2.1 Chemicals and reagents
The promethazine (Cat#46682), chlorpromazine (Cat#31679) were purchased from Sigma-Aldrich (St Louis, MO, USA). The pethidine hydrochloride was purchased from QingHai Pharm (Lot. No 211103-1, China). The commercial kits for creatine kinase (CK)-MB, cardiac troponin 1 (cTnl) and lactic dehydrogenase (LDH) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The commercial kits for ROS, MDA, ATP, SOD, cell and tissue mitochondria isolation were purchased from Beyotime Biotech (Shanghai, China). The hematoxylin-eosin (HE) solution and triphenyl tetrazolium chloride (TTC) staining were purchased from KangLang Biotech (Shanghai, China). DMEM/F12 culture medium and foetal bovine serum (FBS) were from Gibco (Grand Island, NY, US). A human cardiomyocyte cell line (AC16) was obtained from the American Type Culture Collection (ATCC, USA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimid azolcarbocyanine iodide) kit (Cat#CS-7539) and H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) probe (Cat#CS-1148) were purchased from MedChemExpress (MCE, New Jersey, US). The antibodies of anti-Bcl-2 (Cat#15071), anti-Bax (Cat#5023), anti-cleaved-caspase-3 (Cat#9661), anti-Cyt-C (Cat#4280), anti-β-actin (Cat#3700) and HRP-labeled goat anti-Rabbit/Mouse IgG (H + L) were purchased from Cell Signaling Technology (CA, USA). The anti-SLC25A10 antibody (Cat#12086) and were purchased from Proteintech Group, Inc (Wuhan, Hubei, China).
2.2 MI/RI rat model
Adult Sprague-Dawley (SD) rats (male, 220–250 g) were provided by the Experimental Animal Center of Fudan University. SD rats were housed at 25℃ under 60% relative humidity with a 12 h light/12 h dark cycle. SD rats were acclimatized for one week before the experiment with ample drinking water and food. All procedures were approved by the ethics committee of Department of Experimental Animal Science, Fudan University (2022JS Huashan Hospital-070).
The MI/RI rat model was established by ligating the left anterior descending (LAD) coronary artery for 30 min followed by 24 h of reperfusion. Briefly, SD rats were anesthetized by sodium pentobarbital (50 mg/kg) intraperitoneally. After the completion of tracheal intubation, 6 − 0 silk was used to ligate the LAD for 30 min and then reperfusion was allowed for another 24 h by relieving the LAD ligation. SD rats in sham group were treated with the same surgical procedures without the ligation of LAD.
2.3 Experimental groups
According to previous reports [15], SD rats were randomly divided into three groups (n = 12 per group, 6 rats for TTC staining and 6 rats for other examinations): (1) Sham; (2) I/R; (3) I/R + MTH. Sham, rats receiving vehicle (0.9% NaCl) without being subjected to I/R; I/R, rats receiving vehicle (0.9% NaCl) with being subjected to I/R; I/R + MH, rats receiving the injection of a dose of 3ml/kg mild hypothermia mixture (50 mg promethazine + 50 mg chlorpromazine + 100 mg pethidine hydrochloride dissolved in 100 ml normal saline, IP) after the onset of ischemia. A second injection with one third of the original dose was added at 1 h after reperfusion to enhance the drugs’ effects. SD rats in the Sham group and I/R group were given equal volumes of vehicle at the same time. Rectal temperature of rats in each group was measured once an hour to confirm the cooling effects of hypothermia mixture. After receiving first injection for 2 h, the average temperature of rats in the MTH group kept at 36.5℃, indicating the successful cooling effect of MTH treatment.
2.4 Assessment of cardiac function and myocardial injury
Ventricular parameters of rats including left ventricular internal dimension at end-diastole (LVIDd) and left ventricular internal dimension at systole (LVIDs) were assessed using the Vevo2100 High-Resolution Imaging System (VisualSonics, Canada).
Serum samples were collected for the assessment of myocardial injury markers. CK-MB, LDH and cTnl in serum were determined by commercial assay kits according to instructions.
2.5 Histopathological analysis
For HE staining, rat hearts were fixed in 10% formalin solution. After embedding in paraffin wax, the tissues were sliced to a 4-µm thickness, and then were stained with HE solution.
For Evans blue/TTC staining, following reperfusion, the thread around the LAD was re-ligated, and 0.5 mL of 1% Evans blue solution was reversely injected into the aortic root. The hearts were then extracted, hardened at − 80°C and cut vertically into 2 mm slices along the heart's longitudinal axis, and placed in 1% TTC solution for 30 min at 37°C. The red part represented area at risk, the blue part represented normal non-ischemic myocardial tissue and the white part represented myocardial tissue with ischemic infarct. Therefore, the ischemic area at risk was expressed as percentage of total ventricular weight, whereas infarct size was expressed as percentage of area at risk.For immunohistochemistry, 4-µm thickness slide was deparaffinized in xylene, and then rehydrated in ethanol and deionized water. After heat-mediated antigen retrieval, slides were incubated with anti-SLC25A10 antibody at 4℃ overnight and then incubated with HRP-conjugated secondary antibody. Stained by haematoxylin, the slides were then stained with 3,3′-diaminobenzidine and photographed under a light microscope (Leica Microsystems, USA).
