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Brugada Syndrome (BrS) is a rare inherited cardiac arrhythmia causing potentially fatal ventricular tachycardia or fibrillation, mainly occurring during rest or sleep in young individuals without heart structural issues. It increases the risk of sudden cardiac death, and its characteristic feature is an abnormal ST segment elevation on the ECG. While BrS has diverse genetic origins, a subset of cases can be conducted to mutations in the SCN5A gene, which encodes for the Nav1.5 sodium channel. Our study focused on three novel SCN5A mutations (p.A344S, p.N347K, and p.D349N) found in unrelated BrS families. Using patch clamp experiments, we found that these mutations disrupted sodium currents: p.A344S reduced current density, while p.N347K and p.D349N completely abolished it, leading to altered voltage dependence and inactivation kinetics when co-expressed with normal channels. We also explored the effects of mexiletine treatment, which can modulate ion channel function. Interestingly, the p.N347K and p.D349N mutations responded well to the treatment, rescuing the current density, while p.A344S showed a limited response. Structural analysis revealed these mutations were positioned in key regions of the channel, impacting its stability and function. This research deepens our understanding of BrS by uncovering the complex relationship between genetic mutations, ion channel behavior, and potential therapeutic interventions.

Introducción: el Síndrome de Brugada es una canalopatía en los canales de sodio que dificulta el transporte de sodio a la membrana celular, produce arritmias ventriculares y en el peor de los casos la muerte súbita cardíaca. El gen más influyente es el SCN5A. Objetivos: el objetivo de este trabajo es el análisis bibliográfico sobre las estrategias de prevención, diagnóstico precoz y tratamiento del Síndrome de Brugada. Resaltar los genes influyentes en el síndrome, importancia del factor genético, determinar etnia, edad y género más afectados. Metodología: se realizó una revisión sistemática en diferentes bases de datos como PubMed, SCOPUS y Web of Science (WOS). De los 2.259 artículos resultantes, se han seleccionado 18. Resultados: la prevalencia del síndrome de Brugada es de 0’01-0’05% a nivel mundial, un 40% de los enfermos padecen de eventos arrítmicos. El diagnóstico se realiza mediante la identificación de complejos QRS fragmentados, ECG espontáneo de tipo I, historia familiar de muerte súbita cardíaca y padecimiento de síncopes prediagnóstico. El tratamiento es farmacológico mediante antiarrítmicos o el uso de un desfibrilador automático implantable cuando el riesgo de muerte súbita cardíaca es elevado (más de 1’2% de posibilidades por año). Hay >20 genes asociados con esta enfermedad, pero el más común es el SCN5A que se encuentra presente en un 11-30% de los casos. El síndrome de Brugada es una enfermedad autosómica dominante con patrón hereditario. La etnia más afectada es la del sudeste asiático, la edad y el sexo es en varones menores de 40 años. Conclusión: la evidencia muestra unas consecuencias fatales en el caso de no estar tratado ni diagnosticado el síndrome ya que el primer síntoma puede ser la muerte súbita, de ahí la importancia de un diagnóstico precoz y tratamiento adecuado, Introdución: a Síndrome de Brugada é unha canalopatía nas canles de sodio que dificulta o transporte de sodio cara a membrana celular, produce arritmias ventriculares e no peor dos casos a morte súbita cardíaca. O xene máis influente en dita patoloxía é o SCN5A. Obxectivos: o obxectivo deste traballo é a análise bibliográfica sobre as estratexias de prevención, diagnóstico precoz e tratamento da Síndrome de Brugada. Resaltar os xenes influentes na síndrome, importancia do factor xenético, determinar etnia, idade e xénero máis afectados. Metodoloxía: realizouse unha revisión sistemática en diferentes bases de datos como PubMed, SCOPUS e Web of Science (WOS). Dos 2.259 artigos resultantes, seleccionáronse 18. Resultados: a prevalencia da síndrome de Brugada é de 0’01-0’05% a nivel mundial, un 40% dos enfermos padecen de eventos arrítmicos. O diagnóstico realízase mediante a identificación de complexos QRS fragmentados, ECG espontáneo de tipo I, historia familiar de morte súbita cardíaca e padecemento de síncopes prediagnóstico. O tratamento é farmacolóxico mediante antiarrítmicos ou o uso dun desfibrilador automático implantable cando o risco de morte súbita cardíaca é elevado (máis de 1’2% de posibilidades por ano). Hai >20 xenes asociados con esta enfermidade, pero o máis común é o SCN5A que se atopa presente nun 11-30% dos casos. A síndrome de Brugada é unha enfermidade autosómica dominante con patrón hereditario. A etnia máis afectada é a do sueste asiático, a idade e o sexo é en varóns menores de 40 años. Conclusión: a evidencia mostra unhas consecuencias fatais no caso de non estar tratada nin diagnosticada a síndrome xa que o primeiro síntoma pode ser a morte súbita, de aí a importancia dun diagnóstico precoz e tratamento axeitado, Introduction: Brugada syndrome is a canalopathy in sodium channels that makes it difficult to transport of sodium to the cell membrane, produces ventricular arrhythmias and in the worst of cases sudden cardiac death. The most influential gene is SCN5A. Objectives: the objective of this work is the bibliographic analysis on the strategies of prevention, early diagnosis and treatment of Brugada syndrome. Highlight the influential genes in the syndrome, importance of the genetic factor, determine ethnicity, age and most affected gender. Methodology: a systematic review was carried out in different databases as PubMed, SCOPUS and Web of Science (WOS). Of the resulting 2,259 articles, 18 have been selected. Results: Brugada syndrome worldwide prevalence is 0.01-0.05%, 40% of patients suffer from arrhythmic events. Diagnosis is made by identifying fragmented QRS complexes, spontaneous type I ECG, history of sudden cardiac death families, and prediagnostic syncope disease. The treatment is pharmacological by using antiarrhythmics or the use of an implantable cardioverter defibrillator when the risk of sudden cardiac death is high (more than 1.2% of chances per year). There are >20 genes associated with this disease, but the most common is SCN5A, which is present in 11-30% of cases. Brugada syndrome is an autosomal dominant disease with hereditary pattern.The most affected ethnic group is Southeast Asian people, age and sex is in males under 40 years. Conclusion: the evidence shows fatal consequences in the case of not being treated or diagnosed the syndrome since the first symptom may be sudden death, hence the importance of early diagnosis and appropriate treatment

