| Background:Cardiomyopathy is characterized by mechanical and/or electrical dysfunction that is not explained by ischemic or valvular heart disease,which is often associated with increased risks for heart failure,cardiac arrhythmia,sudden cardiac death(SCD),and stroke.The causes of cardiomyopathy are various.Genetic pathogenesis is an important cause to cardiomyopathy.CAV3-encoded caveolin-3(Cav-3),as a key component of dystrophin complex,is specifically expressed in cardiomyocytes and skeletal muscle.Accumulating evidence has demonstrated the Cav-3 in cardiac excitability is likely,but so far,little evidence has been provided for their involvement in cardiomyopathy.Myocyte hypertrophy is a hallmark feature of DCM,which involves changes in the balance between protein synthesis and protein degradation.Autophagy,a ubiquitous cellular process in cardiomyocytes,plays a critical role in intracellular degradation,providing nutrients and energy for cellular homeostasis.In recent years,accumulating pieces of evidence have indicated an important role of autophagy in controlling the hypertrophic response.Moreover,emerging research findings have demonstrated that caveolins are involved with various diseases by disturbed autophagy.However,whether the role of Cav-3 in cardiac hypertrophy remains unclear.Objective:To explored whether mutations in CAV3 could alter cardiac hypertrophy by modulating the autophagy in heart,we transfected adenovirus Mut-CAV3 and adenovirus WT-CAV3 into neonatal rat cardiomyocytes(NRCMs).We validate the above hypothesis and further explore its underlying mechanisms,providing evidence that autophagy related 3(Atg3)may be a key regulator underlying the development of cardiac hypertrophy induced by the CAV3 p.W101 C mutation.Methods:1.The potential pathogenic mutation was predicted by multiple bioinformatics tools.2.WT-CAV3 adenovirus and Mut-CAV3 adenovirus were constructed and transfected into NRCMs.Western blot,quantitative RT-PCR,and TRITC phalloidin were used to determine whether CAV3 p.W101 C mutation can induce hypertrophy in NRCMs.3.An immunoprecipitation-mass spectrometry assays was performed to screen the potential mechanisms of cardiomyocyte hypertrophy induced by CAV3 p.W101 C mutation.4.The effect of CAV3 p.W101 C mutation on autophagy flow was observed by Western blot,transmission electron microscopy,and confocal microscopy.5.To clarify the specific steps of autophagy flow involved in CAV3 p.W101 C mutation,3-MA and chloroquine were used to intervene the NRCMs transfected with Mut-CAV3 adenovirus.6.Immunoprecipitation,immunofluorescence analysis,Glutathione-S –transferase(GST)pulldown assay were performed to determine the interaction between Cav-3 protein and Atg3 protein.7.After down-regulating the expression of Atg3 in primary cardiomyocytes of the Mut-CAV3 group,The effects on autophagy flow and cardiomyocyte hypertrophy in the NRCMs transfected with Mut-CAV3 adenovirus were detected after Down-regulation the expression of Atg3.Result:1.The W101 C residue of Cav-3 is absolutely conserved across species.A high pathogenicity possibility was predicted by multiple bioinformatics tools.2.We transfected adenovirus Mut-CAV3 and adenovirus WT-CAV3 into NRCMs,and performed an immunoprecipitation-mass spectrometry assays in the transfected NRCMs after 48 hour.Transfection with the Mut-CAV3 profoundly influenced the signal pathways of DCM and hypertrophic cardiomyopathy.Higher levels of atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP)in protein level and m RNA level were observed in the Mut-CAV3 group compared with WT-CAV3 group.And besides,the effects of the CAV3 p.W101 C mutation on cardiac hypertrophy were further validated using confocal microscopy.3.The up-regulation of LC3II/I protein ratio and increased autophagosome accumulation detected by transmission electron microscopy were found in Mut-CAV3 group compared with that in WT-CAV3 group.The p62 protein expression was down-regulated in Mut-CAV3 group.Increased autophagic flux in Mut-CAV3 group also obtained by confocal microscopy.4.No significant difference in the level of PI3 K class III or phosphorylated m TOR was observed between Mut-CAV3 group and WT-CAV3 group.Increased ratio of LC3II/I induced by CAV3 p.W101 C mutation was aboslihed by 3-MA(an inhibitor of PI3 K in the initial stage of autophagy)instead of chloroquine.Those data means the process of autophagy membrane extension was promoted by CAV3 mutation.5.The upregulation of ANP and BNP in protein level and m RNA level were relieved the upregulation of ANP and BNP in NRCMs transfected with Mut-CAV3 by comparison with that in absence 3-MA.Additionally,the cell area of NRCMs transfected with Mut-CAV3 obviously decreased following 3-MA treatment.6.The protein level of Atg3 obviously increased in the NRCMs transfected with CAV3 p.W101 C mutation compared with those transfected with WT-CAV3.A bind was observed betwwen Cav-3 and Atg3 in NRCMs bu IP.The results showed that purified GST-Cav-3 was not able to bind to purified His-tagged Atg3 under cell-free conditions.7.The up-regulation of LC3II/I ratio,ANP,and BNP were substantially abrogated following downexpression ATG3 in NRCMs transfected with Mut-CAV3.The autophagosome accumulation detected by transmission electron microscope transmission and cell size measured by confocal microscopy were significantly alleviated in NRCMs transfected with Mut-CAV3 after reduction of ATG3 expression.Conclusion:1.A high pathogenicity possibility was predicted by multiple bioinformatics tools in CAV3 p.W101 mutation.2.CAV3 p.W101 mutation induce cardiomyocytes hypertrophy,and enhance autophagic flux by promoting the process of membrane extension.3.Cardiomyocytes hypertrophy induced by CAV3 p.W101 mutation was substantially abrogated after inhibiting autophagic flux by 3-MA.4.There is indirect binding between Cav-3 and atg3 protein,which can be enhanced by CAV3 p.W101 mutation.5.Atg3 as a crucial effector of CAV3 p.W101 C mutation induces hypertrophic cardiomyocytes,which promote the process of autophagosome formation. |
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