Radiation-induced heart energy metabolism dysfunction leads to heart brosis

Thoracic radiotherapy increases the risk of radiation-induced heart disease (RIHD), but its molecular mechanisms are not fully understood. We aimed to explore the effects of radiation on the mouse heart using high-throughput proteomics. An RIHD mouse model was established by exposing the whole heart to 16 Gy high-energy X-rays, and cardiac injuries were veried by cardiac echocardiogram, serum BNP, HE and Masson staining 5 months after irradiation. Proteomics experiments were performed using the whole heart tissue of the irradiated mice and the control mice not exposed to irradiation. The proteomics data were subjected to bioinformatics analysis, and they indicated that irradiated mouse hearts showed alterations in cardiac brosis and energy metabolism proteins. Then, we conrmed the cardiac brosis and energy metabolism changes by IHC staining and WB analysis. Extracellular matrix proteins such as Col1a1, Col3a1, Vimentin and CTGF, along with metabolism-related proteins such as Fans and Slc25a1, were overexpressed after exposure to ionizing radiation. Additionally, myocardial mitochondria inner membranes presented with injury, ATP declined and lactic acid accumulated in the irradiated heart tissues. This study suggests that high doses of ionizing radiation lead to structural remodeling, functional injury and brosis alterations in the mouse heart. Radiation-induced mitochondrial damage and metabolic alterations of the cardiac tissue may be one of the pathogenic mechanisms of RIHD.


Introduction
Thoracic radiotherapy is an important cancer treatment for patients diagnosed with malignant tumors such as breast cancer, lung cancer, Hodgkin's lymphoma and esophageal cancer [1][2][3][4]. Radiotherapy improves overall survival in thoracic cancer patients, but it involves some inevitable complications, especially RIHD, which has gradually become a concern to oncologists and cardiologists [5,6].
The incidence of RIHD has increased due to the overall survival of cancer patients being prolonged, and RIHD usually only occurs 5 to 10 years after radiation therapy [7,8]. It has been reported that RIHD comprises a number of heart diseases, including cardiomyopathy, coronary artery disease, pericarditis, valvular disease and conduction system abnormalities [9][10][11]. The rates of major coronary events increase as the heart mean dose is increased by 7.4% per gray, and these coronary events usually occurred 5-20 years after radiotherapy among breast cancer survivors [12]. The risk of coronary heart disease is increased 2.5-fold in the 20 Gy mean heart dose (MHD) group compared with Hodgkin lymphoma patients treated without radiation [2]. In our previous study, we identi ed a cardiac biomarker, serum ST-2, that was increased among cancer patients after thoracic radiotherapy, and it was positively associated with the radiation parameters V 5 , V 10 , V 20 and MHD [13].
High-energy X-rays could cause cardiac injury, but the underlying mechanisms of RIHD have not been fully investigated. Our understanding of the mechanisms involved in RIHD progression begins with energy deposition and reactive oxygen species (ROS) generation followed by molecular changes, damage to DNA, lipids and proteins, as well as the activation of early response transcription factors, cytokines and signal transduction pathways [14][15][16][17]. In addition, it is also believed that RIHD is associated with endothelial cell injury [18,19]. Irradiated endothelial cells are surrounded by large amounts of proin ammatory cytokines and then cellular degeneration occurs; cardiac dysfunction could be a result of many years of persistent in ammatory stimulation [19]. Our previous study also indicated that microvascular endothelial cell dysfunction may be a predominant mechanism of RIHD [20].
The latest advances in the high-throughput technologies of multiple "combinatorial data", such as genomics and proteomics, may explain the molecular mechanism of diseases more directly and more precisely [21,22]. Notably, proteomics are used to provide a functional context to interpret genomic abnormalities and can present a novel paradigm for understanding cancer biology [23,24]. Proteomics has gradually become an important technique in the elds of disease diagnosis, drug research and development, while also playing a predominant role in the much larger eld of the molecular basis of diseases and biological processes at the protein level [25,26]. The proteomics of radiation injury may provide novel evidence for the study of the mechanism of radiation heart injury, so we tend to use proteomics analysis to reveal the changes of cells or tissues by detecting the alterations of proteins.
This study aimed to use a novel high-throughput proteomics technology to explore the effects of radiation on heart proteins through establishing an RIHD mouse model to better understand the molecular mechanisms of RIHD.

