Myocardial Ischemic Postconditioning Promotes Autophagy Against Ischemia Reperfusion Injury Via The Activation Of The NNOS/AMPK/mTOR Pathway

Background:Acute myocardial infarction(AMI)is one of the high mortality diseases throughout the world.Timely restoration(reperfusion)of the blood supply to the ischemic myocardium is the standard treatment for these patients.However,reperfusion in the early stage will cause further and more serious damage to myocardial tissue,this is called ischemia reperfusion(I/R)injury.According to research,ischemic preconditioning(IPre)has a significant cardioprotective effect,but due to the unpredictability of AMI,its clinical application is limited.In recent years,ischemic postconditioning(IPostC)has been widely concerned because of its similar myocardial protection as IPre and clinical operability.However,its mechanism is not yet fully understood,it is necessary to further improve the study.It is reported that autophagy is involved in the development of myocardia I/R.Studies have also shown that autophagic activity is regulated by IPostC.Autophagy promoted by reactive oxygen species(ROS)which is produced during reperfusion may also contribute to the reduction of oxidative stress.According to our preliminary laboratory study,neuronal nitric oxide synthase(nNOS)was involved in myocardial protection of IPostC in reducing oxidative stress and calcium overload.NO synthesized by nNOS will affect Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)activity,thereby affecting the mammalian target of rapamycinm(mTOR),which is a key regulator of autophagy.Therefore,we hypothesize that nNOS may be involved in the regulation of autophagy in IPostC,but the relationship between autophagy,postconditioning and nNOS has not been studied before.We hypothesize that IPostC promotes autophagic activity against reperfusion injury during early reperfusion,partially via the activation of nNOS related pathways.Objective:To investigate the role of nNOS in the regulation of autophagy in IPostC and the role of autophagy in the cardioprotection of IPostC.Methods:C57/B6 male mice were randomly selected to establish the model of myocardial I/R injury and IPostC in vivo;The model of hypoxia/reoxygenation(H/R)injury and hypoxic postconditioning(HPostC)model of H9c2 cardiomyocytes were established with hypoxia tank;The expression of LC3-II,LC3-I,p62,p-AMPK(Thr172),p-mTOR,p-nNOS(Ser852)and nNOS protein in tissues and cell samples were detected by Western Blot;The number of autophagosomes and mitochondrial morphology were observed by transmission electron microscopy.The number of autophagosomes in each group of cells transfected with GFP-LC3 adenovirus was observed by confocal microscopy.The area of myocardial infarction was measured by TTC kit;The myocardial tissue was observed by HE staining;The expression of ROS in the cells was measured by ROS kit;The apoptotic rate was measured by Annexin V-FITC/PI apoptosis detection kit.Cell viability was determined by CCK-8 kit;Mitochondrial membrane potential of each group was determined by mitochondrial membrane potential detection kit.Results:1)Compared with I/R,the activity of nNOS in IPostC group was restored;2)IPostC increased the formation of autophagosome,restored the autophagic flux and enhanced autophagic activity.The nNOS inhibitor L-VNIO abolished the effect of IPostC.3)Compared with I/R group,IPostC increased AMPK phosphorylation and decreased mTOR phosphorylation.The nNOS inhibitor L-VNIO abolished the effect of IPostC.4)IPostC reduced the area of myocardial infarction,preserved most of the normal mitochondrial and myocardial structures,autophagy inhibitor 3-MA abolished these effects.5)IPostC reduced the generation of ROS and apoptosis,improved cell viability and mitochondrial membrane potential,whereas nNOS inhibitor L-VNIO inhibited the effect of IPostC.Conclusion:IPostC promotes autophagy against I/R injury partially via the activation of nNOS/AMPK/mTOR pathway during early reperfusion.