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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Doisne, Grauso, Mougenot, Clergue, Souil, Coulombe, Guicheney and Neyroud.)

Background: Genetic variants in voltage-gated sodium channels (Na v ) encoded by SCNXA genes, responsible for I Na , and K v 4.3 channels encoded by KCND3 , responsible for the transient outward current (I to ), contribute to the manifestation of both Brugada syndrome (BrS) and spinocerebellar ataxia (SCA19/22). We examined the hypothesis that K v 4.3 and Na v variants regulate each other's function, thus modulating I Na /I to balance in cardiomyocytes and I Na /I (A) balance in neurons., Methods: Bicistronic and other constructs were used to express WT or variant Na v 1.5 and K v 4.3 channels in HEK293 cells. I Na and I to were recorded., Results: SCN5A variants associated with BrS reduced I Na , but increased I to . Moreover, BrS and SCA19/22 KCND3 variants associated with a gain of function of I to , significantly reduced I Na , whereas the SCA19/22 KCND3 variants associated with a loss of function (LOF) of I to significantly increased I Na . Auxiliary subunits Na v β1, MiRP3 and KChIP2 also modulated I Na /I to balance. Co-immunoprecipitation and Duolink studies suggested that the two channels interact within the intracellular compartments and biotinylation showed that LOF SCN5A variants can increase K v 4.3 cell-surface expression., Conclusion: Na v and K v 4.3 channels modulate each other's function via trafficking and gating mechanisms, which have important implications for improved understanding of these allelic cardiac and neuronal syndromes.