Sprague Dawley rats were purchased from Beijing HFK Bioscience Co, Ltd (Beijing, China). TTC was purchased from Sigma. LDH Release Assay Kit, CK-MB ELISA Assay kit, cTnI ELISA Assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ROS Fluorescent Probe-DHE and JC-1 Mitochondrial Membrane Potential Detection Kit was purchased from Beyotime Biotechnology Institute (Wuhan, China), Terminal-deoxynucleoitidy transferase mediated nick end labeling (TUNEL) kit was purchased from Roche (Switzerland). Caspase-3 colorimetric assay kit was purchased from Applygen Technologies Inc (Beijing, China). Rabbit anti-rat cytochrome c antibody, Akt and p-Akt antibody, GSK3β and a p-GSK3β antibody, HRP-conjugated goat anti-rabbit secondary antibodies were purchased from Cell Signaling Technology. RNA reverse transcription kit was purchased from Thermo. Bradford Protein Assay kit was purchased from Bio-Rad. Dulbecco's Modified Eagle Medium (DMEM) was purchased from Solarbio Technology Co Ltd (Beijing, China). MedLab-U/4C501H Biological Signal Acquisition and Processing System were purchased from Medease Science and Technology Co, Ltd (Nanjing, China). Small animal ventilator was purchased from Friends Honesty Life Sciences Company Limited (Hong Kong).
Establishment of rat myocardial I/R model
Adult male SD rats were fasted overnight, and then anesthetized via intraperitoneal (IP) administration of 0.3 g/kg chloral hydrate. A poly ethylene 50 (PE50) catheter was inserted into the left ventricle through the right carotid artery to measure the left ventricular pressure. Then a left thoracic incision was made to explore the heart, and myocardial ischemia model was produced by ligature the left anterior descending coronary artery (LAD) approximately 2–3 mm from its origin using a 6 − 0 silk slipknot. The change of ST segment on electrocardiogram was used to prove and monitor myocardial ischemia and reperfusion. After 30 minutes ischemia, the slipknot was released to recover the bloodstream. After 2 hours’ reperfusion, the rats were executed for next experiments. MedLab-U/4C501H Biological Signal Acquisition and Processing System was used to record the biological signals, such as blood pressure, heart rate, left ventricular systolic pressure, left ventricular end-diastolic pressure, ±LVdP/dtmax, and so on.
Rats were randomly divided into four groups with the following treatments (n = 8): 1) Sham group, received vehicle IP injection and operative procedures without coronary ligature; 2) I/R group, received vehicle IP injection 30 minutes prior to coronary I/R; 3) I/R + β2GPI group, received 1 mg/kg body weight β2GPI IP injection;and 4) I/R + R-β2GPI group, received 1 mg/kg body weight R-β2GPI IP injection.
TTC staining and HE staining
After 2 hours reperfusion, rats were executed and the hearts were taken off rapidly. The atriums and right ventricles were removed, then the left ventricles were frozen at -20℃ for about 20 minutes and cut into 1 mm thick slices perpendicular to the base-apex. Slices were incubated in 1% TTC dissolved in phosphate buffer at 37℃ for 10 minutes (pH 7.4). Pictures were taken and measurements of infarct area were performed by image analysis software Image-Pro Plus. Myocardial infarct size was expressed as percentage of infarct area over total area. On the other hand, some cardiac tissues were made into paraffin-embedded sections and stained with Haematoxylin-Eosin (HE).
Determination of CK-MB and cTnI using ELISA
Creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) levels in plasma were used to evaluate the degree of myocardial cellular damage and necrosis. After 2 hours reperfusion, 1 ml blood samples were drawn from aorta and centrifuged at 3000 rpm for 15 min. Then the upper serum was collected into another tube and stored at -20℃. Enzyme Linked Immunosorbent assay (ELISA) was used to detect the serum CK-MB and cTnI levels in rats. The detailed experimental procedure was performed per the manufacturer’s instruction of CK-MB ELISA Assay kit and cTnI ELISA Assay kit.
Determination of myocardial apoptosis
Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method was used to determine myocardial apoptosis. After 2 hours reperfusion, the left ventricles were made into paraffin-embedded sections. Then the sections were stained per the manufacturer’s instruction of TUNEL assay kit and analyzed by optical microscope. TUNEL-positive cardiomyocytes in ischemic myocardium were counted in double-blinded method. The percentage of TUNEL-positive cells was determined by dividing the number of positive-staining nuclei by the total number of nuclei in a given field of view (at 200 microscopic magnifications).