Methods And Materials
Animal model and local cardiac irradiation C57BL/6 male mice aged 8 weeks were purchased from the Shanghai Institute of Biochemistry and Cell Biology. The mice were irradiated at the age of 8-9 weeks. All mice lived in a 12:12 light:dark cycle environment with free access to food and water. The mice were sacri ced at 1 month, 3 months, or 5 months after irradiation or sham-irradiation. Each cohort included 3-6 mice. All animal procedures in this study were approved by the Animal Care and Use Committee of China. Ethical approval was obtained from the Institutional Review Board of the Second A liated Hospital of Nanchang University.
The whole heart was locally irradiated with a dose of 16 Gy by a precise small-animal radiation research platform (SARRP, XStrahl Medical and Life Sciences, USA) in the Zhejiang Key Radiation Laboratory. Mice were anesthetized by intraperitoneal injection of 75 gm/kg pentobarbital sodium and then were placed in the supine position in the irradiation area of the small animal X-ray radiometer; the laser system was used to establish a three-dimensional coordinate system.
Cone-beam computed tomography (CBCT) using 50 kV and 0.8 mA photons ltered with aluminum (1 mm) was performed for each mouse to visualize the tomographic scanning of the thorax. Heart, lung and spinal cord were drawn by the same physicist on the tomographic scanning of the thorax, then the physicist designed and evaluated the radiotherapy plan, and limited the irradiated volume of the lung tissue and spinal cord in the mice as much as possible. A dose-volume histogram (DVH) of the heart, lung and spine could be obtained. The whole heart was irradiated using 220 kV and 13 mA X-ray beams ltered with a copper lter (0.15 mm). Control mice received sham irradiation (0 Gy).

Cardiac Echocardiogram
Transthoracic echocardiography was performed using the Vevo 2100 ultrasound system (Visualsonics, Toronto, Canada) according to our previous study [20]. Two-dimensional guided M-mode echoes were obtained at the level of the largest left ventricle (LV). The left ventricular posterior wall at the end of diastole was measured from the M-mode image. The LV ejection fraction (EF) and fractional shortening (FS) were calculated from the measured ventricle dimensions.

Measurement of Serum BNP
Mouse blood samples were collected in tubes with EDTA and the serum was separated by centrifugation for 10 min at 600 × g. BNP was determined using a high sensitivity Enzyme-linked Immunosorbent assay (ELISA) kit (Presage BNP assay, USCNK, China) according to the manufacturer's instructions. BNP levels were evaluated after determining the optical density of the samples at 450 nm (Thermo Scienti c Microplate Reader, Varioskan LUX, Finland).

HE staining and immunohistochemistry
Whole mouse hearts were quickly excised, immersed in 10% paraformaldehyde and embedded in para n. The whole heart was cut into 5 µm thick sections. The slides were stained with hematoxylin and eosin (HE). Immunohistochemical (IHC) staining was performed as described previously [27]. Sections were incubated with primary antibodies mouse anti-Col1a1 or mouse anti-Col3a1 (Proteintech, Wuhan, China) at 4 °C overnight and were then incubated with a secondary antibody (ZSJQ-Bio, Beijing China), which was followed by DAB staining.

