Smurf2 Enhances Oxidative Stress-induced Cardiomyocyte Injury By Interacting With Septin4

Objective:The imbalance between reactive oxygen species（ROS）production and antioxidant defense can cause oxidative stress.The damage caused by oxidative stress is the basis of many cardiovascular diseases,such as atherosclerosis,ischemia-reperfusion injury and heart failure.Septin4,which is located in the mitochondrial membrane,has been shown to be a pro-apoptotic protein involved in myocardial cell injury.However,the regulatory mechanism of Septin4 in oxidative stress-induced H9c2 cell injury remains unclear.Ubiquitin-proteasome system（UPS）is an essential pathway to regulate the levels of protein.Misfolded and injured proteins in vivo can be degraded by the ubiquitin-proteasome pathway.Continuous studies have shown that ubiquitination plays an important role in myocardial oxidative stress injury.The HECT-type E3 ubiquitin ligase Smurf2 has been identified as a protein that can regulate the physiological functions of cardiomyocytes.Therefore,we hypothesized that Smurf2-Septin4 signaling pathway is involved in oxidative stress-induced cardiomyocyte injury.The aim of this study is to investigate the role of Smurf2 in oxidative stress-mediated cardiomyocyte injury and the mechanism of Smurf2 in oxidative stress-mediated cardiomyocyte injury through regulation of Septin4.Methods:1.H9c2 cells transfected with Flag-Vector or Flag-Smurf2 plasmids and then stimulated with H2O2（150μmol/L,120min）,and the cell viability was detected by CCK8.The protein levels of Septin4 and apoptosis-related protein Cleaved-caspase3 were detected by Western Blot.The apoptosis rate of H9c2 cells was detected by flow cytometry.The levels of oxidative stress damage were detected by the ROS reactive oxygen species kit.2.H9c2 cells transfected with Control-si RNA and Smurf2-si RNA plasmids and then stimulated with H₂O₂（150μmol/L,120min）,and the changes of cell viability were detected by CCK8.The protein levels of Septin4 and Cleaved caspase3 were detected by Western Blot.Cell apoptosis was detected by flow cytometry.The oxidative stress damage of cells was detected by ROS reactive oxygen species kit.3.H9c2 was selected as the research object in this experiment.The endogenous co-immunoprecipitation assay was used to investigate the interaction between Smurf2 and Septin4.Through plasmid transfection,we overexpressed Flag-Septin4 or Flag-Smurf2 in H9c2 cell line,and semi-exogenous co-immunoprecipitation assay was performed to verify the binding between Smurf2 and Septin4.H9c2 cells overexpressing Flag-Septin4 or Flag-Smurf2 were treated with or without H₂O₂（150μmol/L,120min）to observe the binding between Flag-Smurf2 and Septin4 or between Flag-Septin4 and Smurf2.After transfection of Flag-Smurf2 or silencing of Smurf2 in H9c2 cells overexpressing HA-UB followed by the treatment of MG132,the ubiquitination level of Septin4 was observed.4.In H9c2 cells,overexpression of Flag-Smurf2（0,2,4,6μg）or Smurf2 knockdown was performed,and the protein level of Septin4 was detected by Western Blot.In the cells overexpressing or knocking down Smurf2,the protein level of Septin4 was detected by Western Blot after stimulation with MG132 or CHX for 0,3,and 6 hours,respectively,to observe the changes in the degree of accumulation or half-life of Septin4.In H9c2 cells transfected WWP2,Smurf2 was overexpressed or knocked down,and the binding strength of WWP2 and Septin4 was observed by co-immunoprecipitation method.Results:1.Compared with the empty group,the overexpression of Smurf2 attenuated cell viability under H₂O₂ stimulation in the H9c2 cells.Meanwhile,overexpression of Smurf2significantly increased Septin4 and cleaved caspase3 protein levels,aggravated apoptosis and increased intracellular reactive oxygen species（ROS）production.2.Compared with the empty group,the knockdown of Smurf2 enhanced cell viability under H₂O₂ stimulation in the H9c2 cells.Meanwhile,knockdown of Smurf2 significantly increased Septin4 and cleaved caspase3 protein levels,alleviated apoptosis,and reduced intracellular reactive oxygen species（ROS）production.3.Co-immunoprecipitation assay confirmed that the interaction between Smurf2 and Septin4 and their binding was significantly enhanced under oxidative stress.Meanwhile,overexpression of Smurf2 significantly increased the ubiquitination level of Septin4,and Smurf2 knockdown significantly reduced the ubiquitination level of Septin4.4.The protein level of Septin4 increased gradually when Smurf2 was overexpressed in a gradient manner.Meanwhile,the expression of Septin4 protein was decreased correspondingly when Smurf2 was knocked down.We found that Septin4 protein levels are regulated by Smurf2 and maintain consistent changes.In Flag-Vector or Flag-Smurf2transfected cells,the expression of Septin4 protein in the overexpression group was higher,and the increase degree was lower after MG132 treatment（0,3,6h）and its degradation was slower after CHX treatment（0,3,6h）.In the cells transfected with Control-si RNA or Smurf2-si RNA,the expression of Septin4 protein in the knockdown group was lower,and the accumulation was more obvious after MG132 treatment（0,3,6h）and the degradation was faster after CHX treatment（0,3,6h）.Meanwhile,the binding of WWP2 and Septin4was significantly reduced in H9c2 cells overexpressing Smurf2,while significantly enhanced in H9c2 cells knocking down Smurf2.Conclusions:1.Smurf2 aggravates oxidative stress-induced H9c2 cardiomyocyte injury through regulating Septin4 stability.2.E3 ubiquitin ligase Smurf2 interacts with Septin4 and the interaction between Smurf2and Septin4 was enhanced under H₂O₂stimulation in the H9c2 cardiomyocyte.Meanwhile,Smurf2 could promote the ubiquitination level of Septin4.3.Smurf2 stabilizes Septin4 protein levels,which is closely related to the inhibition of WWP2-mediated Septin4 proteasomal degradation.