The survival rate of childhood cancer has increased from 60% to more than 85%, thanks to advanced diagnosis, treatment, and care models (1). Indeed, there are more than 500,000 childhood cancer survivors in the United States and this number is expected to increase. Although the increased survivorship is a cause for celebration, up to 73% of childhood cancer survivors suffer from long-term health complications (2). Cardiovascular disease is one of the most common long-term complications in survivors and the second leading cause of death in childhood cancer survivors after secondary malignancy (2). The high burden of cardiovascular diseases in childhood cancer survivors is mainly attributed to cardiotoxic cancer treatments such as anthracyclines and radiation therapy (3). Doxorubicin (DOX) is an anthracycline chemotherapeutic agent widely used in the treatment of lymphoma, leukemia, and other pediatric cancers, despite its known cardiotoxic effects (4). Since the severe cardiotoxic effects of DOX are dependent on the cumulative dose, the current treatment protocols usually do not exceed this threshold. Therefore, the rates of severe cardiovascular complications have declined in recent years. However, it has also been shown that low cumulative doses of DOX cause subclinical cardiotoxicity in childhood cancer survivors (5–7).

DOX-induced subclinical cardiotoxicity predisposes the survivors to adult-onset cardiovascular risk factors in a two-hit manner (8, 9). Given the expected long survivorship life in childhood cancer survivors, many of them would develop multiple cardiovascular risk factors later in their adult life, which can be considered as “second hits.” Since hypertension is the most significant cardiovascular risk factor for all adverse cardiac events, including heart failure and cardiac death, in anthracycline-treated childhood cancer survivors (10), we have recently developed a two-hit mouse model of juvenile DOX-induced latent cardiotoxicity that is exacerbated by adult-onset angiotensin II (ANGII)-induced hypertension (11). Nevertheless, it is still not known how juvenile DOX-induced latent cardiotoxicity would predispose the heart to other cardiovascular pathologic stimuli that do not cause hypertension. In the current study, we characterize the detrimental synergy in the DOX/ANGII model in parallel to a new model wherein juvenile DOX exposure is followed by adult-onset catecholamine stress by daily injections of isoproterenol (ISO). ISO is a non-specific beta-adrenoceptor agonist that is commonly used to induce a dose-dependent cardiac pathology without elevating blood pressure (12–15). Characterizing both DOX/ANGII and DOX/ISO models is critical to understanding why hypertension is the most significant risk factor for cardiovascular morbidity and mortality in anthracycline-treated childhood cancer survivors and thereby devising effective therapeutic strategies against this significant clinical problem.

Childhood cancer survivors have a considerably increased risk for premature cardiovascular diseases (20), with an estimated 15 times higher risk of heart failure than their siblings who did not have cancer (2). Nearly 50% of pediatric cancer patients receive anthracyclines such as doxorubicin (DOX), which are known to cause cardiotoxicity (21). Although the risk of anthracycline-induced cardiotoxicity increases with a higher anthracycline cumulative dose (22), latent (subclinical) cardiotoxicity occurs in children who receive low doses of anthracyclines (5–7). Anthracycline-induced subclinical cardiotoxicity is characterized by reduction in the left ventricular mass, mild cardiac fibrosis, and modest decline in ejection fraction (5–7). This latent cardiotoxicity can be unmasked and overt cardiomyopathy precipitated by other cardiovascular risk factors in adulthood, in a two-hit manner (10). We designed the current experimental protocol to mimic the scenario in cancer survivors that undergo DOX treatment at young age. With this protocol, we are able to show that latent cardiotoxicity caused by juvenile exposure to DOX is exacerbated when adult mice undergo a hypertensive “second-hit” on the heart. We used ANGII and ISO as two pharmacological agents that both increase the stress on the heart through distinct mechanisms. In this report, we show that the combination of DOX and ANGII causes the most changes to heart size, cardiac function and fibrosis. While the combination of DOX and ISO shows modest changes in heart size, cardiac function and fibrosis are not affected (Figure 8).

