Role Of Mitochondrial Fssion In Cardiac Microvascular Endothelial Cells After Ischemia/reperfusion

BackgroundCoronary heart disease (CHD) is one of the most common causes that lead to heartfailure and death, of which the foundamental pathophysiology is myocardial ischemiadue to coronary artery stenosis or occlusion. The most effective therapeutic approach tosalvage the ischemic myocardium is to establish blood perfusion quickly and efficiently.In recent years, researchers have found that some local myocardial ischemia patients willget worse after restoring the normal reperfusion, which indicates that the reperfusion willtrigger additional injury. The reinjury, caused by reperfusion on the basis of ischemia, iscalled ischemia reperfusion（I/R） injury. Among the numerous studies focusing onmyocardial ischemia/reperfusion injury (MI/R), there are increasing evidences showingthat cardiovascular microvascular injury plays a critical role in MI/R injury. It has beenreported that coronary artery endothelial and microvascular dysfunctions are closelyassociated with the―no reflow phenomenon‖. In addition, in the early stage ofreperfusion, the apoptosis of myocardial microvascular endothelial cells (CMECs) occursprior to the cardiac myocytes. More importantly, the injured microvasculature could release various inflammatory mediators，which further stimulate the apoptosis of cardiacmyocytes. Hence, cardiac microvascular injury is believed to act as the initiator of MI/Rinjury, and protection of CMECs against I/R injury may offer a promising strategy in thetreatment of MI/R.The morphological and functional alteration of mitochondria plays an important rolein the cellular response to I/R injury. It has been recently documented that the balancebetween mitochondrial fusion and fission participates in the regulation of mitochondrialfunction. Existing data showed that mitochondrial fission increased rapidly in the I/Rmyocardium, and inhibiting mitochondrial fission in cardiomyocyte could effectivelyprotect heart from I/R injury. Mdivi-1is a newly-discovered mitochondrial fissioninhibitor, which has been suggested to be protective in cardiomyocyte following I/Rinjury. However, so far there is no data available regarding whether Mdivi-1possessesthe similar protective effect on the ischemia/reperfused CMECs.ObjectivesThis study is aimed to establish a simulated ischemia/reperfusion (SI/R) model incultured CMECs from adult rat, and investigate the role of mitochondrial fission inCMEC I/R injury and the underlying mechanisms.Experimental MethodExperiment I: To establish CMECs I/R injury model and investigate the role ofmitochondrial fission in I/R injury1. CMECs were isolated from adult rat ventricles and exposed to simulatedischemia/reperfusion (SI/R). CMECs were randomly divided into two groups: controlgroup and SI/R group;2. Cell viability was measured through the MTT(methyl thiazolyl tetrazolium) assay;3. Migration ability was detected by the Transwell method;4. Flow cytometry based on Annexin V-FITC/PI double staining and TUNEL stainingwere used to detect apoptotic rates;5. Mitochondrial morphology of CMECs was observed by fluorescence microscopefollowing Mito-Tracker Green staining; 6. Protein levels of Drp1and Fis1were analyzed by Western blot;7. Intracellular ROS levels were tested by a reactive oxygen species assay kit;8. Mitochondria ROS level was measured using mitochondria superoxide markerMitosox;9. Mitochondrial membrance potential was detected by a mitochondrial membrancepotential assay kit (JC-1staining).Experiment II: to examine the effects of inhibiting mitochondrial fission on CMECs I/Rinjury. and the underlying mechanisms1. CMECs were isolated from adult rat ventricles and exposed to simulatedischemia/reperfusion (SI/R). CMECs were randomly divided into three groups: controlgroup, SI/R group, SI/R+Mdivi-1(a mitochondrial fission inhibitor) group andSI/R+NAC (an reactive oxygen species scavenger) group;2. Cell viability was measured with MTT assay;3. Migration ability was detected by the Transwell method;4. Flow cytometry based on Annexin V-FITC/PI double staining and TUNEL stainingwere used to detect apoptotic rates;5. Mitochondrial morphology of CMECs was observed by fluorescence microscopefollowing Mito-Tracker Green staining;6. Protein levels of Drp1and Fis1were analyzed by Western blot;7. Intracellular ROS levels were tested by a reactive oxygen species assay kit;8. Mitochondria ROS level was measured using mitochondria superoxide markerMitosox;9. Mitochondrial membrance potential was detected by a mitochondrial membrancepotential assay kit (JC-1staining).Statistical Analysis:All values in the text and figures are presented as means±SEM. All data wereanalyzed by one-way analysis of variance (ANOVA) or an unpaired two-tailed t test. Pvalues <0.05were considered statistically significant. All analyses were performed usingSPSS11.0statistical software. Results:1. Compared with control group, both cell viability and migration ability in the SI/Rgroup were impaired, and apoptosis index was significantly increased. Mitochondrialfission was also enhanced following SI/R, as evidenced by increased mitochondrialfragments using fluorescence microscope. Cells in the SI/R group exhibited significantlyhigher expression of Drp1and Fisl protein than that in the control group.2. Comparedwith SI/R group, both cell viability and migration ability in the SI/R+Mdivi-1group wereincreased, with apoptosis index decreased by Mdivi-1administered before reperfusion.Besides, mitochondrial fission was significantly confined. However, Drp1and Fislprotein level showed no significant changes, and both cellular and mitochondrial ROSlevels decreased remarkablely, along with increased mitochondrial membrance potential.3. Antioxidant NAC reversed the SI/R-induced increase in both cellular andmitochondrial ROS levels, and further inhibited the SI/R-stimulated mitochondrialfission.Conclusion:Increased mitochondrial fission play an important role in CMECsischemia/reperfusion injury. Underlying mechanisms involves its interaction withcellular and mitochondrial ROS generation and the maintenance of mitochondrialmembrance potential. Thus inhibiting mitochondrial fission may represent a newpromising strategy in protecting CMECs from I/R injury, hence the salvage of I/R heart.