Mechanism Of Autophagy Regulated By DJ-1 In Restoring The Sensitivity Of Diabetic Myocardium To Ischeamic Post-conditioning In Rats

BackgroundIschaemic heart disease(IHD)is a major cardiovascular complication and remains a leading cause of death in.patients with diabetes.Clinical studies demonstrated that diabetic patients are more vulnerable to myocardial ischaemia-reperfusion injury(IRI)than non-diabetic patients,their risk of post-myocardial infarction death is higher and their prognosis is poorer than that of non-diabetic patients.Myocardial ischaemic post-conditioning(IPO)is regarded as a promising approach to cardioprotection;however,IPO-induced cardioprotection seems to be weakened or absent in subjects with diabetes,the mechanisms underlying this phenomenon are largely unknown.The DJ-1 was initially discovered as a novel oncogene,recent evidence suggests that it plays pivotal roles in anti-oxidative,anti-apoptotic processes and regulates autophagy.Autophagy is indispensable for cell homeostasis under normal conditions and facilitates cytoprotective responses to adverse conditions,it participates in the pathogenesis of myocardial IRI;moreover,one of the mechanism of IPO-induced cardioprotection is autophagy motivation.Previous studies demonstrated that DJ-1 expression,as well as autophagy activity is markedly down-regulated in diabetic myocardium,which may compromise or abolish the effectiveness of IPO-induced cardioprotection in diabetes;however,the exact molecular mechanisms underlying have not yet been elucidated.ObjectiveIn the current study,we hypothesized that DJ-1 inhibition,which results in autophagy impairment,is responsible for the absence of IPO-induced cardioprotection in myocardial tissue of diabetic rats.We aimed to determine whether DJ-1 overexpression could restore IPO-induced cardioprotection in diabetic rats and to explore the roles of autophagy and AMPK/mTOR signalling in this process.MethodsIn vivo study:SPF-grade health male Sprague-Dawley(SD)rats were employed in this study.Type 1 diabetes model was induced via a single intraperitoneal injection of 60 mg/kg streptozocin(STZ),rats exhibiting hyperglycaemia(blood glucose level>16.7 mmol/L)were considered diabetic.After 8 weeks of diabetes,both diabetic(DM)and age-matched non-diabetic(N)rats were randomly assigned to the following 6 groups(n=12 in eachgroup):① N+ sham(S),② N+IR,③ N+IPO,④ DM+S,⑤DM + IR and⑥ DM + IPO.Furthermore,to evaluate the effects of autophagy induction or cardiac DJ-1 overexpression on IPO-induced cardioprotection,a second set of experiments was performed on groups treated as follows:① CDM + S +rapamycin(R),② DM + IR + R,⑧ DM + IPO + R,④ DM + IR + DJ-1 overexpression(AAV9-CMV-DJ-1)and ⑤ DM + IPO+AAV9-CMV-DJ-1.The IR injury model was achieved by occluding the LAD(left anterior descending artery)for 30 min,followed by 2 h of reperfusion.IPO was completed after 30mim of ischaemia,it comprised 3 cycles of 10s of reperfusion and 10s of ischaemia,and then followed by 2h of reperfusion.Sham-operated rats underwent the same surgical procedures without LAD occlusion.Cardiac function was continuously monitored during operation.Myocardium ischaemia post-infarct size was measured by Evans Blue and TTC staining.Autophagosomes were observed by transmission electron microscopy.Serum creatine kinase isoenzyme-MB(CK-MB)and myocardium malonaldehyde(MDA)levels were detected by assay kits.The expression of cardiac DJ-1,adenosine monophosphate activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),protein kinase B Akt),phosphorylated Akt(p-Akt),p70 Ribosome S6 protein kinase(p70S6K),phosphorylated p70S6K(p-70S6K),microtubule-associated proteinl light chain 3 Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ),p62/SQSTM1 and autophagy effecter Beclin-lwere assayed by Western Blotting.In vitro study:Rat cardiomyocyte-derived H9c2 cells were maintained in Dulbecco’s modified Eagle medium containing 10%FBS and 100 μg/mL penicillin/streptomycin under an atmosphere of 10%C02 and 90%air at 37℃.The cells were randomly divided into the following 8 groups:① low-glucose(LG,5.5 mM)medium,② LG+ hypoxia-re-oxygenation(HR),③LG+ hypoxic post-conditioning(HPO),④ high-glucose(HG,30 mM)medium,⑤ HG+HR,⑥ HG+HPO,⑦HG+HR+R,and ⑧ HG+ HPO + R.To determine whether DJ-1 overexpression preserves HPO-induced