Effects And Mechanisms Of S-nitrosylated ANT1 On Pathological Cardiac Hypertrophy

Background and Objectives:Pathological cardiac hypertrophy is one of the common features of various clinical diseases such as hypertension,acute myocardial infarction,and a variety of congenital heart diseases,Which is an independent risk factor for cardiovascular disease.In recent years,studies have shown that nitric oxide(NO),as an important gas signaling molecule,plays an important role in the regulation of cardiovascular function.It can play a biological effect by regulating various proteins'S-nitrosylation.Mitochondrial damage and energy metabolism dysfunction contributes to cardiac hypertrophy.Adenine nucleotide translocase(ANT)is a vitial mitochondrial protein in the inner mitochondrial membrance,Which involved in the exchange of cytosolic ADP for ATP within the mitochondrial matrix.It is also associated with the energy generation and consumption.The adenine nucleotide translocase(ANT)plays an important role in the regulation of the intracellular energetic balance.However,the role and mechanism ofs-nitrosylated ANTI in the pathogenesis of myocardial hypertrophy are not clear.The main purpose of this study is to investigate the effect of s-nitrosylated ANTI on myocardial hypertrophy,which was stimulated by Ang? and TAC.Meanwhile,We predicted mechanism of s-nitrosylated ANTI in cardiac hypertrophy.Methods and Results:We used biotin-switch method to detect the protein s-nitrosylation level of heart tissues in SHR and TAC hypertrophy model and myocardial cell stimulated with Angll.The results showed that s-nitrosylated ANTI increased significantly in the cardiac hypertrophy model.Then liquid chromatography tandem mass spectrometry(LC/MS/MS)technology was used to screen the exact cysteine sites of s-nitrosylated ANTI.We found that ANTI can be s-nitrosylated at cys57,cys129,cys160 and cys257.Cysteine was transformed into alanine to construct the ANTI-specific cysteine site mutation plasmid.The recombinant plasmid was transfected into HEK293 cells and biotin-switch was used to detect the cysteine sites of ANT1.We finally made sure that cys160 was the key cysteine site of s-nitrosylated ANTI.We mutated cys160 of ANTI into alanine and constructed adenovirus.Neonatal rat ventricular myocytes were infected with adenovirus,followed with Angll of 10-7 M for 48h.Expression levels of ANP and BNP were detectd by real time PCR,and the area of cardiac myocytes were detectd by immunofluorescence.The results showed that the cys160 site mutation can significantly ruduce the degree of cardiomyocyte hypertrophy mediated by Angll stimulation.The oxidative rate(OCR)or mitochondrial oxidative respiration was measured by Seahorse bioassay analyzer.MitoSox fluorescence staining was used to detect mitochondrial reactive oxygen species(ROS),and MitoCMX Ros staining was used to detect mitochondrial membrane potential(??m).It can be founded that in the cardiomyocytes Ang?stimulation can significantly reduce the mitochondrial respiratory ability,increasing the production of mitochondrial reactive oxygen species and reducing the mitochondrial membrane potential.But after the cys160 sites mutation,mitochondrial respiratory capacity recovery,the production of ROS decreased and mitochondrial membrane potential recovery too.That is to say 160 site mutation can protect from cardiac hypertrophy.We also detected the GSNOR protein level in the cardiac hypertrophy model and what we found was that the protein level of GSNOR reduced.After overexpression of GSNOR,given stimulation of hypertrophic factors,we analyzed the level of s-nitrosylated ANTI and the degree of hypertrophy.The results showed the level of s-nitrosylated ANTI was decreased and the degree of cardiac hypertrophy was decreased too.Conclusion:The above results indicate that under the stimulus hypertrophy factor,s-nitrosylation of 160 cysteine residue in ANTI,which may reduces the mitochondrial respiratory capacity,increasing the production of mitochondrial active oxygen species and decreasing the mitochondrial membrane potential.It finally leads to mitochondrial dysfunction and promotes cardiac hypertrophy.GSNOR plays an important role in the regulation of s-nitrosylation of ANTI in pathologic cardiac hypertrophy.Overexpression of GSNOR can decrease the level of s-nitrosylated ANTI and resist cardiac hypertrophy.