure occurrence many years after the end of anthracycline-chemotherapy, and the percentage of patients with cardiac dysfunction, as well as the severity of the dysfunction itself, increased in parallel with time elapsed from the end of anthracycline administration (14). However, the clinical relevance of such a classification at present is uncertain, especially when referred to adult populations.

In particular, recent findings challenge this old classification, suggesting that anthracycline-induced cardiotoxicity is potentially a continuous phenomenon, starting at the myocardial cell level, followed by progressive functional decline, progressively leading to overt heart failure. (**Figure 2**) (5, 8, 15). To be practical, anthracycline-associated cardiotoxicity is now thought to occur at the time of first exposure, a hypothesis supported by the finding of troponin release after anthracycline administration (16). Clinical presentation may occur years later the initial damage (16–18). Looking at symptoms, the diagnosis may take years ("late" cardiotoxicity). Considering LVEF reduction, it may take months ("early" cardiotoxicity). With the use of circulating biomarkers, such as troponin (pre-clinical myocardial cell damage), prompt identification of cardiotoxicity is possible, allowing for an "acute" form. So far, we are probably observing the evolving stages of the same phenomenon and not three distinct diseases (15, 17, 18).

# DIAGNOSIS AND DEFINITION

The diagnosis of anthracycline-induced cardiotoxicity has remained the same over the last 60 years. It has always been based on heart failure symptoms, and, later, also on evidence of LVEF drop (echocardiography or multi-gated acquisition scans) (4, 18). A former definition adopted was an LVEF absolute decrease higher than 10% points, associated with a decline <50% (5). More recently, the consensus [Plana et al. (19)] defined it

as an LVEF decrying >10% points, with a final value <53% (19). In patients at low risk—i.e., without risk factors or a negative cardiovascular history, with an indication to receive a low dose of anthracyclines (total cumulative dose ≤240 mg/m²) or standard dose followed by trastuzumab-based regimens cardiac monitoring is not suggested by the American Society of Clinical Oncology guidelines. Moreover, they suggest a diagnosis of cardiotoxicity based on clinical symptoms (20). Reasons comprise "medicalization, the possibility of causing stress and anxiety, and costs" to be incurred (20, 21). Otherwise, the international cardiological guidelines recommend monitoring of cardiac function by serial LVEF measurements, but do not provide an accurate indication on timing, frequency, modalities, and long-term schedule (7). Moreover, a diagnosis based on symptoms or asymptomatic decrease of LVEF is not only delayed, but also potentially prevents any form of effective prevention, as the cardiac damage may be no longer reversible (17, 18).

A recent study evaluating a significant (n = 2,625) population scheduled for anthracycline therapy showed that close monitoring of LVEF after chemotherapy allowed nearly all (98%) cases of cardiotoxicity to be identified within the first 12 months of follow-up (15). In addition, early treatment with angiotensin-converting enzyme (ACE)-inhibitors (enalapril) and beta-blockers (carvedilol or bisoprolol) enabled normalization of cardiac function in most cases (82%), but only 11% of patients who had renormalized LVEF had full recovery—i.e., the same LVEF value as before the start of anthracyclines—while the final LVEF value in 71% of patients remained below the baseline value (**Figure 3**).



These findings confirm that this approach is limited in identifying reversible cardiotoxicity, probably because left ventricular compensation mechanisms have been exhausted (8). Of great importance, the evidence of a normal LVEF does not exclude the risk of future deterioration of cardiac function.

# TREATMENT

The historical concept that anthracycline-induced cardiotoxicity is irreversible, with a reported mortality rate up to 60% within 2

From Cardinale et al. (8).

years of diagnosis, is now reconsidered. In particular, this belief is based on seminal studies in which heart failure therapeutic strategies were limited (i.e., digoxin, diuretics), or on studies with small populations, retrospective design, short follow-up, or on case reports (22–30).

Up until 2010, the response to heart failure therapy of patients with anthracycline-induced cardiotoxicity hadn't been thoroughly investigated. Moreover, these kind of patients have been excluded from large randomized trials evaluating the impact of current heart failure therapies (8).

The effectiveness of ACE-inhibitors and beta-blockers has been prospectively assessed in two extensive papers (15, 31). In 201 patients with anthracycline-induced cardiotoxicity, an inverse relationship in terms of LVEF improvement has been found between the time interval from the end of chemotherapy and the beginning of heart failure therapy (**Figure 4A**) (31). LVEF recovery rate was 64% in those treated early (i.e., within 2 months after the end of chemotherapy); later on, however, this percentage rapidly decreased, with no complete recovery after 6 months. After 12 months, obtaining even partial LVEF improvement was almost impossible (**Figure 4B**) (31). It emerges that cardiotoxicity is not irreversible, but that reversibility is a matter of time, depending on early diagnosis, allowing prompt treatment. Furthermore, these findings, based on standard cardiac symptoms surveillance, might miss this change (8).

On the contrary, close monitoring and timely treatment with HF therapies have reported that they are critical for functional recovery in a non-selected population treated with anthracycline, allowing early detection of cardiotoxicity in the

vast majority of cases during the first year after chemotherapy, with normalization of LVEF (final value of LVEF >50%) in 82% of cases (15). However, only 11% of patients had a complete restoration (i.e., final LVEF equal to baseline). This highlights the need for detection methods able to identify early cardiotoxicity and for strategies aimed at preventing the development and the progression of left ventricular dysfunction.

# PRECLINICAL EARLY DETECTION

Today, at an early preclinical stage, we can detect cardiotoxicity long before symptoms of heart failure occur and before an asymptomatic drop in LVEF. Most data relate to cardiac biochemical markers: mainly troponins and echocardiography of tissue Doppler and strain (5, 7, 8).

# Troponin Assessment in Anthracycline-Treated Patients

Troponin may be considered the gold standard biomarker for myocardial injury and cardiotoxicity from different causes/etiologies (32). Troponin has many advantages: elevated cardiac specificity, high sensitivity, availability, and costs respective to imaging methods. Moreover, there are limited variability issues. In this field, several studies have demonstrated that troponins may detect cardiotoxicity in patients treated with anthracyclines (**Table 3**) (33–56).

The most extensive study included 703 cancer patients, in whom Troponin I (TnI) was assessed before and during the first 72 h after chemotherapy (early TnI), and after 1 month (late TnI) (38). Three different troponin release patterns were recognized: Troponin I remained within the normal interval in 72% of patients, rose at only early evaluation in 21%, and increased at early and late assessments in 9%. Patients with no rise in troponin showed little difference in LVEF and had a good prognosis, with a low incidence of significant adverse heart events (MACE) (1%) during follow-up. Alternatively, TnIpositive patients had a higher rate of MACE: In particular, severe cardiac dysfunction and a higher rate of MACE were associated with a persistent TnI elevation compared to patients with only a temporary rise (p < 0.001). Based on the high negative predictive value (99%), TnI has been able to safely identify low-risk patients, limiting the need and subsequent costs of close long-term cardiac monitoring (34, 35, 38). Conversely, TnI-positive patients deserve more stringent monitoring, mainly those showing a persistent TnI increase.

In summary, we can assert that troponin evaluation in patients treated with anthracyclines allows for:


On the other hand, the identified weakness points are:


# Other Circulating Biomarkers

Although patients with pre-treatment levels of natriuretic peptides (BNP and N-terminal prohormone) tend to experience cardiac events (including cardiac dysfunction), the results are sparse (44, 57, 58).

More recently (57), BNP levels were shown to be significantly higher after every anthracycline cycle in


TABLE 3 | Clinical studies demonstrating Troponins as predictor of anticancer drug-induced left ventricular dysfunction (33–56).

AC, anthracycline-containing chemotherapy; ALL, acute lymphoblastic leukemia; CT, chemotherapy; Cycl, cyclophosphamide; HD, high-dose; LAP, lapatinib; lipoAC, liposomial anthracycline; NA, not available; I, troponin I; T, troponin T; TRZ, trastuzumab; HS, high-sensitive; US, ultra-sensitive \*, pediatric population.

subjects following cardiac events, while another study demonstrated an association between an increased BNP at 72 h after chemotherapy and a decrease of LVEF at 1 year (59).

Actually, studies of other biomarkers, including microRNAs (miRNAs), C-reactive protein (CRP), growth differentiation factor-15 (GDF-15), myeloperoxidase (MPO), and galectin-3 (Gal-3) have not demonstrated an association between pretreatment biomarker levels and cardiovascular outcomes (60, 61).

Regarding monitoring for cardiovascular toxicity during therapy, CRP has shown conflicting findings (50).

More recently, some reports emerged in the field of microRNAs, in particular for miR-1, showing a trend to earlier detection of cardiotoxicity respective to troponin (62, 63).

Another study of patients during a period of 10 years after anthracycline therapy did not find an association with Gal-3 and LV dysfunction.

A separate study that included Gal-3 and ST2 found no association with these biomarkers and LVEF 1 year after therapy (64, 65).

# Tissue Doppler and Strain Echocardiography

Novel echocardiographic methods have emerged as sensitive parameters in the early identification of cardiotoxicity. In particular, introduction of tissue Doppler and strain imaging techniques can detect early subclinical changes in cardiac function, before LVEF falls (4, 7, 19, 51). In this respect, myocardial deformation (strain imaging) has emerged as a sensitive marker for earlier detection of myocardial dysfunction. In particular, 2D (and more recently, 3D) speckle tracking imaging, allowing the evaluation of global myocardial deformation in the longitudinal axis (global longitudinal strain, GLS, %), has become a clinical standard. Several papers demonstrated the value of GLS in detecting subclinical myocardial dysfunction, with prognostic relevance in terms of overt LV dysfunction in cancer patients (66–69).

The recent ASE/EACVI consensus defined a relative decrease in GLS of >15% from baseline as an indicator of subclinical LV dysfunction and appropriate use criteria for multi-modality imaging include strain for the evaluation of patient candidates for chemotherapy (19, 70). Finally, the SUCCOUR trial (first randomized controlled trial of GLS-guided therapy introduction) will better define the role of GLS for surveillance for chemotherapy-related cardiac dysfunction (71).

However, these methodologies are not always readily available in all laboratories and seldom used in the routine evaluation of patients receiving anthracyclines (8).

# An Integrated Approach to Biomarkers and Cardiac Imaging

Breakthroughs in laboratory technology have allowed for the introduction of more specific and sensitive troponin assay methods (55), which are able to measure minimal amounts (highsensitivity [HS] dosing systems) of a biomarker that were not detectable with previous methods. This is of pivotal importance, since troponin release as a consequence of anthracycline cardiotoxicity may be minimal, and it is essential to use highprecision dosing systems (72).

The first HS troponin trial enrolled 45 breast cancer patients who were treated with anthracyclines, taxanes, and trastuzumab (44). International and regional myocardial function was assessed at baseline, every 3 months, with tissue Doppler and strain imaging, combined with troponin. A reduction in the longitudinal strain and an increase in HS troponin were predictive of late left ventricular dysfunction after the end of anthracyclines. Notably, the combined assessment of imaging methods and changes in troponin resulted in an increased specificity (93% combined vs. 73% for each single method). Ky et al. tested a multi-marker approach in a similar population of breast cancer patients receiving the same anti-cancer therapy regimen (50). All levels of the markers increased significantly from baseline (except for NT-proBNP and Galectin-3). However, at the end of anthracycline therapy, only HS troponin absolute values and changes in troponin and myeloperoxidase levels resulted as predictors of further development of left ventricular dysfunctions.

# PRIMARY PREVENTION: REDUCTION OF THE DIRECT CARDIOTOXIC EFFECT (FIGURE 2)

# Limitation of the Maximum Dose of Anthracyclines

Present oncologic guidelines recommend limiting the total cumulative dose of anthracyclines to 450–550 mg/ml (4, 8). However, this may limit the effectiveness of anti-cancer treatment. Moreover, significant variability exists in terms of proneness to anthracycline cardiotoxicity, suggesting that genetic variation might modulate the risk (5, 7, 8).

# Use of Less Cardiotoxic Anthracycline Analogs

Epirubicin, idarubicin, and mitoxantrone are analogs of anthracyclines that are less cardiotoxic than conventional anthracyclines. Epirubicin cardiotoxicity occurs after higher doses of doxorubicin. However, to obtain the same clinical response, higher doses must be given. In preclinical studies and animal models, idarubicin and mitoxantrone also showed a less cardiotoxic profile than doxorubicin (5, 7).

# Use of Liposomal Anthracyclines

In the heart, liposomes cannot get out from the vascular space because capillaries have tight junctions. As the tendency to accumulate in the heart cells is limited, this may reduce the risk of cardiotoxicity. On the contrary, the liposomes reach high concentrations in the tumor site, leaving the circulatory system where tumor growth damages the capillaries itself (73, 74).

# PRIMARY PREVENTION: PHARMACOLOGIC PREVENTION (FIGURE 2)

# Lifestyle Measures

Before pharmacologic strategies, primary prevention starts indeed with lifestyle corrective measures.

Since a strong link exists between cancer and cardiovascular risk factor, addressing smoking and sedentary habits (potentially leading to obesity, with a detrimental role especially in the postmenopausal women), as well as high alcohol intake, is pivotal. A healthy diet has been associated with a protective effect in terms of cancer relapses and cardiovascular disease, while smoking has an ominously detrimental effect. While light to moderate alcohol intake has shown a protective impact in terms of cardiovascular disease, the results in terms of risk of developing cardiotoxicity are conflicting (75–77).

Of notice, several pieces of evidence emerged on the protective role of exercise training (and eventually, cardiac rehabilitation) against cardiotoxicity (78).

# The Use of Cardioprotection

The use of cardioprotective drugs to reduce the direct cardiotoxic effect is a potential alternative to anthracycline treatment modifications, dosage limitations, or interruptions (4, 5, 8).

The hypothesis that iron chelators may reduce the cardiotoxicity induced by anthracyclines suggests that dexrazoxane may be a clinically useful cardioprotective agent (9, 79). Doxorubicin is a potent Top2 inhibitor. In the clinical scenario, many studies demonstrated that dexrazoxane significantly reduces cardiotoxicity in adults and pediatric populations: Patients treated with dexrazoxane had a significantly lower incidence of heart failure than untreated patients. Apart from patients with metastatic breast cancer treated with doses of doxorubicin >300 mg/mq and despite previous findings, dexrazoxane is not routinely used in clinical practice, because suspected of interfering with the anti-tumor effects and by the occurrence of secondary malignancies. In September 2011, the outcome of a referral (80) that recommended several restrictions on dexrazoxane use in both children and adults with cancer was published. However, several new trials on the benefit-risk of dexrazoxane have been published from then (81–83). So far, dexrazoxane results an effective cardioprotector when administered with anthracycline chemotherapy being not associated with a reduction in anti-tumor efficacy or survival or a relevant increased risk of second primary malignancies, and can

be recommended as a cardioprotector particularly for children and adolescents for whom the development of anthracyclineinduced cardiotoxicity could have a crucial prognostic impact. These studies contributed to the CHMP's decision to remove the contraindication on Cardioxane (84).

Macedo et al. recently published a systematic review and metaanalysis of nine trials (seven randomized and two retrospective non-randomized trials) on the efficacy of dexrazoxane in patients with breast cancer treated with anthracyclines (with or without trastuzumab). Despite the quality of available evidence remaining low, dexrazoxane was shown to reduce the risk of heart failure and cardiac events, independently from previous exposure to anthracyclines. The oncological response and survival rates were not affected by dexrazoxane (85).

Other potentially cardioprotective agents have been studied in animal models and small clinical studies. Preliminary data are promising, but they need to be ratified by further extensive studies (2, 5, 7, 8).

# The Use of Cardiovascular Agents

Several heart failure drugs have been shown to be effective in terms of cardioprotection against anthracylines (**Table 4**) (86–98).

Overall, a recent meta-analysis of randomized clinical trials of adult patients that underwent chemotherapy and cardiovascular therapies vs. placebo with follow-up (17 trials, 1,984 patients) showed higher (although with small changes) LVEF values at follow-up in cancer patients receiving neurohormonal therapies (99).

#### Beta-Blockers

The non-cardioselective beta-blocker carvedilol is cardioprotective against anthracyclines toxicity. In vitro studies and a small randomized clinical trial, the drug was able to prevent the development of ventricular dysfunction (86). In breast cancer patients, carvedilol blunted strain abnormalities and the increase in troponin, preserving diastolic function, after anthracycline use (100). However, the drug failed to prevent an LVEF reduction >10% (101). It appears that carvedilol's efficacy is linked to its antioxidant activity rather than its beta-blocking action. Indeed, a comparative study of carvedilol and atenolol, a selective β1 antagonist with no antioxidant properties, showed that carvedilol—but not atenolol—prevented mitochondrial damage and mitigated the ultrastructural changes associated with doxorubicin (8, 102).

Nebivolol, a selective β1 antagonist with vasodilatory properties, started 7 days before anthracyclines and continued for 6 months in 27 patients with breast cancer prevented a significant decrease of LVEF and an increase of NT-proBNP (87). In a retrospective study including 106 breast cancer patients, a reduced incidence of heart failure over a 5-year follow-up period was associated with the continuation of beta-blocker therapy during oncology treatment—including anthracyclines (88). Existing data indicate, from preclinical studies, that

TABLE 4 | Cardiovascular drugs showing a prophylactic effect against anticancer therapy-induced LVD in adult cancer populations.


ACEI, angiotensin-converting enzyme inhibitor; ANP, atrial natriuretic peptide; ARB, angiotensin receptor blocker; BNP, brain natriuretic peptide; HD CT, high-dose chemotherapy; LVD, left ventricular dysfunction; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic diameter; HF, heart failure; MACE, major adverse cardiac events; NHL, non Hodgkin lymphoma; NT-proBNP, N-terminal-proBNP; QT, QT interval; PO, prospective observational; RCT, randomized controlled trial; Tx, taxanes; TNI, troponin I; TRZ, trastuzumab.

cardio-specific beta blockers offer superior protection against anthracycline damage than non-cardioselective ones (8).

#### ACE-Inhibitors and Sartans

Experimental data demonstrated a crucial role of the reninangiotensin system (RAS) in the development and progression of cardiomyopathy induced by anthracyclines (90). Valsartan, administered in combination with anthracyclines, blunted natriuretic peptides increase, the increase in chamber size in patients with non-Hodgkin's lymphoma treated doxorubicin (91). The authors hypothesized a direct inhibition of the drug, independent from hemodynamic effects (8, 90).

Telmisartan, started before epirubicin, was able to prevent strain reduction and inflammatory markers increase because of its RAS blocking action, but also because of its anti-inflammatory and anti-oxidant properties (92).

In the PRADA (Prevention of Cardiac Dysfunction during Adjuvant Breast Cancer Therapy) trial candesartan—but not metoprolol—administrated with adjuvant chemotherapy including anthracyclines, with or without trastuzumab, can protect against an early decline in LVEF, assessed with cardiac MRI (93).

