Effects And Mechanisms Of S-nitrosylated MLP On Myocardial Hypertrophy

Background:Myocardial hypertrophy>as one of the main performance of cardiac remodeling,constitutes an independent risk factor for myocardial ischaemia,myocardial infarction and heart failure.Nitric oxide(NO)is an important gas signal molecule in the body,and play important role in regulation of cardiovascular function.It could play a biological effect through regulating protein s-nitrosylation.MLP(muscle lim protein)is expressed in striated muscle,which plays an important role in a variety of cardiovascular diseases.However,the role of s-nitrosylated MLP in myocardial hypertrophy is not clear.Objective:The main purpose of this study is to test the effects of s-nitrosylated MLP on myocardial hypertrophy and clarify the underlying mechanisms.Methods and Results:We detected the s-nitrosylation level of heart tissues protein of WKY,SHR,sham and TAC group with biotin-switch method,the results showed that s-nitrosylated MLP increased significantly in the cardiac hypertrophy model.10-7 M Angiotensin II was applied to neonatal rat cardiomyocytes(NRCMs)for 24h,biotin-switch method was used to extract s-nitrosylated protein of cardiac myocytes,and the results in vitro were corresponding to the results in vivo.These results are both verified that MLP can be s-nitrosylated in cardiac hypertrophy model.Then we used liquid chromatography tandem mass spectrometry(LC/MS/MS)technology to screen the s-nitrosylated sites of MLP,we found that MLP can be s-nitrosylated at cys25 and cys79.We mutated cys25,cys79 of MLP into alanine and constructed adenovirus,then infected neonatal rat cardiomyocytes,followed with Angll of 10-7M for 24 h.ANP and BNP mRNA level were detectd by real time PCR,and a-actinin were detectd to observe the area of cardiac myocytes by immunofluorescence.The results showed that mutantion of cys79 but not cys25 can reduce the degree of myocardial hypertrophy caused by Ang II stimulation.Next,we infected neonatal rat cardiomyocytes with Ad C79A and Ad WT,and detected the combination of MLP and calcineurin(CnA),the results showed that mutantion of cys79 can affect the combination of MLP and CnA,which further decrease the dephosphorylation of NFAT3 and the level of luciferase reporter gene.Conclusion:The above results show that under the stimulus hypertrophy factor,s-nitrosylation of 79 cysteine residue in MLP may promote the interaction between MLP and CnA,activate the downstream of CnA/NFAT3 signaling pathways in the end promotes myocardial hypertrophy.