Mutant SCN5A Research Articles

Requirement of β subunit for the reduced voltage-gated Na+ current of a Brugada syndrome patient having novel double missense mutation (p.A385T/R504T) of SCN5A.

Mutations within the SCN5A gene, which encodes the α-subunit 5 (NaV1.5) of the voltage-gated Na+ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the NaV1.5 current (INa) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of INa was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the coexpression of SCN1B with p.A385T/R504T revealed significant reduction of INa and slower recovery from inactivation, consistent with the loss-of-function in Na+ channels. The SCN1B dependent reduction of INa was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of NaV1.5, respectively.

Functional analysis of novel SCN5A mutations related to Brugada syndrome

Abstract Brugada syndrome (BrS) is an arrhythmogenic disorder that has been linked to mutations in SCN5A, the gene encoding for the pore-forming α-subunit of the cardiac Na+ channel. Recently, novel SCN5A missense mutations (A385T and R504T) were identified in a BrS patient. Since the mutations are in the loop connecting transmembrane segments 5 and 6 in domain 1 (S5-S6 in DI) and segments 6 and 1 between domain 1 and 2 (DI-DII linker), it can lead to dysfunctional property of the Na+ channel. Here we aimed to characterize the electrophysiological properties of A385T and R504T. The wild-type (WT) and mutant SCN5A were transiently transfected in HEK293 cells, and the Na+ channel was analyzed using the whole-cell patch-clamp technique. WT, A385T, R504T, and double mutant (A385T/R504T) showed no significant differences in the current density and the voltage-dependent activation. Unexpectedly, a rightward shift of the voltage-dependent inactivation was identified in the three groups of mutation. Besides, the recovery from inactivation of double mutant was faster than that of WT. These results suggest that, contrary to the expected mechanism of BrS, the mutations cause a gain-of-function of NaV1.5. However, the current densities of R504T and double mutant transfected with β-subunit SCN1B were significantly suppressed but A385T was not different from WT. The voltage dependent activation and inactivation of all mutants were not significantly different from WT. The recovery from inactivation of all mutants were slower than that of WT. These results suggest that R504T mutation of α-subunit SCN5A interacting with β-subunit SCN1B is responsible to pathophysiological function of novel BrS. Funding Acknowledgement Type of funding sources: None.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes.

Simple SummaryIn this study, the induced pluripotent stem cell-derived cardiomyocyte model from a patient with long QT syndrome harboring a heterozygous Nav1.5 R1623Q mutation exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). FPDcF was shortened with mexiletine treatment and increased the frequency of arrhythmia-like EAD events following E4031, an Ikr blocker, administration. These characteristics partly reflect the patient phenotypes. As the R1623Q mutation is related to severe congenital LQT syndrome in fetuses and neonates, the effect of the neonatal variants on the electrophysiological properties of the R1623Q mutant was examined using an automated patch-clamp system. Our results demonstrated that both R1623Q and neonatal R1623Q delayed inactivation of INa and increased late Na current. We speculated that neonatal Nav1.5 ameliorates QTc prolongation. Developmental switching of neonatal/adult Nav1.5 isoforms might play a role in the mechanisms underlying severe long QT syndrome in fetuses and neonates.The SCN5A R1623Q mutation is one of the most common genetic variants associated with severe congenital long QT syndrome 3 (LQT3) in fetal and neonatal patients. To investigate the properties of the R1623Q mutation, we established an induced pluripotent stem cell (iPSC) cardiomyocyte (CM) model from a patient with LQTS harboring a heterozygous R1623Q mutation. The properties and pharmacological responses of iPSC-CMs were characterized using a multi-electrode array system. The biophysical characteristic analysis revealed that R1623Q increased open probability and persistent currents of sodium channel, indicating a gain-of-function mutation. In the pharmacological study, mexiletine shortened FPDcF in R1623Q-iPSC-CMs, which exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). Meanwhile, E4031, a specific inhibitor of human ether-a-go-go-related gene (hERG) channel, significantly increased the frequency of arrhythmia-like early after depolarization (EAD) events. These characteristics partly reflect the patient phenotypes. To further analyze the effect of neonatal isoform, which is predominantly expressed in the fetal period, on the R1623Q mutant properties, we transfected adult form and neonatal isoform SCN5A of control and R1623Q mutant SCN5A genes to 293T cells. Whole-cell automated patch-clamp recordings revealed that R1623Q increased persistent Na+ currents, indicating a gain-of-function mutation. Our findings demonstrate the utility of LQT3-associated R1623Q mutation-harboring iPSC-CMs for assessing pharmacological responses to therapeutic drugs and improving treatment efficacy. Furthermore, developmental switching of neonatal/adult Nav1.5 isoforms may be involved in the pathological mechanisms underlying severe long QT syndrome in fetuses and neonates.

