Effects of adenosine monophosphate activated protein kinase regulated autophagy on restoring the sensitivity of diabetic myocardium to ischemic postconditioning

Objective To investigate the effects of adenosine monophosphate activated protein kinase(AMPK) regulated autophagy on restoring the sensitivity of diabetic myocardium to ischemic postconditioning(IPO). Methods Healthy male Sprague-Dawley rats(weighed 220-280 g) were used in this study. Thirty healthy rats were randomly divided into: sham group (N+S group), ischemia reperfusion group (N+IR group) and ischemia postconditionig group(N+IPO group); type 1 diabetic rat models were induced by a single intraperitoneal injection of streptozotocin at the dose of 60 mg/kg and confirmed by blood glucose concentration≥16.7 mmol/L; all rats were raised for 8 weeks. Fifty-four diabetic rats were randomly assigned into diabetic sham group (D + S group), diabetic ischemia group(D + IR group), diabetic ischemia postconditionig group(D+IPO group), diabetic ischemia+AMPK activtor A-769662 group(D+IR+A group), diabetic ischemia postconditionig+A-769662 group(D+IPO+A group). There were 6 rats in N+S and D+S group and 12 rats each in the other groups. Myocardial ischemia reperfusion model was completed by occlusion of the anterior descending branch of left coronary artery for 30 min followed by 120 min of reperfusion. IPO was induced by 3 cycles of 10 second of reperfusion and ischemia at the onset of reperfusion. AMPK agonist A-769662(6 mg/kg) was given as intraperitoneal injection 30 min before ischemia. Myocardium infarct size (IS) was detected by triphenyltetrazolium chloride staining. Transmission electron microscopeserum was used to observe autophagosomes. Arterial blood was collected to detect creatine kinase-MB(CK-MB) and lactate dehydrogenase(LDH) release. The phosphorylation of AMPK (p-AMPK) and mammalian target of rapamycin (p-mTOR), the ratio of microtubule-associated protein 1 light chain 3B/A(LC3B/A) and ubiquitin binding protein p62 in heart tissues were examined by Western blotting. The t test was adopted to detect the differences between two groups, one-way analysis of variance was used to analyze the differences between groups. Results Diabetic rats characterized with lower body weight, p-AMPK expression, autophagosomes number and LC3B/A ratio and higher CK-MB, LDH, p-mTOR, p62 and blood glucose when compared with nondiabetic rats(t=4.15- 34.65, all P 0.05). Compared with D+IR group, the IS(28%±5% vs 45%±6%), CK-MB((1 047±178) vs (2 050±325) U/L), LDH((1 303±365) vs (2 558±189) U/L), p-mTOR(0.48±0.24 vs 1.29±0.25), p62(0.74±0.12 vs 1.04±0.24) decreased significantly, and the p-AMPK level(1.81±0.19 vs 0.61±0.15), autophagosomes number(9.3±1.4 vs 3.8±0.9) and LC3B/A ratio (1.43±0.23 vs 0.65±0.13) increased significantly in D+ IPO+ A group (t=5.26- 11.82, all P<0.05). Conclusion AMPK/mTOR activation by A-769662 can restore the sensitivity of diabetic hearts to IPO-induced cardioprotection through autophagy promotion. Key words: Diabetes mellitus, type 1; Adenosine monophosphate activated protein kinase; Autophagy; Myocardial reperfusion injury; Ischemic postconditioning