Effects And Mechanisms Of S-nitrosylated Hsp90 On Cardiac Fibrosis

Background:Myocardial fibrosis is a development to a certain stage of various heart disease common pathological change,which can lead to reduced myocardial compliance,arrhythmia,etc.,resulting in heart failure.As a new kind of REDOX mechanism of signal transduction,s-nitrosylation participates in a variety of tissues and organs functions.Heat shock protein 90(Hsp90)as a molecular chaperone protein,plays an important role in cell protection,cytoskeleton formation,anti-apoptosis and signal transduction.It has been reported that Hsp90 can be s-nitrosylated in other models;however,whether the s-nitrosylation of Hsp90 plays an important regulating role in the cardiovascular system,especially myocardial fibrosis,is not clear.Therefore,the effects of s-nitrosylation of Hsp90 on myocardial fibrosis and its exact mechanism need in-depth exploration and research.Objective:The main purpose of this study is to test the effects on s-nitrosylation of Hsp90 on myocardial fibrosis and clarify the underlying mechanisms.Methods and Results:We detected the s-nitrosylation level of heart tissue proteins of WKY,SHR,sham and TAC group with biotin-switch method,the results showed that s-nitrosylated Hsp90 increased significantly in different cardiac fibrosis models.100 nM Angiotensin II was applied to neonatal rat myofibroblast for 24h,biotin-switch method was used to extract s-nitrosylated protein of fibroblast,and the results in vitro are in agreement with the results in vivo.These results are both verified that Hsp90 may be involved in process of myocardial fibrosis.Then we used liquid chromatography tandem mass spectrometry(LC/M S/M S)technology to screen the s-nitrosylated sites of Hsp90,we found that Hsp90 can be s-nitrosylated at cys587 and cys588.On this basis,we mutated cys587 and cys588 of Hsp90 into alanine and constructed adenovirus,then infected neonatal rat myofibroblasts,followed with Ang? of 100 nM for 24 h.Type ? collagen and collagen type ?mRNA level were detected by real time PCR,and ?-SMA were detected to observe the degree of myocardial fibrosis by immunofluorescence.These results showed that the s-nitrosylation of cys587 of Hsp90 plays an important role in myocardial fibrosis caused by Ang ? stimulation.Next,we infected neonatal rat myofibroblasts with Ad C587A and Ad WT,and detected the combination of Hsp90 and T?R?,the results showed that mutantion of cys587 can affect the combination of Hsp90 and T?R?,which further affect the degree of fibrosis.Conclusion:In summary,under stimulating of promoting fibrosis factor,Hsp90 can be S-nitrosylated at cys587,which improves the combining capacity of Hsp90 and T?R?,leading to the occurrence and development of myocardial fibrosis.