Multiple arrhythmic and cardiomyopathic phenotypes associated with an SCN5A A735E mutation

BackgroundSCN5A mutations are associated with multiple arrhythmic and cardiomyopathic phenotypes including Brugada syndrome (BrS), sinus node dysfunction (SND), atrioventricular block, supraventricular tachyarrhythmias (SVTs), long QT syndrome (LQTS), dilated cardiomyopathy and left ventricular noncompaction. Several single SCN5A mutations have been associated with overlap of some of these phenotypes, but never with overlap of all the phenotypes. ObjectiveWe encountered two pedigrees with multiple arrhythmic phenotypes with or without cardiomyopathic phenotypes, and sought to identify a responsible mutation and reveal its functional abnormalities. MethodsTarget panel sequencing of 72 genes, including inherited arrhythmia syndromes- and cardiomyopathies-related genes, was employed in two probands. Cascade screening was performed by Saner sequencing. Wild-type or identified mutant SCN5A were expressed in tsA201 cells, and whole-cell sodium currents (INa) were recorded using patch-clamp techniques. ResultsWe identified an SCN5A A735E mutation in these probands, but did not identify any other mutations. All eight mutation carriers exhibited at least one of the arrhythmic phenotypes. Two patients exhibited multiple arrhythmic phenotypes: one (15-year-old girl) exhibited BrS, SND, and exercise and epinephrine-induced QT prolongation, the other (4-year-old boy) exhibited BrS, SND, and SVTs. Another one (30-year-old male) exhibited all arrhythmic and cardiomyopathic phenotypes, except for LQTS. One male suddenly died at age 22. Functional analysis revealed that the mutant did not produce functional INa. ConclusionsA non-functional SCN5A A735E mutation could be associated with multiple arrhythmic and cardiomyopathic phenotypes, although there remains a possibility that other unidentified factors may be involved in the phenotypic variability of the mutation carriers.

Reduced current density, partially rescued by mexiletine, and depolarizing shift in activation of SCN5A W374G channels as a cause of severe form of Brugada syndrome.

Background SCN5A‐related Brugada syndrome (BrS) can be caused by multiple mechanisms including trafficking defects and altered channel gating properties. Most SCN5A mutations at pore region cause trafficking defects, and some of them can be rescued by mexiletine (MEX).ObjectiveWe recently encountered symptomatic siblings with BrS and sought to identify a responsible mutation and reveal its biophysical defects.MethodsTarget panel sequencing was performed. Wild‐type (WT) or identified mutant SCN5A was transfected into tsA201 cells. After incubation of transfected cells with or without 0.1 mM MEX for 24–36 hr, whole‐cell sodium currents (INa) were recorded using patch‐clamp techniques.ResultsThe proband was 29‐year‐old male who experienced cardiopulmonary arrest. Later, his 36‐year‐old sister, who had been suffering from recurrent episodes of syncope since 12 years, was diagnosed with BrS. An SCN5A W374G mutation, located at pore region of domain 1 (D1 pore), was identified in both. The peak density of W374G‐INa was markedly reduced (WT: 521 ± 38 pA/pF, W374G: 60 ± 10 pA/pF, p < .01), and steady‐state activation (SSA) was shifted to depolarizing potentials compared with WT‐INa (V1/2‐WT: −39.1 ± 0.8 mV, W374G: −30.9 ± 1.1 mV, p < .01). Incubation of W374G‐transfected cells with MEX (W374G‐MEX) increased INa density, but it was still reduced compared with WT‐INa (W374G‐MEX: 174 ± 19 pA/pF, p < .01 versus W374G, p < .01 versus WT). The SSA of W374G‐MEX‐INa was comparable to W374G‐INa (V1/2‐W374G‐MEX: −31.6 ± 0.7 mV, P = NS).ConclusionsReduced current density, possibly due to a trafficking defect, and depolarizing shift in activation of SCN5A W374G are underlying biophysical defects in this severe form of BrS. Trafficking defects of SCN5A mutations at D1 pore may be commonly rescued by MEX.

