Effects And Mechanisms Of S-nitrosylated JNK1 On Cardiac Fibrosis

Background:Myocardial fibrosis(MF)is one of the main manifestations of myocardial remodeling,with the proportion of various types of collagen imbalance and disorder.Nitric oxide(NO),as a kind of gasotransmitter,is one of the important neurohumoral factors in the cardiovascular system,and it can regulate proteins to be S-nitrosylated,which affects protein expression,biological activity and molecular interaction,subcellular localization etc..C-Jun N-terminal kinase protein(JNK)is an important branch of MAPK pathway,which plays an important role in many physiological and pathological processes,such as cell cycle,proliferation,apoptosis and cell stress.Research shows that JNK 1 can be S-nitrosylated,but whether S-nitrosylation of JNK 1 participate in cardiac fibrosis has not been reported.Objective:The main purpose of this study is to elucidate the JNK1 S-nitrosylation involved in cardial fibrosis,and explore its specific sites and mechanism of S-nitrosylation.Methods and results:Animal level:We detected the s-nitrosylated JNK1 level of heart tissue proteins of sham and TAC,WKY,SHR group with biotin-switch method,and then detected the enzyme which involved in this S-nitrosylation modification.The results showed that in the animal models of cardiac fibrosis,S-nitrosylation of JNK1 level is significantly increased,and only the expression of iNOS obviously increased.8-10 weeks healthy male iNOS-/-mice which were randomly divided into TAC and sham two group,using biotin-switch methods to detecte the level of JNK1 S-nitrosylation and the myocardial fibrosis,the results showed that in iNOS-/-group,compared with WT group,after TAC,JNK1 S-nitrosylation level and cardiac fibrosis markers expression is no longer obviously increased.Cell level:SD rat myocardial fibroblasts were treated with angiotensin II(Ang II,100 nM,24h),using Biotin-switch method to detect JNK1 S-nitrosylation level,and the results in vitro are in agreement with the results in vivo,giving Ang II induces JNK1 to be S-nitrosylated.Then we transfected JNK1 cysteine mutation(MT)and wild type(WT)plasmids into HEK293T cells to detecte the S-nitrosylated modifiction sites,and transfected plasmids into HT1080 cells and then under the Ang II sitimulation to detect the expression and activity of activator protein-1.The results showed that giving Ang II treatment can induce JNK1 to be S-nitrosylated at cysteine 116 and cysteine 163(Cys116,Cys163),and then activite the downstream AP-1,which can promote the occurrence of myocardial fibrosis.Conclusion:Under stimulating of promoting fibrosis factors(TAC/AngIIsf),the expression of iNOS is increased and then JNK1 can be S-nitrosylated at Cys116 and Cys163,which improves the AP-1 activity and the expression of profibrotic genes,leading to the occurrence and development of cardiac fibrosis.