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 sacrificed 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 Affiliated 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 filtered 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 filtered with a copper filter (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 Scientific Microplate Reader, Varioskan LUX, Finland).
HE staining and immunohistochemistry
Whole mouse hearts were quickly excised, immersed in 10% paraformaldehyde and embedded in paraffin. 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 first 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 flow 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 fixed 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 first-level scanning, the first 20 peptide parent ions with the highest signal strength were selected to enter the high-energy C-trap 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. (Hangzhou, China).
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 influence of contaminating proteins in the identification 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 first 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.
Annotation Methods
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 classified by the GO annotation based on the biological process, cellular component and molecular function.
Subcellular Localization
We used ‘Wolfpsort’ (http://www.genscript.com/psort/wolf_psort.html), a subcellular localization predication program, to predict the subcellular localization of the proteins.
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 identified proteins. A GO analysis with p < 0.05 is considered significant.
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 significant. These pathways were classified according to the hierarchical classification 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 significant.
Transmission electron microscope (TEM)examination
Fresh heart apical portions of the mice at 5 months was quickly cut into 1 mm cubes and fixed in paraformaldehyde (Solarbio, China) for 2 h at 4 °C, and then fixed in 1% osmium tetroxide for 2 h at room temperature, followed by stepwise dehydration in graded acetone, and then it was infiltrated, 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 fluid, 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 Scientific 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.
Western Blot Analysis
Western blotting was carried out according to standard methods, as described previously[28], using Col1a1, Col1a3, GTGF, Vimentin, Fasn, Slc25a1 and anti-α-tubulin antibodies. All primary antibodies (Boster, Wuhan, China) were diluted 1:3000. Goat anti-rabbit (1:20000, Abcam, USA) or goat anti-mouse secondary antibodies conjugated with horseradish peroxidase were used. ECL reagent (Thermo Scientific, USA) was used for chemiluminescence detection.
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 significant. All experiments were performed with at least three biological replicates.