More Severe Loss-of-Function Sodium Channel Induced by Compound SCN5A R965C and R1309H Mutants Lead to Complex Familial Arrhythmia Syndrome

Objective: A complex familial arrhythmia syndrome was identified in a Chinese Han family, characterized by sick sinus syndrome, progressive conduction block, atrial fibrillation, atrial standstill and Brugada syndrome. The underlying mechanism associated with the genetic mutation was investigated. Methods: Genetic analysis was conducted for probands in the coding and splicing regions of genes susceptible to cardiac arrhythmia diseases. The stable cell lines overexpressing wild-type (WT) or mutant SCN5A gene were generated in HEK293T cells and electrophysiological recordings were performed to evaluate the functional changes of mutant NaV1.5 channels. Results: We identified a rare double R965C and R1309H mutations in NaV1.5 channel, as a heterozygous and compound pattern, locating in the same chromosome. Compared to WT, a single mutation of R965C or R1309H, the compound mutations of R965C-R1309H most dramatically reduced the peak current of NaV1.5 channel with testing potentials ranging from -35 mV to 25 mV. Notably, the maximal peak current of NaV1.5 channel carrying R1309H only and R965C-R1309H mutations displayed significant decreases by 31.5% and 73.34%, respectively, compared to WT. Additionally, compared to single mutations of R965C or R1309H, the R965C-R1309H mutations resulted in more obviously depolarization-shifted activation and hyperpolarization-shifted inactivation, also caused most significantly changed time constant, V1/2 and slope factor of activation and inactivation. Conclusions: The compound mutations of R965C-R1309H led to more dramatically reduced current density of Nav1.5 channel, as well as more depolarization-shifted activation and hyperpolarization-shifted inactivation than single mutation of R965C or R1309H, which may provide more understanding on the molecular mechanisms underlying the familial arrhythmia syndrome. Funding Statement: The study was supported by the Science and Technology Project of Zhuhai (20191210E030072), the Natural Grant from National Natural Science Foundation of China (81870869 and 81870019), the National Key R&D Program of China (2017YFA0104704), the Medical Science and Technology Research Project of Guangdong Province (A2018043), the Science and Technology Program of Guangdong Province (2014B070705005), the General Project of Natural Science Foundation of Guangdong Province (2018A030313554), the Hundred People Plan Scientific Research Launch Project of Sun Yat-sen University. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: This study was approved by the Guangdong Medical Institutional Review Board and Medical Ethics Committees [No.GDREC2016001H (R1)]. Informed written consent was obtained from all patients.

SCN5A variant R222Q generated abnormal changes in cardiac sodium current and action potentials in murine myocytes and Purkinje cells

The cardiac sodium channel (SCN5A) mutation R222Q neutralizes a positive charge in the domain I voltage sensor. Mutation carriers display very frequent ectopy and dilated cardiomyopathy. To describe the effect of SCN5A R222Q on murine myocyte and Purkinje fiber electrophysiology, and identify underlying mechanisms. We generated mice carrying humanized wild-type (H) and mutant (RQ) SCN5A channels. We characterized whole-heart and isolated ventricular and Purkinje myocyte properties. RQ/RQ mice were not viable. INa from RQ/H ventricular myocytes displayed increased "window current" and hyperpolarizing shifts in both inactivation and activation compared to H/H, as previously reported in heterologous expression systems. Surprisingly, action potentials were markedly abbreviated in RQ/H myocytes (action potential durations at 90% repolarization: 12.6± 1.3 ms vs 29.1 ± 1.0 ms in H/H, P < .01, n = 10 each). We identified a large [K+]o-dependent outward gating pore current in RQ/H but not H/H myocytes, and decreasing [K+]o elicited early afterdepolarizations (EADs) and triggered activity in isolated myocytes and ectopic beats in whole hearts. Further, RQ/H Purkinje cells displayed striking, consistent low-voltage EADs. Invivo, however, RQ/H mice displayed little ectopy and contractile function was normal. While SCN5A R222Q increases plateau inward sodium current, action potentials were unexpectedly shortened, likely reflecting an outward gating-pore current. Low extracellular potassium increased this pore current, and was arrhythmogenic invitro and exvivo.