Caspase-3 activity in cardiac tissue was also used to determine myocardial apoptosis. The cardiac tissue from left ventricles was made into homogenate and caspase-3 activity was detected by caspase-3 colorimetric assay kit. In brief, myocardial tissue homogenate was centrifuged at 4℃ and supernatants were collected into new tubes. Protein concentration was measured by Bradford method. To each well of a 96-well plate, supernatant containing 200 mg of protein was loaded and incubated with 25 mg caspase-3 substrate N-acetyl-Asp-Glu-Val-Asp p-nitroanilide at 37℃ for 2 hours. The optical density was measured at 405 nm with a microplate spectrophotometer. Caspase-3 activity was calculated using a standard curve and expressed as fold increase over the mean value of sham group.
Quantitative real-time PCR
Total cellular RNA was isolated using Trizol Regent according to the manufacturer’s instructions. The A260/A280 ratios were around 1.8-2.0. Total RNA was reverse transcribed into cDNA using a Reverse Transcription Kit. The primer sequences for bcl-2 were: forward:5'-GCCTTCTTTGAGTTCGGTGG-3', reverse:5'-CAGGAGAAATCAAACAGAGG-3';bax:forward:5'-TTTGGAGTGGTAGTAAAAAGGGC-3',reverse:5'-TGACATCAGGGACTCAGAGTAG-3';β-actin:forward:5'-TGGAGAAGAGCTATGAGCTGCCTG-3',reverse:5'-GTGCCACCAGACAGCACTGTGTTG-3'. All sample measurements were performed in triplicate. For each experimental sample, mRNA levels were normalized to β-actin mRNA levels.
H9c2 cell Hypoxia/Reoxygenation (H/R) injury model
H9c2 cells were cultured in DMEM with 4500 mg/L glucose supplemented with 10% (v/v) fetal bovine serum at 37℃ in a humidified atmosphere with 5% CO2. Cells were routinely subcultured when grown to subconfluency (> 90% by visual estimate). Cells H/R injury model was established as previously described . In brief, H9c2 cells were harvested and seeded onto 6-well plates, placed into a hypoxic tri-gas incubator (5% CO2/0%O2/95% N2) at 37℃ for 21 hours, and then re-oxygenated in a standard incubator for 6 hours with new medium.
Cells were randomly divided into four groups with the following treatments (n = 8) : 1) Control group; 2) H/R group,; 3) H/R + β2GPI group,; 4) H/R + R-β2GPI group. Cells incubated with β2GPI or R-β2GPI (100ug/ml) in hypoxic tri-gas incubator for 21 hours, and then reoxygenated in a standard incubator for 6hours with new medium.
Determination of intracellular reactive oxygen species (ROS)
Intracellular ROS generation was measured by fluorescent probe DCFH-DA. Cell-permeable non-fluorescent DCFH-DA oxidizes to the highly fluorescent 2,7-dichlorofluorescin in ROS presence. H9c2 cells were plated upon a 6-well plate, and incubated with medium containing β2GPI or R-β2GPI (100ug/ml) in hypoxic tri-gas incubator for 21 hours, and then re-oxygenated in a standard incubator for 6 hours with new medium. Then cells were harvested and washed by PBS. 10 mM DCFH-DA was added for 20 minutes at 37℃ in the dark. Fluorescence intensity was measured by flow cytometry at excitation wavelength 488 nm, and emission wavelength 525 nm.
Measurement of mitochondrial membrane potential
Mitochondrial transmembrane potential was assessed using a sensitive fluorescent dye, a lipophilic cationic probe JC-1. H9c2 cells were collected, incubated with 5 mM JC-1 dye (Molecular Probes) at 37 °C for 15 min, and washed with PBS for three times and analyzed immediately with Flow Cytometry. Some cells were grown on cover slips, stained with JC-1 dye and then analyzed with fluorescent microscope. Red emission indicates membrane potential-dependent JC-1 aggregates in mitochondria. Green fluorescence reflects the monomeric form of JC-1 appearing in cytoplasm after mitochondrial membrane depolarization. The green/red fluorescence intensity rate was used to evaluate the change of mitochondrial membrane potential.
Western blot analysis
Whole cell extracts were prepared as follows: Cultured H9c2 cells were washed twice with cold PBS and immersed in lysis buffer (composition: 50 mM HEPES, pH 7.4, 0.1% Chaps, 5 mM DTT, 0.1 mM EDTA, and 0.1% Triton X-100). Cell lysates were centrifuged. Protein concentrations in the supernatants were determined by BCA method. Equal samples were loaded onto and separated by 12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes by electrophoretic transfer system (Bio-Rad). Membranes were blocked in 5% skim milk for 2 hours at room temperature and incubated with primary antibody (rabbit anti-Akt, rabbit anti-GSK-3β) overnight at 4℃, followed by secondary antibody conjugated to horseradish peroxidase for 2hours. Immunoblot was visualized with DAB method and analyzed with Image-Pro Plus software.
All data are expressed as means ± SEM unless indicated otherwise. Differences among groups were determined by ANOVA. Differences between groups were determined by Student’s t-test with P < 0.05 considered statistically significant.