Materials and methods
Animals
Male Sprague-Dawley rats (250±10 g body weight, aged 8 weeks) were supplied by the Laboratory Animal Service Center of Wuhan University and had free access to food and water before the start of the experiments. All rats were maintained under specific pathogen-free conditions and were housed in a laboratory room with a 12-hour dark and 12-hour light cycle, a room temperature of 22°C–24°C, and a humidity of 50%–60%. Water intake and food consumption were recorded daily, while blood glucose and body weight were monitored weekly.
Diabetic model of rats
As previously described,13 diabetes was induced by a single tail vein injection of streptozotocin (Sigma-Aldrich, S0130) at the dose of 60 mg/kg body weight in 0.1 M citrate buffer (pH 4.5) or citrate buffer alone as the control. After 3 days injection, blood glucose was measured using a glucose meter (LifeScan) and rats with fasting blood glucose levels over 16.7 mM at least three times were considered diabetes.
Myocardial IPostC model in vivo and SSO treatment
Eight weeks after the onset of diabetes, a well-established model of myocardial IPostC in vivo was used.13 Rats, after induction by intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg), were intubated with an endotracheal tube and connected to a rodent ventilator with a tidal volume of 1.0 mL/100 mg body weight and 70 breaths/min. Anesthesia was maintained with inhalational sevoflurane at a concentration from 2.0% to 3.0% until the end of the experiments. During the experiment, all rats were kept warm by a lamp and the vital signs were measured. Cardiac ischemia was achieved by occluding the left anterior descending (LAD) coronary artery by 6–0 silk suture using a snare occluder for 0.5 hours. Ischemia was verified by myocardial discoloration of the ischemic zone and elevation of ST-segment in limb lead II. IPostC was performed by three cycles of 10 s of reperfusion and 10 s of ischemia after myocardial ischemic, then followed by reperfusion for 2 hours. SSO was obtained from Santa Cruz Biotechnology (sc-208408) and freshly dissolved in pure dimethyl sulfoxide (DMSO) to obtain a concentration of 4 mM, then freshly diluted with normal saline to prepare a 0.5 mM for working solution. SSO was applied intravenously at a rate of 0.2 mL/hour from 4 min before ischemia to the end of reperfusion in diabetic rats based on the previous study11 and our preliminary experiments. Plasma was extracted from blood samples and stored at −80°C until assay. The ventricular tissue was removed and immediately frozen in liquid nitrogen and stored at −80°C for further analysis.
Echocardiographic assessment of cardiac function
Transthoracic echocardiography was performed non-invasively at experiment termination with Vevo 770 high-resolution imaging system equipped with a 17.0 MHz transducer (RMV-716, Visual Sonics), and left ventricular (LV) dimensions and LV diastolic and systolic functions were assessed by M-mode and Doppler echocardiography. The ratio of the peak velocity of early (E) and late (A) diastolic filling (E/A), left ventricular internal dimensions at end-systole and diastole (LVIDs and LVIDd), LV end-diastolic volume and end-systolic volume (LVVd and LVVs) were monitored. Ejection fraction (%)=(LVVd–LVVs)/LVVd×100%, fractional shorting (%)=(LVIDd–LVIDs)/LVIDd×100%. All derived measures by echocardiography were obtained by averaging the readings of three consecutive beats.
Hemodynamic monitoring
The hemodynamic monitoring of diabetic animals was performed as we described.13 Briefly, a saline-filled catheter was inserted into the left ventricle via an incision on the right common carotid artery and then connected to a pressure transducer (SCW Medicath, DPT-248). The left ventricular systolic pressure, left ventricular maximum rate of increase of left ventricular developed pressure (dp/dtmax), maximum rate of decrease of left ventricular developed pressure (dp/dtmin), and heart rate were monitored (Bene View T5, Xi’an Jutian Medical Equipment, China) at 10 min before ischemia (baseline) and 2 hours after reperfusion.
