Junctophilin-2 Is Required For Stabilizing Junctin And Protection Against Cardiomyocyte Death And Heart Dysfunction In Lipid Overload-induced Cardiomyopathy

Background Lipid overload has been implicated in cardiomyocyte death,myocardial remodeling and dysfunction,participating in the development of various cardiac diseases;however,the underlying mechanisms remain obscure.We recently reported that lipid overload results in a lower level of junctophilin-2(JPH2)protein in palmitate(PA)-loaded cardiomyocytes and heart tissues from mice fed a high fat diet(HFD),implying a potential role of JPH2 in lipid overload-induced cardiomyopathy.Objective To determine the role of JPH2 in maintaining junctin(JCN)stability and its implications in lipid overload-induced cardiomyocyte death and myocardial dysfunction.Methods1.To establish lipid overload model and determine the role of JPH2 in lipid overload-induced myocardial injury.In vivo study,transgenic mice with cardiomyocyte-specific over-expression of human JPH2 and their littermate controls were fed a HFD or normal diet(ND)for 3months.Myocardial function was analyzed by echocardiography.Myocardial histopathological changes were measured by determination of cardiomyocyte cross-sectional areas and collagen deposition.Myocardial apoptosis was detected by measurement of caspase-3 activity.The protein expression of JPH2 and JCN were determined by western blot analysis.In vitro study,neonatal mouse or rat cardiomyocytes were isolated and PA was chosen to induce lipid overload in cardiomyocytes.After PA incubation,we detected the protein expression of JPH2 and JCN,the cytosolic Ca2+ level and the apoptosis of cardiomyocytes.In addition,JPH2 and JCN were over-expressed through adenoviral infection in cardiomyocytes and analyzed their role in PA-induced cardiomyocyte injury.2.To determine the interaction between JPH2 and JCN.To explore whether JPH2 expression influenced the protein level of JCN,JPH2 was knocked down by infection with an adenoviral vector in cardiomyocytes and then the protein expression of JCN was detected.We also over-expressed JPH2 through adenoviral infection in cardiomyocytes and investigated whether JPH2 over-expression protected JCN from PA-induced down-regulation.In addition,JCN was knocked down by adenoviral infection and explored whether JCN knockdown was involved in the regulation of the protective effects conferred by JPH2 in cardiomyocytes.Furthermore,we measured the physical interaction between JPH2 and JCN by mass spectrometry,immunoprecipitation followed by western blot analysis and bimolecular fluorescence complementation(Bi FC)assay.3.To investigate the mechanisms about the down-regulation of JCN To explore the mechanisms of JCN down-regulation,the cardiomyocytes were treated with PA and then the ubiquitination of JCN was detected by immunoprecipitation followed by western blot analysis.We also tried to find out the possible enzymes involved in the ubiquitination of JCN by mass spectrometry.Next,we over-expressed JPH2 in cardiomyocytes through adenoviral infection and investigated the role of JPH2 in the regulation of JCN ubiquitination.4.To confirm the ubiquitination sites of JCN To provide further insights into ubiquitin-mediated proteasomal degradation of JCN,in-silico analysis of JCN protein was performed with Bayesian Discriminant Method to predict the ubiquitination sites of JCN in PA-stimulated cardiomyocytes.Some ubiquitination sites of JCN in PA-treated cardiomyocytes were verified by mass spectrometry.In addition,to determine putative ubiquitination sites which were modulated by lipid overload,we generated a mutant plasmid of JCN by replacing lysine with arginine at 4 different ubiquitination sites.The cardiomyocytes were transfected with the mutant plasmid of JCN and determined whether the mutant JCN was resistant to PA-induced ubiquitination and degradation in cardiomyocytes.Results1.Feeding a HFD to control mice for 3 months induced myocardial remodeling,apoptosis and dysfunction,and reduced cardiac protein levels of JPH2 and JCN.These effects of HFD were attenuated in transgenic mice with JPH2 over-expression.In cultured mouse and rat cardiomyocytes,PA treatment decreased JPH2 and JCN protein levels,elevated cytosolic Ca2+ and induced apoptosis,all of which were prevented in cardiomyocytes over-expressing JPH2 or JCN.These results demonstrated that lipid overload reduced the protein expression of JPH2 whereas JPH2 protected cardiomyocytes against lipid overload-induced cardiomyocyte injury.2.Furthermore,knockdown of JPH2 sufficiently reduced JCN protein level,increased cytosolic Ca2+ and induced apoptosis in cardiomyocytes.However,JPH2over-expression attenuated PA incubation-induced reduction of JCN.Notably,knockdown of JCN offset the protective effects conferred by JPH2 over-expression(inhibiting apoptosis and lowering cytosolic Ca2+)in lipid-overloaded cardiomyocytes.It was further verified the physical interaction between JPH2 and JCN.These results supported the notion that JPH2 interacted with JCN and regulated the expression of JCN.In addition,the protective role of JPH2 was mediated through JCN in lipid-overloaded cardiomyocytes.3.PA incubation significantly increased the ubiquitination of JCN in cardiomyocytes.Mass spectrometry analysis found that PA treatment resulted in higher Mu RF1-JCN interaction in cardiomyocytes.These data indicated that PA incubation induced the interaction of Mu RF1 and JCN,leading to the ubiquitination and subsequent degradation of JCN and resulting in the protein down-regulation of JCN.However,JPH2 disrupted Mu RF1-JCN interaction thereby preventing Mu RF1-mediated JCN ubiquitination and degradation in response to lipid overload.4.Lastly,software prediction and mass spectrometry analysis identified multiple ubiquitination sites on the JCN protein(K8/K102/K107/K140)after incubation with PA.We transfected cardiomyocytes with the mutant plasmid of JCN(K8R/K102R/K107R/K140R)and found that mutant JCN was resistant to PA-induced ubiquitination and degradation.These results implied that PA treatment promoted Mu RF1-JCN interaction,resulting in the ubiquitination of JCN on specific sites and the following proteasome-dependent degradation of JCN.However,JPH2-JCN interaction blocked the ubiquitination sites of JCN thereby disabling JCN ubiquitination and degradation in PA-stimulated cardiomyocytes.Conclusions1.JPH2 over-expression attenuates lipid overload-induced cardiomyocyte death and heart dysfunction;2.JPH2 prevents Mu RF1-mediated JCN ubiquitination and degradation in response to lipid overload.3.JCN is involved in the protective role of JPH2 in lipid-overloaded cardiomyocytes.These findings uncover a novel role of JPH2 over-expression in preventing lipid overload-induced cardiomyocyte death and dysfunction.Lipid overload decreases JPH2 thereby predisposing JCN to ubiquitination and proteasome-dependent degradation,leading to cardiac pathology.Thus,reduced JPH2 and JCN may be important mediators of lipid overload-induced heart dysfunction and potential targets for intervention.