Molecular mechanisms of Brugada syndrome related to p.R211H mutation in RRAD gene

Abstract

Brugada syndrome (BrS) is a rare inherited arrhythmic disorder with increased risk of ventricular fibrillation and sudden death. More than 20% of BrS cases have been linked to mutations in the SCN5A gene, which encodes the cardiac voltage-gated sodium channel Nav1.5. We have previously identified a novel variant (p.R211H) in RRAD gene that co-segregates with a familial form of BrS. RRAD encodes the Rad (Ras associated with diabetes) small monomeric G protein. We have shown that p.R211H RRAD variant induces a reduction of the Na+ current amplitude, cytoskeleton anomalies and morphological defects in cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM). Our objective is to identify the mechanisms linking RRAD mutation to those abnormalities in iPSC-CMs of the index case having the p.R211H mutation, compared to an intrafamilial control, and a knock-in (KI) mouse model carrying the equivalent (p.R210H) mutation. Western Blot and Immunostaining were performed from mouse cardiomyocytes and iPS-CMs. Homozygous KI mice (12 weeks old) showed longer QRS complex duration (12.3 ± 0.2 ms; n = 22) than wildtype (WT; 11.1 ± 0.2 ms; n = 13; P < 0.01) and heterozygous KI (11.7 ± 0.2 ms; n = 22; ns), a defect persisting in 30-week-old mice and exacerbated after ajmaline injection. Protein quantification in the left ventricle showed a lower expression of Nav1.5 (by 49%; P < 0.01) and Connexin 43 (Cx43; by 34%; P = 0.06) in 30-week-old homozygous KI mice compared to WT mice. Rad expression was also decreased by 33% ( P < 0.05). Similar results were obtained in the BrS index case iPSC-CMs, in which preliminary immunostainings also suggest an altered location of Rad and Cx43, compared to control iPSC-CMs. To conclude, p.R211H mutation of RRAD gene affects Rad expression, and leads to a decrease of Nav1.5 and Cx43 expression, explaining both sodium current lower amplitude and conduction defects.