The MANTICORE-101 study (Multi-disciplinary Approach to Novel Therapies in Cardiology Oncology Research) tested the use of perindopril vs. bisoprolol in the prevention of left ventricular remodeling, defined as an increase in end-diastolic diameters and primary study point, and of left ventricular dysfunction in HER2+ breast cancer patients treated with trastuzumab prior to anthracycline (89). Neither drug prevent left ventricular remodeling; however, the use of both drugs was associated with a preserved left-ventricular function in multivariate analysis.

The combination of enalapril and carvedilol have been tested in the OVERCOME study (preventiOn of left-ventricular dysfunction with enalapril and carvedilol). The study involved 90 patients treated with anthracyclines, with malignant hemopathies. LVEF didn't change in the intervention group after 6 months, but decreased significantly in controls. In addition, the intervention group had a lower rate of combined death or heart failure or death, heart failure, and a final LVEF of <45 % (95).

#### Aldosterone Antagonists

A recent randomized trial, including 43 breast cancer patients, evaluated the use of spironolactone vs. placebo. Spironolactone was started 1 week before anthracyclines. Three weeks after the end of chemotherapy, the treated group did not show relevant variations in LVEF and rise in troponin I and NT-proBNP (94). In ELEVATE (Effect of Eplerenone on Left Ventricular Diastolic Function in Women Receiving Anthracyclines for Breast Cancer), a recent randomized placebo-controlled trial, administration of eplerenone for 6 months was not associated with significant differences in ventricular function compared with placebo in patients with breast cancer treated with anthracyclines (103).

#### Statins

The effect of statins on cardiotoxicity of anthracyclines is most likely due to their pleiotropic effect, and in particular, to their antioxidant properties (8). Forty hematologic cancer patients with no history of heart disease were randomized to receive atorvastatin or placebo before the onset of anthracyclines (95). The dosage was 40 mg/day, regardless of the levels of cholesterol, and lasted for 6 months. During the follow-up, a reduction of the high-sensitivity reactive C protein level and no significant changes in LVEF were observed in the statin group.

Conversely, the LVEF value in the control group resulted in a significant reduction from the baseline. The protective effect of statins also emerged when chemotherapy was started in patients already receiving statins for the prevention of cardiovascular disease (96). In a retrospective observational study of 67 breast cancer patients treated with anthracycline, statin therapy continued to be associated with significant reduction in the risk of heart failure and cardiac-related mortality during follow-up. More recently, patients on statin therapy for the prevention of cardiovascular disease reported a smaller drop in LVEF at 6 months in a retrospective observational study, including 51 patients with breast cancer or hematological malignancies treated with anthracyclines (97).

### Perspectives

A recent study identified the molecular and cellular signature of dose-dependent, doxorubicin-mediated cardiotoxicity and provided evidence that prokineticin receptor (PKR-1)-1, acting at myocardial and vascular level, is a promising target to combat cardiotoxicity of cancer treatments (104).

Since G protein-coupled receptors (GPCRs) are a target of 40% of clinically used drugs and newly identified cardioprotective agents that bind GPCRs of adrenalin, adenosine, melatonin, ghrelin, galanin, gpelin, prokineticin, and cannabidiol may further aid in the cardioprotective task (105).

# PREVENTION IN SELECTED HIGH-RISK PATIENTS

Prevention may be an option for all patients who are candidates for cardiotoxic therapy (primary prevention) or restricted to patients with preclinical symptoms of cardiotoxicity, with the advantage of limiting prophylactic therapy to a small number of patients (also reducing the side effects of preventive therapy, i.e., hemodynamic effects) (**Figure 2**).

A randomized trial has tested the cardioprotective capacity of enalapril, involving 473 patients with different types of cancers treated with high-dose chemotherapy (90). 114 patients showed an increase in troponin and were randomized for treatment with or without enalapril. After the end of chemotherapy, enalapril was begun, titrated as tolerated, and continued for 1 year. No patients in the enalapril-treated group showed a decrease in LVEF by 10 absolute points below the value of 50%—the study's primary endpoint—and the incidence of major cardiac events was remarkably small (**Figure 5**). Of note, in the enalapril community, the LVEF value was still the same as the baseline value in 80% of cases after a follow-up duration of 12 months, showing that enalapril can be a very effective drug in the complete preservation of systolic function in this population.

Two studies are currently evaluating the efficacy of carvedilol as a preventive therapy in a selected patient with a deterioration in the strain parameter. For Research NCT02177175 (Carvedilol for the Prevention of Anthracyclines/Anti-HER2 Therapy-Associated Cardiotoxicity between Women with HER2+ breast cancer Using Myocardial Strain), the primary endpoint is the identification of a reduced LVEF value during the 1-year followup. At Northwestern University of Chicago, the research is still hiring.

# PRIMARY VS. SECONDARY PREVENTION

Enalapril, which began early after the increase in troponin during anthracycline chemotherapy and continued for 12 months, is an effective therapy to avoid left ventricular dysfunction and subsequent heart events (90). Repeated assessment, however, is needed to detect an increase in troponin, as the marker may increase at different times after infusion with therapy (dose of anthracycline and schedules). Primary prevention, applied to all anthracycline-treated patients, does not pose this downside. The ICOSONE (International CardioOncology Society-One) randomized trial prospectively compared the efficacy of two different approaches, to test whether enalapril, initiated in all patients before chemotherapy (Prevention Group), was able to prevent troponin rise and further the development of left ventricular dysfunction, and to test whether this strategy was more successful than enalapril initiated only after troponin elevation during chemotherapy (Troponin-triggered Group) (106). The study included 273 patients from 21 different Centers of Oncology. The most-often administered anthracyclines were epirubicin and doxorubicin. During chemotherapy and the 12-month follow-up, no significant reduction in LVEF and a minimal incidence of cardiovascular events were detected in both groups. Only three patients experienced cardiotoxicity defined as a 10% reduction in LVEF, below 50% value.

In brief, the main result of the study was that the two approaches appear to be similarly effective in preventing left ventricular dysfunction and adverse cardiac events, endorsing the use of enalapril in averting anthracycline-induced cardiotoxicity, irrespectively from the strategy used.

Which strategy is best? Secondary prevention (i.e., troponin driven) has the limitation of repeated blood samplings. Nevertheless, considering the high negative predictive value of troponin (34, 35, 37, 38), this strategy appears warranted and cost-effective, as it permits the exclusion of low-risk patients (patients without troponin rise, the vast majority) from longterm monitoring programs based on imaging techniques with a relevant cost–benefit ratio by reducing "medicalization, distress,

TABLE 5 | Pros and Cons of primary prevention vs. secondary prevention with enalapril (83).


LVD, left ventricular dysfunction; MACE, major adverse cardiac events; FU, follow-up; TNI, troponin I.

patients treated with ACEI Group and in Controls. Modified from Cardinale et al. (90).

anxiety, and costs" (21). Primary prevention, although not needing a repeated evaluation of troponin during chemotherapy, can be hard in terms of clinical surveillance during the drug uptitration to include 100% of patients. Finally, it may expose to potential side effects all those low-risk subjects for cardiotoxicity (**Table 5**) (106).

# CONCLUSION

Anthracycline-induced cardiotoxicity is still a significant problem that compromises the quality of life and overall survival of cancer patients. However, recent findings demonstrate that this form of cardiomyopathy is mostly reversible with early detection and prompt therapeutic introduction strategy. Probably, anthracycline-induced cardiotoxicity is a single and continuous phenomenon, from cellular to clinical stage, starting with myocardial cell injury, followed by progressive LVEF decline and, potentially, overt heart failure. The current standard for monitoring cardiac function (periodic assessment of LVEF), detects cardiotoxicity at a late stage when a significant impairment has already occurred, precluding the chance of effectively prevent and treat its development.

The use of troponins to identify patients with subclinical cardiotoxicity combined with early treatment with ACE-

# REFERENCES


inhibitors occurrence appears to be an effective method to prevent anthracycline-related left ventricular dysfunction and cardiac events.

Finally, adoption of internal procedures, shared in a multi-disciplinary team, may actively aid in optimizing patient management. In this respect, a direct relationship with the laboratory medicine service for the assessment of troponin values during chemotherapy and the availability of a cardiologist and a dedicated nurse staff should always mix with an active collaboration with the referral oncologist/hematologist (possibly, surgeon) for updates and remains of pivotal importance (107, 108).

# AUTHOR CONTRIBUTIONS

DC, FI, and CC contributed conception, design of the review, and wrote the first draft. All authors contributed to manuscript revision, read, and approved the submitted version.

# FUNDING

The present paper was funded by the European Institute of Oncology.


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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Cardinale, Iacopo and Cipolla. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cardiovascular Damage Associated With Chest Irradiation

#### Simone M. Mrotzek, Tienush Rassaf and Matthias Totzeck\*

*Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, Medical Faculty, University Hospital Essen, Essen, Germany*

The improvement of anticancer-therapies results in a greater amount of long-term survivors after radiotherapy. Therefore, the understanding of cardiotoxicity after irradiation is of increasing importance. Cardiovascular adverse events after chest irradiation have been acknowledged for a long time but remain difficult to diagnose. Long-term cardiovascular adverse events may become evident years or decades after radiotherapy and the spectrum of potential cardiovascular side effects is large. Recent experimental and clinical data indicate that cardiovascular symptoms may be caused especially by heart failure with preserved ejection fraction, which remains incompletely understood in patients after radiation therapy. Heart radiation dose and co-existing cardiovascular risk factors represent some of the most important contributors for incidence and severity of radiation-induced cardiovascular side effects. In this review, we aim to elucidate the underlying patho-mechanisms and to characterize the development of radiation-induced cardiovascular damage. Additionally, approaches for clinical management and treatment options are presented.

#### Edited by:

*Carlo Gabriele Tocchetti, University of Naples Federico II, Italy*

#### Reviewed by:

*Alessandra Cuomo, Federico II University Hospital, Italy Laura Cella, Italian National Research Council, Italy*

#### \*Correspondence:

*Matthias Totzeck matthias.totzeck@uk-essen.de*

#### Specialty section:

*This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine*

> Received: *27 December 2019* Accepted: *03 March 2020* Published: *20 March 2020*

#### Citation:

*Mrotzek SM, Rassaf T and Totzeck M (2020) Cardiovascular Damage Associated With Chest Irradiation. Front. Cardiovasc. Med. 7:41. doi: 10.3389/fcvm.2020.00041* Keywords: radiation therapy, irradiation, cardio-oncology, cardiotoxicity, cardiovascular damage, cancer therapy

# INTRODUCTION

Radiation therapy is an important part of multimodal treatment strategies in cancer therapy. Fifty to sixty percent of all patients with advanced cancer undergo irradiation (1, 2). The increasing number of cancer survivors also leads to an increase occurrence of late-time adverse events following radiation therapy (3, 4). Although strategies to spare surrounding tissue have been developed in modern radiation therapy techniques, damage of healthy tissue/organs cannot totally be avoided by performing an effective cancer treatment using ionized radiation. Exposure of the heart during chest/ thoracic irradiation occurs in particular during treatment of breast and lung cancer (especially left sided) as well as mediastinal lymphomas (3, 5, 6). With increasing number of long-term survivors of esophageal cancer resulting from the addition of chemotherapy to radiotherapy, the risk for radiation-induced cardiovascular toxicity is now recognized as an issue of major concern also in this patient category (7).

Radiation-induced cardiovascular diseases typically manifest years or decades after cancer therapy. Therefore, a causal relation is often difficult to diagnose. Overall incidence and severity correlates with higher radiation dose, larger exposed volumes, younger age at time of exposure, and greater time elapsed since treatment (8, 9). But it has been shown that even little doses of 0.5 Gray (Gy) can significantly enhance cardiovascular risk for the patients (10) and that not total radiation dose but the "volume of the left ventricle receiving 5 Gy" (LV V5Gy) was an important prognostic dose-volume parameter (11). Moreover, concomitant or sequential treatment with cardiotoxic chemotherapy (e.g., anthracyclines) poses an additional risk for the development of radiation-induced cardiovascular damage (6, 12, 13). Early diagnosis seems to be important to decrease long-term damage, reduce incidence of fatal cardiovascular adverse events and improve quality of life in cancer survivors.

But not only late-time effects are important. An association between higher values of heart dosimetric variables and a worse overall survival at a median follow-up of 2 years was described, suggesting that radiation to the heart could contribute to early mortality in a non-small cell lung cancer population (14). Especially lung cancer patients are also more likely to have preexisting risk factors such as known cardiac diseases (15) and smoking history that may predispose them to cardiovascular events occurring at earlier time points than would be seen in a healthier patient population treated with thoracic radiation therapy (16, 17).

Myocardial tissue was found to be very sensible to cancer therapy due to high metabolic activity (12). Underlying pathomechanisms as well as clinical management of radiation-induced cardiovascular diseases are still incompletely characterized. In this review, we discuss different approaches and cardiooncological strategies after chest irradiation- from bench to bedside.

# SPECTRUM OF CARDIOVASCULAR DISEASES FOLLOWING CHEST IRRADIATION

The relative risk of fatal cardiovascular events in survivors after Hodgkin's lymphoma is 2.2–12.7 (median follow-up 18.7 years) and 2–2.2 after breast cancer (median follow-up 12 years) (18, 19). In survivors of childhood-cancer and single therapy with radiation, over 22% show signs of diastolic dysfunction in echocardiography studies (20). The spectrum of cardiovascular diseases associated with chest irradiation is in its occurrence and appearance manifold. **Figure 1** gives an overview and illustrates the relation between different levels of cardiovascular damage.

Radiation-induced pericarditis has been feared because the acute form often led to a life-threatening constrictive pericarditis. Due to advances in radiation protocols (improved techniques, lower dosages and less volume exposed) the occurrence has become rare nowadays (21). In contrast, chronic pericarditis is still one of the most frequent radiation-induced cardiotoxicities and is characterized by exudation of a protein-rich secretion (10). After chemo- and radiation therapy for locally advanced non-small cell lung cancer an incidence of pericardial effusion of nearly 50% is described with the existing risk for cardiac tamponade (17, 22). The underlying patho-mechanism mainly includes inflammatory processes and fibrin-deposition due to microvascular damage (23).

Radiation-induced vascular damage can be divided into a micro- and marcovascular injury, but both can cause a significant myocardial perfusion deficit. Endothelial cells are describe to be very radiation-sensible and seem to form the initial point for patho-mechanistic changes after heart irradiation. Capillaries have only one layer of endothelial cells and are therefore especially challenged. Reduction in capillary density and a disturbed vascular network contribute particularly to the development of radiation-induced myocardial dysfunction (1). The macrovascular damage of the coronary arteries results in an enhanced development of arteriosclerosis. Possible radiationexposed coronary segments such as the left main coronary artery and the ostial left anterior descending artery and ostial right coronary artery are mainly affected (8). The occurrence of a vascular inflammatory reaction, additional microvascular dysfunction, and subendothelial fibrosis leads to the development of unstable plaques in the large vessels and at the vascular

bifurcations (23, 24). This results in an increased incidence of acute myocardial infarction, coronary heart disease, and the development of ischemic cardiomyopathy (25). In women who underwent radiotherapy for breast cancer an increase of major coronary events started within the first 5 years after radiotherapy and continued into the third decade after radiotherapy (26). A four- to seven-fold increased risk of highgrade coronary artery stenosis in mid and distal left anterior descending artery was investigated when comparing women with irradiated left sided with those with right-sided breast cancer (27, 28). The precise signaling pathways are not fully understood. Also a difference in radiosensitivity due to different structure and subregions of the heart is discussed.

The development of radiation-induced cardiomyopathy is based on a combination of structural changes in myocardial tissue as well as a perfusion deficit resulting from microand macrovascular changes. Clinically, patients usually have a characteristic, diastolic functional impairment and heart failure with preserved systolic ejection function (3). Diffuse, interstitial fibrosis and amyloid deposition have been forwarded as underlying causes (29–31). Arrhythmias can occur as a result of these structural changes and further conduction system abnormalities. Direct damage to critical structures such as the sinoatrial or atrio-ventricular nodes may lead to bradycardia or all types of heart block (23).

In the area of the heart valves, fibrotic processes are most common on aortic and mitral valves and are similar to degenerative changes. Often these changes are hemodynamically irrelevant, however, in patients with radiation in childhood, higher-grade stenosis, or insufficiency of the heart valve can manifest itself clinically in early adulthood and require surgical treatment (21).

# PATHO-MECHANISM AND DEVELOPMENT OF RADIATION-INDUCED CARDIOVASCULAR DAMAGE

Ionizing radiation induces cell death mainly through induction of deoxyribonucleic acid (DNA) single- and double-strand breaks (32). During cancer therapy, ionizing radiation is also associated with increased risk of damage to healthy, cancer surrounding tissue. Cardiomyocytes are described to be radio-resistant (30) but endothelial cells are particularly sensitive to radiation and are suspected to be the initial point for cardiovascular radiation-induced damage due to changes in the surrounding milieu (1, 33). In addition to direct vascular damage, there is also a causal relationship between endothelial dysfunction and the development of muscular, valvular, and arrhythmogenic complications, since the resulting pro-inflammatory environment is a strong initiator of cardiac fibrosis (1).

The mechanisms of endothelial damage primarily base on the induction of apoptosis (acute process) and increased senescence (cell aging, chronic process) (1). As a result, an inflammatory reaction with increased leukocyte recruitment and increased oxidative stress develops through the release of cytokines (1). Depending on the context, radiation-induced DNA damage in endothelial cells can be repaired or trigger apoptosis, which can be p53-mediated or induced by sphingomyelin-produced ceramides (34). In p53-mediated apoptosis, mediation via cytochrome C-induced mitochondrial initiation of apoptotic cell death is leading (intrinsic signaling pathway) (1, 34). Senescence is also triggered by radiation-induced DNA damage. It leads to a change in the cellular phenotype of the endothelial cells and thus to a secretion of cytokines, proteins and other factors (1).

Apoptosis and senescence of endothelial cells together lead to an imbalance between pro- and anticoagulatory as well as pro- and anti-inflammatory factors in the vascular milieu. This leads to an increased adhesion of leukocytes and macrophages, chronic inflammation, a pro-thrombotic status and the increased occurrence of reactive oxygen species (1).

Radiation-induced senescence leads to an inactivation of the phosphoinositide-3-kinase/protein kinase B (PI3k/Akt) signaling pathway and downregulates the serine/threonine kinase mTor (mechanistic target of rapamycin). As a regulator of actin polymerization and the interaction of cell adhesion molecules such as integrins, mTor has a direct influence on the contractility of smooth muscle cells (35, 36). Furthermore, an increased expression of cell surface-located cluster of differentiation 44 (CD44) on endothelial cells has been described. This leads to an increased adhesion of monocytes and ultimately to an increased formation of arteriosclerosis (37).

