Reagents
Streptozocin (STZ), DMBG and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies against ATG5 (Cat. No. 12994, CST, Beverly, MA, USA), BECLIN1 (Cat. No. 3495T, CST), microtubule-associated protein 1 light chain 3 (LC3) (Cat. No. 4108, CST), mammalian target of rapamycin (mTOR) (Cat. No. 2983, CST), phosphorylated (p-) mTOR (Cat. No. 5536, CST), AMPK (Cat. No. 5831, CST), p-AMPK (Cat. No. 2535, CST) and GAPDH (Cat. No. AF1186, Beyotime Biotechnology, Shanghai, China), as well as the secondary antibodies rabbit anti-mouse (A27025, ThermoFisher Scientific, Waltham, MA, USA) and donkey anti-rabbit (SA1-200, ThermoFisher) were purchased.
Experimental animals
Total seventy male Sprague-Dawley (SD) rats with of 12-week-old (160–200 g) acquired from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) were enrolled in this study. The study was approved by the Animal Care and Use Committee of Zhongshan Hospital (Shanghai, China), and all animal protocols were executed following the guidelines accordingly. All rats were housed in a controlled environment of 53% humidity, (23 ± 2˚C) in a 12/12 h dark/light cycle with free access to high calorie diet (60% normal fodder plus 15% animal fat, 20% sucrose and 5% cholesterol) just as previously described [10]. The rats were anaesthetised using 50 mg/kg pentobarbital, and the blood was sampled from caudal vein. The levels of glycated hemoglobin α1 (HbA1c), plasma glucose, lactate dehydrogenase (LDH), creatine phosphokinase (CK), CK isoenzyme MB (CK-MB) and aspartate aminotransferase (AST) were detected biochemical analyser (BK-400, BIOBASE, Jinan, Shandong, China). Echocardiography was conducted as the described below.
For establishment of DM model rats, 65 mg/kg/day STZ dissolved in 0.1 M citrate buffer (pH 4.1) was intraperitoneally injected in the male SD rats for one week prior to surgery. Control animals were administered with an equivalent volume of saline solution. The rat with the level of blood glucose reaching more than 300 mg/dl 10 successive days following STZ treatment was considered as type II diabetes, whereas those with blood glucose levels lower than that were excluded from the experiment. Diabetic and metabolic parameters were measured to ensure that the experimental groups were well-prepared. Rats were sacrificed using cervical dislocation, following with the heart tissues harvest for the next experiment, and the body was transferred to the Animal Department of the school after experiment.
For myocardial IRI model preparation, the male SD rats were anesthetized by 30 mg/kg ketamine and conducted the IRI surgery as described previously [11]. Following 1 h transitory ligation of the left anterior coronary artery, the hearts were subjected to 3 h of reperfusion. The visualization of the colour return in the formerly pale area was used to indicate the IRI and immediately recorded the electrocardiographic variations. The SD rats were administered 30 mg/kg/day DMBG intravenously or 1 IU/kg/day insulin subcutaneously for five days. Five groups of rats (ten rats in each group) were included: (1) negative control (NC); (2) diabetic (DM); (3) diabetic with IRI (DM + IRI); (4) diabetic with IRI and treated with DMBG (DM + IRI + DMBG); (5) diabetic with IRI treated with insulin and DMBG (DM + IRI + I + DMBG).
Echocardiography
Rats were sedated and exposed shaved chests. Rats were placed in a left lateral decubitus position and scanned by a Vivid-i™ ultrasound system (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using a 10S transducer and a cardiac application with 10 MHz, 2.5 cm depth and 225–350 fps of transmission frequency as previously described [12]. The measurements included short-axis cross sections of the apex and papillary muscles levels. The heart rate was calculated from the R-R interval of the electrocardiogram (ECG) signal. The end-systolic and end-diastolic LV areas (ESA and EDA, respectively) were measured from the ultrasound B-scan at the PM level. Enddiastole was defined by the ECG R-wave, and the end-systole defined as the image with the smallest LV area. The fractional area change (FAC) was defined as the ratio of the difference between end-diastolic area and end-systolic area, divided by the end-diastolic area. Left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), interventricular septal depth (IVSD), ejection fraction (EF) and fractional shortening (FS) were calculated as the average values of three cardiac cycles.
Masson’s Trichrome Staining
Myocardial infarct size was measured in order to determine the extent of IRI. 4 µm paraffin slides were conducted Masson’s staining following the manufacturer's instructions (Solarbio, Beijing, China) to assess the myocardial infarct size. The extent of the necrotic area was imaged by an Olympus BX-51 microscope and measured by computerized planimetry (Image J 1.4; National Institutes of Health, Bethesda, MD, USA). Infarct size was expressed as the percentage of the total weight of the area at risk of left ventricle. The silk-like fibres indicated the early phase while the appearance of collagen deposition indicated the late phase of myocardial infarction. The muscle fibres appeared red and the collagen fibres appeared blue.
Western blotting
10 mg tissues were grinded in liquid nitrogen and added the 500 µl RIPA buffer (ThermoFisher) with protease inhibitor cocktail (Beyotime), and quantified the concentration using BCA methods. Aliquots of proteins (40 µg) were added into the lanes of 10% SDS polyacrylamide gel, and the proteins were separated through electrophoresis and transferred onto nitrocellulose membranes. Subsequently, the membranes were congested with 5% nonfat dry milk in 0.01 M PBS buffer (pH 7.4) and 0.05% Tween-20 for 1 h at room temperature (RT). The blocked membranes were then incubated with primary antibodies of LC3 (1:2000), BECLIN1 (1:2000), ATG5 (1:2000), AMPK (1:2000), p-AMPK (1:500), mTOR (1:1000), p-mTOR (1:500) and GAPDH (1:10000) overnight at 4˚C, followed by incubation with the appropriate secondary antibodies (horseradish peroxidase-conjugated rabbit anti-mouse diluted with 1: 10000 and donkey anti-rabbit diluted with 1: 5000) for 30 min at RT. The expression was determined by enhanced chemiluminescence method using Amersham Imager 600 system (GE Healthcare) whereas the density of the immunoblots was measured with Quantity One 4.62 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Transmission electron microscopy
Immunoelectron microscopy was performed according to a previous protocol [13]. In brief, 6 µm sections were crosslinked within the following solution in order: 2.5% glutaraldehyde for 2 h, 2% agarose II for 1 h and 1% osmium tetroxide for 1 h all at RT. Following fixation, dehydration using an ethanol dilution series and infiltration in resin mixture of propylene oxide/Epon812, the sections were stained with lead citrate and uranyl acetate. Autophagosomes were identified by microscopic examination, which was performed using JEOL JEM1230 electron microscope as previously reported [14].
Statistical analysis
GraphPad Prism software 5.1 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analysis. The results of experimental data are expressed as the mean ± standard error of the mean. Student’s t-test and One Way Anova were used for evaluating differences between two and multiple groups, respectively. For significant differences, Student-Newman-Keuls specific post hoc tests were performed for comparisons between treatment groups. P value less than 0.05 was considered to indicate a statistically significant difference.