The Brugada syndrome (BrS) is a rare heritable cardiac arrhythmia disorder associated with ventricular fibrillation and sudden cardiac death. Mutations in the SCN5A gene have been causally related to BrS in 20–30% of cases. Twenty other genes have been described as involved in BrS, but their overall contribution to disease prevalence is still unclear. This study aims to estimate the burden of rare coding variation in arrhythmia-susceptibility genes among a large group of patients with BrS. We have developed a custom kit to capture and sequence the coding regions of 45 previously reported arrhythmia-susceptibility genes and applied this kit to 167 index cases presenting with a Brugada pattern on the electrocardiogram as well as 167 individuals aged over 65-year old and showing no history of cardiac arrhythmia. By applying burden tests, a significant enrichment in rare coding variation (with a minor allele frequency below 0.1%) was observed only for SCN5A, with rare coding variants carried by 20.4% of cases with BrS versus 2.4% of control individuals (P = 1.4 × 10⁻⁷). No significant enrichment was observed for any other arrhythmia-susceptibility gene, including SCN10A and CACNA1C. These results indicate that, except for SCN5A, rare coding variation in previously reported arrhythmia-susceptibility genes do not contribute significantly to the occurrence of BrS in a population with European ancestry. Extreme caution should thus be taken when interpreting genetic variation in molecular diagnostic setting, since rare coding variants were observed in a similar extent among cases versus controls, for most previously reported BrS-susceptibility genes.

Aims: The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. Methods and results: Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. Conclusion: This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6–9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2^{p.Q300*/p.Q300*} in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4–5 weeks, Bcat2^{p.Q300*/p.Q300*} mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs—leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2^{p.Q300*/p.Q300*} mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure.

Selective inhibition of cardiac late sodium current (I_NaL) is an emerging target in the treatment of ventricular arrhythmias. We investigated the electrophysiological effects of GS-458967 (GS967), a potent, selective inhibitor of I_NaL, in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from both human SCN5A-1795insD^+/− induced pluripotent stem cells (hiPSC-CMs) and mice carrying the homologous mutation Scn5a-1798insD^+/−. On patch-clamp analysis, GS967 (300 nmol/l) reduced I_NaL and action potential (AP) duration in isolated ventricular myocytes from wild type and Scn5a-1798insD^+/− mice, as well as in SCN5A-1795insD^+/− hiPSC-CMs. GS967 did not affect the amplitude of peak I_Na, but slowed its recovery, and caused a negative shift in voltage-dependence of I_Na inactivation. GS967 reduced AP upstroke velocity in Scn5a-1798insD^+/− myocytes and SCN5A-1795insD^+/− hiPSC-CMs. However, the same concentration of GS967 did not affect conduction velocity in Scn5a-1798insD^+/− mouse isolated hearts, as assessed by epicardial mapping. GS967 decreased the amplitude of delayed after depolarizations and prevented triggered activity in mouse Scn5a-1798insD^+/− cardiomyocytes. The I_NaL inhibitor GS967 decreases repolarization abnormalities and has anti-arrhythmic effects in the absence of deleterious effects on cardiac conduction. Thus, selective inhibition of I_NaL constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced I_NaL. Our findings furthermore implement hiPSC-CMs as a valuable tool for assessment of novel pharmacological approaches in inherited sodium channelopathies.