LC-MS/MS
Heart tissue protein extraction, trypsin digestion and TMT labeling The heart tissue samples were ground in liquid nitrogen and then mixed with lysis buffer, followed by sonication three times on ice (Scientz). The supernatant was collected after centrifugation and we measured the protein concentration. The protein solution was reduced with dithiothreitol and alkylated with iodoacetamide in the dark. The protein sample was diluted by adding tetraethylammonium bromide (TEAB). Finally, trypsin was added at a trypsin-to-protein mass ratio for the rst digestion overnight, repeated for a second 4 h-digestion. After the trypsin digestion, the peptides were desalted with a Strata X C18 SPE column (Phenomenex) and vacuum-dried. The peptides were reconstituted in 0.5 M TEAB and processed following the manufacturer's instructions for the TMT kit.

HPLC Fractionation
A high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 µm particles, 4.6 mm ID, 250 mm length) was used to fractionate the tryptic peptides into fractions. First, the peptides were isolated by using a gradient of 8-32% acetonitrile (ACN, pH 9.0) to separate them into 60 fractions over 1 h. Then, the peptides were merged into 18 fractions and dried by vacuum freeze-drying.

LC-MS/MS Analysis
The tryptic peptides were dissolved in solvent A (0.1% formic acid in 2% ACN) and separated by an EASY-nLC 1000 UPLC system. The liquid gradient setting consisted of an increase from 9-25% solvent B (0.1% formic acid in 90% ACN) over 24 min, 25-36% over 30 min, and increasing to 36 ~ 80% over 32 min, then holding at 80% for the last 36 min, and all of the above settings were maintained at a continuous ow rate of 350 nL/min. The peptides were subjected to an NSI source, which was followed by tandem mass spectrometry (MS/MS) in Q Exactive TM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. Secondary fragments of the peptides were detected and analyzed by a high-resolution Orbitrap. The scanning range of the primary mass spectrometry was set to 350-1800 m/z, and the scanning resolution was set to 700000; the scanning range of the secondary mass spectrometry was set to a xed starting point of 100 m/z, while the secondary scanning resolution was set to 17500. The data acquisition mode used a data-dependent scanning (DDA) program; that is, after the rst-level scanning, the rst 20 peptide parent ions with the highest signal strength were selected to enter the high-energy Ctrap dissociation (HCD) collision pool in turn to use 31% fragmentation energy for fragmentation, and the second-stage mass spectrometry analysis was also carried out in turn. To improve the effective utilization of the mass spectrometry, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 10000 ions/s, the maximum injection time was set to 200 ms, and the dynamic exclusion time for the tandem mass spectrometry scanning was set to 30 seconds to avoid repeated scanning of the parent ions. LC-MS/MS was conducted and analyzed by Jingjie PTM Biolab Co. Ltd.

Database Search
Maxquant search engine (v.1.5.2.8) was used to process the MS/MS data results, and the tandem mass spectra were analyzed through the SwissProt Mouse database concatenated with the reverse decoy database to calculate the false positive rate (FDR) caused by random matching. In addition, common pollution databases were added to the database to eliminate the in uence of contaminating proteins in the identi cation results. Trypsin/P was regarded as the cleavage enzyme, allowing up to 2 missing cleavages, and the minimum length of the peptides was 7 amino acid residues. The mass error tolerance of the primary parent ion of the rst search and main search were 20 ppm and 5 ppm, respectively. The mass error tolerance of the secondary fragment ion was 0.02 dalton (Da). The FDR was adjusted to < 1% and the minimum score for the peptides was set to > 40.

GO Annotation
The Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (www. http://www.ebi.ac.uk/GOA/). First, we used UniProt ID to match the GO ID, and then we retrieved the corresponding information from the UniProt-GOA database according to the GO ID. InterProScan software would be used to annotate the protein's GO function if the proteins were not annotated by the UniProt-GOA database. Then, the proteins were classi ed by the GO annotation based on the biological process, cellular component and molecular function.

Functional Enrichment
Enrichment of Gene Ontology analysis GO annotations can be divided into three categories: Biological Process, Cellular Component and Molecular Function, which categorize the biological functions of proteins based on different features. A two-tailed Fisher's exact test was employed to test the enrichment of the differentially expressed proteins against all identi ed proteins. A GO analysis with p < 0.05 is considered signi cant.

