Enhanced Autophagic Flux Contributes To Cardioprotection Of Remifentanil Postconditioning After Myocardial Ischemia/Reperfusion Injury

Background:Ischemic heart disease,which can cause irreversible myocardial damage and ultimately lead to heart failure,is the leading cause of death worldwide.Timely restoration of blood flow can salvage ischemic myocardium;however,reperfusion can also cause myocardial ischemia/reperfusion(I/R)injury.Remifentanil is a potent synthetic opioid receptor agonist which has some unique characteristics,with a rapid onset,extremely short half-life,rarely cumulative effects and minimally altered by age,and is widely used in clinical anesthesia,especially cardiovascular surgery.Extensive preclinical and clinical evidence shows that remifentanil postconditioning(RPC),which can mimic ischemic postconditioning,can provide powerful cardioprotection against I/R injury.Recently,autophagy has been found to be extensively involved in I/R injury.Furthermore,autophagy plays different roles during ischemia and reperfusion,most believe that moderate autophagy may be protective during ischemia,whereas excessive autophagy and impaired autophagy flux may be detrimental during reperfusion.Therefore,improving autophagic flux during reperfusion becomes a potential therapeutic target to alleviate myocardial I/R injury.Previous studies have confirmed that ischemic postconditioning can activate autophagy and enhance impaired autophagic flux during early reperfusion,thereby exerting a protective effect against I/R injury.Since RPC can mimic ischemic postconditioning,we hypothesized that autophagy may also be involved in the cardioprotective effect of RPC following I/R injury.The purpose of this study was to investigate whether RPC can improve impaired autophagic flux during reperfusion and play a protective role after cardiac I/R injury.Purpose:1.To study the effect of RPC on autophagic flux during reperfusion following I/R injury in rats and to verify whether autophagy involved in the cardioprotective effect of RPC.2.To observe the effect of RPC on autophagy induced by hypoxia/reoxygenation(H/R)injury in H9c2 cardiomyocytes and to further verify whether autophagy involved in the protective effect of RPC by adding autophagy inhibitors and using ATG7 gene knockdown technique.Methods:The experimental research is divided into two parts.Part ?:To investigate whether RPC exerts a protective effect against myocardial I/R injury by activating autophagy and promoting autophagic flux.Healthy adult male SD rats were established in vivo myocardial I/R injury model.The left anterior descending coronary artery was ligated and opened to achieve myocardial ischemia and reperfusion.RPC was achieved by continuous infusion of remifentanil 10 ?g/kg/min from 25 min of ischemia to the first 5 min of reperfusion.In order to investigate whether RPC can activate autophagy and promote autophagic flux at the early stage of reperfusion,the LC3 dot aggregation and the fusion of LC3 with LAMP2 were observed by immunofluorescence technique,the protein expression levels of LC3? and p62 were detected by Western blot at 30 min of reperfusion.To determine whether the protective effect of RPC is related to autophagy,CQ was administrated intraperitoneal 1 h before surgery,immunofluorescence was used to detect the LC3 dots and Western blot was used to detect the expression of LC3?,p62,Beclin 1,LAMP2 and Cleaved caspase 3 at120 min of reperfusion;at the end of reperfusion,the concentration of troponin I(c Tn I)in serum was detected by chemiluminescenc and the myocardial infarct size(IS)was detected by TTC staining.Part ?:To investigate whether RPC can play a role in myocardial protection against H/R injury by activating autophagy and promoting autophagic flux during the reoxygenation stage.H9c2 cardiomyocytes were cultured and H/R injury model was established.Remifentanil was given immediately after reoxygenation to achieve RPC.The LC3 dot aggregation and the fusion of autophagosomes with lysosomes were observed by immunofluorescence at 1 h of reoxygenation.The expression of LC3? was detected by Western blot at different time of reoxygenation.To further verify whether the cardioprotection of RPC is related to autophagy,lysosomal inhibitors Baf and CQ were given at the same time at the onset of reperfusion,the LC3 dot aggregation was observed by immunofluorescence,the protein expression of LC3?,p62,Beclin 1,LAMP2 and Cleaved caspase 3 were detected by Western blot,the cell viability was detected by CCK-8,and the cell apoptosis was detected by HO/PI staining.In addition,cardiomyocytes