Enhanced closed-state inactivation of mutant cardiac sodium channels (SCN5A N1541D and R1632C) through different mechanisms

BackgroundSCN5A variants can be associated with overlapping phenotypes such as Brugada syndrome (BrS), sinus node dysfunction and supraventricular tachyarrhythmias. Our genetic screening of SCN5A in 65 consecutive BrS probands revealed two patients with overlapping phenotypes: one carried an SCN5A R1632C (in domain IV-segment 4), which we have previously reported, the other carried a novel SCN5A N1541D (in domain IV-segment 1). ObjectiveWe sought to reveal whether or not these variants are associated with the same biophysical defects. MethodsWild-type (WT) or mutant SCN5A was expressed in tsA201-cells, and whole-cell sodium currents (hNav1.5/INa) were recorded using patch-clamp techniques. ResultsThe N1541D-INa density, when assessed from a holding potential of -150 mV, was not different from WT-INa as with R1632C-INa, indicating that SCN5A N1541D did not cause trafficking defects. The steady-state inactivation curve of N1541D-INa was markedly shifted to hyperpolarizing potentials in comparison to WT-INa (V1/2-WT: –82.3 ± 0.9 mV, n = 15; N1541D: –108.8 ± 1.6 mV, n = 26, P < .01) as with R1632C-INa. Closed-state inactivation (CSI) was evaluated using prepulses of −90 mV for 1460 ms. Residual N1541D-INa and R1632C-INa were markedly reduced in comparison to WT-INa (WT: 63.8 ± 4.6%, n = 18; N1541D: 15.1 ± 2.3%, n = 19, P < .01 vs WT; R1632C: 5.3 ± 0.5%, n = 15, P < .01 vs WT). Entry into CSI of N1541D-INa was markedly accelerated, and that of R1632C-INa was weakly accelerated in comparison to WT-INa (tau-WT: 65.8 ± 7.4 ms, n = 18; N1541D: 13.7 ± 1.1 ms, n = 19, P < .01 vs WT; R1632C: 39.5 ± 2.9 ms, n = 15, P < .01 vs WT and N1541D). Although N1541D-INa recovered from closed-state fast inactivation at the same rate as WT-INa, R1632C-INa recovered very slowly (tau-WT: 1.90 ± 0.16 ms, n = 10; N1541D: 1.72 ± 0.12 ms, n = 10, P = .41 vs WT; R1632C: 53.0 ± 2.5 ms, n = 14, P < .01 vs WT and N1541D). ConclusionsBoth N1541D-INa and R1632C-INa exhibited marked enhancement of CSI, but through different mechanisms. The data provided a novel understanding of the mechanisms of CSI of INa. Clinically, the enhanced CSI of N1541D-INa leads to a severe loss-of-function of INa at voltages near the physiological resting membrane potential (~–90 mV) of cardiac myocytes; this can be attributable to the patient's phenotypic manifestations.

Pathophysiology of R222Q mutant SCN5a channels

Consistent with the role of sodium channels in initiating action potentials throughout myocardial tissue, mutations in SCN5a are associated with arrhythmias and cardiomyopathy. The present studies aim to advance understanding of the mechanism underlying the arrhythmias and dilated cardiomyopathy (DCM) observed in patents with an SCN5a mutation (c.665G>A/R222Q) located in one of the voltage sensor regions. Induced pluripotent stem cells (iPSCs) were generated from a patient heterozygous for the R222Q mutation. Isogenic controls were generated by corrected the mutation. Directed differentiation techniques were used to produce ventricular CMs characterized by the expression of cTnT (Control: 85.10±2.90%, Mutant: 83.14±4.46%) and ventricular-specific MLC2v (Control: 45.62±7.02%, Mutant: 43.92±3.86%). The SCN5a gene undergoes extensive alternative splicing, which is developmentally regulated, resulting in the R222Q loci being expressed exclusively in adult SCN5a mRNA. Consistent with an immature phenotype in our iPSC-derived CMs, about half of the SCN5a mRNA is the adult isoform (Control: 54.40±9.29%, Mutant: 48.45±6.21%, n=4), indicating that ~25% of the SCN5a channels expressed in mutant CMs contain the R222Q mutation. Optical mapping of our 2-dimentional tissue model (monolayers) reveal slower conduction velocities in mutant compared to control monolayers (13.14±0.74cm/s, n=10, vs. 25.09±1.99cm/s, n=7, p<0.05). Treatment with flecainide (10 μM), a sodium channel blocker associated with arrhythmia reduction and DCM reversal in R222Q patients results in conduction slowing (Control: 25.09±1.99cm/s to 18.47±3.20cm/s, n=7, p<0.05, Mutant: 13.14±0.74cm/s to 8.92±0.64cm/s, n=10, p<0.05) and a reduction in maximum capture rates (Control: 3.22±0.14Hz to 1.42±0.13Hz, n=5, p<0.05, Mutant: 3.43±0.18 to 2.38±0.22Hz, n=10, p<0.05). We are currently performing electrophysiological studies to further assess the mechanisms underlying R222Q-associated cardiomyopathy and arrhythmias and advancement of therapeutic options for R222Q patients.