In the past 20 years, genetic variants in SCN5A encoding the cardiac voltage-gated sodium channel Nav1.5 have been linked to a range of inherited cardiac arrhythmias: variants resulting in loss-of-function of Nav1.5 have been linked to sick sinus syndrome, atrial stand still, atrial fibrillation (AF) impaired pulse generation, progressive and non-progressive conduction defects, the Brugada Syndrome (BrS), and sudden cardiac death. SCN5A variants causing increased sodium current during the plateau phase of the cardiac action potential is associated with Long QT Syndrome type 3 (LQTS3), Torsade de Pointes ventricular tachycardia and SCD. Recently, gain-of-function variants have been linked to complex electrical phenotypes, such as the Multifocal Ectopic Purkinje-related Premature Contractions (MEPPC) syndrome. MEPPC is a rare condition characterized by a high burden of premature atrial contractions (PACs) and/or premature ventricular contractions (PVCs) often accompanied by dilated cardiomyopathy (DCM). MEPPC is inherited in an autosomal dominant fashion with an almost complete penetrance. The onset is often in childhood. The link between SCN5A variants, MEPPC and DCM is currently not well understood, but amino acid substitutions resulting in gain-of-function of Nav1.5 or introduction of gating pore currents potentially play an important role. DCM patients with a MEPPC phenotype respond relatively poorly to standard heart failure medical therapy and catheter ablation as the PVCs originate from all parts of the fascicular Purkinje fiber network. Class 1c sodium channel inhibitors, notably flecainide, have a remarkable positive effect on the ectopic burden and the associated cardiomyopathy. This highlights the importance of genetic screening of DCM patients to identify patients with SCN5A variants associated with MEPPC. Here we review the MEPPC phenotype, MEPPC-SCN5A associated variants, and pathogenesis as well as treatment options.

