Sodium Channel Function Change In The Inherited Long Qt Syndrome Pathogenesis Research

BackgroundThe inherited long QT syndrome (LQTS) is a hereditary cardiac disorder characterized by prolonged QT interval on the surface electrocardiogram (ECG) and increased risk for sudden death due to ventricular tachyarrhythmia. It is one of the common causes of unexplained syncope especially in young, seemingly healthy individuals. To date more than1000mutations in13genes have been identified to associate with LQTS, in which6genes encode cardiac ion channels (KCNQ1, KCNH2, SCN5A, KCNJ2, CACNA1C and KCNJ5), and7encode ion channel subunits or channel-interacting proteins (ChIPs)(ANKB, KCNE1, KCNE2, CAV3, SCN4B, AKAP9and SNTA1).SCN5A encodes the α subunit of voltage-gated cardiac sodium channel hNav1.5also denoted SCN5A that is responsible for large peak inward sodium current (/Na) in the heart. The a subunit has four homologous domains (DI-DIV) and each domain contains six transmembrane segments (S1-S6). LQTS-associated mutations in SCN5A cause LQT3, a "gain-of-function" leading to prolonged cardiac action potential duration, lengthened QT interval, and increased risk of arrhythmia.a1-syntrophin (SNTA1), a dystrophin-associated protein, is the dominant syntrophin isoform in skeletal and cardiac muscle. As a scaffolding adapter, SNTA1binds to neuronal nitric oxide synthase (nNOS), which is constitutively expressed in the heart, and the cardiac isoform of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA4b) to form a complex in which PMCA4b acts as a potent inhibitor of NO synthesis. SNTA1also interacts directly with the PDZ domain-binding motif formed by the last three residues (serine-isoleucine-valine) of Navl.5C terminus. Previous study revealed a novel mutation A390V-SNTA1in a LQTS patient disrupted the association with PMCA4b and antagonized the inhibition of nNOS, resulting in augmentation of both peak and late/Na, suggesting SNTA1as a novel LQTS-susceptibility gene (LQT12).ObjectivesThe present study was designed to identify potentially novel mutations which would be responsible for LQTS in a Caucasian pedigree, to further characterize electrophysiological features and to elucidate a plausible pathogenic arrhythmia mechanism for LQTS.MethodsA Caucasian family with syncope and prolonged QT interval was identified. Genomic DNA was extracted from peripheral blood lymphocytes and was screened for the entire open-reading frames of13LQTS-susceptibility genes by polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (HPLC) and direct DNA sequencing. Identified mutations were created with the aid of site-directed mutagenesis. Wild-type or mutant channels were heterologously expressed in HEK293cells by transient co-transfection. Macroscopic voltage-gated/Na was measured24hours after transfection with the standard whole-cell patch clamp technique in HEK293cells.ResultsThe proband was a37-year-old woman who had experienced recurrent bouts of syncope for30years. Her resting12-lead ECG showed a marginal prolongation of corrected QT interval (QTc>480ms). Her10-year-old son was suffered from supraventricular tachycardia and the ECG showed moderately prolonged QTc (500ms). Genetic analysis revealed they both harbored the R800L mutation in SCN5A and A261V mutation in SNTA1. Other family members harboring either mutation had weaker clinical phenotype. Functional assays showed Peak/Na densities were unchanged for WT and for mutant channels containing R800L-SCN5A, A261V-SNTA1or R800L-SCN5A plus A261V-SNTA1. However, late/Na for either single mutant was moderately increased2-3fold compared to WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1significantly enhanced the/Na late/peak ratio by5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The "gain-of-function" effect could be blocked by the NG-monomethyl-L-arginine (L-NMMA), a nNOS inhibitor.ConclusionsWe conclude that novel mutations in SCN5A and SNTA1jointly exert a nNOS dependent "gain-of-function" on SCN5A channels (i.e., increased late/Na as well as slowed current decay), which may consequently prolong the action potential duration and lead to LQTS phenotype. BackgroundSCN5A encodes the voltage-dependent sodium channel α-subunit protein SCN5A, also called hNav1.5, found predominantly in human heart muscle. This channel is responsible for large peak inward sodium current (/Na) that underlies excitability and conduction in working myocardium (atrial and ventricular cells) and special conduction tissue (Purkinje cells and others), and also for late/Na that influences repolarization and refractoriness. SCN5A in humans has two splice variants, one lacking a glutamine at position1077(Q1077del) and one containing Q1077. Mutations in SCN5A can cause a broad variety of pathophysiological phenotypes, such as long QT syndrome type3(LQJ3), Brugada syndrome (BrS), cardiac conduction disease (CCD), or sick sinus syndrome (SSS)./Na dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed, and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in1of295healthy white controls.ObjectivesHere, we determined the in vitro biophysical phenotype of S1787N-SCN5A in an effort to further assess its possible pathogenicity.MethodsWe engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position1077(Q1077del) and the minor isoform which contains Q1077in the pcDNA3.1vector, and expressed them in HEK293cells for eletrophysiological study. Macroscopic voltage-gated/Na was measured24hours after transfection with the standard whole-cell patch clamp technique. We applied two kinds of intracellular solution varied in pH.ResultsAfter24h transfection, S1787N in Q1077background had WT-like/Na including peak/Na density, activation and inactivation parameters as well as late/Na in both pHi7.4and pHi6.7. However, with S1787N in the Q1077del background, the percentage of/Na late/peak was increased2.1fold compared to WT in pHi7.4(n=7-9, p<0.05) and was increased2.9fold compared to WT in pHi6.7(n=6-8, p<0.03).ConclusionsA LQT3-like biophysical phenotype for S1787N is both SCN5A isoform and intracellular pH dependent. These findings provide further evidence that the splice variant and environmental factors could affect the molecular phenotype with implications for the clinical phenotype and may provide insight into acidosis-induced arrhythmia mechanisms.