Enrichment of the pathway analysis
The Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/kegg/) was used to study the enriched pathways by using a two-tailed Fisher's exact test. The enrichment of pathway analysis with a p < 0.05 was considered signi cant. These pathways were classi ed according to the hierarchical classi cation method of the KEGG website.

Enrichment of the protein domain analysis
InterPro (http://www.ebi.ac.uk/interpro/, a database providing functional analysis of protein sequences and predicting the presence of domains and important sites, was applied and a two-tailed Fisher's exact test was used to test the enrichment of the differentially expressed proteins. Protein domains analysis with a p < 0.05 was considered signi cant.

Transmission electron microscope (TEM)examination
Fresh heart apical portions of the mice at 5 months was quickly cut into 1 mm cubes and xed in paraformaldehyde (Solarbio, China) for 2 h at 4 °C, and then xed in 1% osmium tetroxide for 2 h at room temperature, followed by stepwise dehydration in graded acetone, and then it was in ltrated, embedded and polymerized. The pieces were sectioned with an ultramicrotome into 1-2 µm pieces and then stained with a toluidine blue dye solution. The myocardial ultrastructure of the hearts were observed on a HITACHI-7700 electron microscope (Hitachi, Japan).

ATP and Lactic Acid Assays
Heart tissue samples were mixed with lytic uid, homogenized with a homogenizer, and then centrifuged at 12000 g/min for 5 min. The ATP production was determined according to the manufacturer's instructions (Beyotime, China). Relative light unit (RLU) values were collected using a multimode microplate reader with the Luminometer mode (Thermo Scienti c Microplate Reader, Varioskan LUX, Finland). The lactic acid was detected using a Lactic Acid Kit (Nanjing Jiancheng Bioengineering, China) following the manufacturer's instructions. The OD value was collected using a multimode microplate reader at 530 nm. The protein concentration was detected using the BCA method. Absolute ATP and lactate levels were calculated from the corresponding standard curve and normalized by the total protein concentration. Each group contained 3 mice.

Statistical analysis
All data are presented as the mean ± standard deviation (SD). Statistical differences were determined by one-way ANOVA or Student's t test using SPSS 20 (IBM Corporation, Armonk, NY, USA). P < 0.05 was considered signi cant. All experiments were performed with at least three biological replicates.

High-dose ionizing radiation causes cardiac structural remodeling and functional injury to the hearts of mice
To investigate the effects of high-energy X-rays on mouse heart tissue, we rst established an RIHD animal model with local 16 Gy heart irradiation. Cardiac echocardiography, serum myocardial biomarkers, and HE and Masson staining were used to verify the cardiac injury at different time points. The results showed that LVEF, systolic thickness of the left ventricular posterior wall and diastolic thickness of the left ventricular posterior wall at 5 months after radiation ionizing were signi cantly changed (p<0.05) compared with those of the control mice (Fig. 1A). Pericardial effusion was not present in all groups (Fig. 1A). In addition, the serum cardiac biomarker BNP (Fig. 1A) was obviously increased 5 months after exposure to ionizing radiation (p<0.05).
Moreover, the HE staining sections (Fig. 1B) indicated that the cardiomyocytes had degenerated and the myo lament boundary was blurred after radiation, and the myocardium changes were more obvious as the follow-up time increased. In addition, compared with the control mice, collagen bers in the 5-month irradiated heart tissues had obviously increased (Fig. 1C). Cardiomyocyte degeneration, brosis deposition, ventricular wall remodeling, BNP elevation, and left ventricular systolic dysfunction were evident in the 5-month irradiated mice. This indicated that myocardial damage and cardiac remodeling manifested in the 5-month mice after local heart radiation exposure.

