Mechanisms Of Cardioprotective Effects Recovery Of Sevoflurane Postconditioning In High Glucose Status By Mitochondrial Morphological And Functional Regulation

Objective: Cardiovascular disease is a serious threat to the perioperative anesthesia and surgical safety.Ischemia reperfusion injury(IRI)is the major cause of perioperative cardiac adverse events.The sevoflurane usage at early reperfusion stage to achieve myocardial protection is known as sevoflurane postconditioning(SPostC).SPostC produces myocardial protection similar to ischemic/hypoxic preconditioning,and has been proved good myocardial protective effect on IRI in young and healthy myocardium,which is becoming a potential strategy for myocardial injury.However,the protective effect of SPostC in diabetes mellitus was impaired and weakened,which extremely limited the application in clinic.Furthermore using insulin for diabetic rats didn’t restore the myocardial protective effect of SPostC.Thus how to restore the protective effect of existing protection methods on the diabetic myocardium or other pathological conditions is an important clinical problem to be solved.This study aims to find the cause of impaired SPost C protective effect on diabetic myocardium by investigating the SPostC protection mechanism on mitochondria and by observing the influence of high glucose on mitochondrial fusion and fission regulated by SPostC.Finally,regulating the balance of mitochondrial fusion and fission was to restore the protective effect of SPost C in high glucose status.Methods: Rats in vivo IRI and Langendorff ischemia/reperfusion models were established according to previous study.In rats IRI model,myocardial infarct size,ATP content and phosphorylation level of JAK2 and STAT3 were measured after 30 min ischemia and 2h reperfusion.Mitochondrial state 3 respiratory rate,respiratory control ratio(RCR),enzyme activities of cytochrome C oxidase(CcO),NADH oxidase(NADHO)and succinate oxidase(SUCO)were measured at the end of reperfusion in isolated Langendorff ischemia/reperfusion model.The mitochondrial ultrastructure of cardiomyocytes was observed by transmission electron microscopy.Primary cultured neonatal rat cardiomyocytes were cultured with low glucose and high glucose conditions for 48 h,and then exposed to hypoxia/reoxygenation injury and SPostC(inhalation of 2.4%sevoflurane at the beginning of reoxygenation).Inhibitor YC-1(final concentration 100μmol/L),2ME2(2μmol/L),cyclosporin A(final concentration 0.2μmol/L)and Mdivi-1(final concentration 50μmol/L or 100μmol/L)were given before hypoxia or ischemia.Cell viability,LDH level,cell death,mitochondrial morphology,mitochondrial membrane potential,mitochondrial permeability transition pore(mPTP)opening level,as well as fission-and fusion-related proteins were measured after H/R injury.Western blotting and immunofluorescence were used to detect the expression levels of key proteins(Drp1,Fis1,Mfn1,Mfn2 and Opa1)and HIF-1α.Results: In the in vivo IRI model,SPostC reduced the infarct size after reperfusion by increasing the JAK2/STAT3 phosphorylation level(P<0.05,SPost C group vs I/R group).In Langendorff perfusion model,SPost C restored mitochondrial respiratory function and respiratory enzyme activity by stabilizing HIF-1α expression(P<0.05,SPost C group vs I/R group).In vitro experiment,cardiomyocyte H/R injury resulted in increased LDH release and cell death which is concomitant with reduced cell viability and mitochondrial interconnectivity(mean area/perimeter ratio)and mitochondrial elongation,and concomitant with reduced mitochondrial membrane potential and increased mPTP opening as well.All these changes were significantly attenuated by SPostC,which achieved protective effect on the cultured cardiomyocytes following H/R by increasing mitochondrial interconnectivity and mitochondrial elongation,inhibiting mPTP opening and maintaining mitochondrial membrane potential thus leading to increased cell viability and decreased LDH level and cell death(P<0.05,SPostC group vs H/R group).HIF-1α inhibitor eliminated this cardioprotective effect(P < 0.05,YC-1 group vs SPostC group).High glucose concentration abrogated SpostC cardioprotection via promotion of mitochondria fragmentation(evidenced by decreased mitochondrial interconnectivity and mitochondrial elongation)and facilitation of mPTP opening.CsA didn’t reduce the LDH level and cell death after hypoxia/reoxygenation injury(P>0.05),and there was no significant difference in the mean area/perimeter ratio of mitochondria and mitochondrial membrane potential(P>0.05).Mdivi-1(100μmol/L)inhibited excessive mitochondrial fission and restored the cardioprotective effect of Spost C in high glucose conditions.The regulation effect of SPostC on mitochondrial morphology,LDH release,cell death and cell viability were restored by Mdivi-1 under high glucose concentration.Conclusion: SPost C protect myocardium by activating JAK2-STAT3 signaling pathway in rats with ischemia/reperfusion injury in vivo.SPostC regulate the expression level of mitochondrial fusion and fission proteins(Opa1,Mfn2 and Drp1),maintain the balance of mitochondrial fusion and fission,thereby increase mitochondrial connectivity(average area/perimeter)and elongation,inhibit mPTP opening and stabilize mitochondrial membrane potential.HIF-1 is the key molecule for SPostC to maintain the mitochondrial fusion and fission balance,and to improve myocardial mitochondrial respiratory function and respiratory enzyme activity.In vitro experiments,high glucose decreased mitochondrial connectivity and elongation,promoted mitochondrial fission,facilitated mPTP opening and decreased the mitochondrial membrane potential,thus eliminated the myocardial protective effect of SPostC,and this effect was related to the Drp1 increasing and Opa1 lacking.Application of cyclosporin A before hypoxia to inhibit the opening of mPTP was not able to restore the protective effect of SPostC.Inhibiting excessive mitochondrial fission under high glucose condition with Drp1 inhibitor(Mdivi-1)restored the SPostC myocardial protective effect.