In the past 20 years, genetic variants in SCN5A encoding the cardiac voltage-gated sodium channel Nav1.5 have been linked to a range of inherited cardiac arrhythmias: variants resulting in loss-of-function of Nav1.5 have been linked to sick sinus syndrome, atrial stand still, atrial fibrillation (AF) impaired pulse generation, progressive and non-progressive conduction defects, the Brugada Syndrome (BrS), and sudden cardiac death. SCN5A variants causing increased sodium current during the plateau phase of the cardiac action potential is associated with Long QT Syndrome type 3 (LQTS3), Torsade de Pointes ventricular tachycardia and SCD. Recently, gain-of-function variants have been linked to complex electrical phenotypes, such as the Multifocal Ectopic Purkinje-related Premature Contractions (MEPPC) syndrome. MEPPC is a rare condition characterized by a high burden of premature atrial contractions (PACs) and/or premature ventricular contractions (PVCs) often accompanied by dilated cardiomyopathy (DCM). MEPPC is inherited in an autosomal dominant fashion with an almost complete penetrance. The onset is often in childhood. The link between SCN5A variants, MEPPC and DCM is currently not well understood, but amino acid substitutions resulting in gain-of-function of Nav1.5 or introduction of gating pore currents potentially play an important role. DCM patients with a MEPPC phenotype respond relatively poorly to standard heart failure medical therapy and catheter ablation as the PVCs originate from all parts of the fascicular Purkinje fiber network. Class 1c sodium channel inhibitors, notably flecainide, have a remarkable positive effect on the ectopic burden and the associated cardiomyopathy. This highlights the importance of genetic screening of DCM patients to identify patients with SCN5A variants associated with MEPPC. Here we review the MEPPC phenotype, MEPPC-SCN5A associated variants, and pathogenesis as well as treatment options.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch-clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.

Genetic testing in Brugada syndrome (BrS) is still not considered to be useful for clinical management of patients in the majority of cases, due to the current lack of understanding about the effect of specific variants. Additionally, family history of sudden death is generally not considered useful for arrhythmic risk stratification. We sought to demonstrate the usefulness of genetic testing and family history in diagnosis and risk stratification. The family history was collected for a proband who presented with a personal history of aborted cardiac arrest and in whom a novel variant in the SCN5A gene was found. Living family members underwent ajmaline testing, electrophysiological study, and genetic testing to determine genotype-phenotype segregation, if any. Patch-clamp experiments on transfected human embryonic kidney 293 cells enabled the functional characterization of the SCN5A novel variant in vitro. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.5000T>A (p.Val1667Asp) in the SCN5A gene, and demonstrate its segregation with a severe form of BrS and multiple sudden deaths. Functional data revealed a loss of function of the protein affected by the variant. These results provide the first disease association with this variant and demonstrate the usefulness of genetic testing for diagnosis and risk stratification in certain patients. This study also demonstrates the usefulness of collecting the family history, which can assist in understanding the severity of the disease in certain situations and confirm the importance of the functional studies to distinguish between pathogenic mutations and harmless genetic variants.

Aims : Brugada syndrome (BrS) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia/fibrillation (VT/VF) in young, otherwise healthy individuals. Despite SCN5A being the most commonly known mutated gene to date, the genotype-phenotype relationship is poorly understood and remains uncertain. This study aimed to elucidate the genotype-phenotype correlation in BrS. Methods and results: Brugada syndrome probands deemed at high risk of future arrhythmic events underwent genetic testing and phenotype characterization by the means of epicardial arrhythmogenic substrate (AS) mapping, and were divided into two groups according to the presence or absence of SCN5A mutation. Two-hundred probands (160 males, 80%; mean age 42.6 ± 12.2 years) were included in this study. Patients harbouring SCN5A mutations exhibited a spontaneous type 1 pattern and experienced aborted cardiac arrest or spontaneous VT/VF more frequently than the other subjects. SCN5A-positive patients exhibited a larger epicardial AS area, more prolonged electrograms and more frequently observed non-invasive late potentials. The presence of an SCN5A mutation explained >26% of the variation in the epicardial AS area and was the strongest predictor of a large epicardial area. Conclusion : In BrS, the genetic background is the main determinant for the extent of the electrophysiological abnormalities. SCN5A mutation carriers exhibit more pronounced epicardial electrical abnormalities and a more aggressive clinical presentation. These results contribute to the understanding of the genetic determinants of the BrS phenotypic expression and provide possible explanations for the varying degrees of disease expression.

The SCN5A gene encodes the alpha-subunit of the voltage-gated cardiac sodium channel (Na(V)1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.

