Histone Deacetylase (Sirt1) Protects Mouse Against Myocardial Ischemia/Reperfusion Injury

Objective:Ischemic heart disease is one of the critical diseases seriously endangering human health,especially among the elderly.In the past 20 years,coronary reperfusion has been the main therapy for acute myocardial infarction.However,blood flow restoration in ischemic tissue（ie,"reperfusion"）deteriorats the heart tissue damage induced by ischemia only.So far,there is still a lack of effective treatment for the myocardial ischemia/reperfusion injury（MI/R）.Accumulated evidences have shown that the increased levels of oxidative stress are closely related to the myocardial damage caused by reperfusion.Therefore,mechanisms underlying the imbalance between endogenous antioxidants and oxidative stress during MI/R injury has attracted a lot of attention.In the present study,cardiomyocyte oxidative stress injury caused by H₂O₂ or hypoxia/reoxygenation（H/R）in vitro were employed,as well as the MI/R injury in vivo induced by coronary artery left anterior descending artery ligation in mice.The effect of Sirt1 on Nrf2 regulation and mechanisms underlying such action against oxidative stress damage induced by MI/R were explored by biochemistry and molecular biology techniques.Methods:（1）Sirt1 declined the levels of myocardial oxidative stress through Nrf2.cardiomyocyte oxidative stress injury caused by H₂O₂ or hypoxia/reoxygenation（H/R）in vitro were employed,respectively.We detected the cell damage indicators,such as the cell viability and the leakage rate of lactate dehydrogenase（LDH）.The protein expressions of Sirt1,Nrf2,NQO1 and HO-1 in heart tissue were determined by Western Blot,and cellular levels of ROS were examined by flow cytometry.Furthermore,the cardiomyocytes were transfected by the sense or antisense adenoviral vector containing the gene of Sirt1 to up-or down-regulate the protein expression of Sirt1 level.Then,cells were also treated by H₂O₂ or H/R.The protein levels of Sirt1,Nrf2,NQO1 and HO-1 were detected again,and the interaction between Sirt1 and Nrf2,as well as the degree of acetylation of Nrf2 were determined by immunoprecipitation.（2）Sirt1 indeed actives Nrf2 signaling pathway to decrease the oxidative stress induced by MI/R in mice.The ligation of coronary artery left anterior descending artery was employed to induce MI/R injury in mice.Meanwhile,intramyocardial injection of Sirt1 sense or antisense adenovirus was used to up-or down-regulate the protein levels of Sirt1.The electrocardiogram,echocardiography and myocardial infarct size were monitored.Protein expressions of Sirt1,Nrf2,NQO1 and HO-1were analysed by Western Blot.The interaction between Sirt1 and Nrf2 and the degree of acetylation of Nrf2 were detected by immunoprecipitation.The transcriptional activity of Nrf2 was examined by EMSA method.Results:（1）Sirt1 is involved in the regulation of oxidative stress induced by MI/R.Meanwhile,the expressions of Nrf2,NQO1 and HO-1 were increased in H₂O₂ or H/R treated cardiomyocytes.Notably,overexpression of Sirt1 significantly increased the cell viability to 1.5 folds of the control group and significantly decreased the LDH leakage rate to 65%of the control group in cells subjected by H₂O₂ or H/R,respectively.However,downregulation of Sirt1 reversed all these alterations.The mechanisms underlying such action:Sirt1 and Nrf2 could interact with each other,and Sirt1 could active Nrf2,further upregulating the expressions of two-phase detoxification enzymes（ie.NQO1 and HO-1）.（2）After the intramyocardial injection of Sirt1 sense and antisense adenovirus,the MI/R injury in vivo was established.The results showed that the myocardial infarct size was significantly reduced to 35%of the control group and the cardiac function was significantly enhanced in the Sirt1-sense adenovirus group,compared with the MI/R group.However,downregulation of Sirt1 reversed all these alterations.We also demonstrated that Sirt1 could indeed promote the translocation and transcriptional activity of Nrf2,along with the up-regulated protein expressions of NQO1 and HO-1,via decreasing the degree of acetylation of Nrf2.Conclusion:Sirt1 up-regulates the expression of two-phase detoxification enzymes（ie.NQO1 and HO-1）by increasing the deacetylation activity of Nrf2,further enhancing cardiac function and reducing myocardial infarct size,thereby exerting anti-oxidative stress damage in myocardial ischemia/reperfusion.