Proteins quanti cation
In the above results, we proved that mice manifested the RIHD phenotype 5 months after 16 Gy ionizing radiation. Therefore, proteomics was performed in control and 5-month irradiated mice heart tissue ( Fig. 2A). Principal component analysis (PCA) and relative standard deviation (RSD) were used to evaluate the quantitative repeatability of the proteins (Fig. 2B-C). A total of 269637 secondary spectra were obtained by MS. After the secondary spectrum of the MS went through the protein theory database, the available number of available spectra was 64186 and the utilization rate of the spectrum was 23.8%. A total of 29161 peptides were identi ed by the spectral diagram, among which there were 28194 speci c peptides. From these peptides, 3777 proteins were identi ed, and 3274 were quanti able (Fig. 2D). Highthroughput proteomics analysis identi ed 234 proteins in total, and 219 proteins were found to be signi cantly increased (log2>1.2), while only 15 proteins were certi ed as downregulated (log2<1/1.2) in the irradiated mouse heart (Fig. 2E).
According to the GO (Gene Ontology) category results, the proteins related to Biological Process were mainly cellular processes, biological regulation, single organelle treatment, response to stimulation and metabolic related processes; Cell Component was mainly composed of extracellular components and organelle components. Binding proteins and catalases were the main Molecular Functions (Fig. 3B).
The differentially expressed proteins in the heart tissue induced by radiation were classi ed by COG/KOG (Fig. 3C). The results of the COG/KOG functional classi cation showed that the differentially expressed proteins were mainly distributed in the following functions: (W) Extracellular structure, (O) Posttranslational modi cation and protein turnover, (T) Signal transduction mechanism, and (V) Defense mechanism. Moreover, there are also dramatically differently expressed proteins in substance metabolism after radiation, such as (C) energy metabolites and conversion, (E) amino acid metabolism and transport, (F) nucleotide metabolism and transport, (G) glucose transport and metabolism and (I) lipid transport and metabolism. Metabolism-related proteins were signi cantly changed according to their subcellular localization and functional classi cation analysis.

Functional enrichment analysis
To nd the signi cant enrichment trend of the differentially expressed proteins among the functional types, we analyzed the enrichment of the differentially expressed proteins in the cardiac irradiation and control group through three aspects: GO category, KEGG pathway and protein domain.
The biological process enrichment analysis results of the GO secondary classi cation showed that the expression of proteins regulated by protein metabolism, negative regulation of protein metabolism and proteolysis increased after irradiation. Collagen ber tissue, protein activation cascade and invagination of the cytoplasmic membrane were among the most apparent differences (Fig. 4A). Cell component enrichment analysis demonstrated that the proteins related to vesicles, extracellular cellular components, extracellular vesicles, exocrine and extracellular region or the parts of extracellular structures increased signi cantly 5 months after irradiation (Fig. 4B). Molecular functions such as protein complex binding, receptor binding, molecular function regulation and enzyme activity regulated calcium binding proteins were signi cantly increased after irradiation (Fig. 4C).
The domains of these differential proteins were mainly enriched in immunoglobulin subtypes, immunoglobulin folding, EGF-like domains, immunoglobulin-like domains and so on (Fig. 4D). KEGG enrichment analysis found that the radiation-induced altered proteins were mainly enriched in Staphylococcus aureus infection, complement and collagen cascade, protein digestion and absorption, extracellular matrix interaction, folding and adhesion related signal pathways (Fig. 4E). From the three functional enrichment analyses, brosis was considered to be increased after irradiation.