Acute cardiac hypoxia produces life-threatening elevations in late sodium current (ILATE) in the human heart. Here, we show the underlying mechanism: hypoxia induces rapid SUMOylation of NaV1.5 channels so they reopen when normally inactive, late in the action potential. NaV1.5 is SUMOylated only on lysine 442, and the mutation of that residue, or application of a deSUMOylating enzyme, prevents hypoxic reopenings. The time course of SUMOylation of single channels in response to hypoxia coincides with the increase in ILATE, a reaction that is complete in under 100 s. In human cardiac myocytes derived from pluripotent stem cells, hypoxia-induced ILATE is confirmed to be SUMO-dependent and to produce action potential prolongation, the pro-arrhythmic change observed in patients.

Acute cardiac hypoxia produces life-threatening elevations in late sodium current (ILATE) in the human heart. Here, we show the underlying mechanism: hypoxia induces rapid SUMOylation of NaV1.5 channels so they reopen when normally inactive, late in the action potential. NaV1.5 is SUMOylated only on lysine 442, and the mutation of that residue, or application of a deSUMOylating enzyme, prevents hypoxic reopenings. The time course of SUMOylation of single channels in response to hypoxia coincides with the increase in ILATE, a reaction that is complete in under 100 s. In human cardiac myocytes derived from pluripotent stem cells, hypoxia-induced ILATE is confirmed to be SUMO-dependent and to produce action potential prolongation, the pro-arrhythmic change observed in patients.

Background Dysfunction of Na(V)1.5 encoded by SCN5A accounts for approximately half of the channelopathic SIDS cases. We investigated the functional effect of two gene variants identified in the same patient, one in SCN5A and one in SCN1Bb. The aim of the study was to risk stratify the proband's family. Methods The family was referred for cardiovascular genetic evaluation to assess familial risk of cardiac disease. Functional analysis of the identified variants was performed with patch-clamp electrophysiology in HEK293 cells. Results A 16-month-old healthy boy died suddenly in the context of nonspecific illness and possible fever. Postmortem genetic testing revealed variants in the SCN5A and SCN1Bb genes. The proband's father carries the same variants but is asymptomatic. Electrophysiological analysis of the Na(V)1.5_1281X truncation revealed complete loss-of-function of the channel. Coexpression of Na(V)1.5 with Na(V)1b significantly increased I-Na density when compared to Na(V)1.5 alone. The Na(V)1b _V268I variant abolished this I-Na density increase. Moreover, it shifted the activation curve toward more depolarized potentials. Conclusions Genetic variation of both sodium channel and its modifiers may contribute to sudden unexplained death in childhood. However, the asymptomatic father suggests that genetic variation of these genes is not sufficient to cause sudden death or clinically detectable SCN5A phenotypes

A variety of life-threating arrhythmias are caused by mutations in the cardiac voltage-gated sodium channel encoded by the SCN5A gene. In this study, we report a novel loss-of-function SCN5A variant, p.lle 343Val (c.4027A>G), identified in a 42-year-old proband who presented with an unusual ECG with abnormal repolarization with biphasic T-waves in anteroseptal leads, persistent atrial fibrillation (AF), intermittent left bundle branch block (LBBB), and reversible cardiomyopathy. The patient did not meet the diagnostic criteria for Brugada syndrome, long QT syndrome, or any other known SCN5A-associated phenotype. Characterization of the biophysical properties of the variant by in vitro patch clamp experiments revealed a reduced Na+ current with no effect on the inactivation kinetics of the channel. This lossof-function of Na+ current could explain the intermittent LBBB as well as the AF. In conclusion, we describe a unique combination of electrical and structural abnormalities associated with a novel SCN5A variant. Our findings broaden the spectrum of cardiac phenotypes associated with SCN5A channelopathy, underlining the complex clinical manifestations of genetic variations within this gene. (C) 2018 S. Karger AG, Basel