A homozygous SCN5A mutation associated with atrial standstill and sudden death.

Atrial standstill is an arrhythmogenic condition characterized by the absence of spontaneous electrical and mechanical atrial activity or in response to stimulation. There are few reported familial cases which have been associated with SCN5A mutations cosegregating with GJA5 or RYR2; however, isolated SCN5A mutations are rare. The purpose of this study was to determine the clinical and biophysical consequence of a novel SCN5A mutation identified in a family with progressive atrial standstill and sudden death. The family of a sporadic case of congenital atrial standstill underwent genetic screening. Human Embryonic Kidney 293 cells were transfected with wild-type (WT) or mutant SCN5A cDNAs. Biophysical properties were studied using whole-cell using patch clamp methods. A novel homozygous SCN5A mutation, p.V1340L, was identified in the proband and her sister. The proband had complete atrial standstill whereas the sister had partial atrial standstill. Heterozygous mutations were identified in the mother, father, and brother. All three had normal sinus rhythm and were asymptomatic. The mutant Nav1.5(V1340L) reduced Nav1.5 current density as well as showed a depolarizing shift in the voltage-dependent steady-state activation (WT: -35.3 ± 1.62mV; V1340L: -22.4 ± 2.59mV; P=0.001). A homozygous loss-of-function SCN5A mutation likely results in atrial standstill and sudden death due to suppression of initiation of action potential.

Effects of SCN5A mutation on intracellular calcium concentration

Objective: To construct the delQKP1507-1509 mutant of SCN5A channel, and to study effects of this mutation on intracellular calcium concentration. Methods: SCN5A delQKP1507-1509 mutation was engineered using site-directed mutagenesis. Wild-type (WT), mutant (MT) and mixed (the mixture of wild-type and mutant plasmids at a ratio of 1:1) Na v 1.5 plasmids were transfected into human embryonic kidney 293 (HEK293) cells respectively. Intracellular free calcium concentration were determined by laser confocal scanning microscope (LCSM) after the cells loaded with the Ca 2+ indicator dye Fluo-3/AM. Results: Gel electrophoresis and DNA direct sequencing proved a deletion of nine base pairs in the SCN5A gene at position 1507-1509. Protein expression and localization of wild-type and mutant SCN5A gene in HEK293 cells were detected under the LSCM. Fluorescence intensity in wild-type group, mutant group and co-expression group (1:1 expression of WT and MT sodium channel) mimicking the heterozygous state were 79.3683±3.051, 94.6165±8.383, 90.7463±6.421 respectively, and there was no significant difference among the three groups (P>0.05). Conclusions: SCN5A delQKP1507-1509 mutant is successfully constructed and transfected into HEK293 cells. The mutation does not affect calcium concentration and further study is required to describe the possible pathophysiological mechanisms of dilated cardiomyopathy phenotype in sodium channel overlap syndromes caused by this SCN5A delQKP1507-1509 mutation.

Sick sinus syndrome with HCN4 mutations shows early onset and frequent association with atrial fibrillation and left ventricular noncompaction

