A total of 30 adult male Sprague-Dawley rats (250-300 g) were provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. All surgical and experimental procedures were performed according to the guidelines of the Huazhong University of Science and Technology Guide for the Care and Use of Laboratory Animals (TJ-A20150804). The rats were maintained and habituated under controlled conditions (12-h light-dark cycles, 22°C±0.5°C, relative humidity, 40-60%, with free access to food and drinking).

Rats were randomly divided into sham and model groups (n=9 in each group). To induce myocardial IR injury, a previously reported procedure was followed (37,42). rats were anesthetized with pentobarbital sodium (30 mg/kg, intraperitoneal). Before intratracheal intubation, rats were maintained with 2% isoflurane in 100% oxygen in an anesthetic chamber until losing righting reflex. After intubation, continuous 2% isoflurane in 100% oxygen were given and a small animal ventilator (tidal volume at 2.5 ml/100 g and a respiration rate 80/min) was used to control the respiration of the animal during the surgical procedure. The chest was opened via the third intercostal space, then the left anterior descending artery (LAD) was ligated with 6-0 silk suture via a silicon tube. A paleness in the appearance in the ischemic myocardium was one proof of a successful LAD ligation. After 30-min ischemia, the ligation was released and the silicon tube was removed, then the reperfusion for 2 h was initiated. Sham rats were operated as the model group but without LAD ligation. The serum cardiac troponin I (cTnI) concentration was used to detect myocardial injury and values are presented as the mean ± standard error of the mean (SEM; n=4 rats per group, Mann-Whitney U test).

According to Hickman's method (43), rats were decapitated before tissue harvest, which was considered a common physical method for euthanasia. Normally, pulse oxygen saturation, respiration rate or heartbeats were used to verify death, prior to tissue collections. Spinal cord segments between T1 and T4 were obtained from the operated spinal cord area of the IR rats and sham rats. Tissues were washed in PBS, directly frozen in liquid nitrogen and stored at −80°C until further use.

For proteomics experiments, tissues from each rat were homogenized individually with a douncer in RIPA buffer containing various protease inhibitors (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 1 mM Na₃VO₄, with an added protease inhibitor cocktail mixture, COMPLETE; Roche Applied Science). The homogenates were held at 4°C for 30 min and then centrifuged at 12,000 × g for 30 min at 4°C. The supernatants were collected for the following in-solution digestion.

The in-solution digestion was performed as described previously with minor modifications (40). Briefly, proteins were precipitated and re-suspended with 8 M urea/4 mM CaCl₂/0.2 M Tris-HCl, pH 8.0, and then reduced with 10 mM DTT at 37°C for 30 min and alkylated with 40 mM iodoacet-amide in the dark for 30 min. The resultant proteins were digested with trypsin at a ratio of 1:50 (trypsin/protein w/w) at 37°C for overnight and then desalted using a SepPak C18 cartridge (Waters Corporation) and dried with a SpeedVac.

Desalted peptides were re-suspended in 0.1 M sodium acetate, pH 6.0. Next, 4% formaldehyde (CH₂O, which serves as 'light labeled'), 4% deuterated formaldehyde (CD₂O, which serves as 'Heavy labeled') were added to the peptides extracted from IR rats and sham rats, respectively. After mixing, 0.6 M sodium cyanoborohydride (NaBH₃CN) was added and the mixtures were incubated at room temperature (20±2°C) for 1 h. The samples were then quenched by adding 1% ammonium hydroxide, followed by the addition of 5% formic acid. After labeling, the peptides were mixed at a ratio of 1:1 and desalted prior to separation via SCX chromatography as describe previously (44).

All ESI-based LC-MS/MS experiments were performed on a TripleTOF 5600+ System coupled with an Ultra 1D Plus nano-liquid chromatography device (SCIEX) as previous reported (45). Dried peptides were dissolved in 0.1% formic acid, 2% acetonitrile and 98% H₂O. Subsequently, samples were loaded onto a C18 trap column (5 µm; 5x0.3 mm; Agilent Technologies, Inc.) at a flow rate of 5 µl/min and eluted from the trap column over the C18 analytic column (75 µm x150 mm; 3 µm particle size, 100 Å pore size; Eksigent Technologies) at a flow rate of 300 nl/min using a 100 min gradient. The mobile phase consisted of two components: Component A comprised 3% DMSO and 97% H₂O with 0.1% formic acid; acomponent B contained 3% DMSO and 97% acetonitrile with 0.1% formic acid. Data was acquired using a spray voltage of 2.3 kV, curtain gas of 20 psi and an interface heater temperature of 150°C. The information dependent acquisition mode was used to acquire mass spectrometric data. Each scan cycle consisted of one full-scan mass spectrum (with m/z ranging from 350-1,500; ion accumulation time, 250 msec) followed by 40 MS/MS events (m/z ranging from 100-1,500; ion accumulation time, 50 msec). The threshold for MS/MS acquisition activation was set to 120 cps for +2-+5 precursors. Former target ion exclusion was set for 18 sec. The generated raw MS spectra were analyzed with ProteinPilot 4.5 software (SCIEX) using the Paragon algorithm. The uniprot database [Rattus norvegicus (Rat), UP000002494] was used. The false discovery rates of the peptide-spectra matches determined by a decoy database search were set to 1.0% for all experimental datasets. Proteins were considered to be successfully identified when at least two correct assigned peptides (95% confidence) were obtained.

