It is, nowadays, commonly acknowledged that the exposure of the heart to ionizing radiation, as in radiation therapy for breast cancer, Hodgkin's disease, or other cancers of the chest, increases the risk of heart disease (1, 2). This has become a growing problem with the advancements in cancer therapy that have successfully reduced both mortality rates and the recurrence, expanding the life expectancy of the survivors (3, 4).

Manifestations of radiation-induced heart disease include pericarditis, pericardial fibrosis, diffuse myocardial fibrosis, coronary artery disease, microvascular damage, and stenosis of the valves (5, 6). Considering causal biological processes in the development of the disease, persistent inflammation and oxidative stress, fibrosis, and pre-mature endothelial senescence are thought to be salient (7–9). Recently, the role of mitochondrial dysfunction and related metabolic perturbations has become more and more evident (10–12).

Detecting cardiac toxicity by assessing left ventricular function often requires a large amount of myocardial damage, characteristic of irreversible heart injury (13, 14). There is increasing emphasis on the use of biomarkers to detect cardiotoxicity at a stage before it becomes irreversible.

The most important blood biomarkers of heart injury are cardiac troponins T (cTnT) and I (cTnI), heart proteins controlling the calcium-mediated interaction between actin and myosin filaments (15). While cTnT is expressed to a small extent in skeletal muscle, cTnI has been found only in the myocardium. A previous study by Skyttä et al. showed that cTnT levels increased during adjuvant whole-breast radiation therapy in one out of five patients. Moreover, the increase in cTnT release was positively associated with cardiac radiation dose and with minor changes in the left ventricular diastolic function (16). A sustained irreversible leakage of cardiac troponins to the blood stream is due to the degradation of the myofibrils after heart damage (17).

Since irradiation tends to stimulate inflammatory processes, C-reactive protein (CPR), an acute phase protein, could be an additional potential predictive marker of cardiotoxicity after irradiation. Increased CRP level was associated with the severity of radiation-induced cardiomyopathy after radiation therapy of lung or breast cancer (18). We have shown elevated levels of inflammatory cytokines such as interleukin (IL)-1, IL-6, and tissue necrosis factor alpha (TNF-α) in serum after local cardiac irradiation in mice (19), but data on their role in the prediction of myocardial changes in clinical trials are lacking to date (20, 21).

Although cardiac troponins and CRP are established sensitive biomarkers of myocardial injury and inflammation, respectively, there is no specificity for radiation-associated heart disease. In fact, there are no biomarkers available to identify radiotherapy patients who are in the process of developing radiation-associated heart disease, although the current data suggest that certain blood biomarkers may be associated with myocardial dysfunction (20, 22, 23).

Proteomics represents a promising global technology to discover new types of biomarkers for radiation-induced cardiac injury (11, 24). However, identification and quantification of serum/plasma proteins remains an analytical challenge, mainly due to the dominance of albumins and immunoglobulins and the high dynamic range of protein abundances (25). This is particularly true for disease-specific biomarkers that are mainly low-abundance proteins (26, 27). The newly established quantitative proteomics technology based on the data-independent acquisition (DIA) mass spectrometry (MS) was introduced recently to overcome the limitation of previous approaches (26). The aim of this study was to identify biomarkers of cardiac toxicity in the serum proteome of mice after local heart irradiation by using DIA-MS.

The serum proteome is a reliable mirror of the individual's healthy and diseased states (33). In this study, we used global serum proteomics analysis as a starting point to predict radiation effects outside the target tissue. Applying a multivariate analysis on the data, in this case principal component analysis and hierarchical clustering, we could separate the control group from the irradiated groups. Although the analysis could even differentiate between the two irradiated groups based on the radiation dose, it also highlighted a panel of proteins being differentially expressed in both irradiated groups. This panel, rather than one single protein, can be considered as a radiation biomarker in the serum proteome.

This analysis also clearly showed that local heart irradiation is able to induce systemic inflammation and hypercholesterolemia in mice. These two responses are similar to those found in a multiomics study comparing atherogenic and dyslipidemic mice with wild-type mice and, more importantly, when comparing familial hypercholesterolemia patients with healthy controls (34).

The degree of this systemic inflammatory and dyslipidemic effect was dose-dependent and thereby presumably also related to the degree of the heart damage. The dose of 8 Gy was only partly able to induce similar proteome changes as the 16-Gy dose, and the proteome was, in general, altered to a lesser extent. Furthermore, the lower radiation dose was not able to alter the cytokine or lipid profile of the serum as strongly as the higher dose.

The pathological changes in the locally irradiated heart tissue of these mice have been described in our previous study (35) where we showed radiation-induced elevation of inactive phosphorylated PPARα and increased expression levels of proteins involved in SMAD-dependent and SMAD-independent TGF-β signaling. Furthermore, we showed enhanced levels of proteins involved in fibroblast to myofibroblast conversion and inflammation at 16 Gy. Some, but not all, of these protein expression changes were also present at 8 Gy (35). Histological examination in similarly treated C57BL/6J mice revealed a significant increase in epicardial thickness (8 and 16 Gy), enhanced levels of inflammatory cells, and iron-containing macrophages (16 Gy) after 20 weeks (36). These changes are in line with the alterations found in the serum of irradiated mice in this study.

We have shown previously that, particularly, cardiac endothelial cells respond to high-dose radiation by secreting proinflammatory cytokines in vivo and in vitro (19, 37–39). TNF-α that we found significantly elevated at the 16-Gy dose modulates the inflammatory response by activating the expression of IL-1 and IL-6 (40). These cytokines that also were upregulated in the serum of irradiated animals serve as significant predictors of cardiovascular disease (40, 41). In agreement with our data, elevated levels of IL-1 and IL-6 were found in patients after radiation therapy for lung cancer (42).

