A total of 120 male Sprague-Dawley rats of specific pathogen-free level, weighing 250±10 g (age, 6–8 weeks) were provided by Hunan SLAC JD Laboratory Animal Co., Ltd. (Hunan, China). All rats were housed at 24°C, with a fixed light/dark cycle (12 h light/12 h dark) and with ad libitum access to food and water. All of the experimental protocols were in accordance with the principles of Animal Care of Wuhan University (Wuhan, China), and approved by the Committee for the Use of Live Animals in Teaching and Research. Diabetic rats were induced by a single intraperitoneal (i.p.) injection of streptozotocin (60 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as previously described, and rats exhibiting hyperglycemia (blood glucose ≥16.7 mmol/l) were considered to be diabetic (4,5). The body weight, blood glucose and food and water intake of all rats were observed and recorded.

A well-established myocardial IR injury model was used in the present study (4). All rats were anesthetized (sodium pentobarbital; 50 mg/kg i.p.; Sigma-Aldrich; Merck KGaA) with tracheotomy and ventilation. The IR injury model was achieved by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion. IPO was established by 3 cycles of 10 sec of reperfusion and ischemia at the onset of reperfusion. Sham-operated rats were subjected to the same surgical procedures without ligation. Ischemia was confirmed by elevation of the ST segment with limb lead II and discoloration of the ischemic zone.

A total of 8 weeks subsequent to the onset of diabetes, diabetic (D) and age-matched non-diabetic (N) rats were randomly divided into 10 groups (n=12/group) as follows: 1, N+sham (S); 2, N+IR; 3, N+IPO; 4, D+S; 5, D+IR; 6, D+IPO; 7, D+IR+A-769662; 8, D+IPO+A-769662; 9, D+IPO+3-MA+A-769662; and 10, D+IR+3-MA. A-769662 (6 mg/kg; catalogue no. S2697) (21) and 3-MA (catalogue no. S2767; 15 mg/kg) (both from Selleck Chemicals, Houston, TX, USA) (22) were given as i.p. injections 30 min prior to myocardial ischemia.

Invasive hemodynamic monitoring was performed to evaluate cardiac function. Left ventricular systolic pressure (LVSP), maximal rates of increase and decrease in LVSP (± dP/dtmax), and heart rate (HR) were intermittently monitored using an electrophysiolograph (MH150; BioPAC Systems, Inc., Goleta, CA, USA) and the data were analyzed using AcqKnowledge software (version 5.0; BioPAC Systems, Inc.).

Myocardial infarct size was measured using 3% Evans blue dye and 1% 2,3,5-triphenyltetrazolium chloride (both from Sigma-Aldrich; Merck KGaA) staining, and scanning (v30; Seiko Epson Corporation, Nagano, Japan) and image analysis using Image-Pro Plus software (version 3.0, Media Cybernetics, Inc., Rockville, MD, USA), as described previously (4). The risk areas were stained red, while the infarct areas remained pale.

Blood samples were centrifuged (1,200 × g for 10 min at 4°C), and the serum was collected to measure CK-MB using commercial kits (catalogue no. 1327c; Elabscience Biotechnology Co., Ltd., Wuhan, China), according to the manufacturer's protocol.

Myocardial tissue and H9c2 cells were homogenized and centrifuged (2,400 × g for 15 min at 4°C) to obtain the supernatants. The activity of superoxide dismutase (SOD) was detected using a SOD assay kit, which employed the hydroxylamine method (catalogue no. A001-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The expression of malondialdehyde (MDA) was determined using an ELISA assay kit (catalogue no. 0060c; Elabscience Biotechnology Co., Ltd.), according to the manufacturer's protocol.

Observation of the number of autophagosomes under a transmission electron microscope (TEM) is a direct qualitative measure of autophagy (23). Ischemic heart tissue samples of ~1 mm³ were removed and pre-fixed in a solution of 2.5% glutaraldehyde at 4°C for 24 h, and subsequently post-fixed in 1% OsO₄ at 4°C for 30 min, dehydrated in an ascending series of alcohol, and embedded in epoxy resin. The slide was stained by uranyl acetate and lead citrate at 4°C for 0.5–1 h, and observed under a TEM (HT7700; Hitachi, Ltd., Tokyo, Japan).