Total RNA from T1-4 segments of the spinal cord tissue was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA samples were quantified using a spectrophotometer (BioPhotometer; Eppendorf) and then synthesized to cDNA via reverse transcription using the PrimeScript™ RT reagent kit (Takara Bio, Inc.). The temperature protocol of reverse transcription was as follows: 15 min at 37°C, 5 sec at 85 and 4°C. cDNA was quantified by quantitative PCR using SYBR-Green Master Mix (Takara Bio, Inc.). The thermocycling conditions for PCR were as follows: 30 sec at 95°C, followed by 40 cycles of 15 sec at 95°C, 15 sec at 60°C and 45 sec at 72°C. Relative gene expression was determined by normalization to GAPDH using comparative (2^-ΔΔCq) method (46). The specific forward and reverse primer sequences (Table I) were designed. The experiments were performed in triplicate. Data are expressed as the mean ± SEM, Mann-Whitney U test compared with the sham group.

Total protein was extracted from T1-4 spinal cord tissues with ice-cold RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein concentrations were determined using the bicinchoninic Protein Assay kit (Wuhan Boster Biological Technology, Ltd.). A total of 30 µg protein samples were separated by SDS-PAGE (5% stacking gel and 10% separating gel) and transferred onto PVDF membranes (EMD Millipore). Then the membrane was blocked in 5% skimmed milk in 0.1% TBST for 1.5 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: SOS1 (1:500; cat. no. A3272; ABclonal Biotech Co., Ltd.), Coro2b (1:1,000; A16670; ABclonal Biotech Co., Ltd.), Gephyrin (Gphn; 1:500; cat. no. A8572; ABclonal Biotech Co., Ltd.), Rptor (1:200; cat. no. 20984-1-AP; ProteinTech Group, Inc.) and Decorin (1:1,000; cat. no. 14667-1-AP; ProteinTech Group, Inc.). After incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (cat. no. A5014; ABclonal Biotech Co., Ltd.) at 1:5,000 concentration for 1.5 h at room temperature. This was followed by protein detection using the ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.). The mean intensities of selected areas and the areas of these images were calculated using ImageJ software (v.1.8.0_112; National Institutes of Health) and normalized to values of GADPH (1:5,000; ProteinTech). Values were expressed as means ± SEM, Mann-Whitney test compared to the sham group.

Each myocardial tissue sample was cut transversely. Hematoxylin and eosin (H&E) staining was used to observe myocardial pathology. After deparaf-finization, 4 µm thick sections were immersed in hematoxylin (cat. no. H9627; Sigma-Aldrich; Merck KGaA) for 5-7 min at room temperature, differentiated in 1% acid alcohol for 2-5 sec and stained with 0.5% eosin (cat. no. 71014544; Sinopharm Chemical Reagent Co., Ltd.) for 2 min at room temperature. After rinsing with distilled water for 30 sec, sections were dehydrated with graded alcohol and cleared in xylene. Infarct size was assessed by TTC staining 2 h after reperfusion. After surgery, the hearts were removed and frozen for 20 min at -20°C, then transversally cut into sections with thickness of ~1 to 2 mm. Tissue sections were incubated in 2% TTC for 10 min in dark conditions at 37°C and then fixed in 10% formaldehyde overnight at 4°C. The infarct area was white, while the normal tissues were red.

Statistical analysis of the MS results derived from all the experimental groups was performed using Microsoft Office Excel. Pearson correlation analyses was performed to assess the correlation among experimental replicates. Biological Networks Gene Ontology (BiNGO) 3.03 (http://apps.cytoscape.org/apps/bingo) was used to calculate the gene ontology term enrichment of differentia proteins. The analyses were conducted using the default BiNGO Rattus norvegicus database. For protein interaction network analysis, the Uniprot functional annotations (http://www.uniprot.org/uniprot) were used to classify the proteins into several clusters. Proteins matched in any cluster of pathways were extracted and submitted to the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 9.0; https://string-db.org/) to qualify the physical and functional interactions. The proteins and their interactions were then uploaded to Cytoscape (www.cytoscape.org/) for data visualization.