Aims: To clarify the clinical characteristics and outcomes of children with SCN5A-mediated disease and to improve their risk stratification.Methods and results: A multicentre, international, retrospective cohort study was conducted in 25 tertiary hospitals in 13 countries between 1990 and 2015. All patients ≤16 years of age diagnosed with a genetically confirmed SCN5A mutation were included in the analysis. There was no restriction made based on their clinical diagnosis. A total of 442 children {55.7% boys, 40.3% probands, median age: 8.0 [interquartile range (IQR) 9.5] years} from 350 families were included; 67.9% were asymptomatic at diagnosis. Four main phenotypes were identified: isolated progressive cardiac conduction disorders (25.6%), overlap phenotype (15.6%), isolated long QT syndrome type 3 (10.6%), and isolated Brugada syndrome type 1 (1.8%); 44.3% had a negative electrocardiogram phenotype. During a median follow-up of 5.9 (IQR 5.9) years, 272 cardiac events (CEs) occurred in 139 (31.5%) patients. Patients whose mutation localized in the C-terminus had a lower risk. Compound genotype, both gain- and loss-of-function SCN5A mutation, age ≤1 year at diagnosis in probands and age ≤1 year at diagnosis in non-probands were independent predictors of CE.Conclusion: In this large paediatric cohort of SCN5A mutation-positive subjects, cardiac conduction disorders were the most prevalent phenotype; CEs occurred in about one-third of genotype-positive children, and several independent risk factors were identified, including age ≤1 year at diagnosis, compound mutation, and mutation with both gain- and loss-of-function.

BackgroundBrugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder.ObjectivesThe objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).MethodsThis study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs.ResultsBrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression.ConclusionsPatient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.

BackgroundBrugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder.ObjectivesThe objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).MethodsThis study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs.ResultsBrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression.ConclusionsPatient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.

Heart rhythms arise from electrical activity generated by precisely timed opening and closing of ion channels in individual cardiac myocytes. Opening of the primary cardiac voltage-gated sodium (NaV1.5) channel initiates cellular depolarization and the propagation of an electrical action potential that promotes coordinated contraction of the heart. The regularity of these contractile waves is critically important since it drives the primary function of the heart: to act as a pump that delivers blood to the brain and vital organs. When electrical activity goes awry during a cardiac arrhythmia, the pump does not function, the brain does not receive oxygenated blood, and death ensues. Perturbations to NaV1.5 may alter the structure, and hence the function, of the ion channel and are associated downstream with a wide variety of cardiac conduction pathologies, such as arrhythmias.

Background Long QT syndrome type 3 (LQT3) is a lethal disease caused by gain-of-function mutations in the SCN5A gene, coding for the alpha-subunit of the sodium channel NaV1.5. Mexiletine is used to block late sodium current and to shorten QT interval in LQT3 patients. Objectives The aim of this study was to determine whether mexiletine prevents arrhythmic events (arrhythmic syncope, aborted cardiac arrest, or sudden cardiac death) in LQT3 patients. Methods The endpoint of this retrospective cohort study, which studied consecutive LQT3 patients who were referred to our center and treated with mexiletine, was to evaluate the antiarrhythmic efficacy of mexiletine by comparing the number of arrhythmic events per patient and the annual rate of arrhythmic events during observation periods of equal duration before and after the beginning of therapy with mexiletine. Results The study population comprised 34 LQT3 patients, 19 (56%) of whom were male. The median age at beginning of treatment with mexiletine was 22 years, and median QTc interval before therapy 509 ms. The median duration of oral mexiletine therapy was 36 months, at an average daily dose of 8 ± 0.5 mg/kg. Mexiletine significantly shortened QTc (by 63 ± 6 ms; p < 0.0001) and reduced the percentage of patients with arrhythmic events (from 22% to 3%; p = 0.031), the mean number of arrhythmic events per patient (from 0.43 ± 0.17 to 0.03 ± 0.03; p = 0.027), and the annual rate of arrhythmic events (from 10.3% to 0.7%; p = 0.0097). Conclusions Besides shortening QTc interval, mexiletine caused a major reduction of life-threatening arrhythmic events in LQT3 patients, thus representing an efficacious therapeutic strategy.

The cardiac voltage-gated sodium channel, Na(V)1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca(2+))/calmodulin(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of Na(V)1.5 function through phosphorylation of multiple residues including S516, T594, and S571, and these phosphorylation events may be important for the genesis of acquired arrhythmias, which occur in heart failure. However, phosphorylation of full-length human Na(V)1.5 has not been systematically analyzed and Na(V)1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human Na(V)1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC-MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of Na(V)1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human Na(V)1.5.