protection and to examine the role of AMPK-regulated autophagy in this process,experiments were performed under the following conditions:① HG + HR +p-EX-2-EGFP-DJ-1(D),② HG+HPO+D,③HG+HR+DJ-1siRNA,④ HG+HPO+DJ-1siRNA,⑤ HG+HPO+D+ compound C(CC)and ⑥ HG+HPO +D + 3-MA.HR was established by 4 h of hypoxia(10%C02 and 90%N2)followed by 2 h of re-oxygenation(10%C02 and 90%air).HPO was achieved after 4 h of hypoxia,which comprised 3 cycles of 5min of re-oxygenation and hypoxia,then followed 2 h re-oxygenation.Cell viability was detected by CCK-8 assay kit.Lactic dehydrogenase(LDH)and MDA levels were measured by assay kits.Monodansylcadaverin(MDC)staining was used to detect autophagic vacuole.Fluorescent cationic JC-1 dye was used to detect mitochondrial membrane potential.Cell apoptotic rate was assayed using flow cytometry.Western Blotting was used to exam the expression of DJ-1,AMPK,p-AMPK,m-TOR,p-mTOR,Akt,p-Akt,p70s6k,p-p70s6k,LC3Ⅱ/Ⅰ,p62 and Beclin-1.ResultsIn vivo study:1.Compared with non-diabetic myocardium,less cardiac autophagy(decrease in autophagosome number,LC3 II/I ratio and Beclin-1 expression,increase in p62 expression),lower AMPK(adenosine monophosphate-activated protein kinase)/mTOR(mammalian target of rapamycin)pathway activity(decrease in p-AMPK and p-Akt,increase in p-mTOR and p-70s6k)and DJ-1 expression were observed in diabetic myocardium2.Diabetic rats subjected to myocardial IR exhibited more severe myocardial injury(increase in myocardium infarct size,LDH releases and MDA levels),less cardiac autophagy,lower DJ-1 expression and AMPK/mTOR pathway activity than non-diabetic rats.IPO significantly attenuated myocardial injury and up-regulated cardiac DJ-1 expression,AMPK/mTOR activity and autophagy in non-diabetic rats but not in diabetic rats.3.Autophagy inducer rapamycin restored IPO-induced cardioprotecction,as evidenced by the smaller myocardial post-ischaemia infarct size,the lower levels of CK-MB and MDA releases,as well as the improvement in cardiac function.Those protective effects were accompanied by the enhancement in cardiac autophagy.There was no significantly alterations in DJ-1 expression and AMPK/mTOR pathway activity in this process.4.Adeno-associated virus 9(AAV9)-mediated cardiac DJ-1 overexpression restored IPO-induced cardioprotection in diabetic rats,an effect accompanied by AMPK/mTOR activation and autophagy up-regulation.In vitro study:1.Compared with LG group,less autophagy,lower activity of AMPK/mTOR signaling and higher DJ-1 expression were detected in H9c2 cells with HG exposure;After HR insult,lower cell viability,higher levels of LDH and MDA releases were observed in H9c2 exposed to HG condition,accompanied by less DJ-1 expression,AMPK/mTOR activity and autophagy.HPO markedly increased cell viability,decreased LDH and MDA releases,up-regulated DJ-1 expression,AMPK/mTOR activity and autophagy level in H9c2 cells cultured in HG conditions,but not in HG conditions.2.Rapamycin restored the protective effects of HPO in H9c2 cells exposed to HG conditions,which accompanied by significantly autophagy enhancement.There was no significantly changes in DJ-1 expression and AMPK/mTOR pathway activity in the process.3.Combining HPO with DJ-1 overexpression markedly attenuated HR injury in H9c2 cells with HG exposure,accompanied by AMPK/mTOR signalling activation and autophagy up-regulation.The DJ-1 overexpression-mediated preservation of HPO-induced cardioprotection was completely inhibited by the AMPK inhibitor compound C and the autophagy inhibitor 3-MA.ConclusionIn vivo study:1.Diabetic myocardium exhibited decreased DJ-1 expression and autophagy activity,which may major mechanisms underlying the loss of IPO-induced cardioprotection in diabetes.2.Autophagy activation could restore IPO-induced cardioprotection in diabetic hearts.3.Overexpression of DJ-1 could restore IPO cardioprotection possibly via motivating AMPK/mTOR signaling pathway,ultimately activating cardiac autophagy in diabetic IR hearts.In vitro study:1.Down-regulation of autophagy may confer to the impairment of HPO protective effects in H9c2 cells with HG exposure.Autophagy activation restores HPO-induced protection.2.DJ-1 overexpression restores HPO-induced protective effects through activating AMPK/mTOR cascades and its downstream autophagy inH9c2 cells exposed to HG condition.