The Role And Molecular Mechanisms Of SENP3 In H9C2 Suffered H/R Injury

Objective:Cardiovascular disease is the most common cause of death in the worldwide,among which the myocardial infarction accounts for the majority of disease.Great efforts have been made in the amelioration of acute myocardial infarction(AMI)reperfusion therapy,but the mortality rate is still high.Patients suffering from AMI have a high risk of developing heart failure(HF)which is resulted from myocardial ischemia reperfusion(MIR)injury.Apoptosis of cardiomyocyte after MIR inhibits the recovery of cardiac function during revascularization treatment.In the early stage of reperfusion,reactive oxygen species(ROS)is accumulated as a result of the loss of energy substrates in myocardial ischemia.In pathological conditions,ROS eventually leads to oxidative stress damage caused by excessive accumulation of ROS production,or due to the inhibition of its own degradation pathway.Signal transducer and activator of transcription(STAT3)is an important regulator of cell-to-cell communication in the heart involved in a variety of mechanisms of myocardial protection,which can be regulated by SENP3,a sentrin/SUMO2/3-specific protease.However,the relationship between STAT3 and SENP3 in H9C2 cells suffered to hypoxia/reoxygenation(H/R)injury has not been elucidated.Our preliminary data have demonstrated the accumulation of SENP3 in H9C2 cell when suffered H/R.The present study was designed to verify whether SENP3 contributes to protect H9C2 cell from apoptosis induced by H/R through STAT3 pathway.Methods Firstly,the model of myocardial ischemia/reperfusion injury(IRI)in male C57/BL-mice(8-10 weeks old)was used to check the expression of SENP3 and STAT3 in myocardial tissue suffered IRI operation of mice,then RT-PCR was used to detect m RNA level of SENP3.Then we established hypoxia/reoxygenation(H/R)models in H9C2 cell line.After H9C2 cells were exposed to 1,2,4,8 h reoxygenation respectively followed by 12 h hypoxia,the expression of SENP3 was determined by Western blot,and the ROS generated during H/R was detected by DCFH kit.To investigate the effect of SENP3 on H/R injury in H9C2,we knocked down the expression of SENP3 by using si RNA,then detected apoptosis rate by flow cytometry using an Annexin V-FITC/PI kit,the expression of cleaved-caspase3(c-caspase3),Bcl2,and Bax measured by western blot and Immunofluorescence was used to detect the intracellular localization of SENP3 under H/R in H9C2 cells.Tofurther determine whether the JAK2/STAT3 pathway is involved in antiapoptotic effect mediated by SENP3 in H/R injury,H9C2 cells were stably overexpressed of SENP3,JAK2 / STAT3 pathway-related protein is detected by western blot.In addition of AG490,test the above indicators to confirm the protective effect of SENP3.Results The expression of SENP3 and p-STAT3 were increased in myocardial tissue of mice following I/R and the m RNA level of SENP3 did not change significantly in I/R group.In vitro,the expression level of SENP3 was increased,accompanied by elevated ROS,in H9C2 cells following H/R and the increase of SENP3 expression induced by H/R is mediated by ROS.Then knockdown of SENP3 reduced the activity of STAT3,and overexpression of SENP3 enhanced the activity of STAT3.Knockdown of SENP3 increased apoptosis of H9C2 cell line induced by H/R,tested by flow cytometry,affected the expression of related apoptotic proteins,such as Bax and Bcl2,and also inhibited the activity of STAT3.Meanwhile,we found SENP3 mainly located in the nucleus and also existed in the cytoplasm in the H/R process by immunofluorescence analysis.Overexpression of SENP3 during the H/R process could increase the activity of STAT3.Application of AG490 could reverse the effect of SENP3 in protecting H9C2 cells against apoptosis.Conclusions The current findings demonstrated that SENP3 was a crucial mediator of the cellular response during H/R.The accumulation of SENP3 in the cell blamed to the stimulation of the ROS produced during H/R procedure,which exerted antiapoptotic effects.Our results showed that SENP3 induced phosphorylation of STAT3,with upregulation of the Bcl-2/Bax expression and inactivation of caspase-3.These findings provided novel mechanistic insights into SENP3 induced cardioprotection,which may help in expanding the therapeutic utility of target drug in limiting myocardial infarction and apoptosis following I/R injury.