were infected with ATG7 sh RNA lentiviral vector to knock down the expression of ATG7 gene,and further verified whether the myocardial protection of RPC is related to autophagy.Results:Part ?: Effect of RPC on autophagy induced by myocardial I/R injury in rats1.Compared with the Sham group,the LC3 dots and the protein expression of LC3?and p62 were increased,but the fusion rate of autophagosomes with lysosomes was decreased in the I/R30 min group.In comparison with the I/R30 min group,the LC3 dots and the protein expression of LC3? were increased further,the fusion rate of autophagosomes with lysosomes was increased,but the level of p62 was decreased significantly in the I/R+RF30min group;indicating that RPC can initiate autophagy and promote autophagic flux in the early stage of reperfusion.2.Compared with the I/R group,the LC3 dots and the protein expression of LC3?,p62 and Beclin 1 were decreased,whereas the expression level of LAMP2 was significantly upregulated in the I/R+RF group.Although the LC3 dots,and the expression levels of LC3?,p62,Beclin 1 and LAMP2 in the I/R+CQ group were not different from those in the I/R group.But in comparison with the I/R+RF group,the LC3 dots and the protein expression of LC3?,p62 and Beclin 1 were markedly increased,while the protein level of LAMP2 was obviously decreased in the I/R+RF+CQ group.3.In comparison with the I/R group,the myocardial IS was reduced,the concentration of serum c Tn I was decreased and the expression of Cleaved caspase 3 was down-regulated in the I/R+RF group.The myocardial IS,the concentration of c Tn I and the expression of Cleaved caspase 3 in the I/R+CQ group did not change significantly compared with the I/R group.However,CQ could cancel the cardioprotection of RPC,which was manifested as myocardial IS enlargement,c Tn I concentration increased and the level of Cleaved caspase 3 up-regulated.The above results indicate that autophagy is involved in RPC mediated myocardial protection against I/R injury.Part ?: Effect of RPC on autophagy induced by H/R injury in H9c2 cardiomyocytes1.The LC3 puncta and the expression of LC3? in HR1 h and HR+RF1h cells were significantly higher than that in HR0 h and HR+RF0h cells,respectively.More importantly,the LC3 puncta and the level of LC3? protein in HR+RF1h cells were markedly increased compared with HR1 h cells.In comparison with HR0 h cells,the fusion rate of LC3 with LAMP2 was decreased in HR1 h cells;However the fusion rate was increased in HR+RF1h cells compared with HR+RF0h cells.Furthermore,the fusion rate in HR+RF1h was significantly increased compared with HR1 h cells.2.The expression of LC3? was significantly increase at 1 h,but decrease at 4 h after reoxygenation in RPC cells.Increased LC3? levels did not change over time in HR cells.Additionally,LC3? expression differed between HR and HR+RF cells at 1 h and4 h after reoxygenation.These results show that H/R injury blocked autophagic flux,but that RPC reversed the blockage.3.Compared with the CON group,the number of LC3 dots and the expression levels of LC3?,p62 and Beclin 1 were obviously increased,while the expression level of LAMP2 was markedly downregulated in the HR group.The above indicators in the HR+Baf and HR+CQ group changed similarly to the HR group.In comparison with the HR group,the increased LC3 dots and the up-regulation of LC3?,p62 and Beclin 1were reduced,while the down-regulation of LAMP2 was increased in the HR+RF group.But Baf and CQ can cancel the reversal effect of RPC on the above indicators.4.Cell death was dramatically increased and cell viability was markedly decreased in the HR group compared with the CON group,but HR+RF reduced cell death and increased cell survival significantly following H/R injury.However,Baf and CQ administration during reperfusion eliminated the effects of RPC.Additionally,Western blot analysis revealed that Cleaved caspase 3 levels increased in the HR group,whereas RPC down-regulated the expression of the protein;this process was prevented by Baf and CQ.5.The protective effect of RPC on H/R injury was also attenuated by ATG7 sh RNA,including that cell viability was reduced,while cell apoptosis and the expression of Cleaved caspase 3 were increased in the HR+RF group compared with the HR group.Conclusions:1.RPC could activate autophagy and promote the fusion of autophagosomes with lysosomes during eraly reperfusion;2.RPC could up-regulate the expression of LAMP2 protein and enhance the impaired autophagic flux following I/R injury;3.Autophagy involved in the RPC mediated myocardial protection against I/R injury.