Po-04-164 molecular mechanisms of brugada syndrome related to p.r211h mutation in gene

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 novel 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. We have shown that p.R211H RRAD 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 characterize the cardiac phenotype of a knock-in (KI) mouse model carrying the equivalent (p.R210H) mutation and identify the mechanisms linking RRAD mutation to INa decrease and conduction defects. Mouse phenotype was determined using ECG recordings, and patch-clamp technique on ventricular cardiomyocytes. Western blot and immunostaining were performed on mouse ventricular tissue and iPSC-CMs. Co-immunoprecipitation experiments were performed on mouse ventricle and on HEK cells transfected with plasmids encoding Nav1.5 and either wild type or p.R211H-Rad. Homozygous KI mice (12 weeks old) showed longer QRS complexes (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) mice, a defect persisting in older mice. Homozygous KI mice showed a 38% decrease in INa density versus WT mice (P<0.01; n=22 & 19). This was associated with a lower ventricular expression of Nav1.5 (by 49% in left ventricle; P < 0.01) and Connexin 43 (Cx43; by 34%; P = 0.06) in homozygous KI mice versus WT mice. Rad expression was also decreased by 33% (P < 0.05). Similar results were obtained in the BrS index case hiPS-CMs, 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, and of Rad with Cx43. Nav1.5 interaction with Rad has been confirmed in HEK cells, which are used to determine the effect of the mutation on this interaction. To conclude, BrS-related RRAD p.R211H mutation affects Rad expression and leads to a decrease of Nav1.5 and Cx43 cardiac expression, explaining both sodium current lower amplitude and conduction defects.