The histopathological and functional validation analysis was performed to ensure that the IR rats used for the experiments showed acute myocardial failure. It was observed that 30 min of ischemia resulted in a pale appearance of the ischemia myocardium (Fig. 1A). Serum cardiac troponin cTnI increased significantly (1.211±0.1815 µg/l in sham group, n=4; 47.94±1.368 µg/l, n=4; P<0.001; Fig. 1B). Also, the IR rats showed damaged myocardial cells in the H&E stained heart sections (Fig. 1C). These results verified IR induced myocardial injury in the rats in the study.

To investigate the mechanisms in the spinal cord that respond to myocardial injury, the present study employed a comparative quantitative proteomics approach, based on the stable isotope dimethy labeling strategy, to study the alterations in the spinal cord proteome after acute myocardial IR injury in rats. An experimental flowchart including tissue homogenization, protein extraction, trypsin digestion, chemical labeling and LC-MS/MS processing is shown in Fig. 2. Among the three biological replicates, the present study quantified 4,149 proteins in total, in which 2,362 proteins were shared (Fig. 3A). The gaussian distribution of the shared quantitative data [as log2 (Ratio)] was further analyzed, showing a reasonable ratio distribution (Fig. 3B). The Pearson correlation coefficient analysis showed good repetitiveness among the three biological replicates, as demonstrated by r=0.68, 0.58 and 0.56 (Fig. 3C). All the quantified proteins are summarized in Table SI.

To identify the differentially expressed proteins in the spinal cord that are regulated by myocardial injury, a filtering rule was set as previously reported (41,45), to screen those differentially expressed proteins: Proteins with 50% change of ratio in each replicate, or P<0.05 in each replicate and additionally 50% change of average ratio, were considered to be differentially expressed. Among the whole quantitative dataset, 33 spinal cord proteins were found to be upregulated in IR rats, whereas 57 proteins were downregu-lated (Fig. 3C and D). All the differentially expressed proteins are summarized in Table SII.

To ascertain the functional representation of these differentially expressed proteins, the data were further analyzed based on the gene ontology term enrichment (Fig. S1). The present study found the terms of biological process including axon projection, neurofilaments assembly, microtubule based transport, amino acid metabolism and cell adhesion were enriched among the differentia proteins (Fig. S1A). Molecular function terms including NADPH activity and protein binding and cellular component terms including spiceosomal complex, endocytic vesicle and neurofilament were enriched. These results indicate diverse cellular functions were involved in the spinal cord after myocardial injury.

Considering the possible regulatory network that exists among the differentially expressed proteins, the present study performed protein interaction network based on the STRING database and Uniprot annotations. As shown in Fig. 4, the differentially expressed proteins constitute multiple functional clusters, including collagen fibril organization, cell junction, microtubule dynamics, amino acid/lipid/purine metabolism, stress-activated protein kinase signaling, endosomal sorting, transcription, protein biosynthesis and apoptosis. Several co-regulations possibly exist among these clusters, as the present study found proteins of stress-activated protein kinase signaling and endosomal sorting showed a consistent down-regulatory tendency in IR rats. While upregulatory proteins were mainly found in clusters of cell junction, microtubule dynamics, transcription and protein biosynthesis. These results indicate a complicated response to myocardial injury in the spinal cord.

To cross-check the reliability of quantitative proteomics, several protein expression levels were further assessed using conventional western blotting and quantitative PCR. A total of five proteins involved in stress-activated protein kinase signaling (Sos1 and Rptor), cell junction (Gphn), collagen fibril organization [Dcn (Decorin)] and microtubule dynamics [Coro2b (Corin2b)], in the regulated protein network, were selected for western blot analysis (Fig. 5). The relative gray value compared with GAPDH of Sos1, Rptor, Gphn, Dcn and Corin2b were 0.6136±0.06747 (n=6), 0.8721±0.1504 (n=4), 1.622±0.2363 (n=5), 1.006±0.1513 (n=5) and 1.245±0.03118 (n=5), respectively. The authors found most of the analyzed proteins were consistent with the MS results except the protein Dcn, which almost did not change in the western blotting results.

Moreover, these five genes and another four genes (Wash2c, Fry, Aspn and Lamc1) were further analyzed by quantitative PCR. As shown in Fig. 6, the results showed that most of the quantitative ratios were consistent with the MS results. These results provide alternative evidences supporting the MS findings.