Irradiation induces alterations in the ECM and metabolism proteins
According to the proteomic analysis results, it was found that irradiation led to the overexpression of extracellular matrix proteins such as Col14a1, Postn, Lgals3, Hpx, Tgfbi, Col2a1, Col5a2, Col3a1, Col1a2, Col1a1, Sparcl1, Col5a1, Vtn, Col6a1, and Lama5 (Table 1). COL1a1 and COL3a1 in the irradiated heart have been shown to be overexpressed by IHC and WB experiments (Fig. 5C, D). Additionally, we con rmed that other typical extracellular matrix proteins (Vimentin and CTGF) were also overexpressed (Fig. 5D). Moreover, the proteomic analysis results indicated that metabolism-related proteins associated with carbohydrate transport and metabolism, energy production and conversion, amino acid transport and metabolism, nucleotides transport metabolism and lipid transport metabolism were increased (Table 2). Metabolism-related proteins such as Fans and Slc25a1 were veri ed to be overexpressed by WB (Fig. 5D).  Subsequently, the electron micrograph results of the irradiated heart mitochondria were found to have myo laments with fuzzy boundaries, swollen and cavitation of the mitochondria, and fewer mitochondria (Fig. 5A). ATP production (Fig. 5B) was decreased after irradiation, from 133.2 µmol/gprot in the control to 103.0 µmol/gprot in the irradiated mice (p<0.05). However, lactate (Fig. 5B) in the irradiated heart tissue was obviously increased (p<0.05), from 110.7 µmol/L in the control to 269.4 µmol/L in the irradiated mice (p<0.05).

