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

Abstract

Brugada syndrome (BrS) is a rare inherited arrhythmic disorder with increased risk of sudden cardiac death. More than 20% of BrS cases have been linked to mutations in SCN5A gene, which encodes the voltage-gated Na+ channel Nav1.5. We have previously identified a variant (p.R211H) in RRAD gene that co-segregates with a familial form of BrS. RRAD encodes the Rad (Ras associated with diabetes) monomeric GTPase. RRAD p.R211H variant induces a reduction of the Na+ current (INa) amplitude and morphological defects in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs). To determine the mechanisms linking RRAD variant to INa reduction and the cardiac phenotype of a knock-in (KI) mouse model carrying the equivalent p.R210H variant. Mouse phenotyping was performed using ECG recordings, and patch-clamp technique on ventricular cardiomyocytes. Western blot and immunostaining were performed on mouse ventricular tissue and hiPSC-CMs. Co-immunoprecipitation experiments were performed on mouse ventricle and on HEK cells transfected with plasmids encoding Nav1.5 and either wild type (WT) or p.R211H-Rad. Twelve-week-old homozygous KI mice showed longer QRS complexes (12.3 ± 0.2 ms; n = 22) than WT (11.1 ± 0.2 ms; n = 13; P < 0.01) and heterozygous KI (11.7 ± 0.2 ms; n = 22) mice, a defect persisting in older mice. In homozygous KI mice INa density was decreased by 38% compared to WT mice (P < 0.01; n = 22 & 19). This was linked to a lower ventricular expression of Nav1.5 (by 49% in left ventricle; P < 0.01), Connexin 43 (Cx43; by 34%; P = 0.06) and Rad (by 33%; P < 0.01) in homozygous KI mice versus WT mice. Similar results were obtained in the hiPS-CMs of the BrS index case, in which an altered location of Rad (more nuclear than cytoplasmic) and Cx43 (lower clusters, but of larger size) was also observed, compared to control hiPS-CMs. Coimmunoprecipitation studies on left ventricular tissue of 30-week-old WT mice showed an interaction of Rad with Nav1.5, a result that was confirmed in HEKs, and of Rad with Cx43. Our results suggest that BrS-related RRAD p.R211H variant affects Rad expression and leads to a decrease of Nav1.5 and Cx43 expression, explaining both sodium current lower amplitude and conduction defects. Our results also show that Rad interacts with Nav1.5, and possibly Cx43.