The cardiac voltage-gated sodium channel, Na(V)1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca(2+))/calmodulin(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of Na(V)1.5 function through phosphorylation of multiple residues including S516, T594, and S571, and these phosphorylation events may be important for the genesis of acquired arrhythmias, which occur in heart failure. However, phosphorylation of full-length human Na(V)1.5 has not been systematically analyzed and Na(V)1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human Na(V)1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC-MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of Na(V)1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human Na(V)1.5.

Background-Sick sinus syndrome (SSS) is a common arrhythmia often associated with aging or organic heart diseases but may also occur in a familial form with a variable mode of inheritance. Despite the identifcation of causative genes, including cardiac Na channel (SCN5A), the pathogenesis and molecular epidemiology of familial SSS remain undetermined primarily because of its rarity. Methods and Results-We genetically screened 48 members of 15 SSS families for mutations in several candidate genes and determined the functional properties of mutant Na channels using whole-cell patch clamping. We identifed 6 SCN5A mutations including a compound heterozygous mutation. Heterologously expressed mutant Na channels showed loss-of-function properties of reduced or no Na current density in conjunction with gating modulations. Among 19 family members with SCN5A mutations, QT prolongation and Brugada syndrome were associated in 4 and 2 individuals, respectively. Age of onset in probands carrying SCN5A mutations was signifcantly less (mean±SE, 12.4±4.6 years; n=5) than in SCN5A-negative probands (47.0±4.6 years; n=10; P<0.001) or nonfamilial SSS (74.3±0.4 years; n=538; P<0.001). Meta-analysis of SSS probands carrying SCN5A mutations (n=29) indicated profound male predominance (79.3%) resembling Brugada syndrome but with a considerably earlier age of onset (20.9±3.4 years). Conclusions-The notable pathophysiological overlap between familial SSS and Na channelopathy indicates that familial SSS with SCN5A mutations may represent a subset of cardiac Na channelopathy with strong male predominance and early clinical manifestations., Circulation: Arrhythmia and Electrophysiology, 7(3), pp.511-517; 2014

Background The debate on the diagnostic value of high intercostal spaces (ICSs) and of the number of diagnostic leads in Brugada syndrome (BrS) has been settled by a recent expert consensus statement. Objective To test the validity, and the underlying anatomy, of the new electrocardiographic (ECG) diagnostic criteria using echocardiographic, molecular, and clinical evidence in 1 clinical study population with BrS. Methods We analyzed 114 patients with BrS and with a spontaneous or drug-induced type 1 ECG pattern recorded in 1 or more right precordial leads in fourth, third, and second ICSs. The right ventricular outflow tract (RVOT) was localized by using echocardiography. All probands were screened on the SCN5A gene. Results The percentage of mutation carriers (MCs) and the event rate were similar regardless of the diagnostic ICS (fourth vs high ICSs: MCs 23% vs 19%; event rate 22% vs 28%) and the number of diagnostic leads (1 vs 2: MCs 20% vs 22%; event rate 22% vs 27%). The concordance between RVOT anatomical location and the diagnostic ICSs was 86%. The percentage of the diagnostic ECG pattern recorded was significantly increased by the exploration of the ICSs showing RVOT by echocardiography (echocardiography-guided approach vs conventional approach 100% vs 43%; P <.001). Conclusion The high ICSs are not inferior to the standard fourth ICS for the ECG diagnosis of BrS, and the interindividual variability depends on the anatomical location of the RVOT as assessed by using echocardiography. This approach significantly increases diagnostic sensitivity without decreasing specificity and fully supports the recently published new diagnostic criteria. © 2014 Heart Rhythm Society.

The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na(+) channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na(+)/H(+) exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Editorial

Background: 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. Objectives: 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. Methods: 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. Results: 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. Conclusion: 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.

Background: 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. Objectives: 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. Methods: 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. Results: 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. Conclusion: 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.

