Role And Mechanism Of Cav-3/eNOS Signaling Pathway In Myocardial Ischemia-reperfusion Injury In Diabetic Rats

Part 1Effects of N-acetylcysteine on Cav-3/eNOS signaling pathway in myocardium of diabetic ratsObjective To observe the changes of myocardial structure and cardiac function in streptozotocin-induced type 1 diabetic rats,and explore the treatment effects of N-acetylcysteine(NAC)on the protein expression of Cav-3 and eNOS and their association in diabetic myocardium.Methods Twenty-four male SD rats were randomly assigned to control group(C),diabetic group(D)and diabetic with NAC treatment group(D+NAC).NAC was given at the dose of 1.5g/kg/day by gavage,and C and D group were given the same amount saline for 4 weeks.The cardiac function of experimental rats was evaluated by echocardiography,morphological changes of cardiac myocytes were observed under light microscope with hematoxylin-eosin staining,and the levels of ROS production in situ were detected by DHE staining.Commercialized kits detected the levels of plasma and cardiac 15-F2t-isoprostane and cardiac nitrotyrosine and NO production.CO immunoprecipitation and cofocal analyzed the relationship between Cav-3 and eNOS in myocardium.The protein expression of Cav-3,eNOS and p-Akt were analyzed by Western blot.Results(1)The levels of plasma glucose,food intake,water intake and the ratio of heart weight and body weight in D group were much higher than that in C group,NAC treatment food intake,water intake and the ratio of heart weight and body weight in diabetic rats.(2)The heart rate(HR),left ventricular posterior wall thickening rate(LVPW%),ventricular septal thickening rate(IVS%)and E/A ratio in D group were significantly decreased as comared with that in C group,and diabetic rats showed disordered myocardial tissue structure,and the cross-sectional area of cardial myocytes were significantly increased.After 4 weeks of NAC treatment,these changes were significantly attenuated except for no significant effects on HR.(3)The plasma and cardiac 15-F2t-isoprostane production,ROS production in situ and cardiac nitrotyrosine production in diabetic rats were significantly increased,but the NO production were significantly decreased as compared with that in control rats.All these alterations were significantly attenuated by NAC treatment.(4)Cofocal analysis showed the colocalization of Cav-3 and eNOS,diabetic condition reduces the association of Cav-3 and eNOS.NAC treatment significantly increased the association of Cav-3 and eNOS in diabetic rats,and restored the protein expression of Cav-3 and p-eNOS.Conclusion Cardiac dysfunction may appear in early stage of diabetes,which is associated with impaired Cav-3 expression and eNOS/NO signaling.Hyperglycemia-induced oxidative stress is involved in the development of cardiovascular complications in diabetes.NAC tratment could attenuate the cardiac dysfuction possiblly through restoring Cav-3/eNOS/NO signaling.Part 2Role of Cav-3/eNOS signaling in N-acetylcysteine-mediated cardioprotection against myocardial ischemia-reperfusion injury in diabetic ratsObjective To establish the models of myocardial ischemia-reperfusion injury diabetic rats or high glucose and hypoxia/reoxygenation injury model in cultured H9C2 cells,observe the treatment effects of antioxidant N-acetylcysteine(NAC)on myocardial ischemia-reperfusion injury,and explore the role of Cav-3/eNOS signaling in NAC-mediated cardioprotection against myocardial ischemia-reperfusion injury in diabetic rats.Methods Twenty-four male SD rats were randomly assigned to control group(C),diabetic group(D)and diabetic with NAC treatment group(D+NAC).NAC was given at the dose of 1.5g/kg/day by gavage,and C and D group were given the same amount saline for 4 weeks,then all these rats were subjected to myocardial ischemia-reperfusion injury,which was achieved by occluding the LAD coronary artery for 30 min followed by 2 h.H9C2 cells were treated with low or high glucose for 36 h,then the cells were performed by 4 h of hypoxia followed by 4 h of reoxygenation.The levels of CK-MB,LDH release 15-F2t-Isoprostane were detected by commercial kits.Myocardial Cav-3,eNOS and p-eNOS expression were analyzed by Western blot.Results(1)At the time points of 10 min before ischemia,the levels of HR,LVSP,+dp/dt and-dp/dt in D group were significantly decreased as compared with that in C group,all these alterations were further increased after 2 h reperfusion.NAC significantly increased the levels of HR,LVSP,+dp/dt and-dp/dt in diabetic rats after 2 h reperfusion.(2)The infarct size,CK-MB,and plasma and myocardial tissue 15-F2t-Isoprostane levels in D group were much higher than that in C group,all these alterations were significantly attenuated by NAC treatment.(3)NAC treatment significantly increased the expression of Cav-3 and p-eNOS in diabetes rats subjected to myocardial ischemia reperfusion insult,and also reduced LDH release and 15-F2t-Isoprostane in H9C2 cells exposed to high glucose and hypoxia/reoxygenation.All these beneficial effects were abolished by Cav-3 siRNA and eNOS siRNA.Conclusion Dibetes-induced inhibition of eNOS activity was associated with caveolae dysfunction and impaired Cav-3 expression.Antioxidant NAC could alleviate myocardial dysfunction and myocardial ischemia-reperfusion injury by improving Cav-3/eNOS/NO signaling pathway in diabetic rats.