In addition to adult endothelial cells, endothelial progenitor cells can also be damaged by radiation. This can lead to disturbed vascular remodeling and thus contribute to the development of vascular dysfunction (38). Within the endothelial progenitor cells, ionizing radiation triggers a p53 stabilization, a p21 mediated cell cycle arrest and finally an apoptosis mediated by Bax (Bcl-2-associated X protein) (39).

The development of radiation-induced cardiomyopathy results from an interaction of myocardial remodeling, degeneration and cellular dysfunction. A close connection with endothelial dysfunction due to the creation of a profibrotic and pro-inflammatory environment has been suggested (10). Similar to the mechanisms of cardiac damage caused by anthracyclines, oxidative stress and inflammation lead to structural and functional damage to the cardiomyocytes due to membrane-bound lipid peroxidation (12). The inactivation of peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α), a key player in the regulation of lipid metabolism in the heart, plays a crucial role, too (10, 40). In contrast to endothelial cells, cardiomyocytes no longer undergo cell division postnatal, so they show no morphological changes (41).

The pro-inflammatory environment is furthermore a strong initiator of cardiac fibrosis (1). For example, interleukin-13 mediated fibroblasts are recruited from various sources such as mesenchymal cells and the bone marrow and ensure myocardial collagen storage (especially collagen types I and III) (23). Increased plasma levels of TGFβ (transforming growth factor-β), angiotensin II and aldosterone are also found after cardiac radiation, lead to increased myocardial fibrosis and thus represent possible therapeutic approaches for cardioprotection during and after radiation therapy (42).

# EXPERIMENTAL MODELS FOR CHARACTERIZATION OF FUNCTIONAL CARDIAC IMPAIRMENT AFTER CHEST IRRADIATION

Multiple animal models have been used to characterize radiationinduced cardiomyopathy. Radiation protocols vary between whole thorax and localized heart irradiation as well as single dose and fractionated schedules. Dosages differ between 5 up to 25 Gy (42). For investigation of radiation-induced coronary artery disease, transgenic mouse models are used because wild-type rodents are usually not prone to atherosclerosis (42). In ApoE−/<sup>−</sup> mice, the development of fatty streaks in carotid arteries was detected 4 weeks after radiation with 14 Gy and a reduction in vascular cell adhesion protein 1 (VCAM-1) as an indication for the development of atherosclerosis was described (43).

While endothelial cell damage plays a major role in the development of radiation-induced cardiac damage, reduction of microvascular density and cardiac capillary damage was found in mice and rats using different protocols (44, 45). This could be shown by an immuno-histological reduction of CD31 positive cells 40 and 60 weeks after 8 or 16 Gy single whole heart irradiation (45). For detailed evaluation of myocardial microvascular damage in vivo, DE-microCT (computed tomography) scans 4 or 8 weeks after partial heart irradiation with 12 Gy could show a time-dependent increase in accumulation of gold nanoparticles in the myocardium as a sign for extravasation. Perfusion defects have also be visualized using microSPECT (46).

Beside myocardial perfusion deficits through marco- and microvascular damages, late-onset radiation-induced cardiac damages are characterized by development of myocardial fibrosis. Collagen-deposition within the myocardial interstitium was described using histopathological staining with Masson's trichrome (31) and picrosirius red (30). In addition, an amyloid deposition was detected using congo-red staining (29, 45). Also increases in mRNA expression levels of pro-fibrotic genes like fibronectin have been shown after irradiation of rats' hearts (47).

Pre-clinical in vivo models can be used for characterization of cardiac functional impairment after irradiation using echocardiography and pressure-volume catheterization. Normal or even increased left-ventricular systolic function at baseline has been documented (30, 31, 45). In contrast, cardiac radiation exposure caused a diastolic dysfunction expressed by an elevated left ventricular end-diastolic pressure (LVEDP/ filling pressure) and higher Tau (time constant of isovolumentric relaxation) in radiated rats compared to control rats (30). Moreover, a reduced contractile reserve was found using mouse stress transthoracic echocardiography with isoproterenol (31).

Experimental studies also indicate a relevant heart-lunginteraction through thoracic radiation. Studies with irradiated rats showed that heart damage was aggravated if also the lung was irradiated and vice versa (47, 48). In addition, this could be translated to a clinical setting, suggesting an importance of heart and lung irradiation in the prediction of radiation-related valve disease in Hodgkin lymphoma survivors (49, 50).

TABLE 1 | Incidence of cardiovascular disease and mortality following chest irradiation.


*LV, left ventricular; Gy, gray.*

# CLINICAL IMPLICATIONS AND THERAPEUTIC STRATEGIES

The occurrence of cardiovascular side effects after radiation is primarily dependent on the radiation dose and the time interval after the cancer therapy. **Table 1** summarizes information from various clinical studies regarding incidence of cardiovascular diseases.

Mediastinal radiation was identified as an important cardiovascular risk factor, but previously, cardiovascular diagnostics were usually only initiated after clinical symptoms had occurred. This leads to the fact that for example coronary heart disease after radiation manifests in a high proportion as fatal myocardial infarction. Late diagnosis is favored due to damaged peripheral nerve endings after mediastinal radiation whereby patients often present with atypical angina pectoris or even no symptoms (9). Peri-interventional and operative management is also aggravated due to the pronounced pathology at the time of diagnosis and mediastinal adhesions after tumor resection and radiation. The early diagnosis and therapy of radiation-induced heart disease is therefore of great relevance.

Cardiotoxic chemotherapy (e.g., anthracyclines) and chest irradiation is a common combination during treatment of breast cancer (Hodgkin), lymphoma, and childhood cancer which led to a success in the fight against cancer but but also reproduces the occurrence of long-term cardiotoxic side effects (6, 13, 55, 56). This is especially true for the development of heart failure due to a synergistic damage on cardiomyocytes (see also section on patho-mechanism) (12, 57). While resulted cardiomyopathy in patients treated with radiotherapy alone is characterized by diastolic dysfunction, combination of anthracycline therapy and chest irradiation more often leads to an additional clinical relevant systolic dysfunction (57, 58). Beside a simultaneous/sequential cardiotoxic chemotherapy, also patients with existing cardiovascular risk factors have a significantly increased risk of developing radiation-induced heart disease. Therefor an assessment of the individual cardiovascular risk profile should be conducted before starting radiation therapy (55, 59). In case of abnormalities, a cardio-oncological presentation for further diagnostics and development of an interdisciplinary treatment plan is recommended (60– 62). Optimizing existing cardiovascular risk factors and preexisting conditions is particularly important. After mediastinal radiation therapy, a preventive diagnostic approach using an electrocardiogram (ECG) and transthoracic echocardiography are currently recommended 5 years after therapy, and in the following every 2–5 years depending on the individual presentation and risk assessment (3, 9, 59). Patients with childhood cancer are classified as high-risk collective from an average cardiac radiation dose of ≥35 Gy, adults from >30 Gy or at <30 Gy with co-existing history of anthracycline chemotherapy. Patients classified as high-risk should receive cardiac diagnostics with an ECG and echocardiography early (children 2 years, adults 1 year after radiation) (63, 64). The determination of the global longitudinal strain has been shown to be particularly sensitive in the detection of left ventricular dysfunction after mediastinal radiation (20) especially in combination of radiation with anthracycline chemotherapy (13). Cardiac magnetic resonance imaging (MRI) should also be considered in poor echocardiographic conditions. A stress test (e.g., bicycle ergometry/stress ECG) or alternatively a coronary CT should be performed 10 years after radiation (59). These recommendations are currently based primarily on expert opinions and implementation in the guidelines is still pending (65, 66).

The relevance of cardiac biomarkers for prediction of cancer-therapy related cardiovascular toxicity is being discussed (61). Radiation-induced cardiac-cell damage and changes in the left ventricular loading conditions have been linked to several biomarkers including N-terminal pro-B–type natriuretic peptide (NT-proBNP) and troponins (67, 68), but the clinical applicability is still unclear.

Radiation therapy aims to maximize tumor control, while minimize the risk for radiation-induced adverse normal tissue effects (69). Therefore, strategies to reduce heart dose during radiation therapy are crucial. Technical improvements like deep inspiration breath hold gating and particle therapy (59, 70) as well as intensity modulated radiotherapy or volumetric modulated arc therapy, where delivered radiation dose varies between different treatment areas were developed (71). This helps to spare normal tissue but technical and physical strategies reach a natural limit while the main goal is still to perform an effective cancer therapy. Therefore, development of medical concepts to specifically protect normal tissue damage

# REFERENCES


during and after radiation therapy represents an important research topic.

One promising therapeutic approach to reduce radiationinduced cardiovascular damage is the application of angiotensin converting enzyme (ACE) inhibitors (72–74). Studies indicate that for example the preventive administration of captopril in animal models can reduce radiation-induced cardiac damage (72). Additionally, the positive effects of an early initiated therapy with ACE inhibitors and beta-blockers are discussed in the context of other cancer therapies to help prevent heart failure from cancer therapy in general (74). Also lipid-lowering therapies with simvastatin has been observed to reduce radiationinduced cardiac damage (73, 75). Furthermore, medical therapy by interleukin-1 blockade (administration of anakinra) targeting radiation-induced vascular inflammation, has been evaluated recently (76). So far, however, none of the therapeutic approaches have been implemented in clinical practice and further studies are needed.

# CONCLUSION

Cardiovascular disease is the leading cause of non-malignancy related death in cancer survivors (50). Minimizing the cardiac radiation dose is currently the only causal way to prevent radiation-induced heart diseases. Additional, assessment of cardiovascular risk before, during and, after irradiation and early diagnosis of radiation-induced cardiac damage is essential to further improve mortality and morbidity in cancer survivors. Further studies to characterize radiation-induced cardiovascular damage and to evaluate potential treatment option are needed.

## AUTHOR CONTRIBUTIONS

SM was responsible for review analysis, synthesis, and manuscript preparation. TR was responsible for drafting and proofreading the manuscript. MT was responsible for the concept design, synthesis, analysis, and drafting of the manuscript. All authors approve the paper for submission.

## ACKNOWLEDGMENTS

SM acknowledges the following funding source: IFORES research grant from the Medical Faculty, University Duisburg-Essen, Germany.

disease. Int J Cardiol. (2019) 280:163–75. doi: 10.1016/j.ijcard.2019. 01.038


cardiotoxicity in cancer patients: a meta-analysis. Eur J Heart Fail. (2019) 22:350–61. doi: 10.1093/eurheartj/ehz748.0328


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Mrotzek, Rassaf and Totzeck. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Human Pluripotent Stem Cell-Derived Cardiomyocytes for Assessment of Anticancer Drug-Induced Cardiotoxicity

#### Verena Schwach<sup>1</sup> , Rolf H. Slaats <sup>1</sup> and Robert Passier 1,2 \*

<sup>1</sup> Applied Stem Cell Technologies, TechMed Centre, University of Twente, Enschede, Netherlands, <sup>2</sup> Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, Netherlands

#### Edited by:

Canan G. Nebigil, École Supérieure de Biotechnologie Strasbourg, France

#### Reviewed by:

Fabrice Jaffré, Cornell University, United States Alessandra Ghigo, University of Turin, Italy

> \*Correspondence: Robert Passier robert.passier@utwente.nl

#### Specialty section:

This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine

> Received: 28 December 2019 Accepted: 16 March 2020 Published: 08 April 2020

#### Citation:

Schwach V, Slaats RH and Passier R (2020) Human Pluripotent Stem Cell-Derived Cardiomyocytes for Assessment of Anticancer Drug-Induced Cardiotoxicity. Front. Cardiovasc. Med. 7:50. doi: 10.3389/fcvm.2020.00050 Cardiotoxicity is a major cause of high attrition rates among newly developed drugs. Moreover, anti-cancer treatment-induced cardiotoxicity is one of the leading reasons of mortality in cancer survivors. Cardiotoxicity screening in vitro may improve predictivity of cardiotoxicity by novel drugs, using human pluripotent stem cell (hPSC)-derived-cardiomyocytes. Anthracyclines, including Doxorubicin, are widely used and highly effective chemotherapeutic agents for the treatment of different forms of malignancies. Unfortunately, anthracyclines cause many cardiac complications early or late after therapy. Anthracyclines exhibit their potent anti-cancer effect primarily via induction of DNA damage during the DNA replication phase in proliferative cells. In contrast, studies in animals and hPSC-cardiomyocytes have revealed that cardiotoxic effects particularly arise from (1) the generation of oxidative stress inducing mitochondrial dysfunction, (2) disruption of calcium homeostasis, and (3) changes in transcriptome and proteome, triggering apoptotic cell death. To increase the therapeutic index of chemotherapeutic Doxorubicin therapy several protective strategies have been developed or are under development, such as (1) reducing toxicity through modification of Doxorubicin (analogs), (2) targeted delivery of anthracyclines specifically to the tumor tissue or (3) cardioprotective agents that can be used in combination with Doxorubicin. Despite continuous progress in the field of cardio-oncology, cardiotoxicity is still one of the major complications of anti-cancer therapy. In this review, we focus on current hPSC-cardiomyocyte models for assessing anthracycline-induced cardiotoxicity and strategies for cardioprotection. In addition, we discuss latest developments toward personalized advanced pre-clinical models that are more closely recapitulating the human heart, which are necessary to support in vitro screening platforms with higher predictivity. These advanced models have the potential to reduce the time from bench-to-bedside of novel antineoplastic drugs with reduced cardiotoxicity.

Keywords: human pluripotent stem cells, cardiomyocytes, cardio-oncology, organ-on-chip, cardiotoxicity

# INTRODUCTION

Cardiovascular diseases and cancer are the two leading causes of death in industrialized countries. Although in recent years new anti-cancer therapies improved long-term survival rates, this is unfortunately also accompanied by an increased risk of cardiovascular complications because of adverse side effects of these anti-cancer drugs. Anthracyclines are a group of widely used anti-cancer drugs that are known to cause cardiac complications, either early or late after start of treatment (1–7). The history of anthracyclines originates in the 1950s when Daunorubicin was isolated from the bacteria Streptomyces peucetius. Ten years later Doxorubicin, a more effective derivative of Daunorubicin, was identified (8). However, Doxorubicin received the ominous nickname "Red Devil" because of the detrimental side effects in combination with its red color. In approximately 11% of patients, Doxorubicin-induced cardiotoxicity leads to an acute response within 2–3 days of administration (early onset of cardiotoxicity), manifested by chest pain resulting from different forms of arrhythmia (3). Chronic Doxorubicin-induced cardiomyopathy has a lower incidence (ranging from 4 to 36% dependent on the dose) and can occur as late as 10 years after the last dose (late onset of chronic cardiotoxicity), with only a 50% 1-year survival prognosis when cardiomyopathy further develops into congestive heart failure (3, 7). Importantly, anthracycline-induced cardiotoxicity endangers cancer patients since the early 70s (9, 10). At present, almost half a century later, success in preventing or counteracting anthracycline-induced cardiotoxicity has been very limited. Moreover, although novel anti-cancer therapeutic compounds to specific target molecules, such as tyrosine kinase inhibitors, have been developed, drug-induced cardiotoxicity remains a persistent problem. One major reason for this is that current in vitro and animal models fail to show sufficient predictive power and limit extrapolation to patients, which consequently leads to high attrition rates during the process of drug development.

Development of preclinical human cell-based models for drug discovery that mimic human physiology and pathophysiology, has the potential to improve the predictability of adverse drug events (11). In particular, human pluripotent stem cells (hPSCs) are excellent candidates for developing these models since they replicate indefinitely and have the capacity to differentiate to any cell type of the human body, including functional cardiomyocytes. This is especially embraced in the cardiac field, since isolated primary human cardiomyocytes are extremely difficult to obtain and maintain in culture. Advances in differentiation and purification of hPSC-derived cardiomyocytes (hPSC-cardiomyocytes) and their unlimited availability has promoted strategies to use these cells for cardiotoxicity assessment of new drugs (12). Previously, the Food and Drug Administration (FDA), the supreme body worldwide that checks the quality and safety of medical products, initiated the so-called "Comprehensive in vitro Proarrhythmia assay (CIPA)," which represents a paradigm shift and encompasses evaluation of life-threatening proarrhythmic risk for all new compounds in a preclinical assay using hPSC-cardiomyocytes (13). This CIPA initiative signifies the enormous potential of hPSC-derived cardiomyocytes for preclinical drug screening. Here, we describe underlying mechanisms of anthracyclineinduced cardiotoxicity, as well as therapeutic approaches for cardioprotection. Furthermore, we will discuss the potential to use hPSC-derived cardiac models for improved safety assessment of anti-cancer drugs and strategies to overcome current limitations for developing in vitro drug testing platforms with a higher predictivity.

# Mechanism of Anthracycline-Induced Cardiotoxicity in hPSC-Cardiomyocytes

The potent therapeutic anti-cancer effect of Doxorubicin is mediated primarily via inhibition of topoisomerase IIa, an enzyme responsible for unwinding DNA before replication or transcription. This inhibition leads to DNA damage and consequently death of highly proliferating cancer cells, but also affects healthy proliferating cells, such as hematopoietic precursors, epithelial lining of the intestine and hair follicle cells. Fully mature cardiomyocytes are typically quiescent and do not express topoisomerase IIα, however studies performed in hPSC-cardiomyocytes demonstrated that Doxorubicin may bind to the enriched topoisomerase IIβ in cardiomyocytes, and knock-out of this isotype improved cell viability significantly when exposed to Doxorubicin (14, 15). A comprehensive review on damage mechanisms of anthracycline-induced cardiotoxicity has been published elsewhere (16). The underlying mechanisms of Doxorubicin-induced cardiotoxicity is not yet completely understood. However, several studies in hPSC-cardiomyocytes have shown that mitochondrial dysfunction, disruption of calcium homeostasis, as well as altered gene and protein expression levels triggering apoptotic cell death, play important roles in anthracycline-induced cardiotoxicity (**Figure 1**). Increased oxidative stress by elevated production of reactive oxygen (ROS) and nitrogen species (NOS) in the mitochondria of cardiomyocytes are major factors of Doxorubicin-induced cardiotoxicity, since cardiomyocytes are more sensitive to these molecules because they possess lower levels of antioxidant enzymes than other cell types (1, 3, 4, 17–20). As a consequence, higher stress levels in cardiomyocytes under oxidative stress circumstances can lead to cardiotoxicity (20). An increase in intracellular levels of ROS and NOS cause mitochondrial dysregulation, lipid peroxidation, DNA damage, and protein carbonylation. Cardiac mitochondria are the major site of Doxorubicin-induced ROS/NOS levels due to the localization of the major redox cycling enzymes such as NAD(P)H. Additionally, Doxorubicin becomes nearly irreversibly bound to cardiolipin. Cardiolipin is an essential phospholipid, almost exclusively expressed on the inner mitochondrial membrane. It has been shown that pathological changes in cardiolipin trigger ROS production and impair mitochondrial function (21). In addition, Doxorubicin also increases mitochondrial iron accumulation which further increases ROS production in the mitochondria. Indeed, cellular and mitochondrial (as measured by cellular H2O<sup>2</sup> production and intra-mitochondrial O2<sup>−</sup> levels) ROS production in hPSC-cardiomyocytes increases already 24 h after exposure to Doxorubicin at concentrations as low as 0.01µM (patient serum levels of Doxorubicin vary in

the range of 5–10µM after single injection). Moreover, short term exposure to 5µM Doxorubicin induces dramatic reduction of the mitochondrial transmembrane potential indicating mitochondrial dysfunction (14, 22). To meet the high energy demand required to sustain contractile function, cardiomyocytes rely on efficient mitochondrial oxidative metabolism for energy production instead of anaerobic glycolysis. Thus, a disturbed energy metabolism is a high risk for cardiomyocyte survival. Repeated Doxorubicin exposure reduces ATP levels which cannot be restored after Doxorubicin removal suggesting a prolonged effect of Doxorubicin on energy generation in hPSC-cardiomyocytes. Together these effects pinpoint to the fact that Doxorubicin induces mitochondrial dysfunction in hPSC-cardiomyocytes (22, 23). Importantly, structural and functional disturbances are more pronounced after repeated dosing of Doxorubicin (mimicking chronic exposure) (23–25).