Objectives: The purpose of this study was to identify risk markers in patients with Brugada syndrome. Background: Patients with Brugada syndrome who experience syncope or aborted sudden death are at high risk for recurrent lethal arrhythmias. The prognosis and therapeutic approaches in asymptomatic individuals with a Brugada-type ECG (asymptomatic Brugada syndrome) are controversial. Methods: We genetically screened 30 asymptomatic probands (29 men and 1 woman; mean age 47.1 years) exhibiting a spontaneous Brugada-type ECG. Family members of patients with Brugada syndrome were excluded from the study. Results: Twenty-nine of 30 patients (96.7%) remained symptom-free for at least 3 years. One patient (case 1) with a family history of sudden death died suddenly during sleep. Ventricular fibrillation was induced by programmed electrical stimulation in 14 of 18 subjects (78%), but none of these 18 subjects developed spontaneous ventricular arrhythmias. Genetic screening failed to identify SCN5A mutations in most cases but demonstrated a novel double missense mutation (K1527R and A1569P) located on the same allele in another asymptomatic subject (case 2). Heterologously expressed mutant Na channels exhibited a negative shift of steady-state inactivation (9.2 mV) and enhanced slow inactivation, suggesting this individual harbors a subclinical channel dysfunction compatible with symptomatic Brugada syndrome. Conclusions: Asymptomatic individuals with a Brugada-type ECG generally have a better prognosis than their symptomatic counterparts, but a subgroup of these individuals may have a poor prognosis. Severe Na channel dysfunction as a result of SCN5A mutations may not be sufficient to cause symptoms or arrhythmias in patients with Brugada syndrome, suggesting unknown factors or modifier genes influence arrhythmogenesis.

The novel SCN5A mutation L212P was identified in the proband (age 11 years) and his father. The father was in normal sinus rhythm. The proband had no P waves on surface ECG, and his right atrium could not be captured by pacing. The recombinant L212P Na channel showed a large hyperpolarizing shift in both the voltage dependence of activation (WT: −48.1 ± 0.9 mV; L212P: −63.5 ± 1.5 mV; P < .001) and inactivation (WT: −86.6 ± 0.9 mV; L212P: −95.6 ± 0.8 mV; P < .001) and delayed recovery from inactivation. Further screenings for genetic variations that might mitigate L212P dysfunction revealed that the proband, but not his father, carries Cx40 polymorphisms inherited from his asymptomatic mother.

The novel SCN5A mutation L212P was identified in the proband (age 11 years) and his father. The father was in normal sinus rhythm. The proband had no P waves on surface ECG, and his right atrium could not be captured by pacing. The recombinant L212P Na channel showed a large hyperpolarizing shift in both the voltage dependence of activation (WT: −48.1 ± 0.9 mV; L212P: −63.5 ± 1.5 mV; P < .001) and inactivation (WT: −86.6 ± 0.9 mV; L212P: −95.6 ± 0.8 mV; P < .001) and delayed recovery from inactivation. Further screenings for genetic variations that might mitigate L212P dysfunction revealed that the proband, but not his father, carries Cx40 polymorphisms inherited from his asymptomatic mother.

Objectives: The purpose of this study was to identify risk markers in patients with Brugada syndrome. Background: Patients with Brugada syndrome who experience syncope or aborted sudden death are at high risk for recurrent lethal arrhythmias. The prognosis and therapeutic approaches in asymptomatic individuals with a Brugada-type ECG (asymptomatic Brugada syndrome) are controversial. Methods: We genetically screened 30 asymptomatic probands (29 men and 1 woman; mean age 47.1 years) exhibiting a spontaneous Brugada-type ECG. Family members of patients with Brugada syndrome were excluded from the study. Results: Twenty-nine of 30 patients (96.7%) remained symptom-free for at least 3 years. One patient (case 1) with a family history of sudden death died suddenly during sleep. Ventricular fibrillation was induced by programmed electrical stimulation in 14 of 18 subjects (78%), but none of these 18 subjects developed spontaneous ventricular arrhythmias. Genetic screening failed to identify SCN5A mutations in most cases but demonstrated a novel double missense mutation (K1527R and A1569P) located on the same allele in another asymptomatic subject (case 2). Heterologously expressed mutant Na channels exhibited a negative shift of steady-state inactivation (9.2 mV) and enhanced slow inactivation, suggesting this individual harbors a subclinical channel dysfunction compatible with symptomatic Brugada syndrome. Conclusions: Asymptomatic individuals with a Brugada-type ECG generally have a better prognosis than their symptomatic counterparts, but a subgroup of these individuals may have a poor prognosis. Severe Na channel dysfunction as a result of SCN5A mutations may not be sufficient to cause symptoms or arrhythmias in patients with Brugada syndrome, suggesting unknown factors or modifier genes influence arrhythmogenesis.