Familial sick sinus syndrome (SSS) is often attributable to mutations in genes encoding the cardiac Na channel SCN5A and pacemaker channel HCN4. We previously found that SSS with SCN5A mutations shows early onset of manifestations and male predominance. Despite recent reports on the complications of atrial fibrillation (AF) and left ventricular noncompaction (LVNC) in patients with SSS caused by HCN4 mutations, their overall clinical spectrum remains unknown. The purpose of this study was to investigate the clinical and demographic features of SSS patients carrying HCN4 mutations. We genetically screened 38 unrelated SSS families and functionally analyzed the mutant SCN5A and HCN4 channels by patch clamping. We also evaluated the clinical features of familial SSS by a meta-analysis of 48 SSS probands with mutations in HCN4 (n = 16) and SCN5A (n = 32), including previously reported cases, and 538 sporadic SSS cases. We identified two HCN4 and three SCN5A loss-of-function mutations in our familial SSS cohort. Meta-analysis of HCN4 mutation carriers showed a significantly younger age at diagnosis (39.1 ± 21.7 years) than in sporadic SSS (74.3 ± 0.4 years; P <.001), but a significantly older age than in SCN5A mutation carriers (20.0 ± 17.6 years; P = .003). Moreover, HCN4 mutation carriers were more frequently associated with AF (43.8%) and LVNC (50%) and with older age at pacemaker implantation (43.5 ± 22.1 years) than were SCN5A mutation carriers (17.8 ± 16.5 years; P <.001). SSS with HCN4 mutations may form a distinct SSS subgroup characterized by early clinical manifestation after adolescence and frequent association with AF and LVNC.

A novel lamin A/C gene missense mutation (445 V > E) in immunoglobulin-like fold associated with left ventricular non-compaction.

Two LMNA mutations (R644C and R190W) have been associated with familial and sporadic left ventricular non-compaction (LVNC). However, the mechanisms underlying these associations have not been elucidated. Genomic DNA was isolated from peripheral blood leucocytes and analysed by direct sequencing. Human embryonic kidney 293 cells were transfected with either wild type or mutant LMNA and SCN5A for whole-cell patch-clamp experiment and fluorescence microscopy. Point mutation modeling for mutant LMNA was also performed. One novel LVNC-associated mutation (V445E) in β2 sheet of immunoglobulin (Ig)-like fold was found in the proband and his father. We also found that the peak current of sodium channel was markedly reduced in mutant LMNA compared with WT while the activation, inactivation, and recovery curves were not significantly altered. The mutant lamin A/C were aggregated into multiple highlighted particles. Three β sheets and multiple side chains in Ig-like fold were altered due to the replacement of a valine by glutamic acid. Our data associated a novel lamin A/C mutation (V445E) with a sudden death form of familial LVNC. The reduced sodium current in mutant LMNA may account for the advent of malignant ventricular arrhythmias. The altered structures of three β sheets and side chains may partially explain the aggregation of lamin A/C protein subjacent to the nuclear envelope.

Modeling type 3 long QT syndrome with cardiomyocytes derived from patient-specific induced pluripotent stem cells

BackgroundType 3 long QT syndrome (LQT3) is the third most common form of LQT syndrome and is characterized by QT-interval prolongation resulting from a gain-of-function mutation in SCN5A. We aimed to establish a patient-specific human induced pluripotent stem cell (hiPSC) model of LQT3, which could be used for future drug testing and development of novel treatments for this inherited disorder. Methods and resultsDermal fibroblasts obtained from a patient with LQT3 harboring a SCN5A mutation (c.5287G>A; p.V1763M) were reprogrammed to hiPSCs via repeated transfection of mRNA encoding OCT-4, SOX-2, KLF-4, C-MYC and LIN-28. hiPSC-derived cardiomyocytes (hiPSC-CMs) were obtained via cardiac differentiation. hiPSC-CMs derived from the patient's healthy sister were used as a control. Compared to the control, patient hiPSC-CMs exhibited dominant mutant SCN5A allele gene expression, significantly prolonged action potential duration or APD (paced CMs of control vs. patient: 226.50±17.89ms vs. 536.59±37.1ms; mean±SEM, p<0.005), an increased tetrodotoxin (TTX)-sensitive late or persistent Na+ current (control vs. patient: 0.65±0.11 vs. 3.16±0.27pA/pF; n=9, p<0.01), a positive shift of steady state inactivation and a faster recovery from inactivation. Mexiletine, a NaV1.5 blocker, reversed the elevated late Na+ current and prolonged APD in LQT3 hiPSC-CMs. ConclusionsWe demonstrate that hiPSC-CMs derived from a LQT3 patient recapitulate the biophysical abnormalities that define LQT3. The clinical significance of such an in vitro model is in the development of novel therapeutic strategies and a more personalized approach in testing drugs on patients with LQT3.