Another mechanism by which anthracyclines fight cancer is the induction of apoptosis. Doxorubicin effectively induces apoptotic cell death via activation of so-called death receptors (DRs), such as TNF receptor 1 (TNFR1), Fas receptor, DR4 and DR5, in many cancer types. Activation of these DRs induces the assembly of death inducing signaling complex (DISC), which starts the caspase cascade to mediate cleavage of cellular proteins and ultimately apoptosis of the cell. This DRmediated apoptosis machinery has been shown to be conserved in human cardiomyocytes. Indeed, a Doxorubicin concentration– dependent increase of caspase 3 and 7 and Annexin V, a marker of early apoptosis, and 7-aminoactinomycin D (7–AAD) or propidium iodide as markers of late apoptosis or necrosis, suggest a strong contribution of programmed cell death to reduced cell survival after exposure to Doxorubicin. Interestingly, in hPSC-cardiomyocytes Doxorubicin induces the expression of all four DRs in a dose-dependent fashion with p53 being the key upstream activator. This suggests a p53-regulated DRmediated apoptotic pathway a key mechanism involved in early Doxorubicin-induced cardiotoxicity. In accordance with these findings, Doxorubicin-induced apoptosis was effectively blocked by pretreating hPSC-cardiomyocytes with a DR5 neutralizing antibody. This cardiotoxic effect was reversible since expression of DR4 and 5 proteins decreased after recovery for 7 days following washout of Doxorubicin (14, 15, 22, 26, 27). In contrast to these studies with acute exposure to Doxorubicin, Li et al. showed that p53 can protect against chronic Doxorubicin exposure by counteracting mitochondrial DNA depletion after chronic exposure of hPSC-cardiomyocytes (and mice) to low doses of Doxorubicin (28).

Disturbed calcium homeostasis in cardiomyocytes hampers proper cardiac contraction. Doxorubicin has been shown to induce accumulation of intracellular calcium release from the sarcoplasmic reticulum (SR) causing calcium overload with sarcomeric disarray and myofibril deterioration in hPSCcardiomyocytes. Moreover, Doxorubicin exposure led to downregulation of several ion channel genes, including genes that encode for calcium channels CACNs (14, 22, 26).

In summary this data shows that mitochondrial dysfunction, apoptosis, as well as disturbed calcium homeostasis are the main mechanisms of anthracycline-induced cardiotoxicity. However, in 2018, a new mechanism of Doxorubicin-induced cardiotoxicity via downregulation of the RNA-binding-protein Quaking has been identified (29). Downregulation of Quaking regulates a set of circular RNAs involved in Doxorubicin-induced apoptosis suggesting that other mechanisms cannot be excluded.

# Protective Mechanisms to Prevent or Reduce Anthracycline-Induced Cardiotoxicity

To increase the dose window of effective treatment without severe adverse side effects (therapeutic index) of conventional Doxorubicin therapy, several protective mechanisms have been developed, including modifications/analogs of Doxorubicin, targeted delivery or protective agents (**Figure 2**).

#### Analogs of Doxorubicin

The cardiotoxic nature of Doxorubicin pushed the development of Doxorubicin analogs into overdrive in the 1970s and 1980s, with over a 1000 analogs manufactured or discovered since (30). However, most of them failed to reach anti-tumor efficacy comparable to Doxorubicin and others were considered too toxic based on trials in rodents (30). Only a handful of analogs reached clinical trial phase, such as epirubicin, pirarubicin (also known as THP), idarubicin, mitoxanthrone, and others (31–42). The success of these analogs varies per clinical setting and malignancy, and a reduction in cardiotoxicity was not always achieved. In patients, cardiotoxic events are classically detected through loss of the left ventricular ejection fraction (LVEF), irregularities in ECG readings (in particular ST-T changes), changes in systolic time intervals (STI, indicating heart muscle failure) and in more recent work, the release of biomarkers in blood, such as cardiac Troponin-T and brain natriuretic peptide (BNP) (31, 33, 35– 38, 41–45). Due to the year of discovery, early Doxorubicin analogs have not been tested in vitro using hPSC-cardiomyocytes to evaluate their cardiotoxicity, since these models were not available at the time. Whereas, biomarker release can also

be assessed in vitro using hPSC-cardiomyocytes, other clinical read-outs are more difficult to evaluate in vitro. Nevertheless, measurement of changes in action potential, contractility, and cell survival in vitro may be associated with ECG/STI changes and impaired LVEF and therefore it will be of interest to test Doxorubicin analogs in hPSC-cardiomyocytes for cardiac safety.

### Targeted Delivery Strategies

An alternative to reducing cardiotoxic properties of anthracyclines is specifically delivering anthracyclines at the location where cancer cells reside. This can be achieved by carriers that encapsulate the compound and release it at the desired location. For example, liposomal encapsulation of anthracycline inhibits transfer from the circulation to tissues with a tight endothelial barrier (e.g., the heart), but allow transfer to tissues with leaky or fenestrated vasculature, such as in tumors, but also in organs such as lung, bone marrow, lymph nodes and liver (39, 46, 47). In the last two decades, numerous trials have been conducted to assess the safety and benefit of liposome-encapsulated Doxorubicin. Success of liposomal encapsulated anthracyclines is inconsistent. Liposomal encapsulation could abolish cardiotoxicity in some clinical trials, allowing a higher cumulative dose for treatment (39, 48–50). However, in some clinical settings, liposomal Doxorubicin did not reduce cardiotoxicity compared to free anthracyclines (51), suggesting that liposomal encapsulation only has limited benefit. Unmasked liposomes such as described thus far are relatively quickly cleared from the circulation by the reticulo-endothelial system (RES), which limits the therapeutic index of so-called non-PEGylated liposomal (NPL) (39).

PEGylated liposomal Doxorubicin (PLD) features an anchored polyethylene glycol (PEG) group on the exterior lipid surface that masks them from the RES, thus reducing uptake from the circulation (52). Caelyx (Doxil), a commercialized PLD, showed significant reduction in cardiotoxicity compared to free Doxorubicin in several studies, even when used for patients who have increased risk of cardiac complications (53–55). Unfortunately, PLDs are limited in their use because of increased incidence and severity of non-cardiac side effects such as palmar-plantar erythrodysesthesia (PPE), mucositis and haematogenic disorders (54, 56). Liposomes (PEGylated or not) cannot prevent severe adverse effects of Doxorubicin entering off-target tissues. The mechanism of specific delivery of classical liposomes depends greatly on the difference in endothelial barrier permeability, which is equally high in tumors and several healthy tissues alike, and not all malignancies cohere to the "tumor with a leaky vasculature" phenotype. In an attempt to increase specific targeting of cancer cells, liposomes were conjugated with antibodies that are directed against the human epidermal growth factor receptor 2 (HER2), which is highly expressed in breast cancer cells. In vitro, these liposomes were effective in killing HER2-positive cancer cells, but did not affect hPSC-cardiomyocytes, highlighting their specificity (47). The HERMIONE phase II trial is now being conducted to test the safety and efficacy in patients (57). Another recent development is the encapsulation of anthracyclines in nanocages, constructed of organic (virus-like particles (VLPs), protein, DNA or carbon-based particles) or inorganic particles (supramolecular nanosystems, hybrid metal-organic, gold, or silica-based systems). Advantages of this encapsulation method are: (1) a large carrier storage capacity, (2) a targeted release of the drug at the site of interest, (3) combined with a porous structure and (4) a low immunogenic surface (58). Nanocages have not been tested in clinical trials yet, however in vitro data suggests improved drug targeting of cancer cells and killing efficacy, and a lower cardiotoxicity, when administered in rodents (59–63). Unfortunately, nanocages have not been specifically tested in vitro on hPSC-cardiomyocytes to test their cardiotoxicity.

In addition to Doxorubicin encapsulation, latest modifications to Doxorubicin are being tested on cell cultures prior to animal testing to assess their safety for the heart, using currently available hPSC-cardiomyocyte in vitro models. These modifications of Doxorubicin are aimed at preventing the anthracycline from exerting its toxicity on cardiomyocytes by making the drug delivery more specific. For example, DTS-201, a Doxorubicin prodrug that is injected in a stable, cell-impermeable state, is cleaved by endopeptidases that are released by tumors, enabling entry into nearby cells and specific anti-tumor activity. In a phase I trial, DTS-201 could be administered at a cumulative dose equivalent to three times the recommended dose of free Doxorubicin, without triggering a significant drop in LVEF (64). Another promising technique is the development of Doxorubicin-dendrimer conjugates with acetylgalactosamine attached to the dendrimer surface. These conjugates are specifically taken up by hepatic cancer cells, increasing therapeutic index and reducing cardiac exposure and thus toxicity in mice. In addition, in vitro, these conjugates did not affect hPSC-cardiomyocyte viability and electrophysiology, or induce apoptosis, demonstrating the specificity for drug uptake by the target cell type (65).

### Protective Agents to Alleviate Cardiotoxicity

Cardioprotection during anthracycline-based therapies can also be offered by combining the anthracycline with a protective compound. As the cardiotoxic mechanism of anthracyclines involves inducing oxidative stress and mitochondrial dysfunction, protective agents that are able to lower the production of ROS or lower the workload of the heart during anthracycline treatment may be beneficial to reduce anthracycline-induced cardiotoxicity. Factors that may reduce oxidative stress in the heart, such as statins and natural antioxidants, and compounds that reduce the workload of the heart, such as angiotensin-converting-enzyme (ACE) inhibitors and beta-blockers, have been combined with anthracyclines in clinical trials, with mixed success (66–77). As far as known, these compounds have never been tested in hPSC-cardiomyocytes for assessment of their direct cardioprotective potential, even though reducing oxidative stress in particular can be readily modeled in vitro. Since both ischemic heart disease and anthracycline-mediated cardiotoxicity share common pathological pathways, therapies for one disease could also be beneficial for the other. Trials with more original approaches, such as remote ischemic conditioning or physical exercise and caloric restriction, strategies that were beneficial in the setting of ischemic heart disease, have been started with the purpose to reduce cardiotoxicity caused by chemotherapy (78, 79). Results of these trials are not yet available.

In hPSC-cardiomyocytes it has been shown that Doxorubicin causes arrhythmic contractions by inducing the accumulation of calcium in the cell and thereby disturbing calcium homeostasis (14, 22). This would suggest that calcium antagonists can help to reduce Doxorubicin-induced cardiotoxicity. Interestingly, a calcium antagonistic compound called prenylamine, was able to reduce cardiotoxic effects when co-administered with Doxorubicin, before it was withdrawn from the market 1 year later (80). Reason for withdrawal was the pro-arrhythmic properties of prenylamine, inducing long QT syndrome (81). Other calcium agonists are currently primarily used at lower doses to reduce Doxorubicin resistance of cancer cells, by inhibiting their capacity to pump out drugs via the Pglycoprotein ATP-dependent efflux pumps (82). Low doses of these calcium antagonists could be repurposed to reduce Doxorubicin-induced cardiotoxicity.

Neuregulin-1β, an ErbB receptor (HER2) family ligand, has been proven effective against Doxorubicin-induced cardiotoxicity, but is also pro-neoplastic in many cancers via formation of ErbB2/3 interactions (83–86). Even if neuregulin, as key mediator of endothelial–cardiomyocyte crosstalk, is able to protect ventricular cardiomyocytes from anthracyclineinduced apoptosis, its use might thus be controversial in the clinic. Nevertheless, phase I, II and III clinical trials have been completed or are ongoing (NCT01251406, NCT1214096, and NCT01541202) (84–86). Patients with symptomatic heart failure (HF) and left ventricular dysfunction showed improved cardiac function with increased LVEF after treatment with recombinant human neuregulin 1. In contrast to native neuregulin-1β, engineered bivalent neuregulin-1β has reduced pro-neoplastic potential because of a shift toward ErbB3 homotypic interactions. Bivalent neuregulin-1β is antineoplastic or cytostatic in cancer cells. Importantly, bivalent neuregulin-1β showed similar cardioprotective properties as neuregulin in hPSC-cardiomyocytes and in mice with chronic cardiomyopathy (87). Especially the reduced pro-neoplastic potential of bivalent neuregulin-1β offers translational potential for cardioprotection after anthracycline therapy, however to date, bivalent neuregulin-1β has not been taken into clinical trial.

Dexrazoxane, a catalytic topoisomerase inhibitor and iron chelator, is the only marketed cardioprotective agent that is used specifically to counteract the adverse effects of anthracyclines (88). In children with acute lymphoblastic leukemia, leukemia or lymphoma, the combined treatment with Doxorubicin and Dexrazoxane significantly reduces release of troponin (as biomarker of cardiotoxicity) and Doxorubicin-induced cardiac remodeling. Importantly, the combination with Dexrazoxane improves left ventricular function compared to Doxorubicin alone (89, 90). Dexrazoxane is also effective in relapsed patients with leukemia when administered prior or after a high cumulative dose (91). In breast cancer patients with increased risk of cardiotoxicity because of an enhanced cumulative dose of anthracycline, Dexrazoxane also robustly reduced the incidence of cardiac events, such as decreased ejection fraction, and incidence and severity of CHF (92, 93). Even in a retrospective 2–20 year follow-up, Dexrazoxane demonstrated late-clinical and subclinical cardioprotective effect (94).

Treatment of cardiomyocytes with Dexrazoxane is therefore expected to prevent cardiotoxicity. However, pre– and co– treatment of hPSC-cardiomyocytes with Dexrazoxane could not alleviate Doxorubicin-induced cardiotoxicity and increased cardiotoxicity at concentrations as low as 0.1µM (22, 95). This discrepancy between in vivo and in vitro may be related to the relative immature character of hPSC-cardiomyocytes. Early stage hPSC-cardiomyocytes may be more sensitive to Doxorubicin because of higher expression levels of topoisomerase IIα and are thus more prone to severe DNA damage (as mentioned earlier: Doxorubicin primarily affects topoisomerase IIα), whereas in mature cardiomyocytes topoisomerase IIα switches to the IIβ isoform. Importantly, Dexrazoxane might exert its protective effects via depletion of topoisomerase IIβ, the major topoisomerase isoform in more mature cardiomyocytes (95, 96). This suggests that more advanced models are needed to assess cardiotoxicity in vitro.

# HPSC-Cardiomyocyte Models in Drug Screening of Tyrosine Kinase Inhibitors (TKis)

Not only anthracyclines, but also safety assessment of other classes of anti-cancer drugs may benefit from hPSC-based in vitro models. For example, malignancies caused by hyperactive receptor tyrosine kinases (RTKs), which drive proliferation and survival, are treated with FDA-approved tyrosine-kinase inhibitors (TKis). However, a number of TKis have been associated with adverse cardiac side effects following treatment of cancer patients, such as reduced LVEF, myocardial infarction, arrhythmias and heart failure (97). While animal models have often failed to predict cardiotoxic effects during safety assessment (98), TKis have shown to inhibit viability, contractility, electrophysiology, calcium handling and cardiac oxidative phosphorylation of hPSCcardiomyocytes (99–101). Different TKis show cell type specific cytotoxicity, either in hPSC-derived cardiomyocytes, endothelial cells and fibroblasts or hPSCs, suggesting that TKis differently affect cardiovascular and non-cardiovascular cell types (99).

TKis can be subdivided into small molecules, including crizotinib, sunitinib, or sorafenib, and monoclonal antibodies. Importantly, different TKis have distinct toxicity profiles and not all TKIs induce cardiotoxicity (97). The broad-range TKi sunitinib, for example, decreased hPSC-cardiomyocyte viability, as well as AMP-activated protein kinase (AMPK), increased lipid accumulation and induced arrhythmic events in sunitinib-treated hPSC-cardiomyocytes. Crizotinib, an ALK/MET inhibitor, increased ROS production and caspase activation, induced cholesterol accumulation and an irregular beat pattern. Similarly, Nilotinib, a second generation Bcr-Abl inhibitor, increased ROS generation and caspase activation and induced arrhythmic beating. Interestingly, compared to the TKis sunitinib, crizotinib and nilotinib, the relatively cardiac-safe TKi erlotinib only displayed minor effects on hPSC-cardiomyocyte health in the same study (98) which suggests that hPSC-cardiomyocytes may represent a reliable in vitro model for predicting cardiotoxic side effects.

RTK activity may also be reduced by blocking the human epidermal growth factor receptor 2 (HER2), which activation is a common feature of a subset of malignant diseases, particularly breast cancer (102). While trastuzumab (TZM), a TKi monoclonal antibody blocking HER2, greatly improves treatment against HER2 positive malignancies, inhibition of HER2 signaling was found to be detrimental for cardiac function (102–104). In a subset of breast cancer patients, treatment with TZM led to mild to severe decrease in LVEF without apparent myocardial tissue damage or cell loss. Using patient-derived hPSC-cardiomyocyte 2D monolayer cultures it was shown that TZM affected metabolic and mitochondrial processes, of which the severity depended on genetic variation. Moreover, this disease phenotype could be rescued using metabolism-stimulating agents, such as AMPK (102). Additionally, HER2 activation triggered by exogenous neuregulin-1 could reduce hPSC-cardiomyocyte damage and cell loss during Doxorubicin exposure, an effect that was lost upon inhibition of the HER2 receptor by TZM (103, 104). These findings indicate that hPSCcardiomyocyte 2D models may serve as a valuable tool to identify cardioprotective compounds in a high throughput system, which can be further validated in more advanced human-based 3D cardiac models.

