NNOS Is Involved In Cardioprotection Of Ischemic Postconditioning Through Improving Intracellular Ca²⁺ Cycling

Myocardial ischemia reperfusion?I/R?injury is a common pathophysiological process in many clinical scenarios in patients with some heart diseases and therapies.Many experiments indicated that myocardial ischemia reperfusion could induce cardiomyocyte death and cardiac dysfunction,resulting in cardiac arrhythmias,heart failure and death.However,there is currently no effective therapy to reduce I/R injury.Previous evidence suggested that ischemic postconditioning?IPostC?had cardiac protection by alleviating oxidative stress,inhibiting cardiomyocyte death,reducing infarct size and preserving endothelial function.But IPostC is limited in clinic because of its unclear mechanisms.The researchers indicate the one of protective mechanisms of IPostC is to recruit the reperfusion injury salvage kinases?RISK?pathways at reperfusion.As distal signaling elements of the RISK pathway,NO and its synthetases have already been research focuses in cardioprotection.Nitric oxide synthases?NOS?have three isoforms and express in distinct subcellular locations in cardiac myocytes.The differences in subcellular localization,differential regulation,and activity rate of the different NOS isoforms in the same cell explain at least some of the often opposing roles within one single cell.The subcellular localization of neuronal nitric oxide synthase?nNOS?is important to intracellular Ca²⁺ handling.Many studies suggested nNOS was localized to the sarcoplasmic reticulum?SR?,where it associated with the ryanodine receptor?RyR2?and xanthine oxidoreductase?XOR?.Moreover,nNOS could impact diastolic function by regulating phospholamban?PLB?phosphorylation and thus its interaction with sarcoplasmic reticulum calcium ATPase?SERCA?.The proteins mentioned above are all relevant with Ca²⁺ handling.Therefore,nNOS is likely to involve in intracellular Ca²⁺ handling by regulating these proteins.In addition,some literatures identified translocation of nNOS from the sarcoplasmic reticulum to the sarcolemma in different heart disease models.Then the translocated nNOS increased the L-type Ca²⁺ channel?LTCC?S-nitrosylation leading to the reduced influx of Ca²⁺ through this channel,and increased RyR oxidation inducing increased diastolic Ca²⁺ leak.In recent years,several studies investigated neuronal nitric oxide synthase?nNOS?was cardioprotective in different disease states,but its role in IPostC had not been mentioned.Our previous study demonstrated that IPostC attenuated oxidative stress,reduced infarct size and improved cardiac function.However,L-VNIO,a selective nNOS inhibitor,abolished the protective effect of IPostC.In other words,IPostC protected hearts against I/R injury via,at least in part,nNOS-mediated pathway,but how nNOS plays a part in IPostC has been unknown.Our study focused on intracellular Ca²⁺ cycling,with emphasis on the subcellular localization of nNOS and its putative targets relevant in Ca²⁺ handling.We also investigated the effect of IPostC on nNOS translocation and the possible mechanism of nNOS translocation.Aims: To investigate what the role of nNOS plays in Ca²⁺ handling in IPostC and what effect of IPostC has on nNOS translocation.Methods: Ischemia reperfusion models were established using isolated hearts from mice and rates with langendorff perfusion system.Cardiomyocytes hypoxia and reoxygenation?H/R?models were established with a hypoxia tank.Intracellular concentration of Ca²⁺ was determined by Fluo-3/AM as a fluorescent signals using a flow cytometer.The intracellular Ca²⁺-transients in isolated cardiomyocytes were detected by confocal microscopy.Western blot and RT-PCR were used to measure the expression of SERCA after H/R.Myocardial sarcoplasmic reticulum was isolated from refused rat hearts and SERCA activity was detected with a kit.Immunogold labeling was used to determine the distribution of nNOS.CO-IP was used to determine the association between nNOS and RyR,Cav-3,or HSP90.XO activity was carried out with the Amplex Red Xanthine Oxidase Assay Kit.Oxidative stress level in the RyR2 was assessed by using the fluorescent probe for cysteines monobromobimane?mBB?.Results: nNOS was involved in the postconditioning protection of decreasing intracellular Ca²⁺ level and improving SR Ca²⁺ load.IPostC could improve intracellular Ca²⁺ cycling by upregulating the expression of SERCA,enhancing the SERCA activity and decreasing oxidative stress level of RyR2.However,additional of nNOS inhibitor L-VNIO abolished the protective effects.The translocation of nNOS was depended on protein HSP90 from the sarcoplasmic reticulum to the sarcolemma in I/R hearts,and that could be inhibited by IPostC.nNOS was also involved in attenuated XO activity of IPostC.Conclusions: nNOS was involved in IPostC protection of improved Ca²⁺ cycling by regulating relevant proteins in Ca²⁺ cycling.The translocation of nNOS was depended on protein HSP90,and that could be inhibited by IPostC.