2.6 Cell treatments
To mimic myocardial I/R injury in vitro, AC16 cells were grown in glucose-free medium in an anaerobic chamber (95% N2 and 5% CO2) at 37℃ for 8 h. Then, AC16 cells were returned to normal glucose-containing medium (4.5 mg/mL) and grown in a normal incubator with 95% air and 5% CO2 for another 12 h. Based on previous reports [16], to establish MTH (33.5℃) treatment for H/R cell, incubation temperature was decreased to 33.5℃ after the onset of hypoxia and maintained during simulated reperfusion. Control group and hypoxia/reperfusion (H/R) groups were maintained at 37℃ throughout the duration of the experiment.
2.7 Nano-UHPLC-MS/MS Analysis
Total proteins of harvested hearts were extracted by lysis buffer. In brief, after calculation of concentration and purity of the samples, the samples were then digested with trypsin and the peptides were re-dissolved in solvent A (A: 0.1% formic acid in water), which were analyzed by Orbitrap Fusion LUMOS mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nanoLC 1200 system (Thermo Fisher Scientific, MA, USA)[17]. Raw Data of DIA were processed and analyzed by Spectronaut 14 (Biognosys AG, Switzerland) with dDIA default settings. Retention time prediction type was set to dynamic iRT. The average top 3 filtered peptides which passed the 1% Qvalue cutoff were used to calculate the major group quantities. After one-way ANOVA test, different expressed proteins were selected if p < 0.05 and absolute fold change > 1.5.
2.8 Small interfering RNA transfection
AC16 cells were transfected with siRNA targeting SLC25A10 (GenePharma, Shanghai, China) according to the manufacturer's instructions. At 48 h post-transfection, the cells were subjected to H/R and then used for further examinations.
2.9 MTT assay
The AC16 cells of each group were seeded in a 96-well culture plate at a density of 104/well. After grown for 48 h and treatment, the supernatant of cells was replaced by MTT solution. The OD value at 490 nm was measured by multiscan spectrum. The cell viability rate was calculated as: (OD value of experimental group / OD value of control group) × 100%.
2.10 Detection of Cyt-C secretion
To investigate the secretion of Cyt-C from mitochondria into the cytoplasm, mitochondria was removed by using the Cell Mitochondria Isolation Kit or the Tissue Mitochondria Isolation Kit (Beyotime, Shanghai, China) to obtain cytoplasm proteins in cardiomyocytes or myocardium according to the manufacturer's instructions. Then Cyt-C in cytoplasm was measured by western blot analysis as described below.
2.11 Western blot
The collected myocardial tissues or cardiomyocytes was placed into RIPA lysate containing a protease inhibitor, homogenized, and centrifuged. After collecting the supernatant, the protein was quantified by BCA kit and was used for SDS-PAGE. After the protein was transferred to the PVDF membrane, the membrane was blocked by 5% milk followed by incubation with primary antibody against SLC25A10 at 4℃ overnight. Next, after the membrane was incubated with corresponding HRP-linked secondary antibodies, the blots were developed using an enhanced chemiluminescence system, and Image J was used to analyze the density of blots.
2.12 Measurement of cellular ATP and Oxidative stress
The cellular contents of ATP, ROS, MDA and SOD activity were measured using commercial kits. All procedures were performed according to the manufacturer’s instructions. To visualize the generation of ROS, AC16 cells were stained with H2DCF-DA probe.
2.13 JC-1 staining
Mitochondrial membrane potential (MMP, Δψm) was determined using the JC-1 kit. AC16 cells were seeded on sterile cover glasses in 6-well plates and then incubated with JC-1 staining solution (2 µM) in PBS for 15 min at 37℃. After staining, cells were visualized and photographed by a fluorescence microscopy.
2.14 Immunofluorescence staining
AC16 cells were seeded on glass slides in 12-well plates and incubated as indicated for 48 h. After treatment, the slides were fixed in 4% paraformaldehyde for 30 min at 37℃, and then permeabilized with 0.3% Triton X-100 for 15 min. Cells were then blocked with 3% BSA for 1 h at 37℃ followed by incubation with a primary antibody SLC25A10 at 4℃ overnight. Slices were incubated with a FITC-conjugated IgG for 1 h at 37℃, and then labeled with DAPI for 5 min. Finally, slices were photographed under a fluorescence microscopy.
2.15 Statistical analysis
All the experimental data are expressed as the means ± SD and analyzed by Prism Graphpad 6.0 (San Diego, USA). The t-test was performed to measure the differences between the two groups and one-way analysis of variance (ANOVA) followed by a Dunnett’s test was performed to compare the differences among three or more groups. p-values < 0.05 were considered statistical significance.