# Generation of Advanced hPSC-Derived in vitro Models for Accurate Assessment of Cardiac Safety

Although cardiotoxic effects could be observed in hPSCcardiomyocyte based models, not all aspects of in vivo cardiotoxicity are recapitulated in current human in vitro models. One of the important shortcomings of hPSC-cardiomyocyte models is their relative immature phenotype. Reviews on the topic of the immaturity of hPSC-cardiomyocytes, including strategies to increase the level of maturity have already been described elsewhere (105–107). **Figure 3** shows a description of current in vitro models utilizing hPSC-cardiomyocytes, including their advantages and limitations. It is therefore crucial to develop innovative advanced human models that approximate the adult human heart. In order to achieve this, defined multicellular models, consisting of the main cell-types of the heart, such as atrial or ventricular cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts, need to be constructed.

Cardiac tissue formation in 3D tissues, such as cardiac microtissues, more closely mimics native heart tissue which might allow studying more physiologically relevant dosing profiles and deciphering acute vs. chronic cardiotoxicity (25). For generating other 3D advanced cardiac models, so-called engineered heart tissues (EHTs), hPSC-cardiomyocytes are mixed with fibroblasts (or other cardiac cells) in a hydrogel and poured into a casting mold around silicon posts, leading to beating structures within the first week under continuous mechanical strain. Previously, it has been shown that 3D EHTs display a higher degree of maturation based on sarcomeric organization, formation of T-tubules and functional characterization (e.g., contraction force, electrophysiology) (108–110). Cardiotoxicity of anti-cancer agents, other than anthracyclines, such as the TKi sunitinib, has already been assessed in EHTs, which resulted in triggered activation of apoptosis and loss of contractile force, spontaneous beating and mitochondrial membrane potential (111).

monolayers to 3D advanced models) are available for assessment of drug-induced cardiotoxicity utilizing hPSC-cardiomyocytes. Whereas 2D monolayer cardiomyocyte cultures can be used for high throughput-screening for specific cellular responses, they lack the physiology, multicellular interactions and tissue organization, which can be provided by organ-on-chip, co-culture or 3D tissue models (for instance engineered heart tissues). Advanced cardiac models, such as an engineered mini heart tube with the capacity to pump fluid, mimic clinically-relevant function(s) of the heart.

Until now studies did not consider subtype-specific cardiomyocytes for cardiotoxicity testing. This would be of high importance, as different cardiac subtypes may exhibit different susceptibility to Doxorubicin. Especially, because of the well-established arrhythmogenic potential of Doxorubicin (31, 112, 113), it is highly relevant to assess arrhythmogenic effects of Doxorubicin on hPSC-derived atrial and ventricular cardiomyocytes. Moreover, in a trial comparing Doxorubicin to Epirubicin in treatment of breast cancer, a quarter of all cardiac side effects of Doxorubicin was identified as sinus tachycardia, for Epirubicin this was half of the cardiotoxic responses in the trial population (31). In addition, a recent study showed that genetic loci are associated with both increased sensitivity to Doxorubicin-induced hPSC-pacemaker cell death and a higher risk of arrhythmia in patients (114), which justifies to perform cardiotoxicity testing on pacemaker cells. Previously, we and others have shown efficient production of atrial, ventricular and pacemaker cardiomyocytes from hPSCs (115–118).

In the cardiac microenvironment in vivo, each cardiomyocyte is surrounded by 3–4 capillaries with a distance between each cardiomyocyte and an endothelial cell of about 1µm (119). This delicate build-up allows for defined cardiomyocyte-endothelium crosstalk via paracrine signaling and cell-cell contact (119–122). This data suggests that the crosstalk of human cardiomyocytes with highly metabolic active cardiac endothelial cells is very important. Recent advances in micro-engineering and stem cell technologies enabled development of microfluidic devices in which living cells (mostly several cell-types) are cultured in channels or small compartments and perfused in a controlled manner, mimicking the microenvironment and responses of tissues or organs (123–125). These so-called organ-on-chips can be combined with integrated sensors (for example for biochemical and electrical readouts) and are compatible with live imaging (for example using stem cell-based fluorescent reporter lines) (126) and human cell and tissue sampling. Very recently, Weng et al. (127) generated a multiple chambered tissue chip, a so-called organ-on-a-chip, which allowed simultaneous culturing of tumor tissue and hPSC-cardiomyocytes while being connected via a microfluidic channel, lined with endothelial cells. This design enables simultaneous testing of potential anti-tumor effects and cardiotoxicity of drugs administered via the endothelial layer. Another important aspect of drug efficacy and safety assessment is to predict pharmacokinetic parameters, determined by absorption, distribution, metabolism and excretion (ADME) of drugs, which are difficult to measure or model in vitro. As mentioned before, organ-on-chips mimic organ-like function and responses in perfusable mircophysiological systems and may offer an opportunity to overcome current limitations related to pharmacokinetic modeling. Multiple organ-on-chip devices with organ-level functionality could be connected to each other with vascularized, endothelium-lined channels mimicking blood circulation and recapitulating tissue-tissue interfaces, thus modeling organ crosstalk and metabolism of compounds in a single system (128, 129). For predictive physiologically based pharmacokinetic (PBPK) modeling, it is of paramount importance to combine defined hPSC culture methods and 3D tissue engineering or organoid formation with technical advances in the field of organ-on-chip technology, ultimately leading to interconnected multiple human organ-on-chip platforms (for example, combining heart-, liver- and gut-on-chip models).

# FUTURE OUTLOOK

Because of the previously mentioned shortcomings, a full recapitulation of clinical cardiotoxic manifestations is beyond the capacity of current in vitro models. Interestingly, recent developments have shown that it is feasible to build human in vitro models that are more closely resembling functional human hearts for assessment of clinically relevant parameters, which may greatly enhance the predictability of these in vitro models for safety pharmacology and disease phenotypes. Recently, Li et al. created for the first time a human ventricular-like cardiac organoid chamber (hvCOC) with hPSC-cardiomyocytes and dermal fibroblasts (130). Similarly, Macqueen and colleagues generated a one cell layer thick ventricular-like tube from hPSCcardiomyocytes (131). Both HvCOCs and ventricular tubes were able to pump fluid, which allowed measuring ejection fraction and pressure-volume loops as readouts for cardiac function. Although these developments are promising, combination with other cardiac cells, such as endothelial and smooth muscle cells and fibroblasts, may lead to further maturation and improvement of function. Future studies will be required in order to evaluate the predictive values of these human advanced 3D cardiac models in preclinical drug testing, their potential to reduce time for bringing new drugs from bench-to-bedside and repurposing of drugs.

Moreover, state-of-the-art multiple organ-on-chip platforms will also advance predictability of efficacy and toxicity of combinatorial drug (cardioprotective) treatment and specific target delivery.

It is evident that interpatient variability further complicates development of predictive in vitro models for drug screening. This is also true for anthracycline-induced cardiotoxicity, which varies tremendously from patient to patient. These variations may be caused due to environmental factors, life-style or genetic variance. Patient-derived (or genetically modified) hPSCs provide the possibility to include diseaseassociated genetic risk factors in these in vitro models, which can be directly compared to isogenic control hPSC lines. Interestingly, studies with hPSC-cardiomyocytes have shown that cardiotoxic effects of Doxorubicin were dependent on the genetic background of patient-derived hPSC-cardiomyocytes (22). Similarly, characterization of hPSC-cardiomyocytes from 45 individuals showed inter-individual variation in transcriptional response after 24 h exposure to different concentrations of Doxorubicin, which were predictive of in vitro cell damage as measured by cardiac troponin release of cardiomyocytes (132). These findings highlight the importance of personalized medicine and risk stratification, which will allow us to predict for which patients new therapies will be effective and safe and for which not. This will reduce the attrition rate, costs and time of drug development and facilitate repurposing of drugs that have shown a lack of efficacy or risk of toxicity. Consequently, successful implementation of these advanced hPSC-based models will lead to better and safer drugs.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# REFERENCES


# FUNDING

This work was supported by ERA-CVD 2016T092 and the Dutch Heart Foundation.

# ACKNOWLEDGMENTS

We would like to thank Christian B. Schwach for illustrations.


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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Schwach, Slaats and Passier. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# High Density Lipoprotein and Its Precursor Protein Apolipoprotein A1 as Potential Therapeutics to Prevent Anthracycline Associated Cardiotoxicity

George E. G. Kluck <sup>1</sup> , Kristina K. Durham<sup>2</sup> , Jeong-Ah Yoo<sup>1</sup> and Bernardo L. Trigatti <sup>1</sup> \*

<sup>1</sup> Department of Biochemistry and Biomedical Sciences, Thrombosis and Atherosclerosis Research Institute, McMaster University and Hamilton Health Sciences, Hamilton, ON, Canada, <sup>2</sup> Faculty of Health Sciences, Institute of Applied Health Sciences, School of Rehabilitation Sciences, McMaster University, Hamilton, ON, Canada

Cardiovascular disease and cancer are the leading causes of death in developed societies. Despite their effectiveness, many cancer therapies exhibit deleterious cardiovascular side effects such as cardiotoxicity and heart failure. The cardiotoxic effects of anthracyclines such as doxorubicin are the most well-characterized of cardiotoxic anti-cancer therapies. While other anti-neoplastic drugs also induce cardiotoxicity, often leading to heart failure, they are beyond the scope of this review. This review first summarizes the mechanisms of doxorubicin-induced cardiotoxicity. It then reviews emerging preclinical evidence that high density lipoprotein and its precursor protein apolipoprotein A1, which are known for their protective effects against ischemic cardiovascular disease, may also protect against doxorubicin-induced cardiotoxicity both directly and indirectly, when used therapeutically.

#### Edited by:

Canan G. Nebigil, École Supérieure de Biotechnologie Strasbourg (ESBS), France

#### Reviewed by:

Alessandra Ghigo, University of Turin, Italy John David Horowitz, University of Adelaide, Australia

> \*Correspondence: Bernardo L. Trigatti trigatt@mcmaster.ca

#### Specialty section:

This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine

> Received: 15 December 2019 Accepted: 06 April 2020 Published: 28 April 2020

#### Citation:

Kluck GEG, Durham KK, Yoo J-A and Trigatti BL (2020) High Density Lipoprotein and Its Precursor Protein Apolipoprotein A1 as Potential Therapeutics to Prevent Anthracycline Associated Cardiotoxicity. Front. Cardiovasc. Med. 7:65. doi: 10.3389/fcvm.2020.00065 Keywords: HDL, ApoA1, anthracyclin, chemotherapy, cardiotoxicity, cardioprotective, doxorubicin

# INTRODUCTION

Advances in cancer treatment over the past few decades have led to substantial increases in cancer survivorship (1, 2). As cancer-related survival has improved, an unexpected increase in premature cardiovascular events, including myocardial ischemia, myocardial infarction, congestive heart failure (HF), QT interval prolongation, hypertension, and stroke has occurred (3, 4). A major contributing factor to cardiovascular outcomes is cardiotoxicity related to antitumor drugs, which may become apparent acutely during treatment, or often, well after treatment has ended (4, 5). In general terms, cancer therapy related cardiotoxicity ultimately leads to pathological alterations in the cardiac muscle tissue (5). Many different classes of chemotherapeutic agents have cardiotoxic effects. Anthracyclines such as doxorubicin (DOX) are, perhaps, the most well-studied cardiotoxic chemotherapeutic agents. They have a number of direct cardiotoxic effects, including DNA damage, interfering with mitochondrial function, induction of reactive oxygen species (ROS), alterations in autophagy and induction in apoptosis. Mechanisms of anthracycline induced cardiotoxicity will be discussed in more detail below. Other cardiotoxic chemotherapeutic agents/treatments include fluoropyrimidines, such as 5 fluorouracil, biologicals, such as trastuzumab and radiation therapy, all of which impact cardiomyocyte survival by triggering apoptosis through differing, but overlapping pathways (summarized in **Figure 1**). For example, fluoropyrimidines, such as 5-fluorouracil (5-FU) and capecitabine, are used to treat different types of tumors, especially those that appear in the head, neck and breast (10, 11). In the case of these agents, cardiotoxicity appears to be due to both direct toxic effects of these drugs on cardiomyocytes, through the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to both oxidative and nitrosative stress, and activation of apoptosis and autophagy (12). Furthermore, these agents also appear to have indirect cardiotoxic effects through interaction with the coagulation system and autoimmune responses (8). Trastuzumab, an antibody targeting the Human epidermal growth factor 2 receptor (HER/ErbB2), and used as first choice therapy against breast cancer (13, 14), also inhibits this receptor in cardiomyocytes, affecting myocardial structure and survival pathways, triggering cardiomyocyte apoptosis and leading to asymptomatic decreased left ventricular ejection fraction, and eventually heart failure, particularly when it is used in combination with other agents, such as anthracyclines (15, 16). Radiation therapy also triggers cardiotoxicity through induction of DNA damage as well as ROS in cardiomyocytes, again affecting cell survival pathways and triggering apoptosis (17). Radiation therapy induced cardiovascular effects may manifest as pericarditis, coronary artery disease, myocardial infarction, valve heart disease, changes in rhythm, silent myocardial ischemia and damage to the conduction system (18). The risk of heart disease is mainly related to the total radiation dose and the volume of the heart receiving radiation (19).

As a result of these cardiotoxic effects, the mode of administration, time and dose of these treatments and the presence of pre-existing comorbidities such as cardiovascular and liver disease, diabetes mellitus and hypertension are

FIGURE 1 | Direct cardiotoxic effects of anti-cancer therapies in cardiomyocytes. Doxorubicin cardiomyocyte cytotoxicity through diverse pathways including DNA damage, accumulation of ROS and RNS, mitochondrial dysfunction, apoptosis and impaired autophagy. Inhibition of ErbB2/4 receptor complex with trastuzumab impacts several signaling pathways resulting in impaired protein synthesis and myocardial structure via the Src/Fak pathway (6). Trastuzumab mediated supression of ErbB2/4 stimulated PI3K-Akt signaling impacts cell survival via mTOR pathway as well as increases apoptosis via BAD/BcL-xL pathway (7). Chemotherapy with fluoropyrimidines, such as 5-fluorouracil (5-FU) cause increase in formation of ROS and RNS leading to mitochondrial dysfunction and activation of caspase-3 and apoptosis (8). Radiotherapy leads to ROS and RNS formation, mitochondrial dysfunction and single-stranded DNA breaks leading to apoptosis activation (9).

important factors to be taken into consideration upon treatment (20). Furthermore, long term follow-up for cardiovascular complications is important, particularly in those patients with pre-existing cardiovascular risk factors (21–23). Understanding the mechanisms by which chemotherapeutic agents cause cardiotoxicity, as well as the identification of pathways that can be targeted in the cardiomyocyte to selectively mitigate chemotherapy induced cardiotoxicity is of fundamental importance in decreasing the undesirable impact on normal tissues and improving cancer treatment outcomes.

High density lipoproteins (HDL) have long been associated with cardioprotection, with a major focus being on arteriosclerosis-associated ischemic heart disease and stroke (24–28). This has largely been associated with its role in the transport of cholesterol from the artery wall to the liver for excretion or recycling, a process called reverse cholesterol transport (RCT) (29). However, recent advances in our understanding of HDL properties and biological functions have revealed HDL associated functions extending beyond cholesterol transport, including direct cytoprotective effects on a number of cell types including cardiomyocytes (30, 31). These advances provide key insights into the potential of exploiting these cytoprotective properties for therapeutic approaches to mitigate chemotherapy associated cardiotoxicity.

Here-in, we will first review the mechanisms by which anthracyclines, the most well-characterized of the cardiotoxic chemotherapeutic agents, induce cardiotoxicity, focusing on DOX as a well-studied anthracycline. While other anti-neoplastic drugs also induce cardiotoxicity, often leading to heart failure, they are beyond the scope of this review. We will then review recent advances in our understanding of HDL functions and properties that can be exploited to mitigate DOX-associated cardiotoxicity. These include understanding the mechanisms by which HDL induces cytoprotective responses in cells including cardiomyocytes, the ability of HDL and synthetic particles based on it to encapsulate DOX and serve as delivery vehicles, and findings that therapeutic treatment with HDL's major structural protein, apolipoprotein A1 (ApoA1) appears to have direct antineoplastic effects in preclinical tumor models (32–34).

# MECHANISMS OF ANTHRACYCLINE INDUCED CARDIOTOXICITY

Anthracyclines are a class of chemotherapeutics commonly prescribed to both adult and pediatric populations (35) and can be used alone or in combination with other cancer treatments. Since its discovery in the late 1960's, DOX (also called adriamycin, the prototypical anthracycline) has become widely prescribed due to its efficacy in treating cancers of both hematologic and solid origin (36, 37). Despite their widespread use, anthracyclines such as DOX are not specific in their cell target and exhibit cytotoxic effects in cardiomyocytes thereby limiting their long-term use due to dose-dependent cardiotoxicity (38). Immediate cardiac side effects of DOX infusion are detectable in the form of arrhythmias (39), and cardiotoxic outcomes can be measured following termination of treatment in both early (weeks to months) and late phases (years). These outcomes range from asymptomatic left ventricular dysfunction, to problematic arrhythmias and severe symptomatic congestive heart failure (37, 38). In a retrospective analysis of three trials of DOX therapy for breast or small cell lung cancers, the estimated cumulative percentage of patients with congestive heart failure was 5% in patients receiving a cumulative DOX dose of 400 mg/m<sup>2</sup> , which increased to 26% at a dose of 550 mg/m<sup>2</sup> , and 48% at 700 mg/m<sup>2</sup> (40). The incidence of cardiotoxicity is highest within the first year following the termination of chemotherapy in adults, although in cases of childhood cancer, onset of cardiotoxicity has been observed to be delayed in survivors by 4–20 years (40–43).

A number of pathways have been implicated in DOXmediated cardiomyocyte cytotoxicity. These include the direct and indirect induction of oxidative stress, DNA damage, and mitochondrial dysfunction. These pathways, along with alterations in homeostatic processes such as autophagy directly and indirectly lead to induction of cell death pathways, including apoptosis and necrosis (44–46) (**Figure 2**). These pathways have been the subject of recent comprehensive reviews (51–55) and will be summarized in the following sections.

