In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant

Loss-of-function mutations in the cardiac Na(+) channel α-subunit Na(v)1.5, encoded by SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by sudden cardiac death due to ventricular fibrillation. We previously evidenced in vitro the dominant-negative effect of the BrS Na(v)1.5-R104W variant, inducing retention of wild-type (WT) channels and leading to a drastic reduction of the resulting Na(+) current (I(Na)). To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAVs). METHODS: Due to the large size of SCN5A, a dual AAV vector strategy was used combining viral DNA recombination and trans-splicing. Mice were injected with two AAV serotypes capsid 9: one packaging the cardiac specific troponin-T promoter, the 5′ half of hSCN5A cDNA, a splicing donor site and a recombinogenic sequence; and another packaging the complementary recombinogenic sequence, a splicing acceptor site, the 3′ half of hSCN5A cDNA fused to the gfp gene sequence, and the SV40 polyA signal. Eight weeks after AAV systemic injection in wild-type (WT) mice, echocardiography and ECG were recorded and mice were sacrificed. The full-length hSCN5A-gfp expression was assessed by western blot and immunohistochemistry in transduced heart tissues and the Na(+) current was recorded by the patch-clamp technique in isolated adult GFP-expressing heart cells. RESULTS: Almost 75% of the cardiomyocytes were transduced in hearts of mice injected with hNa(v)1.5 and ∼30% in hNa(v)1.5-R104W overexpressing tissues. In ventricular mice cardiomyocytes expressing R104W mutant channels, the endogenous I(Na) was significantly decreased. Moreover, overexpression of R104W channels in normal hearts led to a decrease of total Na(v)1.5 expression. The R104W mutant also induced a slight dilatation of mice left ventricles and a prolongation of RR interval and P-wave duration in transduced mice. Altogether, our results demonstrated an in vivo dominant-negative effect of defective R104W channels on endogenous ones. CONCLUSION: Using a trans-splicing and viral DNA recombination strategy to overexpress the Na(+) channel in mouse hearts allowed us to demonstrate in vivo the dominant-negative effect of a BrS variant identified in the N-terminus of Na(v)1.5.