Dominant-Negative Effect of an Brugada Syndrome Variant.

Unlabelled: Loss-of-function mutations in the cardiac Na channel α-subunit Na1.5, encoded by , cause Brugada syndrome (BrS), a hereditary disease characterized by sudden cardiac death due to ventricular fibrillation. We previously evidenced the dominant-negative effect of the BrS Na1.5-R104W variant, inducing retention of wild-type (WT) channels and leading to a drastic reduction of the resulting Na current ( ). To explore this dominant-negative effect , we created a murine model using adeno-associated viruses (AAVs).

Methods: Due to the large size of , a dual AAV vector strategy was used combining viral DNA recombination and -splicing. Mice were injected with two AAV serotypes capsid 9: one packaging the cardiac specific troponin-T promoter, the 5' half of cDNA, a splicing donor site and a recombinogenic sequence; and another packaging the complementary recombinogenic sequence, a splicing acceptor site, the 3' half of cDNA fused to the 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 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 hNa1.5 and ∼30% in hNa1.5-R104W overexpressing tissues. In ventricular mice cardiomyocytes expressing R104W mutant channels, the endogenous was significantly decreased. Moreover, overexpression of R104W channels in normal hearts led to a decrease of total Na1.5 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 dominant-negative effect of defective R104W channels on endogenous ones.

Conclusion: Using a -splicing and viral DNA recombination strategy to overexpress the Na channel in mouse hearts allowed us to demonstrate the dominant-negative effect of a BrS variant identified in the N-terminus of Na1.5.