# Oxidative Stress

Oxidative stress is the accumulation of oxygen and nitrogen free radicals resulting when their production exceeds the capacity of anti-oxidant enzymes to detoxify them (56). Oxidative stress has been proposed to be a major contributor to cardiomyocyte death and dysfunction following DOX treatment (56) (**Figure 2**). DOX can directly induce oxidative stress. The quinone moiety of DOX can act as an electron acceptor which can be reduced by a variety of enzymes to a semi-quinone, with the generation of oxygen free radicals. These can react with proteins, lipids and DNA, resulting in protein dysfunction, lipid peroxidation and DNA damage (50). DOX can also trigger reductions in the activities and expression of antioxidant enzymes, including matrix manganese superoxide dismutase (MnSOD) and glutathione (GSH) peroxidase (57), thereby reducing the antioxidant capacity of the cardiomyocyte. DOX can also interact with nitric oxide (NO) to generate reactive nitrogen species, contributing to nitrosative stress (56). DOX can also contribute to free radical formation and oxidative stress in cardiomyocytes through complex formation with iron (54). DOX treatment also increases accumulation of mitochondrial iron, once again manifesting in increased reactive oxygen and nitrogen species production (50). Dexrazoxane (DRZ) is an iron chelator which has seen limited clinical use to mitigate DOXassociated cardiotoxicity. On the other hand, pre-clinical studies of anti-oxidants have shown limited effectiveness at reducing DOX-associated cardiotoxicity, leading to doubt regarding the role of DOX-induced ROS formation in DOX associated cardiotoxicity (53).

# DNA Damage

DOX also appears to act within the nucleus to trigger cytotoxic effects in cardiomyocytes (**Figure 2**). DOX can reportedly bind to the cytoplasmic proteasome, which assists in the translocation of DOX to the nucleus by an ATP-dependent nuclear pore-mediated

mechanism (58). Topoisomerase (Top)-2β was identified as a molecular mediator of DOX cardiotoxicity (48). Top-2β is located in both the nucleus and mitochondria, and is an important regulator of DNA topology by catalyzing the breaking and rejoining of DNA in order to allow for strands to pass by one another (59). DOX binding to Top-2β and DNA forms the Top-2β–DOX—DNA cleavage complex (48). By binding to Top-2β, DOX reportedly disrupts the rejoining of DNA leading to the accumulation of double stranded DNA breaks thereby triggering apoptosis (49). The importance of this pathway is highlighted by the finding that cardiomyocyte specific deletion of the gene encoding Top-2β protected cultured cardiomyocytes from DOX-induced cytotoxicity and protected mice from DOXinduced cardiotoxicity and heart failure (48). DOX has also been implicated in affecting mitochondrial DNA integrity via similar mechanism of ternary DOX—DNA—topoisomerase complex formation involving Top-2β residing in mitochondria as well as involving a mitochondrial specific topoisomerase, Top-1mt (60, 61).

# Mitochondrial Dysfunction and Damage

Cardiomyocytes are highly dependent on mitochondrial function for energy production and proper contractile function (62– 64). As outlined above, DOX can lead to the accumulation of iron within mitochondria and to the iron dependent and independent accumulation of reactive oxygen and nitrogen species which, themselves, can impair mitochondrial energy production (50, 65, 66). Furthermore, as mentioned above, through ternary complex formation between DOX, DNA and mitochondrial topoisomerases (Top-2β and Top-1mt), DOX can damage mitochondrial DNA by inducing double strand DNA breaks (48, 59). This can impair mitochondrial biogenesis as well as further impact mitochondrial function leading to insufficient energy production for the needs of the cardiomyocyte (60, 61).

# Autophagy

Autophagy is a homeostatic mechanism whereby damaged or dysfunctional organelles are recycled to generate substrates for energy production or anabolic processes. Autophagy contributes to normal physiology of the heart and under conditions of acute cardiac stress can promote cardiac survival by releasing energy substrates and breaking down damaged organelles (67). Stimulation of autophagy results in the recruitment of autophagy-related proteins (ATGs) to a specific subcellular site and nucleation of an isolation membrane forming a structure called a phagophore and then an autophagosome, surrounding the damaged target organelle (68). Under conditions of cellular stress, such as nutrient deprivation, the serine/threonine kinase master cell growth regulator, mTOR, is inhibited, resulting in autophagy (69, 70). ATGs that were phosphorylated by mTOR under normal conditions now become dephosphorylated and recruited for autophagosome formation (71). In addition, under conditions of prolonged stress increased autophagic activity can lead to atrophy (decreased size) of cardiac muscle and activation of cell death pathways (67). Tumor-bearing mice display signs of dysregulated cardiac autophagy (increased expression of autophagic markers) and atrophy (32–34), and several research groups have highlighted the importance of autophagy in cardiotoxicity resulting from treatment of mice with DOX (46, 51, 72).

Autophagy is orchestrated by a complex set of regulatory proteins which identify the target for autophagic disposal, form the limiting membrane and orchestrate fusion with lysosomes [reviewed in Li et al. and Koleini and Kardami (51, 52)]. Recent research has demonstrated DOX dysregulates autophagy in cardiomyocytes and that this appears to play an important role in DOX mediated cardiotoxicity. This has been the subject of a number of recent comprehensive reviews (51, 52). DOX appears to exert a dose-dependent disruption of the normal regulation of autophagy: low, clinically relevant doses, replicating chemotherapy, appear to suppress normal levels of basal autophagy, whereas high doses appear to induce autophagy above normal basal levels (51, 52). The impaired autophagy in cardiomyocytes, resulting from chronic low-dose treatment of mice with DOX, has been reported to involve DOX-mediated interference with lysosome mediated degradation of autophagosome contents (73). On the other hand, DOX, particularly at higher doses, appears to lead to an accumulation of PTEN-induced kinase (PINK) 1 and parkin (an E3 ubiquitin ligase) within mitochondria (74). PINK1 and parkin play a key role in regulating the balance between mitophagy and mitochondrial biogenesis; DOX appears to trigger a depolarization of the mitochondrial membrane potential resulting in an accumulation of PINK1 and parkin on mitochondria. PINK1 is a serine/threonine kinase which phosphorylates ubiquitin and allows parkin to ubiquitinate a number of mitochondrial outer membrane proteins, targeting the mitochondria for mitophagy (52, 74). This, possibly together with DOX-mediated interference with lysosome mediated degradation, results in an accumulation of autophagosomes containing mitochondria (52, 73, 74). An emerging consensus appears to be that DOX interferes with autophagic flux, as a result of simultaneous induction of early stages of mitophagy (triggering mitochondrial dysfunction/membrane depolarization, the marking of mitochondria for mitophagy and formation of autophagosomes surrounding dysfunctional mitochondria) and interference with later stages of autophagy (51, 52). As mentioned above, this may be through DOX-mediated interference with lysosome acidification, thereby preventing the lysosomal degradation of contents of autophagosomes (73). Alternatively, others have reported that DOX mediated mitochondrial damage leads to maladaptive activation of phosphatidylinositol 3-kinase (PI3K) γ signaling, which blocked autophagy, and that this was alleviated by cardiac specific PI3K γ inhibition in mice (75). The consequence of these effects appears to be a build-up of damaged/dysfunctional mitochondria and reactive oxygen species, exacerbating DOX-induced oxidative stress, and leading to activation of apoptotic pathways, triggering cell death (51).

# Apoptosis

Apoptosis is a form of programmed cell death involving the induction of a caspase proteolytic cascade ultimately leading to nuclear condensation and fragmentation, phosphatidylserine exposure on the cell surface, formation of apoptotic bodies and their clearance by phagocytes by virtue of recognition of exposed phosphatidylserine. Apoptosis is characterized by the activation of caspase proteolytic cascades leading to the activation of effector caspases, such as caspases 3, 6, and 7 (76, 77). These activate nucleases and proteases which degrade DNA, nuclear, cytoplasmic, and cytoskeletal proteins (76, 77). These effector caspases are activated by two main pathways of apoptosis regulation, the extrinsic and intrinsic pathways. In the extrinsic pathway, ligation of "death receptors" such as the tumor necrosis factor (TNF) α receptor by their extrinsic ligands on the cell surface results in receptor dimerization, bringing together "death domains" leading to the formation of a death inducing signaling complex (DISC) that ultimately triggers the activation of caspase 8, which, in turn, goes on to activate the effector caspases 3, 6, and 7 (77).

On the other hand, mitochondria play central roles in the intrinsic apoptosis pathway, which is regulated by a series of soluble proteins that regulate pore formation in the mitochondrial membrane (76, 77). These factors belong to the Bcl-2 family of proteins and comprise 3 groups: the anti-apoptotic Bcl-2 family members, such as Bcl-2 itself, the pro-apoptotic effectors such as Bax and Bak, and the proapoptotic regulators such as Bim. Together these comprise the intrinsic apoptosis pathway. In this pathway, the antiapoptotic family members bind to and sequester the proapoptotic effectors, preventing them from assembling as complexes on the mitochondrial membrane. The pro-apoptotic regulators, in turn, bind to the anti-apoptotic regulators, preventing them from sequestering the pro-apoptotic effectors and, additionally, assist the pro-apoptotic effectors in assembly on the mitochondria, where the pro-apoptotic effectors can form pores in the mitochondrial membrane. This leads to the leakage of mitochondrial cytochrome C and formation of a protein complex, called the apoptosome which serves as a platform for the activation of caspase 9, which can then activate effector caspases 3, 6, and 7 (76). In this intrinsic pathway, the relative amounts of pro-apoptotic effectors, regulators and anti-apoptotic factors determine whether apoptosis proceeds (76). Nuclear DNA damage and induction of oxidative stress and damage to mitochondria are all potent activators of the intrinsic apoptosis pathway (76, 77). In addition, DOX treatment of cardiomyocytes has been reported to result in increased transcription of the proapoptotic regulator Bim, thus enhancing the intrinsic apoptotic pathway (78). Additionally, recent work has identified increased expression of death receptors (TNF receptor 1, Fas, death receptor 4, death receptor 5) in human induced pluripotent stem cell derived cardiomyocytes following DOX treatment (44).

# Atrophy

In addition to causing the death of cardiomyocytes, DOX also triggers cardiomyocyte atrophy, or a reduction in cardiomyocyte size. Ultrastructural changes in the myocardium, including myofibril structural disarray and atrophy due to DOXcardiotoxicity appear well before clinical manifestations (79). The ubiquitin proteasome system regulates cardiomyocyte size by tagging proteins with polyubiquitin chains for subsequent degradation by the proteasome (80). The tagging of target proteins for proteolysis by the ubiquitin-proteasome system involves the assembly of polyubiquitin chains on target proteins mediated by E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases. Atrogin-1 is a muscle specific E3 ubiquitin ligase that facilitates atrophic signaling in cardiomyocytes (80). It and other E3 ubiquitin ligases appear to be upregulated in cardiomyocytes by treatment with DOX and/or other anthracyclines (81, 82). This leads to the degradation of contractile proteins, and reduction in cardiomyocyte size (81). As cardiomyocyte size is related to overall contractile force generation, reduced cardiomyocyte size (atrophy) compounds reduced cardiomyocyte numbers in diseased states and eventually manifests as progressive reduction in cardiac function, as in the case of DOX-induced cardiotoxicity (83).

# THERAPEUTIC STRATEGIES FOR PREVENTING OR TREATING ANTHRACYCLINE CARDIOTOXICITY

At present, monitoring and screening for cardiotoxicity following anthracycline therapy is imperative for timely treatment. In a recent prospective study of anthracycline-treated patients, of the 9% of patients who developed heart failure, 98% of cases occurred within a year following anthracycline treatment (40). Countless prophylactic therapeutics are currently under study in animal models but few have been assessed clinically. Furthermore, only a small number cardioprotective therapeutics that have been tested in humans reduce the cardiotoxic effects of anthracyclines and currently no clear guidelines or worldwide accepted therapies exist. β-blockers, angiotensin converting enzyme (ACE) inhibitors, statins, and dexrazoxane (DRZ) are drugs that have been assessed in small trials for protection against anthracycline cardiotoxicity. Results are promising, but data is limited by small study sizes and variability in study methods such as follow up time. Early identification of reduced cardiac function and immediate treatment with heart failure medication such as enalapril (an ACE inhibitor) alone or in combination with β-blockers (carvedilol, or bisoprolol) provided either full or partial improvement of cardiac function in 82% of patients (40). A small group of non-Hodgkins lymphoma patients receiving ramipril and/or bisoprolol as a prophylactic during anthracycline treatment also exhibited reductions in new symptoms of cardiotoxicity and projected prolonged survival (84).

Statins are commonly prescribed to reduce morbidity and mortality associated with atherosclerosis. Given the cardioprotective nature of statins, and the fact that cancer patients receiving chemotherapy may also be concurrently treated with statins for atherosclerosis, the prophylactic effect of statins on anthracycline cardiotoxicity has been examined in a number of small trials. Breast cancer patients on continuous statin therapy had reduced risk of heart failure following anthracycline treatment compared to those with non-continuous statin therapy (85), and similarly, anthracycline induced decline in left ventricular ejection fraction (LVEF) was reduced by statin treatment as compared to no statin treatment (86). In a small study of 40 patients, those receiving atorvastatin prior to chemotherapy infusion showed no significant change in LVEF at 6 months post therapy compared to a reduction in LVEF in those receiving anthracycline alone (87).

While statins and ACE inhibitors have shown promising results in mitigating resultant anthracycline cardiotoxicity or preventing cardiotoxicity when individuals happen to be taking them concurrently with chemotherapy for treatment of comorbidities, DRZ is currently the only United States Food and Drug Administration, and Health Canada approved prophylactic drug for use in combination with DOX to specifically limit cardiotoxicity in adults (88, 89). DRZ acts as an iron chelator, interferes with ROS production, can bind to Top-2β to inhibit complex formation with DOX, and also reduce Top-2β expression (90–92). Early multi-center randomized double blind trials of breast cancer patients receiving combination chemotherapy which included a cumulative DOX dose of 300 mg/m<sup>2</sup> reported a hazard ratio of cardiac events of 2.63 (placebo vs. DRZ); however, despite this promising effect of DRZ on heart function, patient survival was not improved (93). A systematic review also identified an association of DRZ with reduced risk of cardiovascular complications, but increased risk of secondary malignant neoplasms in children receiving chemotherapy (94). Given these results, Health Canada cautions against use of DRZ in children, as well as in elderly populations with reduced cardiac, hepatic, or renal function.

Identification of a treatment that protects against the cardiotoxic side effects without impacting the chemotherapeutic effects of DOX remains of utmost importance. Potential for development or further study of effective primary prevention therapies exists given the expansion of research uncovering the broad nature of mechanisms in the pathogenesis of anthracycline mediated heart failure.

# HDL AND ROLE IN CANCER AND CANCER THERAPY

HDL has long been associated epidemiologically with reduced risk for cardiovascular disease. The main mechanistic explanation has traditionally been its apparent protection against atherosclerotic narrowing of arteries, thereby combating ischemic cardiovascular disease. However, recent research in pre-clinical models have increasingly suggested that HDL may exert direct cardioprotective effects on the heart itself (24). Furthermore, HDL based nanospheres have been developed as delivery vehicles for a variety of drugs including chemotherapeutic agents. Finally, pre-clinical studies have shown that delivery of supra-physiological amounts of HDL's major apolipoprotein, ApoA1, may itself attenuate tumor growth. In the following sections, we provide an overview of HDL structure, composition, formation and function and then discuss recent findings demonstrating direct cardioprotective effects of HDL and/or its precursor, ApoA1 against DOX-induced cardiotoxicity, advances in the use of HDL based nanospheres for encapsulation of DOX for therapeutic delivery and direct anti-tumor effects of ApoA1.

# HDL Structure and Composition

Lipoproteins are diverse biological particles that provide a means of transport for lipids between cells, tissues, and other lipoproteins, and can activate intracellular signaling pathways (95). Lipoproteins are separated into five classes (chylomicron, VLDL, IDL, LDL, and HDL) based on criteria including density, size, and relative content of lipids (cholesterol and triglyceride), and apolipoproteins (95). HDL represents one of the five major classes of lipoproteins, distinguishable from others based on their small particle size (5–11 nm), high density (1.063–1.21 g/ml), and unique apolipoprotein content (96). Unlike other lipoproteins, HDLs are unique in their cytoprotective actions and initiate antioxidative, anti-apoptotic, and anti-inflammatory effects. HDL can inhibit the oxidation of LDL and enhance endothelial function by inhibiting the expression of endothelial adhesion molecules (97). In addition, HDL can suppress atherosclerosis progression and inflammation by modulating production of monocytes and neutrophils (98).

HDL represents a class of particles of distinct protein and lipid composition. The proteome of HDL is diverse and can contain close to 100 proteins, more than can fit on a single particle, underscoring the notion that HDL represents a class of particles of distinct compositions. The HDL proteome includes apolipoproteins, diverse enzymes, lipid transfer proteins, acute phase proteins, and proteinase inhibitors, and other proteins of distinct functions (99). ApoA1 (243 amino acids, 28 kD) is the most abundant protein in HDL, comprising 70% of the protein carried by HDL (100). ApoA1 has over 90% amphipathic α-helical content, allowing for formation and stabilization of the HDL (101). ApoA1 is linked to several beneficial effects of HDL. Therefore, several research groups have reported that HDL quality is highly dependent on the abundance and function of ApoA1 (102).

# HDL Formation and Function

ApoA1 is secreted by the liver (70%) and small intestine (30%) in a lipid-poor state, and is assembled into HDL by the addition of phospholipids and unesterified cholesterol aided by the ATPbinding cassette transporter A1 (ABCA1) at the cell surface, forming nascent HDL (an immature form of HDL). Next, the nascent HDL particle becomes mature HDL by activating lecithin: cholesterol acyltransferase (LCAT), which converts unesterified cholesterol to cholesteryl esters. The esterification of cholesterol increases its hydrophobicity, resulting in its movement into the core of the HDL particle and the particle itself adopting a spherical shape (103, 104).

HDL particles are continuously remodeled and catabolized by plasma and membrane proteins, thereby giving rise to dynamic subfractions. Membrane receptors including ATPbinding cassette transporter G1 (ABCG1) and the scavenger receptor class B type 1 (SR-B1) promote movement of lipids between cells and HDL, and plasma proteins such as cholesteryl ester transfer protein (CETP) assist in movement of lipids between lipoproteins. SR-B1 is a high affinity receptor for HDL, is highly expressed in liver and steroidogenic tissues, and plays a critical role in tissue uptake of HDL cholesterol, a key step in reverse cholesterol transport, which is the transport of cholesterol from peripheral tissues to the liver for repackaging into nascent lipoproteins or excretion (105). ApoA1 appears to play an important role in this process as adenoviral mediated or transgenic overexpression of human ApoA1 in mice leads to enhanced reverse cholesterol transport (106).

# HDL Targeted Therapeutics

The Framingham Heart Study shows strong relationships between levels of HDL and the incidence of developing heart disease (25). Statins, inhibitors of hydroxymethylglutaryl coenzyme A (HMG-CoA), a key enzyme in the cholesterol biosynthetic pathway, have long been major therapeutic tools in the reduction of cardiovascular events. This is due to their ability to reduce production and increase clearance of VLDL and LDL, thereby reducing blood cholesterol levels. Many statins have also been shown to modestly raise HDL cholesterol levels by between 3 and 15% (107–109). The mechanisms and clinical benefit of statin triggered increases in HDL cholesterol are unclear and still the subject of debate (107–111). In part this may be due to the difficulty in evaluating the contribution of the relatively modest statin-induced increases in HDL-cholesterol levels in the context of dramatic reductions in LDL cholesterol.

