The Role Of Tbx20 In Regulating Voltage-gated Sodium Channel And Its Contribution To Cardiac Arrhythmia

Background and PurposeArrhythmias are common heart diseases,and the abnormal function of the heart electrical conductivity system leads to the arrhythmias.The establishment,homeostasis,and regulation of cardiac rhythmare are derived from gene regulatory networks.The networks first specify the components of the cardiac conduction system（CCS）during cardiogenesis.These components integrate CCS signaling into the working myocardium and allow sympathetic and para sympathetic inputs to modulate heartbeat.Congenital defects of the CCS and dysregulation of CCS homeostasis can lead to CCS dysfunction,causing life threatening arrhythmias and increasing the risk of death in both children and adults.Genome wide association studies in various arrhythmia diseases have revealed a close association between abnormal electrocardiogram（ECG）and many ion channels,gap junction proteins,muscle structural proteins as well as many crucial transcription factors that act on cardiogenesis and the specification and homeostatic maintenance of the CCS,among them includes the T-box transcription factors（Tbx3/5/20）.Tbx20 is expressed in multiple cardiac cell lineages.Mutations in Tbx20 are widely associated with a complex spectrum of congenitial heart defects,which is likely due to the broader expression pattern of Tbx20 during cardiac development.Currently,we have found that Tbx20 likely acts as a negative transcription regulator of cardiac voltage-gated sodium channel gene Scn5a–another human gene associated with QRS variants.Our aim is to explore the mechanism of Tbx20 and Scn5a in CCS development/maintenance or maintaining the excitability of the cardiomyocytes,which can potentially provide innovative clinical diagnosis and treatment programs for arrhythmias.MethodsTwo experimental mouse models were used for the experiment:Tbx20myocardial specific overexpression mouse model（Tbx20-TG）and Tbx20 myocardial conditional knockout mouse model（Tbx20CKO）.（1）Tbx20-TG mouse model:we observe the morphological development of the model mouse heart by immunohistochemistry experiment;we measure the hypertrophy and expansion of the mouse heart by the heart weight/body weight ratio;we evaluate the cardiac function of the mouse model by Doppler echocardiography;we perform surface ECG to analyze the arrhythmia situation;we measure the conduction velocity of the ventricular epicardium surface using an optical mapping system;we observe the expression of Scn5a,Nkx2.5,Cacn1a,Cacn1c and other genes through qRT-PCR experiment.（2）Tbx20CKO mouse model:we evaluate the cardiac function of the mouse model by Doppler echocardiography;we perform surface ECG to analyze the arrhythmia situation.（3）The sodium channel of Tbx20CKO and Tbx20-TG mouse model:we evaluate the Scn5a expression level by molecular biological approach;we use patch clamp to measure the change of sodium current density peak(I_Na).Results（1）Morphological and histological analyses revealed that Tbx20-TG hearts were normal prior to 2 months of age when compared to littermate controls.In contrast,the Tbx20-TG mice developed severely enlarged and dilated hearts at ages greater than 9months,suggests that Tbx20-TG mice die from heart failure.The heart weight to body weight ratio was 11.6±1.5（mg/g）in 9-month Tbx20-TG mice as compared to 5.3±0.37（mg/g）in littermate controls.Tbx20-TG cardiomyocytes were markedly hypertrophic as demonstrated by the increased cardiomyocyte cross-sectional area.（2）M-mode echocardiographic analyses of Tbx20-TG mice demonstrated significantly slower heart rates and reduced systolic function at 2-months of age,and a severely compromised cardiac systolic function at older ages（>9 months）.ECG analyses showed Tbx20-TG mice have a marked sinus bradycardia,first degree AV block,prolonged PQ interval and QRS duration when compared with non-transgenic mice.By 9-months,the majority of Tbx20-TG mice analyzed presented with third degree conduction block.The prolonged and multiphasic QRS complex suggested a ventricular conduction defect.We measured the conduction velocity of the ventricular epicardium surface using an optical mapping system,and results revealed increased anisotropy of conduction in the transgenic heart,resulting in an overall reduced conduction velocity.In addition,the propagation of electrical impulses was also found to be disorganized swirls in Tbx20-TG mouse hearts.It was further confirmed that the conduction function of the heart was abnormal.M-mode echocardiography analysis of Tbx20CKO mice showed that EF%and FS%decreased significantly.Surface electrocardiogram analysis showed that QRS wave prolonged and heart rate accelerated,but no obvious arrhythmia wave appeared.（3）qRT-PCR displayed that Scn5a was dramatically down-regulated by over5-fold in hearts isolated from Tbx20-TG mice when compared to the control hearts,and a down regulation of approximately 50%was seen in the cardiac transcription factors such as Nkx2.5 and Tbx2,while expression of other transcription factors was maintained at normal levels such as Tbx1 and Gata4.In Tbx20CKO mice cardiomyocyte,the mRNA and protein expression of Scn5a are significantly increased while remarkable decreased in Tbx20-TG mice cardiomyocyte.Similarly,immunofluorescence analysis confirmed the up-regulation and down-regulation of Nav1.5 levels in Tbx20CKO and Tbx20-TG cardiomyocytes,respectively.（4）The effect of Scn5a expression changes on the electrophysiology of cardiomyocytes:the mean peak density of I_Na was significantly increased in Tbx20CKO and reduced in Tbx20-TG cardiomyocytes respectively when compared to normal control littermates.Conclusions（1）Myocardial overexpression of Tbx20 leads to cardiac arrhythmia and progresses to dilated cardiomyopathy in older mice.（2）Myocardial gain-and loss-of-function Tbx20 studies have shown altered QRS duration and severe arrhythmia.（3）Tbx20 functions as a negative transcriptional regulator of Scn5a and plays major roles in CCS function as well as cardiomyocyte excitability and conduction.（4）The current data show that Tbx20 has no effect on the development of cardiac conduction system.