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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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Aims Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. Methods and results Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. Conclusion Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases. [ABSTRACT FROM AUTHOR]

Mutations in voltagegated Naα channels have been linked to several channelopathies leading to a wide variety of diseases including cardiac arrhythmias, epilepsy, and myotonia. We have previously demonstrated that voltage- gated Na± channel (Nav)1.5 trafficking-deficient mutant channels could lead to a dominant negative effect by impairing trafficking of the wild-type (WT) channel. We also reported that voltage-gated Na± channels associate as dimers with coupled gating properties. Here, we hypothesized that the dominant negative effect of mutant Na± channels could also occur through coupled gating. This was tested using cell surface biotinylation and single channel recordings to measure the gating probability and coupled gating of the dimers. As previously reported, coexpression of Nav1.5-L325R with WT channels led to a dominant negative effect, as reflected by a 75% reduction in current density. Surprisingly, cell surface biotinylation showed that Nav1.5- L325R mutant is capable of trafficking, with 40% of Nav1.5-L325R reaching the cell surface when expressed alone. Importantly, even though a dominant negative effect on the Na± current is observed when WT and Nav1.5-L325R are expressed together, the total Nav channel cell surface expression was not significantly altered compared with WT channels alone. Thus, the trafficking deficiency could not explain the 75% decrease in inward Na± current. Interestingly, single channel recordings showed that Nav1.5-L325R exerted a dominant negative effect on the WT channel at the gating level. Both coupled gating and gating probability of WT:L325R dimers were drastically impaired. We conclude that dominant negative suppression exerted by Nav1.5 mutants can also be caused by impairing the WT gating probability, a mechanism resulting from the dimerization and coupled gating of voltage-gated Na± channel α-subunits. [ABSTRACT FROM AUTHOR]