The epidemiological association of increased HDL cholesterol with reduced risk for cardiovascular disease has, over the years, led to efforts to increase HDL levels pharmacologically. The focus, however, has been in increasing HDL-cholesterol as opposed to increasing HDL particles or, more subtly, to increasing functional HDL particles. This focus on raising HDL cholesterol levels has led to the development of CETP inhibitors which block the CETP mediated transfer of cholesterol from HDL to triglyceride rich lipoproteins such as VLDL. These drugs increase HDL cholesterol levels by more than 25% (up to 60%), but largely have not reduced cardiovascular events (107, 109, 112–118). More recent attempts at increasing HDL particle number and/or function have focused on infusion of reconstituted HDL, administration of ApoA1 mimetics, or upregulation of ApoA1 production by liver (119–124). CSL112 is a new reconstituted HDL (rHDL) made with human ApoA1. Clinical trials of CSL112 showed that it enhances cholesterol efflux capacity an important measure of HDL mediated reverse cholesterol transport mediated cardiovascular protection (124, 125). Also, the AEGIS-I trial suggests that CSL112 has advantages over other rHDL formulations (e.g., CSL-111, CER-001) or ApoA-I Milano because it is well-tolerated with no side effects in major organs (such as liver or kidney toxicity) or immunogenicity (126–128). However, the potential benefit of CSL112 in reducing major adverse cardiovascular events in this group of high-risk patients still remains to be shown in the large phase III AEGIS-II study that is expected to be concluded in 2022 (124, 125).

# Preclinical Studies of HDL Effects on DOX-Induced Cardiotoxicity

The protective effects of HDL against cardiovascular disease have long been the subject of intensive research. While most of the focus has been on the ability of HDL to protect against atherosclerosis and vascular dysfunction, and the impacts of that on ischemic cardiovascular disease, more recent focus has increasingly been placed on the direct cardioprotective effects of HDL, through its interactions with cardiomyocytes themselves (24). Epidemiological studies, as well as pre-clinical studies in animal models, have demonstrated that HDL can protect against cardiac disease independent of effects on coronary artery atherosclerosis, suggesting that it may also exert direct effects on the heart itself (129–132). For example, HDL treatment has been shown to protect hearts (in vivo and ex vivo) and isolated cardiomyocytes, from ischemia/reperfusion injury and infarction (130–132). In the context of DOX-induced cardiotoxicity, recent studies using isolated cardiomyocytes in culture (133–136) and in preclinical animal models (135, 137) demonstrate that HDL is able to protect against cardiomyocyte apoptosis and myocardial atrophy. For example pre-treatment of cultured cardiomyocytes with HDL prior to subsequent treatment with DOX, protects them against DOX induced apoptosis (137, 138). Similarly, HDL pretreatment of cultured cardiomyocytes protects them against other stresses leading to cytotoxicity, including necrosis resulting from oxygen and glucose deprivation (134), suggesting that HDL may protect cardiomyocytes against diverse forms of cell death. HDL mediated protection against DOX-induced cardiomyocyte apoptosis has been reported in different studies to involve the activation of AKT (134, 137) or the activation of the signal transducer and activator of transcription (STAT) 3 (138). Pharmacological or genetic inhibition of these signaling mediators has been shown to impair HDL mediated protection of cardiomyocytes against DOX-induced apoptosis (137, 138). The importance of the AKT pathway in cardioprotection against DOX has also been demonstrated by the finding that expression of constitutively active AKT1 in the myocardium inhibits DOX induced cardiotoxicity by preventing left ventricular dysfunction and cardiac atrophy (139). Furthermore, cardiac restricted overexpression of STAT3 in mice led to protection against DOX-induced atrophy and congestive heart failure, whereas cardiac specific knockout of STAT3 in mice was accompanied by increased cardiac fibrosis and age-dependent heart failure (140, 141). AKT and STAT3 form the respective cornerstones of the RISK (reperfusion injury salvage kinase) and SAFE (survivor activating factor enhancement) signaling pathways known to play important roles in cardioprotection, for example in the setting of ischemia/reperfusion injury (142, 143). These data also suggest that AKT and STAT3 are critical mediators of cardioprotection against DOX-induced cardiotoxicity (**Figure 3**). HDL dependent activation of AKT and STAT3 and other signaling pathways has been reported in different cell types to involve HDL mediated delivery of the bioactive lipid, sphingosine-1-phosphate (S1P), acting via the S1P receptors (136, 144–148) (**Figure 3**). In the case of cardiomyocytes, HDL and S1P mediated activation of STAT3 appears to be mediated by the S1P receptor 2 (S1PR2) (136). On the other hand, the involvement of S1P/S1P receptors in HDL mediated activation of AKT signaling in cardiomyocytes has not been demonstrated (134, 135, 137). However, HDL mediated activation of AKT signaling in cardiomyocytes and protection of cardiomyocytes against DOX-induced apoptosis appears to require the HDL receptor, SR-B1. SR-B1 is expressed by both mouse and human cardiomyocytes in culture and mouse cardiac tissue (137, 149). The ability of HDL to induce AKT phosphorylation and protection against DOX induced cytotoxicity in cultured mouse or human cardiomyocytes was lost when the gene for SR-B1 was either knocked out or knocked down (135, 137). SR-B1 mediates lipid transport between bound HDL particles and cells via a hydrophobic channel, suggesting that SR-B1 mediated transport of HDL bound, water insoluble S1P molecules from HDL, into the cell membrane, where they can access S1P receptors may be a potential mechanism for the involvement of SR-B1, and HDL associated S1P and S1P receptors in cardioprotection. This, however, remains to be demonstrated experimentally. The potential role for HDL associated S1P in cardioprotection against DOX-induced cardiotoxicity highlights the importance of understanding the role of HDL composition in evaluating HDL function and designing HDL based therapeutics such as reconstituted HDLlike particles (150).

These in vitro studies of HDL mediated protection against DOX-induced cardiotoxicity have recently been extended to in vivo models (135, 137) by examining the effects of increased circulating HDL levels on DOX-induced cardiotoxicity in mice. We first tested the effects of genetic overexpression of human ApoA1, on cardiotoxicity induced by repeated weekly DOX dosing in mice. Overexpression of transgenic human ApoA1 in mice has been shown to trigger dramatically increased circulating HDL levels by seeding the formation of new mature HDL particles (151). In one study, transgenic overexpression of human ApoA1 in mice virtually completely prevented chronic low dose DOX treatment from triggering myocardial apoptosis and atrophy, and protected mice from DOX-treatment induced reduction in left ventricular function (137). A drawback of this study was that although it represented a proof of concept, transgenic overexpression of ApoA1 led to levels of ApoA1 and HDL that were extremely high and therefore not likely to be therapeutically relevant (137). A more recent study, however, demonstrated that intraperitoneal injection of purified ApoA1 similarly prevented cardiotoxicity associated with chronic low dose DOX treatment in mice (135). Mice that were treated with five weekly injections of DOX alone exhibited substantial apoptosis in cardiomyocytes in hearts, and substantially reduced left ventricular function, whereas control mice that did not receive DOX displayed little myocardial apoptosis and normal left ventricular function (135). On the other hand mice that were treated with injection of ApoA1 alongside DOX were virtually completely protected against DOX-induced myocardial apoptosis and left ventricular dysfunction (135). Regardless of

means of HDL increase (ApoA1 transgenic expression or ApoA1 injection) cardioprotection was lost if mice lacked SR-B1 (135, 137). In fact, SR-B1 knockout mice were more susceptible to DOX induced cardiotoxicity than corresponding wild type mice. This effect of SR-B1 appeared to be associated with SR-B1 expression in cardiac tissue, consistent with observations that SR-B1 expression in cultured cardiomyocytes was required for HDL mediated protection against DOX-induced apoptosis (135, 137). These findings clearly demonstrate that in pre-clinical models, HDL-therapies such as injection of the HDL precursor ApoA1 have the potential to protect against DOX induced cardiotoxicity but are dependent on the expression of cardiomyocyte SR-B1 (**Figure 3**).

# HDL Based Delivery of Chemotherapeutics

In addition to HDL's ability to protect cardiomyocytes against cytotoxicity induced by anti-cancer agents, reconstituted HDL (rHDL)-based nanoparticles have also been explored as drug delivery vehicles for chemotherapeutic agents such as DOX. The use of rHDL as a drug delivery system for DOX has been studied using both in vitro and in vivo methods. Yuan et al. showed that DOX encapsulated in HDL particles (rHDL-DOX) is more efficiently taken up by and more effective at inducing apoptosis in hepatocellular carcinoma cells, when compared to DOX alone or encapsulated in liposomes (45). Furthermore, in preclinical mouse tumor models, treatment with rHDL-DOX resulted in greater tumor regression than DOX alone (45). Wang et al. confirmed that incorporation of DOX into rHDL-based particles enhanced the cytotoxic effects of DOX on tumors in vivo and cancer cells in vitro (152). Furthermore, they demonstrated that the HDL receptor SR-B1 was required in tumor cells for rHDL mediated delivery of the encapsulated DOX (152). Interestingly, the authors measured DOX tissue distribution after treating mice with rHDL-DOX and showed that DOX uptake by the heart was low (152). Others have tested the effects of using rHDL to deliver paclitaxel (PTX) either alone or in combination with DOX. Codelivery of PTX and DOX encapsulated in rHDL was shown to improve their anti-cancer effects over co-administration of nonencapsulated PTX and DOX (153). When used to treat preclinical models of liver cancer, the majority of PTX and DOX delivered via rHDL was found in the liver tumors (attributed to uptake via SR-B1) with little accumulation in the heart and very little cardiac damage (153). These findings suggest that, at least for liver cancer rHDL encapsulation can provide a means for targeted delivery of anti-cancer agents to tumor cells, sparing cardiac tissues. Whether the reduced cardiac damage was solely due to targeted delivery of the anti-cancer agents to the hepatic tumor over the heart or whether it also involved induction of survival signaling at the heart (PI3K/AKT and STAT3 signaling as described above) remains to be determined. It also remains to be determined whether rHDL-mediated chemotherapeutic delivery is effective against other types of cancer or against tumor cells which do not express high levels of SR-B1. Nevertheless, these studies suggest the potential for rHDL based drug delivery systems to confer tissue selective delivery to at least some types of tumors, sparing the heart from cardiotoxic damage. More research is required to determine the full potential of this.

# HDL and Cancer

In addition to research showing that HDL can protect cardiomyocytes from chemotherapy-induced cytotoxicity both directly by inducing survival signaling in the cardiomyocytes, and indirectly by acting as a targeted delivery system for anticancer agents, sparing the heart, other research has suggested that HDL and its precursor ApoA1 may also have direct anti-tumor effects themselves.

### Endogenous HDL and Cancer Risk

Results of epidemiological studies of endogenous HDL cholesterol levels and the incidence of cancer are mixed with some studies reporting an inverse correlation between HDL cholesterol and cancer risk and/or mortality, while other studies report minimal association, particularly when corrected for confounding factors (152, 154–159). Contributing to this is uncertainty over the cause-vs.-effect relationship between low HDL-cholesterol and cancer, with some studies suggesting that tumor cells may drive the lowering of HDL-cholesterol levels by utilizing HDL-cholesterol to support tumor growth (159). Complicating matters further are reports that HDL prepared from cancer patients or from patients with other co-morbidities, such as type 2 diabetes or obesity exhibit altered functions as compared to HDL from unaffected individuals, for example, promoting rather than inhibiting migration and invasion of tumor cells in in vitro assays (160–166). Therefore, it is presently unclear what, if any, effects levels of endogenous HDL or variations in those levels have on cancer development.

### Anti-cancer Therapeutic Potential of the HDL Precursor ApoA1

On the other hand, preclinical studies in mouse models have suggested that supra-physiological levels of ApoA1 may have therapeutic potential against tumor growth and metastasis. For example, Zamanian-Daryoush et al. reported that transgenic overexpression of human ApoA1 reduced, while complete knockout of endogenous ApoA1 increased tumor growth and metastasis in mice compared to control mice with normal levels of endogenous ApoA1 (167). They also demonstrated that pharmacological treatment with purified ApoA1 similarly attenuated both primary tumor development and metastasis in mouse models (167). They provided evidence that ApoA1 reduced tumor angiogenesis and recruited tumor cell targeting macrophages and CD8<sup>+</sup> cytotoxic T cells, thereby altering the tumor microenvironment to one less permissive for tumor development (167, 168). By using different tumor cell lines, including a human melanoma cell line (A375), they demonstrated that supra-physiological levels of ApoA1 may have general anti-neoplastic effects including toward human tumors (167, 168). Others have reported that synthetic ApoA1 mimetic peptides, which replicate the amphipathic properties of ApoA1, also exhibit anti-tumor properties, when used at pharmacological concentrations. For example, the ApoA1 mimetic peptide, L-5F was reported to prevent angiogenesis, suggesting that it may have therapeutic potential against angiogenesis associated diseases such as cancer (169). In preclinical studies, the ApoA1 mimetic peptide 4F has been reported to suppress ovarian tumorigenesis (170). Similarly, preclinical studies demonstrated that the recombinant ApoA1 mimetic peptide 6F reduced tumor burden in mouse models of metastatic lung cancer (171). However, ApoA1 mimetic peptides may exert anti-tumor effects via mechanisms distinct from ApoA1. For example ApoA1 mimetic peptides are thought to strongly bind and neutralize lysophosphatidic acid (LPA), which is known to stimulate cell proliferation, oncogenesis, and metastasis (172). On the other hand, neither transgenic overexpression of human ApoA1 nor ApoA1 knockout affected LPA levels in tumor-bearing mice (167). Whether ApoA1 (injected or overexpressed) or ApoA1 mimetic peptides exert anti-tumor effects by driving the increased formation of HDL-like particles or whether their antineoplastic effects are independent of HDL particle formation has not been examined. Other pre-clinical studies have reported that in certain cases, HDL may drive the development of breast cancer, particularly in circumstances when breast tumor cells overexpress the HDL receptor, SR-B1, since this receptor can mediate both survival signaling and uptake of cholesterol fueling tumor growth (173–177). Therefore, direct anti-tumor effects of ApoA1 or ApoA1-mimetic peptides may be restricted to tumors that do not overexpress SR-B1; although SR-B1 overexpression in tumors could be exploited by strategies that encapsulate

FIGURE 4 | Emerging roles of high density lipoprotein (HDL) in cancer and cancer therapy associated cardiotoxicity from preclinical studies. HDL induces cardioprotective effects in cardiomyocytes via SR-B1 receptor and activation of PI3K-Akt pathway as well as via S1P2 receptor leading activation of STAT3-JAK-ERK1/2 pathway (136, 137). rHDL used as a drug delivery system may allow for targeted delivery to at least some types of tumors, sparing cardiomyocytes (45, 152). Furthermore, the major apoliprotein of HDL (ApoA1) directly attenuates tumor growth and metastasis in preclinical models (167). These pathways are not necessarily muatually exclusive.

chemotherapeutic agents like DOX in HDL based nanoparticles (see previous section), which may be readily and preferentially taken up by tumor cells overexpressing SR-B1.

# CONCLUSIONS

Preclinical studies suggest that HDL targeted therapies involving pharmacological treatment with supra-physiological levels of ApoA1, peptides based on ApoA1 (ApoA1 mimetic peptides) or rHDL like particles may show promise in the protection against chemotherapy related cardiotoxicity via a number of mechanisms (**Figure 4**). These include (1) direct HDL mediated survival signaling in cardiomyocytes leading to protection against cytotoxicity, as exemplified by studies using DOX as a cardiotoxic agent; (2) indirect protection afforded to the heart by utilizing rHDL-based nanoparticles as targeted delivery vehicles for chemotherapeutic agents which spare the heart and have the potential to target tumor cells which may overexpress SR-B1; and (3) indirect protection resulting from direct ApoA1 mediated tumor suppression (**Figure 4**). It remains to be demonstrated experimentally whether these mechanisms broadly impact diverse malignancies and chemotherapeutic agents or are specific for those that have been tested to date. Importantly, these mechanisms may not necessarily be mutually exclusive. For example, in the case of treatment with

# REFERENCES


ApoA1 along with chemotherapeutic agents such as DOX, the ApoA1 may be acting by seeding the formation of new HDL particles which may incorporate the chemotherapeutic agent, act as targeted delivery systems for certain types of tumors, directly attenuate tumor growth, and directly induce survival signaling in cardiomyocytes, thus inducing both direct and indirect mechanisms of cardioprotection simultaneously. However, studies need to be designed to test whether these mechanisms do occur simultaneously, in the same preclinical models. Whether or not they do occur simultaneously, much more work remains to be done do determine the full potential for HDL targeted therapies as therapeutic approaches to prevent chemotherapy induced cardiotoxicity in human disease.

# AUTHOR CONTRIBUTIONS

All authors listed above made substantial, direct and intellectual contributions to the work, and have approved the final version.

# FUNDING

Research on HDL in the Trigatti Lab was funded by grants from the Canadian Institutes of Health Research (MOP−74753 and PJT−162272).


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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Kluck, Durham, Yoo and Trigatti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Review on the Role of Epigenetic Modifications in Doxorubicin-Induced Cardiotoxicity

Himani Kumari <sup>1</sup> , Wan-Hong Huang<sup>1</sup> and Michael W. Y. Chan1,2,3 \*

*<sup>1</sup> Department of Biomedical Sciences, National Chung Cheng University, Chiayi, Taiwan, <sup>2</sup> Epigenomics and Human Disease Research Center, National Chung Cheng University, Chiayi, Taiwan, <sup>3</sup> Center for Innovative Research on Aging Society (CIRAS), National Chung Cheng University, Chiayi, Taiwan*

Use of anthracyclines such as doxorubicin (DOX), for the treatment of cancer, is known to induce cardiotoxicity, begetting numerous evaluations of this adverse effect. This review emphasizes the mechanism of how consideration of DOX-induced cardiotoxicity is important for the development of cardioprotective agents. As DOX is involved in mitochondrial dysfunction, enzymes involved in epigenetic modifications that use mitochondrial metabolite as substrate are most likely to be affected. Therefore, this review article focuses on the fact that epigenetic modifications, namely, DNA methylation, histone modifications, and noncoding RNA expression, contribute to DOX-associated cardiotoxicity. Early interventions needed for patients undergoing chemotherapy, to treat or prevent heart failure, would, overall, improve the survival, and quality of life of cancer patients. These epigenetic modifications can either be used as molecular markers for cancer prognosis or represent molecular targets to attenuate DOX-induced cardiotoxicity in cancer patients.