Rat cardiomyocyte-derived H9c2 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 µg/ml penicillin/streptomycin in an atmosphere containing 5% CO₂ at 37°C. The cells were randomly divided into 10 groups: 1, low glucose (5.5 mM) medium (LG); 2, LG+hypoxia/reoxygenation (HR); 3, LG+hypoxia post-conditioning (HPO); 4, high glucose (30 mM) medium (HG); 5, HG+HR; 6, HG+HPO; 7, HG+HR+A-769662; 8, HG+HPO+A-769662; 9, HG+HPO+A-769662+3-MA; 10, HG+HR+3-MA. A-769662 (100 mM) and 3-MA (10 nM) (24) was given 1 h prior to hypoxia, and the cells underwent 4 h of hypoxia followed by 2 h of reoxygenation. HPO was performed by 3 cycles of 5 min reoxygenation and hypoxia. Hypoxic conditions were obtained using a gas incubator (5% CO₂ and 95% N2). Each experiment was performed ≥3 times independently in triplicate. Cells and supernatants were collected for further analysis.

Cell viability was determined using a Cell Counting Kit-8 (CCK-8) assay kit at a wavelength of 450 nm (catalogue no. 04-11; Dojindo Molecular Technologies, Inc., Kumamoto, Japan), and LDH was measured using a cytotoxicity assay kit a wavelength of 490 nm (catalogue no. 0218c; Elabscience Biotechnology Co., Ltd.), according to the manufacturer's protocols.

Western blotting was performed as described previously (4). Tissues or cells were homogenized with radioimmunoprecipitation assay lysis buffer. Equivalent proteins were separated using SDS-PAGE on a 5–15% gel and electro-transferred onto a polyvinylidene fluoride membrane. The membranes were incubated with anti-GAPDH (catalogue no. 2118), anti-microtubule associated protein 1 light chain 3 β/α (LC3B/A, catalogue no. 12741), anti-nuclear pore glycoprotein p62 (p62, catalogue no. 5114), anti-mTOR (catalogue no. 2983), anti-phosphorylated (p) mTOR (ser2448, catalogue no. 5536) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), AMPKα (catalogue no. sc25792) and p-AMPKα (Thr172, catalogue no. sc101630) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary antibodies (1:500-1,000 dilution) overnight at 4°C, followed by Alexa Fluor secondary antibody (1:10,000 dilution, catalogue no. A-21210; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Signals were detected using an Odyssey fluorescence imaging scanner and quantified using Odyssey software v3.0.29 (both from LI-COR Biosciences, Lincoln, NE, USA).

Rats and H9c2 cell culture dishes were randomly assigned to treated or control groups. Western blot analysis was conducted blindly, with samples separated into numbered groups at random. Data are presented as the mean ± standard deviation. An unpaired Student's t-test was used to detect the differences in characteristics between non-diabetic and diabetic rats. Two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni's post-hoc test was used to analyze the differences in left ventricular function data between the groups. All other data were evaluated using one-way ANOVA followed by Bonferroni's post-hoc test. Analysis was performed using Prism software (version 5.0.7; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

No significant difference was observed in body weight and blood glucose prior to diabetes induction. Following 8 weeks of STZ-induced diabetes, the rats were characterized by a decreased body weight, increased blood glucose, and increased food and water intake compared with the age-matched non-diabetic rats (data not presented).

No significant difference was observed in the area at risk as a percentage of the left ventricle (AAR/LV) in all groups (data not presented). As presented in Fig. 1A, diabetic rats exhibited increased myocardial infarct size compared with non-diabetic rats, following IR insult. IPO significantly decreased the infarct size in non-diabetic rats, and not in diabetic rats. Biochemical markers of myocardial injury and oxidative stress were additionally examined. Diabetic hearts exhibited an increased level of MDA and decreased activity of SOD. Compared with non-diabetes, IR significantly increased CK-MB and MDA level and decreased SOD activity in diabetes. IPO caused a significant reversion in non-diabetes, and not in diabetes (Fig. 1B-D).