SCN5A Mutation associated with acute myocardial infarction

Ventricular tachycardia and fibrillation (VT/VF) complicating Brugada syndrome, a genetic disorder linked to SCN5A mutations, and VF complicating acute myocardial infarction (AMI) have both been linked to phase 2 reentry. Because of these mechanistic similarities in arrhythmogenesis, we examined the contribution of SCN5A mutations to VT/VF complicating AMI. Nineteen consecutive patients developing VF during AMI were enrolled. Wild-type (WT) and mutant SCN5A genes were co-expressed with SCN1B in TSA201 cells and studied using whole-cell patch-clamp techniques. One missense mutation (G400A) in SCN5A was detected in a conserved region among the cohort of 19 patients. A H558R polymorphism was detected on the same allele. Unlike the other 18 patients who each developed 1-2 VF episodes during acute MI, the mutation carrier developed six episodes of VT/VF within the first 12 hours. All VT/VF episodes were associated with ST segment changes and were initiated by short-coupled extrasystoles. We describe the first sodium channel mutation to be associated with the development of an arrhythmic storm during acute ischemia. These findings suggest that a loss of function in SCN5A may predispose to ischemia induced arrhythmic storm. These results could be very useful for forensic implications regarding genetic screening in relatives.

Dual Variations in SCN5A and CACNB2b Underlie Cardiac Conduction Disease without Brugada Syndrome

Introduction: Inherited loss of function mutations in SCN5A, the gene that encodes the α-subunit of the human cardiac sodium channel (hNav1.5), have been linked to overlapping syndromes including cardiac conduction disease (CCD) and Brugada syndrome (BrS). The mechanisms responsible for the development of one without the other are poorly understood.Methods: Direct sequencing analysis was performed in a family with CCD. Wild type (WT) and variant channels were co-expressed with CD8 cDNA in TSA201 cells for electrophysiological study. Green fluorescent protein (GFP)-fused WT or mutant SCN5A genes were used for confocal microscopy to assess channel trafficking.Results: A novel SCN5A missense mutation, P1008S, was identified in all family members displaying 1st degree AV block, but not in unaffected family members nor in 430 reference alleles. Peak P1008S current was 11.77% of WT (p<0.001). Confocal microscopy showed that WT channels tagged with GFP were localized on the cell surface, whereas GFP-tagged P1008S channels remained trapped in intracellular organelles. P1008S current and trafficking could be rescued by incubation at room temperature, but not by incubation with mexiletine (300μM) at 37°C. We also identified a novel polymorphism (D601E) in CACNB2b. The variation in the β subunit of the calcium channel caused a slowing of inactivation of the L-type calcium channel current (ICa), significantly increasing total charge, when co-expressed with the α1 and α2δ subunits of the calcium channel in TSA201 cells.Conclusions: Our results suggest that variations leading to a loss of function in INa coupled with a gain of function in ICa may underlie the development of cardiac conduction disease without Brugada syndrome.

Therapeutic Strategies for Long-QT Syndrome

The discovery of genetic defects underlying long-QT syndrome (LQTS) has allowed to identify important genotype-phenotype correlations that are now being used for risk stratification. The next challenge is to exploit the new information on the pathophysiology of the disease derived from molecular genetics to devise more effective therapies. The successful response of LQT1 patients to β-blockers, the QT-shortening action of sodium channel blockers in at least some LQT3 patients, and the importance of maintaining adequate plasma potassium levels in LQT2 patients clearly demonstrate the importance of selecting therapy in the context of the molecular substrate. The hurdles on the road toward the development of novel therapeutic strategies are represented by the variable expressivity of the disease, the high prevalence of “private” mutations, and the role of genetic and nongenetic factors that act as modifiers of the phenotype. Given the large number of mutations identified and their phenotypic complexity, it is clearly impossible to anticipate a scenario in which each mutation will be managed through a specific therapy. However, the evidence that mutations in the LQTS gene may be clustered based on their electrophysiological profile and on their response to specific drugs may provide the rationale for the development of mutation-specific therapies. In this article, we will review the most relevant genotype-phenotype features of LQTS and the strategies explored to develop novel therapeutic approaches. ### Definition and Prevalence The congenital LQTS is an inherited arrhythmogenic disease characterized by abnormally prolonged QT interval leading to life-threatening arrhythmias in the presence of a structurally normal heart.1 Different clinical variants of LQTS have been identified. Romano Ward syndrome, transmitted as an autosomal dominant trait, is the most common form of LQTS and presents only a cardiac phenotype. Less prevalent variants of LQTS, such as Andersen and Timothy syndromes or the recessive Jervell and Lange-Nielsen syndromes, also present …