#### Edited by:

*Carlo Gabriele Tocchetti, University of Naples Federico II, Italy*

#### Reviewed by:

*Valentina Sala, University of Turin, Italy Edoardo Lazzarini, University of Zurich, Switzerland*

> \*Correspondence: *Michael W. Y. Chan biowyc@ccu.edu.tw*

#### Specialty section:

*This article was submitted to Cardio-Oncology, a section of the journal Frontiers in Cardiovascular Medicine*

> Received: *11 December 2019* Accepted: *20 March 2020* Published: *07 May 2020*

#### Citation:

*Kumari H, Huang W-H and Chan MWY (2020) Review on the Role of Epigenetic Modifications in Doxorubicin-Induced Cardiotoxicity. Front. Cardiovasc. Med. 7:56. doi: 10.3389/fcvm.2020.00056* Keywords: cardiotoxicity, chemotherapy, epigenetics, cancer, doxorubicin

# INTRODUCTION

Cardiotoxicity, in simpler terms, is defined as "toxicity which damages the heart," often during or after chemotherapeutic treatment (1). Treatment options for cancer have been improving significantly in recent years, and the rates of survival in several human cancers have increased significantly with reduced recurrences (2). However, the applicability of these drugs is limited by the risk of cardiotoxicity (1). Doxorubicin (DOX)–induced cardiomyopathy can occur within a few days of its administration or delayed until decades after chemotherapy, thus affecting morbidity, mortality, and quality of life of cancer patients (3–6). However, the mechanism of DOX-induced cardiotoxicity is not fully understood.

Epigenetic modifications, including DNA methylation, histone modifications, and noncoding RNA (ncRNA) expression, play an important role in regulating gene expression and are considered as a hallmark of several human diseases, such as cardiovascular disease [review in Kimball and Vondriska (7)]. In this review, we discuss the mechanism of how aberrant epigenetic modifications contribute to DOX-induced cardiotoxicity and possible alternative therapeutic options that could forestall or prevent chemotherapy-induced cardiotoxicity (8).

# CHEMOTHERAPEUTIC-ASSOCIATED CARDIOTOXICITY

In the 1960s, DOX (Adriamycin <sup>R</sup> ), first isolated from Streptomyces actinobacteria, was found as one of the first anthracyclines (9), to be used for several cancer treatments, including breast carcinomas, sarcomas, leukemias, non-Hodgkin and Hodgkin lymphoma, and many other cancers (10, 11). At the molecular level, DOX acts to stabilize topoisomerase DNA isomers and therefore blocks DNA replication and transcription (12, 13). It has been reported in several studies over the last 15 years that despite the successful development of small molecules and targeted therapies, anthracycline-based chemotherapy still plays not only prominent anticancer but also overall detrimental roles in many types of cancer treatment (14). Concerning the latter, DOX causes a cumulative, irreversible, and dose-dependent cardiomyopathy that ultimately leads to congestive heart failure (15). Previous studies have demonstrated that cardiotoxicity is a repercussion of dose-dependent administration of DOX, with those exceeding 500 mg/m<sup>2</sup> greatly increasing the risk of congestive heart failure tremendously (16). Understanding the mechanism involved in DOX is important in developing novel preventive measures, and treatment strategies, against DOX-induced cardiotoxicity.

Cardiotoxicity is one of the major adverse effects of chemotherapy, and a leading cause of increased mortality and morbidity, in cancer patients (6, 17). Cardiotoxicity can occur in the early or late stages of the course of the disease and may vary from subclinical myocardial dysfunction to irreversible heart failure or death (18). Documented reports are limited to the mechanism of the appearance of cardiac dysfunction during chemotherapy and the susceptibility of patients to develop cardiotoxicity (1, 19). However, a proposed clinical study demonstrated that among all cancer patients, the overall occurrence of DOX-induced cardiotoxicity was ∼9%, and most cases occurred immediately during the first year after the completion of chemotherapy and have even been noticed after a follow-up of 4 years (20). Complications emerging from chemotherapy-induced cardiotoxicity are potentially life-threatening, further limiting the clinical use of various chemotherapeutic agents (particularly anthracyclines) (8), thus strongly supporting the need for improved cardioprotective agents.

# MECHANISMS OF DOX-INDUCED CARDIOTOXICITY

One widely accepted mechanism for DOX-induced cardiotoxicity is the generation of reactive oxygen species (ROS) after DOX treatment in cardiac mitochondria; this occurrence marks as the primary initiating event in the cascade of intracellular modifications (21). In mitochondria, DOX is reduced by NADH dehydrogenase and undergoes redox cycling, generating ROS (22). Elevated levels of ROS result in cellular damage, also known as oxidative stress, which is initiated when the delicate balance between the ROS-generating system and antioxidant measures is disrupted (8). Cardiomyocytes are highly susceptible to oxidative stress, as treatment with DOX reduced the levels of antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase (23). Cancer patients receiving DOX treatment also undergo immediate systemic oxidative stress, which is due to a decrease in glutathione and total antioxidant capacity of plasma (24).

Production of ROS also affects the DNA, RNA, proteins, and lipids and can also act as secondary signaling molecules in various pathways that are involved in homeostasis, including cell proliferation and cell death (25, 26). Thus, maintenance of a proper level of ROS in the intracellular and extracellular environment is of vital importance. Hence, it could be inferred that oxidative stress could be a leading cause of cellular hypertrophy in the heart (27), due to gene expression alterations (28), cell death activation (29), extracellular matrix transformation (30), ventricular remodeling (29), and calcium transient perturbation (31), all of which could result in the pathophysiological changes that lead to cardiomyopathy and heart failure.

On the other hand, DOX can also disrupt cellular and mitochondrial metabolism, a phenomenon not fully explored. For example, DOX can reduce mitochondrial NADH accumulation and impair oxidative phosphorylation in heart tissues, events associated with reduced glucose uptake (32). Doxorubicin can also induce the opening of mitochondrial permeability transition pore, resulting in the loss of mitochondrial membrane potential, thus explaining DOX-mediated apoptosis in cardiomyocytes. Moreover, DOX can reduce both the protein level and AMPK phosphorylation, thus contributing to stress and metabolic dysfunction (33, 34). More recently, one study found that the noncanonical function of the tumor suppressor p53 is involved in DOX-mediated cardiotoxicity (35). Doxorubicin treatment of TP53-depleted mice resulted in left ventricular systolic dysfunction, in association with decreased oxidative metabolism, and reduced mitochondrial volume and DNA transcription. Taken together, induction of oxidative stress and disruption of metabolism in mitochondria are crucial to the development of cardiotoxicity by DOX.

# ROLE OF EPIGENETIC ALTERATIONS IN DOX-INDUCED CARDIOTOXICITY

Mitochondrial metabolites constitute a large number of cofactors for several enzymes involved in human biochemical pathways, including epigenetic modifications (36). For example, Sadenosylmethionine (SAM) is the universal substrate for DNA and histone methylation. It is therefore believed that mitochondrial disruption may likely affect cardiomyocyte genomic chromatin (7). Indeed, DNA methylation and histone modifications, as well as non-coding RNA expression, have recently been found to play a role in DOX-induced cardiotoxicity. Furthermore, in vivo experiments also demonstrated that rat deficient in methyl donors developed cardiomyopathy with disrupted mitochondrial alignment in the myocardium (37). This effect was due to the reduced activity of PGC-1α, the master regulator for mitochondrial biogenesis (38). Interestingly, such reduced PGC-1α activity was found to be due to increased acetylation and a decreased methylation of PGC-1α, through downregulation of the histone modifiers, SIRT1 deacetylase, and PRMT1 methyltransferase, thus further supporting the interplay between metabolism and epigenetic modifications (37). The role of DOX in the alteration of gene expression via epigenetic modifications is illustrated in **Figure 1**.

# EPIGENETIC MODIFICATION: DNA METHYLATION

DNA methylation is often referred to as the "fifth" DNA based, because of its ubiquitousness in occurring at the 5′ position of cytosine in CpG dinucleotide (39). 5-Methylcytosine is established, maintained, and removed by several enzymes, including DNA methyltransferases (DNMTs) and Tet (the ten-eleven translocation hydroxylases) family protein. DNA methylation at the promoter region of a gene is associated with transcriptional repression by recruitment of transcriptional repressors and histone modifiers (such as histone deacetylases and histone methyltransferase), resulting in a repressive chromatin. The interplay between DNA methylation and histone modifications has been reviewed elsewhere (40, 41) and will not be discussed here.

DNA methyltransferases and Tet require SAM or αketoglutarate (α-KG) for the formation of 5-methylcytosine or 5-hydroxymethylcytosine (5hMC), in the process of DNA methylation and demethylation, respectively (42). In particular, the metabolic pathway from mitochondria generates SAM and α-KG; mitochondrial dysfunction associated with chronic DOX therapy may affect epigenetic machinery.

Indeed, in one of the studies, mouse cardiomyoblast H9c2 cells were used to analyze the effect of DOX (21). Together with a decrease in glycolytic activity and basal respiration in DOXtreated cells, dysregulation of mitochondrial DNA transcripts was observed. Importantly downregulation of DNMT1 (a maintenance methyltransferase), accompanied by a decrease in global DNA methylation, was also observed. This effect is in agreement with a previous animal study that global DNA hypomethylation, accompanied by a dysregulated expression of mitochondrial gene products encoded from both nuclear and mitochondrial genome, was observed in the hearts of rats treated with DOX (15). It is also interesting to point out that Ferreira et al. (21) found that pre-exposure of DOX can confer resistance to subsequent exposure of DOX in H9c2 cells, probably due to mitochondrial adaptation. As DNA methylation of the mitochondrial genome is maintained by DNMT1, the only DNMT member that can be translocated into the mitochondria (43, 44), downregulation of DNMT1 by oxidative stress may

eventually affect the methylation of mitochondrial genome. Taken together, these studies thus suggest that DOX may affect global DNA methylation via dysregulation of mitochondrial function and related metabolites.

Notably, a recent animal study demonstrated that the involvement of mitochondrial genome was not observed, as genes showing significant differential methylation in DOXtreated rats were all encoded from the nucleus. However, a global DNA hypomethylation in the DOX-treated group was still observed (45). This discrepancy may be due to the methods used in these studies. Study from Nordgren et al. (45) utilized a sequencing-based approach Reduced representation bisulfite sequencing (RRBS) that can only interrogate DNA methylation at the CpG rich region, whereas studies from Ferreira et al. used a candidate gene approach to analyze the change of the mitochondrial genome (21). In this regard, further unbiased experiments are required to analyze the role of DOX in the change of the methylome in mitochondria and nucleus.

# HISTONE MODIFICATION

Besides DNA methylation, histone modifications are also involved in DOX-induced cardiotoxicity (**Table 1**). These modifications can give rise to synergistic or antagonistic interactions with chromatin-associated protein, resulting in dynamic switching between transcriptionally active (accessible euchromatin) and silent (condensed heterochromatin) states (50). For example, histone deacetylase, HDAC6, was found to be upregulated in DOX-treated primary rat cardiomyocytes, in vitro, and mice model, in vivo, resulting in deacetylation of α-tubulin (48). The upregulation of other HDACs (**Table 1**) has also been observed in the heart tissue of mice treated with DOX (46). In this regard, Song et al. (48) demonstrated that genetic or pharmacological inhibition of HDAC6 in mice showed a cardioprotective effect against DOX by restoring autophagic flux.

Furthermore, a recent study using H9c2 cardiac myoblast cells also demonstrated that expression of several histone modifiers was dysregulated in association with downregulation of global acetylation of histone H3 (**Table 1**). In this study, Hanf et al. (47) demonstrated that expression levels of histone deacetylases (SIRT1 and HDAC2) were affected upon DOX treatment. In particular, different isoforms of SIRT1 displayed a contradictory expression level. However, pterostilbene, a natural analog of resveratrol and antioxidant, has been found to alleviate DOXinduced cardiotoxicity both in vitro and in vivo (51). This effect is due to enhanced deacetylation activity of SIRT1, suggesting its cardioprotective effect against DOX. In the case of HDAC2, treatment with low-dose DOX resulted in decreased expression of HDAC2, but no significant changes in highdose treatment, as compared to control. Consistently, HDAC2 downregulation was observed in the heart tissue of mice treated with DOX (46). As most of the HDACs were found to be upregulated in DOX-treated cardiomyocytes, it is reasonable to observe the cardioprotective effect of HDAC inhibitors on DOX (52). Intriguingly, studies found that trichostatin A, a pan-HDAC inhibitor, can enhance DOX-mediated hypertrophy and TABLE 1 | Changes of histone modifications and modifiers in DOX treated cardiomyocytes.


Experimental model: <sup>1</sup>*Mice (C57BL/6);* <sup>2</sup>*H9c2 rat cardiomyocyte;* <sup>3</sup>*HDAC*−/<sup>−</sup> *mice and primary rat cardiomyocyte;* <sup>4</sup>*mice (unspecified);* <sup>5</sup> *long term (48 h treatment);* <sup>6</sup>*high dose and long term (48 h treatment);* <sup>7</sup>*histone eviction;* <sup>8</sup>*downregulation of H34me3 and a shift of peak toward the transcription start site.*

apoptosis in H9c2 rat cardiomyoblasts (53, 54). In one of the studies, Ma et al. (54) found that DOX-induced cardiotoxicity is mediated through Rac1, a GTP-binding protein, and subunit of NADPH oxidase, resulting in the suppression of HDAC activity and upregulation of p53. Importantly, this process is ROS-independent. In this regard, treatment of HDAC inhibitor further enhances the effect of DOX-mediated cardiotoxicity. The involvement of specific HDAC isoforms in this process, however, remains to be determined.

Moreover, the histone lysine demethylase, KDM3A, was significantly upregulated upon DOX treatment of H9C2 cells; however, long-term DOX treatment also significantly decreased the lysine-specific histone demethylase 1 (i.e., LSD1). In parallel, significant upregulation of the histone lysine methyltransferases, SET7 and SMYD1, was only observed in long-term and highdose DOX treatment. Notably, a heart-specific transcriptional alteration was only observed in mice treated with DOX, but not etoposide, a nonanthracycline (49). This event was due to the inhibition of topoisomerase 2β (55), as "eviction" of specific histones from chromatin, resulting in a shift of histone modification (H3K4me3), and chromatin structure, around the promoter region of a gene.

# NONCODING RNA EXPRESSION

Another recognized epigenetic modification is the regulation of ncRNAs, including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs). Noncoding RNAs are involved in numerous human biological processes, as well as human diseases (56). Up to 30% of gene expression in humans is regulated by ∼1,000 known miRNAs, ranging from 18 to 25 nucleotides. MicroRNAs may originate from either independent genes or introns of protein coding genes and are transcribed by RNA polymerase II. Subsequently, these "primary miRNAs" are processed into mature miRNAs and then assembled into argonaute family proteins containing ribonucleoprotein complexes called miRNA-induced silencing complexes. These complexes then bind to their mRNA target sequences in 3′ UTR (untranslated region) of mRNA transcripts, resulting in either translational blockage or mRNA degradation.

Aberrant expression of several miRNAs has been shown involved in DOX-mediated cardiotoxicity (**Table 2**). For example, upregulation of miR-15 was observed in DOX-induced apoptotic H9c2 cardiomyocytes (57). This effect was probably due to suppression of Bmpr1a, a target of miR-15 and BMP receptor, previously found to be involved in cardiac contractility (68). Activation of BMP signaling by Bmpr1a agonist is therefore able to rescue DOX-mediated cardiotoxicity in H9c2 cells (57). Similarly, upregulation of miR-23a (58), miR-34a (61, 62), miR-140 (63), miR-146a (64), and miR-532 (66) were observed either in vitro or in vivo models of DOX-induced cardiotoxicity. Interestingly, upregulation of miR-34a, a well-known tumor suppressive miRNA, could epigenetically suppress SIRT1 (61, 62), thus partially explaining the downregulation of this HDAC, by DOX, in the aforementioned study (47).

Therapeutically, adenovirus-mediated overexpression of miR-212/132 cluster has been shown to prevent DOX-induced cardiotoxicity in a mouse model (65). This effect may be partially due to direct targeting of Fitm2, a transmembrane protein involved in fat storage, by miR-232/132. Moreover, downregulation of miR-29b (59) and miR-30 (60) was also observed in DOX-treated cardiomyocytes in an animal model, leading to de-repression of BAX, a proapoptotic protein, and β-adrenoceptor (β1- and β2AR), involved in myocyte contraction, respectively.

On the other hand, lncRNAs, which are more than 200 nucleotides long, regulate gene expression by diverse mechanisms (69). For example, lncRNAs can serve as a scaffold to recruit activators or repressors to regulate gene expression. The molecular function and clinical application of lncRNAs in cardiovascular disease have been recently reviewed (70, 71). Particularly, several studies have provided evidence to demonstrate that lncRNA can directly "sponge" or bind to miRNAs, thus regulating the activity of those miRNAs through a competing endogenous RNA (ceRNA) mechanism (71–74). For example, DOX can upregulate the lncRNA, LINC00339 (**Table 2**), resulting in the suppressing of miR-484 by ceRNA mechanism, in cardiomyocytes in vitro and in an animal model (67).

# CONCLUSION

Although DOX is still the mainstay anthracyclines (9) for the treatment of several human cancers, a major concern


<sup>1</sup>*Adenovirus-mediated overexpression.*

Kumari et al. Epigenetics and Cardiotoxicity

is the side effect of cardiotoxicity. In this review, we have summarized recent findings that epigenetic modifications were observed in cardiomyocytes treated with DOX, both in vitro and in vivo. Although the causal relationship between cardiotoxicity and epigenetic modifications has not been fully explored, epigenetic modifications may contribute to either a cardiotoxic or cardioprotective process. Whether this process is contributed by DOX-mediated ROS or specific signaling pathways may require further investigation (54). Therapeutically, combinations of chemotherapeutic agents with epigenetic therapies, such as small molecule inhibitor of epigenetic writer/reader/eraser or miRNAs manipulations, may confer protection of patients from cardiotoxicity. However, whether such a potential cardioprotective agent will affect the efficacy of DOX or create other side effects requires further clinical investigation (i.e., the colloquial "double-edged sword"). For example, dexrazoxane, the only Food and Drug Administration– approved cardioprotective agent, has been shown to prevent DOX-mediated cardiotoxicity (75). However, the beneficial effect of dexrazoxane is still debated because of the risk for the

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development of acute myeloid leukemia and myelodysplastic syndrome in children (76, 77). In conclusion, epigenetic modifications may play a role in DOX-mediated apoptosis and atrophy in cardiomyocytes. Delineation of specific epigenetic therapies as detrimental vs. beneficial cardioprotective merits further investigation.

# AUTHOR CONTRIBUTIONS

W-HH and MC performed literature search. HK and MC wrote the manuscript.

# ACKNOWLEDGMENTS

This study was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 106-2923-B-194-001- MY3), and the Center for Innovative Research on Aging Society (CIRAS) from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by Ministry of Education (MOE) in Taiwan.


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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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