The Mechanism Of Cardiac Ischemia Reperfusion Injury And The Progress Of Drug Protection

Background:Treatment of acute myocardial infarction(AMI)by timely reperfusion intervention has greatly improved the survival prognosis of patients with coronary heart disease.However,opening only the epicardial blood vessels sometimes does not improve the patient prognosis.Therefore,the study will focus on the mechanisms of cardiac reperfusion injury and drug interventions.Microcirculation is the most extreme part of the blood supply of the heart and is the most sensitive part of sensing ischemia and hypoxia.Therefore,we will focus on the microcirculatory endothelial cells and cardiomyocytes of this study.Our previous studies suggest that intracellular calcium overload and excessive reactive oxygen species production would trigger a range of injuries.SERCA2a is widely studied as the most important endoplasmic reticulum calcium recycle protein.Previous studies have demonstrated that under IR conditions,SERCA2a activity is reduced,resulting in intracellular overload damage.Other studies have suggested that post-transcriptional modification of SERCA2a is a major factor for its reduced activity.Therefore,in our study it’s the first time to focus on the oxidative modification of SERCA2a under IR conditions.Therefore,a further study will focus on whether SERCA2a-induced cellular calcium overload is directly related to mitochondrial mitotic division and mitochondrial apoptosis induced by mitochondrial calcium overload.In addition,previous study found that melatonin has protective effects on microcirculatory ischemia-reperfusion injury.Therefore,we strive to improve the mechanism of melatonin to protect mitochondrial structure after IR injury.Aim:The aim of this study was to elucidate the molecular mechanism of myocardial ischemia-reperfusion-mediated microcirculation injury,to explore the effect of post-transcriptional modification of SERCA2a on cytoplasmic calcium balance,and to elucidate the effect of SERCA2a as an upstream signaling molecule on mitochondrial division and calcium-dependent cell movement.On this basis,it’s important to explore the new target of melatonin to protect mitochondrial integrity.Methods:We use WT mice、SERCA2aAVV and OPA-1KO to establish cardiac reperfusion injury model.The in vitro experiment was divided into two parts.The first part isolated and extracted myocardial microcirculation endothelial cells(CMEC)from the heart of 5-7d rat,and the second part isolated the cardiomyocytes from the mouse heart,and both established the cell-level hypoxia-reoxygenation injury model.First,in vivo experiments,we used WB,co-immunoprecipitation,immunohistochemistry,HE staining,immunofluorescence,mitochondrial electron microscopy,small animal ultrasound,TUNEL staining,Evans Blue staining,etc.to observe differences in the expression levels of different proteins,the number of microcirculation,myocardial infarction area,cardiac reactive oxygen species,mitochondrial morphology,cardiac function,proportion of myocardial apoptosis,and luminal structure and inflammatory cell infiltration;At the cell level,we used flow cytology,immunofluorescence,WB,cell migration,Transwell,tube formation experiments,co-immunoprecipitation,DCFHDA probe technique,JC-1 staining,cell TUNEL staining,electron microscopy,etc.to observe changes in calcium levels,changes in reactive oxygen species,differences in protein expression.Results:1.Microvascular IR damage leads to post-translational modification and subsequent degradation of SERCA2a.2.Overexpression of SERCA2a reduces the size of the heart infarction,restores systolic function,and maintains microvascular integrity and subsequent inflammation distribution.3.In vitro mechanism studies suggest that SERCA2a dysfunction causes cytoplasmic Ca2+overload and ER stress,and overexpression of SERCA2a can effectively alleviate damage.4.SERCA2a dysfunction not only triggers ER stress,but also causes dephosphorylation and activation of the Ca2+/calmodulin-SSH1L-cofilin signaling cascade induced by Ca2+overload.5.Dephosphorylation of cofilin increases F-actin reduces neovascularization after IR injury.6.Dephosphorylated cofilin induces F-actin disintegration and transfer to the outer membrane of mitochondria,involved in Drpl-mediated pathological mitochondrial division.7.Our results demonstrate that OPA1-related mitochondrial fusion/mtochondrial autophagy is regulated by melatonin under cardiac I/R injury conditions.8.AMPK-OPA1-mitochondrial fusion/mitochondrial autophagy via melatonin may be a novel drug treatment and intervention to reduce cardiac I/R injury.Conclusions:1.Microcirculatory IR damage leads to post-transcriptional modification of SERCA2a,decreased activity and decreased expression,and finally induces intracellular calcium overload damage;overexpression of SERCA2a can alleviate this pathological process.2.Dephosphorylation and activation of the Ca2+/calmodulin-SSH1L-cofilin signaling cascade caused by SERCA2a inactivation leads to endothelial dyskinesia and increased pathological mitochondrial division leading to increased apoptosis.3.Melatonin protects against excessively damaged and dividing mitochondria through AMPK-OPA1-mitochondrial fusion/mitochondrial autophagy,maintaining microcirculatory mitochondrial homeostasis.