Most preclinical models of juvenile DOX-induced cardiotoxicity used high cumulative doses of DOX that were enough to cause immediate or delayed cardiac dysfunction (23–26). Although clinically relevant, animal models for juvenile DOX cardiotoxicity have rarely adopted the two-hit models. Huang et al. demonstrated that low doses of DOX administered to very young mice, at postnatal day 5, did not cause immediate cardiac dysfunction. DOX-exposed mice developed normally and had no obvious cardiac dysfunction as adults. However, juvenile exposure to DOX exacerbated cardiac pathology in response to an adult-onset pathologic stimulus (myocardial infarction) and even a physiologic stimulus (swimming exercise) (27). Since hypertension is the most significant cardiovascular risk factor for all adverse cardiac events, including heart failure and cardiac death, in anthracycline-treated childhood cancer survivors (10), we have recently developed another two-hit mouse model of juvenile DOX-induced latent cardiotoxicity that is exacerbated by adult-onset ANGII-induced hypertension (11). Similar to Huang et al., we demonstrated that low doses of DOX (4 mg/kg/week for 3 weeks) did not cause immediate cardiac dysfunction in juvenile mice, but predisposed to late-occurring detrimental cardiovascular changes when the mice were challenged by ANGII-induced hypertension (11). However, unlike Huang et al., our DOX administration regimen starts at 5 weeks of age, equivalent to 10 years in human life. Therefore, these dosage regimens model latent cardiotoxicity in anthracycline-treated pediatric cancer patients who do not immediately develop overt cardiac dysfunction but are left with “weaker” hearts that predispose them to other cardiovascular insults, corroborating the findings of several clinical studies (5, 28–31).

Nevertheless, it is still not known how this low-dose DOX regimen would predispose the heart to other cardiovascular pathologic stimuli that do not cause hypertension. To answer this question, we subjected control and DOX-treated mice to a regimen of ISO injections (10 mg/kg/day for 14 days). ISO is a non-specific beta-adrenoceptor agonist that is commonly used to induce a dose-dependent cardiac pathology without elevating blood pressure (12–15). We have previously demonstrated that this dosage regimen causes cardiac hypertrophy, mild cardiac dysfunction, and modest cardiac fibrosis in C57BL/6N male mice (12). In the current study, we characterize the DOX/ANGII model in parallel to the DOX/ISO model to better understand why hypertension is the most significant risk factor for cardiovascular morbidity and mortality in anthracycline-treated childhood cancer survivors.

Cardiac atrophy and thinning of the LV ventricular walls are common late effects of anthracycline therapy in childhood cancer survivors (32–35). Intriguingly, a study has shown that reduction in the LV mass is associated with worsening of heart failure symptomatology independent of LV ejection fraction in adult cancer survivors (36), demonstrating the predictive value of LV mass. However, the association between LV mass and heart failure symptomatology has not been determined in childhood cancer survivors. We previously demonstrated that juvenile exposure to DOX prevented the adaptive cardiac hypertrophy in response to ANGII-induced hypertension (11). However, it is not known whether juvenile exposure to DOX would also prevent adaptive cardiac hypertrophy in response to other hypertrophic stimuli. In the current study, juvenile exposure to DOX prevented the adaptive cardiac hypertrophy in response to both ANGII and ISO. Indeed, ANGII and ISO cause cardiac hypertrophy via different pathways. ANGII induces cardiac hypertrophy directly through activating the AT1 receptors on cardiomyocytes and indirectly through elevating the afterload (37). In contrast, ISO activates the beta-adrenoceptors on cardiomyocytes to elicit a direct hypertrophic effect (37). ISO-induced tachycardia may also contribute to its hypertrophic effect indirectly. The ability of DOX to prevent cardiac hypertrophy in response to both pathologic stimuli suggest that DOX interferes with common downstream pathways fundamental to the development of cardiac hypertrophy.

