| Cardiovascular disease,which is hazard to human health,has become "the first killer"in the 21st century and attribute to 42%of global human death.Myocardial hypertrophy is a critical stage in the development of cardiac structure from reversible to irreversible change,and is important risk factor for cardiovascular disease and death,which for now remains as hot study topics for basic experiment.Nitric oxide(NO)is gas signal molecule and has been hotspot of research during recent years.As an important neurohumoral factors in cardiovascular system,NO plays an important role in myocardial hypertrophy.In mammals,NO is mainly generated by nitric oxide synthase(NOS)or chemical compounds such as nitrite compounds degradation.Currently,increasing data supported NO as regulator for S-nitrosylation by post-translational modification.Relative stable S-N bond can be formed through nucleophilic attack to specific cysteine residues of target protein by forming reversible covalent S-nitrosylation,therefore regulates the biological activity of protein.Such process has been considered as a novel signal transduction regulation mechanism.Studies have shown that the S-nitrosylation has important effect on the energy metabolism of myocardial cells.Mitochondrial damage and energy metabolism disorder are two important reasons for myocardial hypertrophy.Adenosine transferase 1(ANT,1)serves as important link for mammals cytoplasmic energy utilization,by providing cellular energy through transporting adenosine triphosphate(ATP).Currently,sever studies showed a correlation between ANTI and heart disease.However,for now it remains unclear whether ANTI is involved in myocardial hypertrophy development by S-nitrosylation,with no previous report.The aim of this study is to clarify whethere S-nitrosylation of ANTI is possible and involved in development of myocardial hypertrophy.Transverse aortic constriction(TAC)operation was utilized to establish mouse model of myocardial hypertrophy,which was confirmed by doppler ultrasound to evaluate the degree of postoperative left ventricular hypertrophy.Results confirmed the thickening of the left ventricular wall thickness in mice at 4 weeks with stable cardiac function.Biotin-switch technique was applied to test S-nitrosylation condition in tissue sample of hypertrophic left ventricle.Flight mass spectrometry was used for screening and identification of ANT1 protein.Results showed that in LV samples,ANT1 was detected with S-nitrosylation at residue cysteine 160.Multiple studies showed that increasing NO content or changing S-nitrosylation regulating enzyme regulated S-nitrosylation.Western blot method was used to detect the S-nitrosylation regulating enzyme with extracted LV tissue protein.Results showed that induced inducible nitric oxide synthase(iNOS)expression increased,no decreased endothelial nitric oxide synthase(eNOS),and stable neuronal nitric oxide synthase(nNOS)expression was detected in the myocardial hypertrophy tissue,Decreased S-nitrosoglutathione reductase(GSNOR)expression and unchanged thioredoxin(Trx)were also observed,indicating that S-nitrosylatio2n may be caused by up-regulation of iNOS and down-regulation of GSNOR.Finally,activity of ANTI of myocardial hypertrophy tissue was measured using fluorescent probe to evaluate the reactive oxygen species(ROS)level.Results showed that ANT1 S-nitrosylation may change protein activity which caused increasing ROS and in vivo oxidative stress level,causing increased mitochondrial ROS and contribute to development of myocardial hypertrophy.Above results showed that:(1)protein S-nitrosylation is of great significance in the myocardial hypertrophy.(2)In TAC myocardial hypertrophy model,up-regulation of iNOS,down-regulation of GSNOR contributed to regulation of ANT1 S-nitrosylation.(3)S-nitrosylation of ANT1 cys160 caused depolymerization of dimer into inactive components and thus affecting the its transfer function,causing mitochondrial ROS levels rise,as well as increasing oxidative stress levels. |
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