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 Stat3 on MnSOD activity independent of its transcriptional activity.We further found co-localization of STAT3 and MnSOD by immunofluorescence in NRVCs,coimmunoprecipitation verified their interaction,and ZYZ451 enhanced this interaction.Similar results were found on H9C2 cardiomyoctyes and knockout of STAT3 attenuated the interaction.Consistent with the in vitro results,ZYZ451 reduced myocardial infarct size,cell apoptosis,LDH and MDA content in myocardial infarction rats.The benefits relied on increased MnSOD activity and enhanced STAT3 and MnSOD interaction,as observed in dangerous area of the infracted hearts.CONCLUSION We are the first to report that STAT3 is involved in MnSOD activity regulation,and that ZYZ451 exerts its cardio-protective effects by enhancing MnSOD and STAT3 interaction.These findings indicate a new role for STAT3 beyond as a transcriptional factor and suggest that ZYZ451 is an effective cardioprotective agent.

A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H_2S production

Most reported fluorescent probes have limitations in practical applications in living systems due to the strong autofluorescence background,construction of probes with near-infrared（NIR） fluorescence emission is an accessible approach for addressing this challenge.We here designed a NIR fluorescent probe for monitoring the endogenous production of H2S in living cells.The designed probe showed significant NIR fluorescence turn-on response to H2S with high selectivity,enabling the sensitive detection H2S.Importantly,the probe could be applied in monitoring the endogenous production of H2S in raw 264.7 macrophages.This study showed that fluvastatin can promote the activity of cystathionineγ-lyase（CSE） for generation H2S.