Although high-dose DOX causes cardiac atrophy due to apoptotic and necrotic cell death and loss of cardiomyocytes (38), experimental studies using low/divided-dose DOX have suggested that DOX-induced cardiomyocyte atrophy is the main culprit leading to cardiac atrophy and reduction of LV mass with minimal apoptotic cell death (39, 40). We demonstrate that mice with juvenile exposure to DOX had the smallest cardiomyocyte surface area after ANGII exposure. These experimental observations have recently been supported by a clinical study reporting that the reduction in LV mass after anthracycline therapy is due to cardiomyocyte atrophy in breast cancer patients (41). Another recent preclinical study has shown that acute DOX administration causes dose-dependent cardiac atrophy that parallels the decrease in contractile function (39). In our current study, we demonstrate that chronic administration of low-dose DOX caused cardiac atrophy without reducing the contractile function of the heart. The contractile function of the heart was only affected when the juvenile exposure of DOX was followed by ANGII-induced hypertension. ANGII cause pathologic cardiac hypertrophy characterized by the induction of fetal gene expression such as ANP and BNP, adverse cardiac remodeling, and reduction in cardiac function parameters. Therefore, in the current work, we determined the effect of juvenile DOX exposure on these parameters. Although we previously showed that juvenile exposure to DOX induced ANP gene expression 1 week after the last DOX injection (11), there is no significant change in ANP or BNP in DOX-exposed mice 7 weeks after the last DOX injection. Only the combination of DOX/ANGII was able to significantly elevate the markers of pathological hypertrophy. Although juvenile exposure to DOX prevented the hypertrophic growth of the heart in response to ANGII, it did not abrogate the molecular determinants of pathological cardiac hypertrophy induced by these stimuli.

We also determined the effect of juvenile exposure to DOX on cardiac remodeling in response to both ANGII and ISO. In our previous study describing the DOX/ANGII model (11), the effect on cardiac fibrosis had not been determined. In the current study, we demonstrate that juvenile exposure to DOX did not cause significant cardiac fibrosis in naïve mice, but it exacerbated ANGII-induced cardiac fibrosis. The exacerbated cardiac fibrosis was associated with a marked upregulation in several inflammatory and fibrotic markers in the DOX/ANGII-treated mice. Importantly, MAC-2 positive cells and the expression of Lgals-3 gene encoding galectin-3 were much higher in hearts of DOX/ANGII-treated mice than in hearts of mice receiving either DOX or ANGII alone. In contrast to cardiac atrophy, which is a consistent feature of anthracycline-induced cardiotoxicity, the prevalence and extent of cardiac fibrosis in anthracycline-treated childhood cancer survivors is controversial. In a cohort of childhood cancer survivors, the prevalence of left ventricular and right ventricular fibrosis was 9 and 38%, respectively; however, these values were not compared to a healthy control group (42). Some studies report anthracycline-treated childhood cancer survivors to show modest myocardial fibrosis as evident by an increased extracellular volume fraction (35). On the other hand, other studies demonstrate the absence of a statistically significant increase in myocardial fibrosis in survivors compared to healthy control subjects (43, 44). Since there is no clinical data reporting the association between myocardial fibrosis and cardiovascular risk factors in anthracycline-treated survivors, it may be possible that the discrepancy in these clinical studies arise from the confounding effect of other cardiovascular diseases, particularly hypertension.

In contrast to the DOX/ANGII model, juvenile exposure to DOX did not exacerbate ISO-induced cardiac fibrosis. Surprisingly, the gene expression of the fibrotic marker collagen 1a1 was lower in DOX/ISO-treated mice as compared to mice treated with ISO alone. DOX-induced cardiotoxicity has been shown to attenuate the acute effects of ISO on the heart including its positive inotropic effect (45), acute decrease of myocardial stiffness (46), and stimulation of adenylyl cyclase (47). Nevertheless, the impact of DOX exposure on the chronic effects of ISO has not been previously reported. ANGII-induced increase in afterload coupled with DOX-induced thinning of the left ventricular walls is expected to markedly increase ventricular wall stress according to the Law of LaPlace. Since ISO does not increase the afterload, its effects on the heart of DOX-exposed mice would be expected to be much milder.

