O-GlcNAcylation Of Nav1.5 Contributes To Arrhythmia In Diabetic Heart Injury

Background:Diabetes mellitus is one of the most challenging global health threats,and cardiovascular complications are the major cause of mortality and morbidity in diabetic patients.Diabetic heart injury(DHI),a common cardiovascular complication of diabetes,is defined as diabetes-induced impairment in myocardial structure and function in the absence of coronary artery disease and hypertension.Myocardial diastolic dysfunction as an early manifestation of DHI,myocardial systolic dysfunction as a late manifestation of DHI.The long-term and possibly irreversible DHI eventually caused a variety of arrhythmias,including ventricular premature,ventricular tachycardia and even cardiac sudden death.Detailed underlying mechanisms of diabetes-associated myocardial pathological changes are complex.One of the most possible mechanisms for DHI-induced arrhythmias might be the endoplasmic reticulum stress,dysfunction in intracellular calcium homeostasis and the function and expression abnormalities of in ion homeostasis.However,its potential pathogenesis of cardiac arrhythmias is still poorly understood.More recent reports show that O-GlcNAc linked myocardial metabolism with cardiomyocyte circadian clock,which gives the possibility that O-GlcNAc to be related with the susceptibility of arrhythmias.In preliminary experiments,we observed that the level of O-GlcNAc modification of the total protein in rats after STZ injection was increased,while the cytoplasmic expression of Nav1.5 was increased.Nevertheless,the specific molecular mechanis ms for DHI induced-arrhythmias,especially the role of O-GlcNAcylated Nav1.5 in the disease progression is unknown.Objective:To investigate the role of Nav1.5 O-GlcNAc modification in DHI-induced arrhythmias,we validate the above hypothesis using both in vivo and in vitro experiments to mimic electrophysiological,mechanical and structural dysfunction of the diabetic heart,and to explore its possible mechanisms.The present findings demonstrate that O-GlcNAc of Nav1.5 protein in DHI induced-arrhythmia may have importantly clinical implications for the development of cardioprotective strategies.Methods:Blood glucose and blood pressure analysis,echocardiography(Vevo2100,Visualsonics),ECG monitoring(TA11CTA-F40,Data Sciences International),programmed electrical stimulation(Model 2352 Programmable Stimulator,Medtronic),quantitative RT-PCR,Western blotting,immunoprecipitation,immunofluorescence analysis and histological staining(Hematoxylin&eosin and Masson’s trichrome staining)were performed on diabetic rats.All rats were divided into two groups: Control and diabetic(STZ)groups.The primary cultures method of neonatal rat cardiomyocytes and HEK-293 T cells,high-glucose treatment,whole-cell patch clamp analysis(EPC10,HEKA),quantitative RT-PCR,Western blotting,immunoprecipitation and immunofluorescence analysis were performed on cells in vitro.All cells were divided into six groups: NG,HG,NG+Th-G,HG+Th-G,NG+Don,HG+Don.Results:1.In the STZ-induced diabetic model,we found that typical myocardial remodeling(the elevated blood glucose(BG),heart/body weight(HW/BW)ratio,atrial natriuretic peptide and brain natriuretic peptide mRNA levels)and cardiac dysfunction(the decreased in ejection fraction,fractional shortening and E/A Ratio)were appeared in 3-months STZ rats.2.To explore the arrhythmia susceptibility in diabetic heart,we performed the DSI implantation and extraepicardial programmed electrical stimulation(PES).The prolonged duration of QT interval(89.88 ± 9.14 vs.126.65 ± 11.57 ms)and QT intervals corrected with RR interval(QTc interval,112.69 ±10.35 vs.152.02 ± 11.26 ms)were observed in 3-months STZ induced diabetic rats compared to control rats.None of control rats but ten of the twelve STZ group rats showed the inducible sustained VT.In addition,compared with the control,3-months diabetic rats showed a remarkable increase in duration time of VT(0.83 ± 0.27 vs.7.32 ± 3.79 s)and arrhythmia score.3.In the STZ-induced diabetic model,the level of O-GlcNAc modification was up-regulated in a time-dependent manner.More,increased global O-GlcNAc level in the diabetic hearts was associated with increased OGT expression and decreased OGA expression in both 2-months and 3-months diabetic rats.In addition,our data clearly showed that nearly 2-fold increase in cytoplasmic expression of Nav1.5 in 2-,3-months STZ group rats.However,cardiac membrane expressions of Nav1.5 were significantly decreased in diabetic rats at 2-and 3-months.Above results suggested that Nav1.5 might participate in the regulation of diabetic-related abnormal cardiac electrophysiological function in vivo.4.High glucose ad ministration significantly elevated the expression of cytoplasmic Nav1.5,whereas clearly reduced the expression of membranous Nav1.5.5.We did further Co-immunoprecipitation assays and discovered that Nav1.5 was indeed O-GlcNAcylated in both the cardiac tissue and NRCMs and that hyperglycemia increases O-GlcNAc modified Nav1.5 levels.6.We found that O-GlcNAc Nav1.5 was subjected to bidirectional regulation [Deoxynorleucine(inhibitor)and Thiamet-G(agonist)in the HBP signaling pathway] and OGT(over-expressing exogenous and RNA-interfered endogenous OGT)played a vital role in regulation of Nav1.5 O-GlcNAc.What’s more,we also found that O-GlcNAc increased Nav1.5 expression and inhibited it traffic to the membrane in vitro.7.To observe the effect of the O-GlcNAc Nav1.5 on the functional properties of sodium channels: In vitro whole cell recording confirmed that hyper-O-GlcNAc of Nav1.5 reduced cardiac sodium channel function and increased late sodium current.8.We examined the expression of Nedd4-2,SAP-97 and MOG1 protein in vivo and in vitro models of DHI.Remarkably,the increased O-GlcNAc-modified Nav1.5 was correlated with the decreased interaction between Nav1.5 and Nedd4-2/SAP-97 both in vivo and in vitro.Conclusion:1.In this work,we found that diabetic heart injury displayed increased susceptibility to ventricular arrhythmias.2.Hyperglycemia increased O-GlcNAc modification of Nav1.5 expression both in vivo and in vitro.3.Hyper-O-GlcNAc of Nav1.5 decreased the interaction between Nav1.5 and Nedd4-2/SAP-97,which led to abnormal expression and distribution of Nav1.5.4.Hyper-O-GlcNAc of Nav1.5 inhibited cardiac fast sodium channel current and increased the late sodium current.