Determination of myocardial infarct size
After the reperfusion was finished, rats were sacrificed by an overdose of anesthetic sevoflurane. Then, the LAD was reoccluded and cannulated just distal to the occlusion site. 10 mL of saline and 10 mL of 0.3% Evans blue dye were injected with equal pressure into the aorta root, respectively. Then, hearts were immediately fibrillated, removed, weighed, and frozen at −20°C for 20 min, underwent horizontal long axis slicing at a thickness of 1–2 mm, incubated at 37°C for 20–30 min in 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining (Sigma, USA) in 0.1 mol/L phosphate buffer adjusted to pH 7.4 and scanned with a digital camera. ImageJ V.1.47 (National Institutes of Health) was used to measure and analyze the infarct size. The unstained region by Evans blue dye was considered as the area at risk (AAR). The unstained region of TTC was identified as the area of infarction. Infarct size was expressed as a percentage of the AAR.
Measurement of fatty acid β oxidation rate
The fatty acid β oxidation (FAO) rate of cardiac tissue was measured using the Fatty Acid β Oxidation Rate Assay kit (HPBIO-JM10629) from HePeng (Shanghai) Biotech (Shanghai, China) according to the manufacturer’s specifications. Briefly, cardiac tissues were homogenized in a lysis buffer (100 mg tissues/1 mL) at 0°C. After incubated at 4°C for 10 min, the homogenate was centrifuged at 1000 g for 5 min at 4°C to obtain the supernatant which was centrifuged at 12 000 g for 10 min at 4°C to obtain the pellet. The pellet was resuspended with 0.5 mL wash buffer. Then, centrifugation process was repeated to obtain the pure mitochondrial fraction. After incubating the reaction mixture at 25°C for 3 min, 50 μL of the mitochondrial lysis suspension was added and immediately put into the spectrophotometer for detection. The absorbance at 420 nm and 470 nm was measured during 5 min to value the rate of FAO.
Measurement of biochemical parameters
Add 1 mL RIPA lysis buffer (Beyotime Biotechnology, P0013B) per 100 mg of heart tissue, homogenized on ice and centrifuged at 12 000 g for 20 min at 4°C. Transfer the supernatant to a clean tube as the total protein sample. Then reserve an aliquot of this supernatant for a protein assay by the BCA protein assay kit (Beyotime Biotechnology, P0012S). Plasma samples were collected via vacutainers containing sodium citrate and EDTA.
The cTn-I ELISA kit (E-EL-R1253c) was obtained from Biotech. The lactate dehydrogenase (LDH) assay kit (A020-2), creatine kinase MB isoenzyme (CK-MB) assay kit (E006-1-1), non-esterified free FA assay kit (A042-2-1), triglycerides (TG) assay kit (F001-1-1), total glutathione/oxidized glutathione (GSH/GSSG) assay kit (A061-1-2), and glutathione peroxidase (GSH-Px) assay kit (A005-1-2) were obtained from Nanjing Jiancheng Bioengineering Institute. The superoxide dismutase (SOD) assay kit (S0101S), glutathione (GSH) assay kit (S0053), and lipid peroxidation malondialdehyde (MDA) assay kit (S0131S) were obtained from Beyotime Biotechnology. The 8-isoprostane ELISA kit (516351) was obtained from Cayman Chemical. The pyruvate dehydrogenase kinase 4 (PDK4) ELISA kit (SEA958Hu) and peroxisome proliferator activated receptor alpha (PPARα) ELISA kit (SEA934Mu) were obtained from Cloud-Clone. All experiments were performed according to the manufacturer’s instructions, respectively.
Western blot analysis
Prestained protein marker (SMOBIO, PM2610) and total protein samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel, and then the proteins were transferred to polyvinylidene fluoride membrane overnight at 4°C. Membranes were blocked with 5% non-fat milk in Tris-buffered saline-Tween for 2 hours and were incubated with primary antibodies against CD36 (53KD, Sigma-Aldrich, SAB2100376, 1:500) and GAPDH (36KD, Abcam, ab8245, 1:1000) overnight at 4°C. After washing with phosphate-buffered saline Tween three times for 30 min, the membranes were then incubated with antirabbit fluorescent secondary antibody (1:15 000, CST, USA) for 2 hours at room temperature before scan in Odyssey imaging system (LI-COR, Nebraska, USA). The protein expression level was calculated from the optical density of the bands and normalized to GAPDH.