Novel mutation in the SCN5A gene associated with arrhythmic storm development during acute myocardial infarction

Ventricular tachycardia (VT) and ventricular fibrillation (VF) complicating Brugada syndrome, a genetic disorder linked to SCN5A mutations, and VF complicating acute myocardial infarction (AMI) both have been linked to phase 2 reentry. Given the mechanistic similarities in arrhythmogenesis, the purpose of this study was to examine the contribution of SCN5A mutations to VT/VF complicating AMI. Nineteen consecutive patients developing VF during AMI were enrolled in the study. Wild-type (WT) and mutant SCN5A genes were coexpressed with SCN1B in TSA201 cells and studied using whole-cell patch clamp techniques. Among the cohort of 19 patients, one missense mutation (G400A) in SCN5A was detected in a conserved region. An H558R polymorphism was detected on the same allele. Unlike the other 18 patients, who each developed 1-2 VF episodes during AMI, the mutation carrier developed six episodes of VT/VF within the first 12 hours. All VT/VF episodes were associated with ST-segment changes and were initiated by short-coupled extrasystoles. Flecainide and adenosine challenge performed to unmask Brugada and long QT syndromes both were negative. Peak G400A and G400A+H558R current were 70.7% and 88.4% less than WT current at -35 mV (P </=.001). G400A current decay was accelerated and steady-state inactivation was shifted -6.39 mV (V(1/2) = -98.9 +/- 0.1 mV vs -92.5 +/- 0.1 mV, P </=.001). No mutations were detected in KCNH2, KCNQ1, KCNE1, or KCNE2 in the G400A patient. We describe the first sodium channel mutation to be associated with the development of an arrhythmic storm during acute ischemia. These findings suggest that a loss of function in SCN5A may predispose to ischemia-induced arrhythmic storm.

Novel SCN5A mutation (Q55X) associated with age-dependent expression of Brugada syndrome presenting as neurally mediated syncope

An association between Brugada syndrome and neurally mediated syncope has been described. Although mutations in SCN5A have been identified in Brugada syndrome, the genetic link between Brugada syndrome and neurally mediated syncope has not been determined. The purpose of the study was to clinically and genetically characterize a man with recurrent syncope that originally was diagnosed as neurally mediated syncope at age 8 years but subsequently manifested as Brugada syndrome at age 17 years. The proband underwent clinical examination, which included head-up tilt test, sodium channel provocation test, and electrophysiologic study. Genetic screening of SCN5A was performed for the proband and his family members. The biophysical properties of a mutant SCN5A channel in a heterologous expression system were studied using whole-cell, patch clamp technique. The proband showed positive head-up tilt test, coved-type ST elevation recorded from the third intercostal space, and positive pilsicainide provocation test. Ventricular fibrillation was inducible at programmed electrical stimulation, consistent with characteristics of both Brugada syndrome and neurally mediated syncope. A novel nonsense SCN5A mutation (Q55X) was identified in the proband, his mother, and his asymptomatic brother. The heterologously expressed mutant channel was nonfunctional. We genetically determined an SCN5A mutation in a patient showing the combined phenotype of neurally mediated syncope and Brugada syndrome. Neurally mediated syncope and Brugada syndrome may share, at least in part, a common pathophysiologic mechanism.

SIDS: genetic and environmental influences may cause arrhythmia in this silent killer

In this issue of the JCI, Bowers et al. show that the common polymorphism of the cardiac voltage-gated sodium channel, type Valpha (SCN5A), designated S1103Y, found in African Americans is associated with an increased risk of sudden infant death syndrome (SIDS). Wild-type and mutant SCN5A channels both functioned typically under normal conditions in vitro, but exposure to acidic intracellular pH levels such as those found in respiratory acidosis--a known risk factor for SIDS--produced abnormal gain-of-function late reopenings of S1103Y channels, behavior that is often associated with cardiac arrhythmias. These pathologic late reopenings were suppressed by low levels of the channel-blocking drug mexiletine. These findings provide an excellent illustration of a causal relationship between the interaction of the environment and genetic background in SIDS and also raise interesting questions about the linkage of a genetic abnormality with a clinical phenotype.