NR4A1 Regulates The Cardiac Microvascular Ischemia Reperfusion Injury Through Modulating Mitochondrial Fission And Mitophagy

Background:The damage inflicted on the myocardium during acute myocardial infarction(AMI)is the result of 2 processes:ischemia and subsequent reperfusion(ischemia-reperfusion injury).Modalities for reperfusion include thrombolysis,percutaneous coronary intervention(PCI)and coronary artery bypass grafting(CABG),which are essential to salvage viable myocardium.Paradoxically,ischemia-reperfusion(IR)injury has been noted in all these situations.Ample evidences have demonstrated that reperfusion insult is the main determinant of infarct size which progressed into the post-infarction cardiac dysfunction.Unfortunately,the overwhelming majority of studies have focused on the prevention and therapy directed toward cardiomyocytes with 30 years of work on IR injury.Micro vascular IR injury has been the neglected component of many strategies that aimed to further reduce cardiac damage after successful PCI.As a result of the essential role of microcirculation in the exchange of nutrients,oxygen and energy between blood and cardiomyocytes,reperfusion injury of the micro vascular bed post-ischemia may induce a no-reflow phenomenon that augments additional myocardial damage and increases 30-day mortality rates.Accordingly,there is an unmet clinical need to explore the exact mechanism and novel therapeutic avenues to better resolve the microvascular IR injury,therefore limiting irrecoverable cardiomyocyte damage.Mitochondrial fission and selective mitochondrial autophagy(mitophagy)form an essential axis of mitochondrial quality control that plays a critical role in the development of cardiac ischemia-reperfusion(IR)injury.However,whether mitochondrial fission and mitophagy are also involved in microvascular IR injury remains unknown.Notably,recent studies have confirmed that mitochondrial fission is primarily regulated by mitochondrial fission factor(Mff)whereas mitophagy is governed by FUN 14 domain containing 1(FUNDC1).Based on our previous study,nuclear receptor subfamily 4 group A member 1(NR4A1)could simultaneously modify mitochondrial fission and mitophagy in the development of fatty liver disease.What remains unknown is whether IR-mediated mitochondrial fission and mitophagy are also controlled by NR4A1.Aim:Accordingly,the aim of our study is to explore the mechanism involving in microvascular IR injury,with a particular focus on mitochondrial fission and mitophgy.Methods:In vivo,cardiac IR injury was performed in WT mice,NR4A1-knock out(NR4A1KO)mice and Mff-knock out(MffKO)mice.In vitro,cardiac micro vascular endothelial cells(CMECs)were isolated from WT mice,NR4A1KO mice and MffKO mice and underwent hypoxia-reoxygenation(HR)injury.Immunohistochemistry,immunofluorescence,electron microscope,western blots were used to analyze the endothelial barrier function,microvascular structural damage,endothelium-dependent vasodilatation and inflammation response.In cells experiments,TUNEL assay,qPCR,western blots,co-immunoprecipitation,immunofluorescence,adenovirus-mediated gene overexpression,mutant plasmid transfection were used to evaluate mitochondrial DNA transcription/expression,energy metabolism,mitochondrial ROS overproduction,mitochondrial cyt-c leakage,cardiolipin oxidation,mitochondrial fission and mitochondrial apoptotic pathway activation.Results:1)NR4A1 expression was primarily upregulated in cardiac microcirculation under IR injury and contributed to mitochondrial perfusion defects,luminal stenosis,endothelial barrier damage,inflammatory cell permeation,and micro-endothelial apoptosis;2)genetic ablation of NR4A1 maintained endothelial barrier,recused endothelium-dependent vasodilatation and reduced myocardial inflammation response;3)at the molecular levels,higher NR4A1 expression was closely associated with Mff phosphorylation and FUNDC1 inactivation,resulting into mitochondrial fission activation and mitopahgy arrest;4)inhibition of fatal mitochondrial fission or reactivation of protective mitophagy sends the pro-survival advantage to the endothelium and microcirculation in response to IR injury;5)function study suggested that Mff-mediated mitochondrial fission caused mitochondrial DNA damage followed by ROS overproduction,eventually contributing to the cardiolipin oxidation and cyt-c leakage into cytoplasm;6)moreover,Mff-induced mitochondrial fission evoked excessive mPTP opening via promoting VDAC1 oligomerization and subsequent HK2 liberation;7)cytoplasmic cyt-c initiates and amplifies mitochondrial apoptotic signal,obligating endothelial cells to undergo the cellular death program;8)ablation of Mff in vivo protected mitochondrion and microcirculation against the IR injury;9)regarding mitophagy,FUNDC1-mediated mitophagy was inhibited by IR injury in microcirculation whereas activation of mitophagy contributed to the removal of mitochondrial fragmentations and thus corrected excessive mitochondrial fission,ultimately blocking mitochondrial apoptosis and sustaining endothelial survival in the context of IR injury.Conclusions:1.IR injury significantly increased the content of NR4A1 in microcirculation and upregulated NR4A1 contributed to mitochondrial damage,endothelial apoptosis and microvascular collapse.2.Higher NR4A1 evoked excessive mitochondrial fission via post-transcriptional phosphorylation of Mff.3.NR4A1 abated mitophagy via inactivating FUNDC1.4.Excessive Mff-activated mitochondrial fission promoted endothelial mitochorial apoptosis via mROS/cardiolipin/cyt-c axis and VDAC1/HK2/mPTP cascade.5.FUNDC1-required mitophagy was able to sweep mitochondrial debris and thus antagonize mitochondrial fission,sustaining mitochondrial structure and preventing microvascular IR injury.