DOX-induced cardiotoxicity has been shown to be more severe in hypertensive experimental animals than in normotensive ones (48–50). An important distinction between these studies and our model is the fact that these studies administered DOX to already hypertensive animals, while in our model DOX is administered to young normotensive mice then challenged by ANGII-induced hypertension in their adult life, 5 weeks after the last DOX injection. In an attempt to determine the mechanism of the detrimental synergy between juvenile exposure to DOX and adult-onset ANGII-induced hypertension, we determined the effect of these experimental conditions on the renin-angiotensin-aldosterone-system (RAAS) genes. In the current study, juvenile exposure to DOX had no significant effect on the expression of Ace, Agtr1a, and Agtr1b genes. Nevertheless, there was a significant upregulation in Ace gene expression in the DOX/ANGII group. Similarly, juvenile exposure to DOX prevented ANGII-induced downregulation of Agtr1a, which encodes the AT1 receptor. ANGII mediates its detrimental effects via the AT1 receptors, while AT2 receptors mediate cardioprotective effects. DOX has been previously shown to alter the RAAS in different ways (19). DOX has been shown to significantly increase the expression of AT1 receptors and reduce that of AT2 receptors in a rat model of DOX-induced heart failure (51). Although there was no significant change in the mRNA expression of RAAS genes in the hearts of rabbits treated with a single dose of DOX (52), the plasma and myocardial levels of ANGII were increased three-fold in a rat model of DOX-induced heart failure (53). DOX treatment has also been shown to increase myocardial ACE activity in the cardiac tissues of hamsters (54). Intriguingly, angiotensin receptor blockers (ARBs) have been shown to ameliorate anthracycline-induced cardiotoxicity in animal models (55–57). Importantly, a recent meta-analysis shows that RAAS antagonists were the most efficient drugs to prevent anthracycline-induced cardiotoxicity with 84% risk reduction (58).

The current study has some limitations that warrant discussion. First, we have not measured the blood pressure in our experimental groups. We previously reported that juvenile exposure to DOX caused an increase in blood pressure, which was further exacerbated by ANGII infusion (11). ISO is a beta-adrenergic agonist that does not increase blood pressure, as previously reported by several other investigators (14). We also did not measure the plasma levels of natriuretic peptides, ANP and BNP. Although the induction of fetal gene expression as a hallmark of pathologic hypertrophy is usually assessed by measuring the gene expression of ANP and BNP (59, 60), measuring plasma levels of these peptides would have strengthened our conclusions.

In conclusion, this study shows that juvenile exposure to DOX differentially exacerbates ANGII—but not ISO-induced adverse cardiac remodeling. There was a marked detrimental synergy between juvenile exposure to DOX followed by ANGII-induced hypertension, which resulted in cardiac dysfunction and adverse cardiac remodeling. This preclinical mouse model highlights the clinical finding that hypertension is the most significant risk factor for heart failure in anthracycline-treated childhood cancer survivors. Since ANGII may cause cardiac damage through direct mechanism beyond elevating blood pressure, future studies are planned to delineate the mechanisms of these deleterious effects by targeting elements of RAAS system.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota (Protocol ID:1807-36187A).

MG, KA, and DY: performed experiments. MG, KA, DS, JB, and BZ: analyzed data. KA, MG, and BZ: wrote the manuscript. AB, JD, and BZ: contributed to conception and design of the study.

This work was supported by the National Heart, Lung, and Blood Institute (NHLBI) grant R01HL151740 (BZ and AB), the St. Baldrick's Foundation for Childhood Cancer (Award ID 638335, BZ and AB); and the National Institutes of Health's National Center for Advancing Translational Sciences, grant UL1TR002494 (BZ). AB was funded by a Minnesota Partnership for Biotechnology and Medical Genomics #18.04. JD is a Canada Research Chair in Molecular Medicine and is funded by a Foundation Grant awarded by the Canadian Institutes for Health Research (CIHR) and by a Women and Children's Health Research Institute (WCHRI) grant, University of Alberta.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.