ZYZ451 Protects cardiomyocytes from hypoxia-induced apoptosis by enhancing MnSOD and STAT3 interaction

OBJECTIVE 3,5-Dimethoxy-4-(2-amino-3-prop-2-ynylsulfanyl-propionyl)-benzoic acid 4-guanidino-butyl ester(ZYZ451)showed excellent cardio-protective effects in our previous work.However,its therapeutic potential in vivo and the mechanism remained to be elucidated.Herein,we evaluated cardiac protective role of ZYZ451 in post-myocardial infarction(post-MI)rats,and elucidated the underlying mechanism.METHODS Neonatal rat ventricular cardiomyoctys(NRVCs)were separated and subjected to pre-deoxidized(1%O2,5%CO2),and serum-free medium for 4h to obtain ischemic model.Mitochondrial ROS,MnSOD activity and cell apoptosis were tested to verify the cardiac protective effects of ZYZ451.Inhibitors and siRNA for Stat3 were used to determine role of Stat3 played in cardio-protective effectes of ZYZ451.Mitochondria were isolated from NRVCs to determine expression of Stat3 and MnSOD.Immunofluorescence and coimmunoprecipitation were conducted to determine the interaction between MnSOD and Stat3.To apply post-MI model in rats,the rats were subjected to ligation of LAD except for control group.The vehicle or ZYZ451(1,2or 5mg·kg-1)or mixture of Leonurine and SPRC(15mg·kg-1)was administered 7dbefore and 3more days after the operation.Area at risk(AAR),apoptosis in AAR,LDH and MDA levels,and MnSOD activity and expression were detected to evaluate the cardiac injury.Tissue mitochondria were isolated to determine MnSOD and Stat3 expression in ischemia,and coimmunoprecipitaion was performed to verify the interaction between MnSOD and Stat3 in vivo.RESULTS ZYZ451 prevented hypoxia induced NRVCs apoptosis via increasing MnSOD activity and inhibiting mitochondrial ROS production.Interestingly,5,15-DPP(STAT3phosphorylation inhibitor)failed to inhibit MnSOD activity,while knockout of STAT3 resulted in significant reduction of MnSOD activity,followed by increased mitochondrial ROS production and cardiomyocytes apoptosis in hypoxia.Moreover,protective effects of ZYZ451 were blunted in Stat3 deficient NRVCs.These results indicated the necessity of