We found in this study changed levels in serum proteins involved in blood clotting in irradiated mice, indicating not only inflammatory but also thrombotic changes. Among these were several serpins, plasminogen, fibronectin, and fibrinogen. Fibrinogen is a serum adhesion molecule identified in individuals with a high risk for cardiovascular disorders (43). IL-1 and IL-6 positively influence the synthesis of fibrinogen (44, 45). Fibrinogens contribute to atherosclerotic plaque formation by inducing endothelial permeability and increase the probability for thrombus formation by enhancing the blood viscosity and platelet aggregation (44, 46, 47). In agreement with our results, previous studies show an induction of thrombotic responses in locally irradiated carotid and saphenous arteries and in the heart (48–50).

The proteomics data in this study predicted radiation-induced activation of TGF-β, and its upregulation in the serum was confirmed at 16 Gy using ELISA. TGF-β is a multifunctional cytokine regulating inflammation and fibrosis in the heart (51, 52). The consequences of cardiac fibrosis are severe including contractile dysfunction, deformation and remodeling of the cardiac structure, and heart failure (53). Enhanced levels of TGF-β mediate also radiation-induced cardiac fibrosis that is characterized by excess fibroblast proliferation and deposition of collagen fibers (36, 54). We have shown previously the activation of TGF-β signaling and induction of fibrosis in the mouse heart exposed to local high-dose radiation (16 Gy) (35, 55).

In contrast to the systemic inflammatory effect, this is, to the best of our knowledge, the first study to show that local heart irradiation has a profound effect on serum lipids. Enhanced levels of free fatty acids and total cholesterol that we find here, especially in the 16-Gy irradiated mice, are strong risk factors for cardiovascular disease (56, 57). Similarly, increased LDL and decreased HDL levels, particularly in combination, are associated with increased risk for cardiovascular disease in humans since, if long lasting, they are known to lead to hardening of the arteries and atherosclerosis (58). The enhancement of oxLDL serum level that we have observed already in a previous study (19) is a strong predictive marker for upcoming coronary heart disease events in healthy men and a potential risk factor for cardiovascular disease (59, 60). OxLDL is involved in the early progression of the atherosclerotic plaque formation including endothelial injury, increased levels of adhesion molecules, leukocyte recruitment and extravasation, and foam cell and thrombus formation (61). Moreover, it activates the inflammatory response and increases the production of cytokines (62).

The transcription factor PPARα that was predicted to be inactivated in irradiated animals, based on the serum proteome profiling, is the main regulator of lipid metabolism (63). Furthermore, it exerts anti-inflammatory effects in the vascular wall and, thereby, protects against initiation and progression of atherosclerosis (64). The PPARα protein is highly expressed in the heart but not excreted in the serum. We have shown previously that cardiac PPARα is inactivated after local heart irradiation in mice (28). More importantly, it was inactivated in a dose-dependent manner in the cardiac left ventricle of Mayak nuclear workers exposed to varying total body doses of external gamma radiation when compared with Mayak workers not exposed to irradiation (10). Both exposed and control workers were diagnosed and died of ischemic heart disease. These data indicate that, although deactivation of PPARα is a common feature in ischemic heart disease and has been observed in human heart failure patients (65–69), it is especially prominent in radiation-induced heart disease and, therefore, a radiation target in the heart. It is particularly interesting that this is reflected in the serum proteome and cytokine and lipid profiles.

Immunoglobulins G and M were significantly upregulated in the serum of irradiated mice. Increased levels of both immunoglobulins in blood have been associated with adverse cardiovascular events, particularly in dyslipidemic men, but the epidemiological data are contradictory (70–73). In contrast, we did not identify cardiac troponins that are immediate markers of cardiac damage in humans as in mice. In mice, cardiac TnI concentrations in serum peaked at 1 to 4 h and declined to baseline by 48–72 h after a single administration of isoproterenol (74). This rapid decline is probably the reason why we did not find it elevated in the mouse serum 20 weeks after local heart radiation. Nevertheless, cardiac troponins seem to stay downregulated in the cardiac tissue a long time after radiation exposure. We have shown previously a dose-dependent decrease in cTnT in the human left ventricle in the Mayak worker study (10) and cTnI in the locally irradiated mouse heart (8 and 16 Gy) (28) suggesting an early leakage of cardiac troponins to the serum after radiation-induced myofibril degradation.

All in all, the data presented here suggest that the serum proteins and lipids function as potential biomarkers of cardiac injury following heart high-dose radiation exposure. They confirm our previous findings in the heart proteome following high-dose irradiation suggesting radiation-associated activation of TGF-β but inactivation of PPARα (35, 56). Especially, PPARα has become an interesting therapeutic target due to its pleiotropic activity in controlling lipid metabolism and energy homeostasis, inhibiting inflammation, reducing oxidative stress and apoptosis, and ameliorating contractile function. However, the clinical trials using PPARα agonists have shown contradictory outcomes so far (75). We suggest that administering such agonists could be particularly beneficial in connection with radiation therapy for thoracic malignancies where the heart may receive considerable radiation doses leading to adverse cardiovascular events (76). Furthermore, the data from this serum study could be beneficial in identifying patients who may develop radiation-associated cardiac toxicity.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

All animal experiments were approved and licensed under Bavarian federal law (Certificate No. AZ 55.2-1-54-2532-114-2014).

The study was designed by OA, VS, MA, GM, and ST. The irradiation was done by WS and VS. Serum collection was done by VS. The proteomics analysis was done by CT and OA. The ELISA experiments were performed by OA. The multivariate analysis was done by OA. OA and ST wrote the draft manuscript. All authors contributed to the revision of the manuscript, read it, discussed, and approved the final version.

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.