Key Role And Molecular Mechanism Of UBC9 In Regulation Cardiac Sodium Channel Na_v1.5 Expression And Cardiac Sodium Current Density

Cardiovascular disease causes sudden cardiac death,which is the major cause of death worldwide.Sudden cardiac death is caused by cardiac arrhythmias,which can be classified into ventricular fibrillation?VF?,ventricular tachycardia?VT?and atrial fibrillation?VF?.Cardiac arrhythmias are mainly caused by abnormal ion flow in cardiac myocytes,which is controlled by ion channels.Genomic mutations in ion channels lead to hereditary arrhythmias.For example,in 1995,mutations in the Na_v1.5 sodium channel were reported to cause long QT syndrome associated arrhythmias and sudden death.Na_v1.5 is a voltage-gated sodium channel and plays a key role in the generation and transmission of the cardiac action potential.Na_v1.5 is encoded by the SCN5A gene.To date,more than 300 genetic mutations in SCN5A have been found to cause Brugada syndrome?BrS?,long QT syndrome?LQTS?,sick sinus syndrome,dilated cardiomyopathy,AF,and other cardiac diseases.The cardiac sodium channel is a large protein complex composed of Nav1.5?the?-subunit?,different?-subunits and many other interacting proteins such as MOG1,CRYAB,SAR1A/SAR1B identified by our laboratory.However,other key components of the Na_v1.5 protein complex and the mechanism of each component in regulating the expression and function of Na_v1.5 needs to be further studied.As a membrane protein,the expression level of Na_v1.5 on the surface of cell membrane is closely related to its function because the electrical activity of a cardiac cell depends not only on its activity but also on its expression level.The ubiquitination of Na_v1.5 plays an important role in controlling its membrane expression.Nedd4-2,the key component of the UPS?ubiquitin-proteasome system?,interacts with Na_v1.5 and regulates the ubiquitination,endocytosis and degradation of Nav1.5.Nedd4-2 is a ubiquitin E3 ligase with HECT catalytic domain.Na_v1.5 includes a PY?xPPxY?motif,which can interact with the WW domain of Nedd4-2.Ubiquitinization is a prerequisite for endocytosis and degradation of plasma membrane proteins.SUMOylation is another post-translational modification process involved in protein quality control.UBC9 is a small ubiquitin-like modifier-conjugating enzyme E2 that helps ligation of SUMO to the substrate during SUMOylation.To date,there was no report on SUMOylation of Na_v1.5.Therefore,we planned to characterize the role of SUMOylation in regulation of Na_v1.5 expression and function.We over expressed UBC9 in HEC293 cells and cardiomyocytes and found that UBC9 indeed significantly reduced the expression of of Na_v1.5 and the density of cardiac sodium current I_Na.Surprisingly,we found that UBC9 regulates Nav1.5 turnover not in a SUMOylation-dependent manner.Instead,we found that UBC9 regulates Na_v1.5 turnover through ubiquitination by interacting with Nedd4-2.The detailed results of our research are described as follows:?1?We co-transfected an overexpression plasmid for UBC9 and the Nav1.5/pCMV-HA plasmid for overexpression of Nav1.5 in HEK293 cells.We also transfected a UBC9 overexpression plasmid or control empty vector in HEK293-Nav1.5cells with stable expresson of Na_v1.5.We found that overexpression of UBC9 decreased the expression level of Nav1.5.The experiment further showed that UBC9 regulated Na_v1.5 expresson in a dose-dependent manner.Na_v1.5 on cell membranes plays the main role in its function.After overexpression of UBC9,membrane proteins were extracted,and Western blot analysis showed that overexpression of UBC9 down-regulated the expression level of Na_v1.5 on cell membranes.?2?Whole-cell sodium currents were recorded in HEK293-Nav1.5 cells transfected with pCMV-UBC9 or pCMV-HA empty vector by patch-clamping.The results showed that overexpression of UBC9 reduced the sodium current density,but did not affect the steady-state activation,inactivation and the time of recovery from inactivation of sodium currents.?3?Endogenous UBC9 was knocked down by using UBC9 siRNA in HEK293-Nav1.5 cells.Western blot analysis showed that after knockdown of UBC9,the expression level of Na_v1.5 was significantly elevated.Further studies showed that after knockdown of endogenous UBC9 in HEK293-Nav1.5cells,the sodium current density increased,but it did not affect the steady-state activation,inactivation and the time of recovery from inactivation of sodium currents.?4?UBC9 was overexpressed or knocked down in neonatal rat cardiomyocytes,and the whole cell sodium current was recorded by patch-clamping.The results showed that overexpression of UBC9 decreased the sodium current density and knockdown of UBC9 increased the sodium current density in neonatal rat cardiomyocytes.?5?We constructed a mutant UBC9 with mutation C93S,which lost the SUMO binding activity.Positive control experiments with p53,a well-known substrate of SUMOylation showed that overexpression of mutant C93S UBC9 affected the SUMOylation of p53:overexpression of UBC9 promoted SUMOylation of p53,but mutant C93S UBC9 inhibited SUMOylation of p53.We then overexpressed wild type UBC9 and mutant C93S UBC9 together with Na_v1.5.Western blot analysis showed that overexpression of wild type UBC9 decreased the Na_v1.5 expression level,but surprisingly,the mutant C93S UBC9 also decreased the Na_v1.5 expression level.The data showed that UBC9 regulated Na_v1.5 expression in a SUMOylation-independent manner.?6?Overexpression of UBC9 in HEK293-Nav1.5cells reduced the level of Nav1.5 protein,however,the reduction was blocked by MG132treatment,suggesting that UBC9 regulates the Na_v1.5 expression level through a proteasomal degradation pathway.Further studies showed that overexpression of UBC9 in HEK293 cells and HEK293-Nav1.5 cells promoted the ubiquitination of Na_v1.5.?7?Nedd4-2 is the E3 ligase involved in protein ubiquitination by interacting with an E2enzyme.Because UBC9 is the E2 enzyme for protein SUMOylation,we hypothesized that it may act as an E2 enzyme for ubiquitination.To test the hypothesis,we performed a protein-protein interaction assay using co-immunoprecipitation?Co-IP?.Our Co-IP experiment showed that UBC9 interacted with Nedd4-2,supporting a potential role of UBC9 as one E2 enzyme for protein SUMOylation of Na_v1.5.In conclusion,we found that UBC9 regulated ubiquitination of Na_v1.5 and cardiac sodium current densities in both a heterologous HEK293 cell expression system and neonatal cardiomyocytes.We found that UBC9 interacted with Nedd4-2 and mediated Nav1.5 degradation through the UPS.Previous studies have defined that Nedd4-2 is the E3 Ub ligase for ubiquitination and degradation of Na_v1.5.We propose that UBC9 acts as an E2 Ub-conjugation enzyme in the Ub-conjugation machinery regulating Nav1.5ubiquitination and degradation.Our findings thus identify a key structural element of the ubiquitin-conjugation machinery for Na_v1.5 and provide important insights into the regulatory mechanism for ubiquitination and turnover of Na_v1.5.The complete dissection of the Ub conjugation machinery will be important to our understanding of the structure,function and regulation of the cardiac sodium channel complex,which is fundamentally important to cardiovascular physiology,health and disease.