In the present study, myocardial autophagy status, and AMPK and mTOR expression and phosphorylation, were observed following IR injury or IPO treatment. As presented in Fig. 2A-D, compared with non-diabetes, a decreased autophagosome number and LC3B/A ratio, in combination with increased p62 expression, were observed in diabetes. IR insult significantly increased the autophagosome number and LC3B/A ratio, and decreased p62 expression, in non-diabetes; these alterations were further increased by IPO. However, IR and IPO did not significantly alter myocardial autophagosome number, LC3B/A ratio and p62 expression in diabetic hearts. No significant difference was detected in the total expression of AMPK and mTOR among all the groups (data not presented). As presented in Fig. 2E and F, in non-diabetes, IR increased AMPK phosphorylation and decreased mTOR phosphorylation, which was further increased by IPO. Compared with non-diabetes, a decrease in phosphorylated AMPK with an increase in phosphorylated mTOR were detected in the diabetic myocardium. IR and IPO were observed to increase AMPK phosphorylation and decrease mTOR phosphorylation.

The present study investigated whether A-769662 is able to restore IPO cardioprotection in diabetes, and whether these effects may be affected by the autophagy inhibitor 3-MA. As presented in Fig. 1, administration of A-769662 alone failed to decrease myocardium infarct size, and CK-MB and MDA level, and to increase SOD activity. By contrast, A-769662 with IPO significantly decreased the infarct size, decreased CK-MB and MDA expression, and elevated SOD activity. All of these effects were reversed by treatment with the autophagy inhibitor 3-MA, although 3-MA alone did not influence the infarct size, CK-MB and MDA release, and SOD activity in diabetic rats following IR insult. Hemodynamic parameters reflecting left ventricular function were analyzed in the present study. Diabetic rats exhibited markedly decreased HR, LVSP, +dP/dt and -dP/dt compared with age-matched non-diabetic animals at baseline (data not presented). As presented in Table I, all of the hemodynamic parameters were decreased in the diabetic and non-diabetic groups following 2 h reperfusion. IPO significantly increased the level of HR, LVSP, +dP/dt and -dP/dt in non-diabetic animals. Treatment with A-769662 alone did not alter the hemodynamic parameters, compared with the untreated group. However, A-769662 treatment with IPO increased the levels of HR, LVSP, +dP/dt and -dP/dt in diabetic rats. Notably, all of the alterations in hemodynamic parameters were reversed by treatment with 3-MA.

In order to investigate the underlying mechanisms, the present study analyzed the effects of A-769662 on myocardial autophagy status and the AMPK/mTOR signaling pathway. As presented in Fig. 2, A-769662 administration or IPO alone did not affect myocardial autophagy status and the AMPK/mTOR signaling pathway in diabetes. However, A-769662 combined with IPO increased the autophagosome number, LC3B/A ratio and AMPK phosphorylation, and decreased p62 expression and mTOR phosphorylation. However, these alterations were reversed by the autophagy inhibitor 3-MA.

In vitro, H9c2 cells were exposed to HG conditions for 48 h to simulate the diabetic myocardium. As presented in Fig. 3, HG insult led to decreased cell viability and SOD activity, and increased LDH and MDA release. These alterations were further increased by HR in the LG and HG groups. HPO significantly increased cell viability and SOD activity, and decreased LDH and MDA release in LG medium cultured cells only. As presented in Fig. 4, a decreased LC3B/A ratio and decreased phosphorylated AMPK expression, with increased p62 expression and phosphorylated mTOR expression, was detected in H9c2 cells exposed to HG, compared with the LG group. Following HR insult, the autophagy level and activity of the AMPK/mTOR signaling pathway were upregulated in the LG group, and were further increased by HPO. In the HG groups, HR and HPO did not significantly affect the autophagy level and the AMPK/mTOR signaling pathway.

In order to confirm whether AMPK activation restores the protective effects of HPO in H9c2 cell lines exposed to HG, cells were pretreated with the AMPK agonist A-769662. As presented in Fig. 3, A-769662 or HPO alone did not confer protective effects to combat HR injury in cells exposed to HG conditions. By contrast, A-769662 with HPO protected H9c2 cells exposed to HG conditions from HR injury, as evidenced by increased CCK-8 and SOD activity, and reduced LDH and MDA release. All of the observed protective effects were reversed by treatment with 3-MA.

As presented in Fig. 4, pretreatment with HPO or A-769662 alone did not affect autophagy status or the AMPK/mTOR signaling pathway. However, HPO with A-769662 significantly activated AMPK/mTOR-regulated autophagy, as evidenced by an increased LC3B/A ratio and increased AMPK phosphorylation, in combination with decreased p62 expression and m-TOR phosphorylation. All of these alterations were reversed by treatment with the autophagy inhibitor 3-MA, which demonstrated that A-769663 and HPO confer their combined protective effects by activating AMPK-regulated autophagy.