S-nitrosylated ANT1 Promotes Cardiac Hypertrophy By Mitophagy And Necropotosis

Background:Cardiac hypertrophy is a common pathophysiological manifestation of various cardiovascular diseases : hypertension,coronary heart disease,valvular heart disease,congenital heart disease and so on;it has compensatory significance in the early stage,but comes to miserable ending such as heart failure and sudden death eventually.Studies have shown that S-nitrosylation of protein,that is the protein cysteine thiol-SH,produces nitrosothiol-SNO under the action of nitric oxide(NO),plays an important role in cardiovascular diseases.Adenine Nucleofide Translocator 1(ANT1)is a transmembrane protein involved in the cytoplasm and mitochondrial matrix ADP/ATP translocation in the mitochondrial inner membrane.It is an important hub for mitochondrial energy production and energy utilization of cardiomyocytes,and it plays an important regulatory role in mitochondrial function.Mitophagy is a specific class of autophagy that cleans out dysfunctional mitochondria in the heart under normal physiological conditions,as well as in response to pathological stresses.Necropotosis is a death-receptor-mediated caspases-independent cell death pattern discovered recently.It usually occurs when apoptosis is inhibited and RIP1-RIP3 complex forms,having the morphological features of necrotic cells.In cardiac hypertrophy,the S-nitrosylation of ANT1 as well as its effect on cardiac hypertrophy and the regulatory mechanism under it have not been reported.Objective:We aimed to ensure the regulatory role of S-nitrosylated ANT1 on cardiac hypertrophy and further explore the mechanism under which S-nitrosylation of ANT1 regulated cardiac hypertrophy.Methods and Results:Animal experiments:(1)Wild Type(WT)or Cysteine 160 point mutated(C160A)ANT1 was specifically overexpressed in cardiomyocytes of C57BL/6 mice with aav-9 adeno-associated virus.Then thoracic aorta constriction(TAC)was performed for 4 weeks to establish a cardiac hypertrophy model.Doppler ultrasound system was used to detect left ventricular thickness and the cardiac function.The results showed that the Interventricular Septum(IVS)and Left Ventricular Posterior Wall(LVPW)were significantly thicker in TAC group than sham in aav-9-ANT1-WT mice;while thinner in aav-9-ANT1-C160 A mice after TAC than aav-9-ANT1-WT,with EF and FS similarly normal.(2)The hearts were harvested,and tissue was used to detected S-nitrosylation level of ANT1 by Biotin-switch combined immunoblot analysis technique.Results show that the S-nitrosylation level of ANT1 significantly increased after TAC in aav-9-ANT1-WT mice while scarcely increased in aav-9-ANT1-C160 A mice.(3)Autophagy markers LC3 II/I were detected in heart tissue extract.We found that LC3 II / I significantly reduced after TAC in aav-9-ANT1 WT mice;while mutation of Cys160 in ANT1 Significantly saved the reduction of autophagy in cardiac hypertrophy.(4)The heart of mice was cryosectioned and stained with DHE fluorescence to detect reactive oxygen species(ROS)levels.ROS levels increased significantly after TAC in aav-9-ANT1-WT mice;after mutation of the Cys160 locus,ROS production was significantly reduced.(5)Finally co-immunoprecipitation and Western blot were used to detect the level of RIP1-RIP3 complex.It came out that the RIP1-RIP3 complex increased in the TAC hypertrophy model of aav-9-ANT1 WT mice,while in aav-9-ANT1-C160 A mice,to a certain extent,activation of the programmed necrosis signaling pathway RIP1-RIP3 complex was inhibited.Cell experiments:(1)wild-type or Cys160-mutated ANT1(Ad-ANT1 WT/C160A)were overexpressed with adenovirus in neonatal rat ventricular myocytes(NRVM).And then the overexpressed NRVM were treated with AngII for 48 h.Immunoblots and Immunofluorescence technique was used to detect mitochondrial autophagy.The results showed that LC3II/I was significantly reduced in cardiac hypertrophy caused by AngII,together with decreased mitochondrial autophagy level(show by co-localization of LC3 II and mitotracker);while mutation of ANT1-Cys160 significantly improved mitophagy in cardiac hypertrophy.(2)The expression level of RIP1-RIP3 complex was detected in the myocardial hypertrophy cell model as well.It also showed that mutation of Cys160 could rescue the increase of RIP1-RIP3 complex expression induced by AngII stimulation significantly.(3)Finally,we used RIP1 inhibitor necrostain-1(Nec-1)to test the effect of RIP1-RIP3 activation on cardiac hypertrophy,detection of hypertrophy-related genes ANP and BNP mRNA expression levels showed that inhibition of the activation of programmed necrosis signaling pathway can significantly mitigate ANT1-SNO-mediated cardiomyocyte hypertrophy,confirmed by representative images of ?-actinin stained myocardial cell size.Conclusion:The above results indicate that the S-nitrosylation of Cys160 in ANT1 regulates cardiac hypertrophy by reducing mitochondrial autophagy and promoting the activation of programmed necrosis signaling pathway RIP1-RIP3.