Discussion
An RIHD animal model was established 5 months after subjecting mice to 16 Gy local heart irradiation. The mice exhibited cardiac structural remodeling and functional injury. High-throughput proteomics was used to investigate the protein alterations of the irradiated mice hearts.
We obtained 29161 peptides, including 28194 speci c peptides and 3272 quantitative proteins from among 3777 identi ed proteins. We found 219 proteins were signi cantly upregulated (log2>1.2) and 15 proteins were downregulated (log2<1/1.2). In contrast, Azimzadeh O et al. investigated the proteomics of 16-weeks irradiated mouse heart (16 Gy) and found that there were 662 myocardial proteins identi ed and 371 quanti ed proteins [29]. Subramanian V et al. studied the proteomics of the mouse heart at 40 weeks after 16 Gy, and 1038 proteins were identi ed and 940 proteins were quanti ed [30]. We screened 3777 proteins and quanti ed 3274 proteins by TMT labeling, which might be the highest ux and labeling rate in the histological data of RIHD. Abundant quanti ed proteins could present more comprehensive and e cient bioinformatics data to investigate RIHD.
Myocardial cell energy metabolism is very important to maintain the autonomic and contractile function of cardiac myocytes [31]. ATP is the direct energy supply and fatty acid β oxidation is a vital method to produce myocardial ATP [32]. Moreover, the mitochondria is the main organelle involved in energy metabolism [33,34]. Therefore, derangement of energy and fatty acid metabolism could be a factor involved in cardiac diseases [35,36]. High-throughput proteomics results of the annotation and functional enrichment indicated that the metabolism and ECM-related proteins were altered.
A large number studies have reported that the mechanism of RIHD may be associated with endothelial cell injury, in ammatory reactions, and reactive oxygen species (ROS) [9,14,19]. Irradiation leads to endothelial cell injury by pro-in ammatory cytokines and DNA or proteins damaged by ROS. Endothelial dysfunction in uences the vascular intimal collagen deposition, which could cause pipe wall thickening and luminal stenosis [18]. A series of pathological effects such as ischemia and hypoxia of the irradiated heart may aggravate the in ammatory reaction [19]. With the continuous release of in ammatory cytokines such as TNF, IL-1, IL-6, and IL-8, cardiac parenchyma cell necrosis and excessive deposition of extracellular matrix could be found in irradiated heart tissue [9]. Radiation-induced myocardial brosis may be a late pathological process of RIHD [14,37]. Fibrosis is an abnormal deposition of extracellular matrix, which can lead to organ dysfunction, morbidity and necrosis [38][39][40]. Radiation-induced myocardial brosis is often regarded as the nal pathological process of RIHD and is the main risk factor for adverse myocardial remodeling and vascular changes [41,42]. Our proteomics data and veri ed experiments also indicated that the mouse heart showed signi cant brosis deposition after receiving 16 Gy radiation, and the common extracellular matrix proteins such as Col14a1, Postn, Lgals3, Hpx, Tgfbi, Col2a1, Col5a2, Col3a1, Col1a2, Col1a1, Sparcl1, Col5a1, Vtn, Col6a1, and Lama5 were signi cantly upregulated.
In brief, the results of electron micrographs, ATP and lactate indicated that high-dose ionizing radiation actually led to cardiac mitochondria structural alterations. Consistent with the histological results, changes in the inner membrane of the mitochondria led to alterations in metabolism-related enzymes. In addition, the mitochondria are usually regarded as a vital energy-producing organelle of cells, which produce ATP via the TCA cycle in the mitochondrial inner membrane. Furthermore, one of the most vital functions of the mitochondria is the generation of ATP by aerobic respiration while lactate is a metabolite produced in hypoxic conditions. Moreover, several ECM and metabolism-related proteins have been proven to be overexpressed after RT by WB veri cation. There are emerging studies about metabolic processes promoting factors involved in brosis pathogenesis [43][44][45]. Glucose metabolism can provide energy for anabolic processes and collagen production. It is well known that not only glucose metabolism but also protein and lipid metabolism are closely correlation with mitochondria [46][47][48]. Indeed, mitochondria are considered to be the major suppliers of cellular energy in the form of adenosine triphosphate (ATP) produced by oxidative phosphorylation and it is the main energetic organelle [49,50].
Vincent AS et al. investigated keloid scars with excessive amounts of collagen production concluded that keloid-associated broblasts consume unusually large amounts of glucose and produce more lactate to ful ll their ATP needs, which means the secretion of collagen relies on ATP produced by glucose, protein and lipids metabolism [51,52].
Meanwhile, metabolic changes are regarded as an important pathogenic process of brosis in various organs, and metabolic targeted therapy may become an important strategy to reduce brosis [33,37,39].
The disturbance of myocardial energy metabolism is an important cause of myocardial heart disease, and mitochondria are the main organelles of energy metabolism [53][54][55].
Similarly, mitochondrial dysfunction may also be an important cause of radiation-triggered heart disease. The myocardial mitochondria of C57BL/6 mice were signi cantly decreased 40 weeks after local 2 Gy radiation, but not in the 0.2 Gy group [56]. Marjan Boerma [57], using a model of radiation injury to the heart of SD rats, found that the mitochondrial permeability transition pore (MPTP) was opened for 6 h and 9 months after SD rats were exposed to different doses of radiation, and further studies con rmed that late application of a palladium lipoate complex (POLY-MVA) could improve myocardial mitochondrial function by reducing the cellular in ammatory microenvironment. However, it had no effect on repairing the damaged mitochondrial structure [58]. In 2019, Chen Tianfeng's team also con rmed that radiation can cause damage to the heart function caused by ROS produced by the mitochondria. The simple Ganoderma lucidum spore oil system they developed can target the myocardial mitochondria and slow down the production of ROS, thus having a preventive and therapeutic effect on RIHD. The above studies showed that mitochondrial damage is an important target of radiation heart disease, and drugs/drug-loaded materials for the repair of mitochondrial damage can reduce radiation-induced heart injury. Therefore, the mitochondria damage caused by ionizing radiation may be a signi cant cause of the pathogenic mechanisms of RIHD.
In this study, we analyzed the differentially expressed proteins in irradiated mice heart tissue and found brosis and metabolic alterations. Next, we may concentrate on the relationship between mitochondrial damage and RIHD to search for further pathogenic mechanisms.

Conclusion
The results of this study suggest that ionizing radiation causes structural remodeling, functional injury and brosis alterations in the heart. Radiation-induced mitochondrial damage and metabolic alterations Page 14/19 in cardiac tissue may be some of the pathogenic mechanisms of RIHD. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests:
The authors declare that they have no competing interests.