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

Abstract

Loss-of-function mutations in the cardiac Na+ channel α-subunit Nav1.5, 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 Nav1.5-R104W variant, inducing retention of wild-type (WT) channels and leading to a drastic reduction of the resulting Na+ current (INa). To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAVs).MethodsDue 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.ResultsAlmost 75% of the cardiomyocytes were transduced in hearts of mice injected with hNav1.5 and ∼30% in hNav1.5-R104W overexpressing tissues. In ventricular mice cardiomyocytes expressing R104W mutant channels, the endogenous INa was significantly decreased. Moreover, overexpression of R104W channels in normal hearts led to a decrease of total Nav1.5 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.ConclusionUsing 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 Nav1.5.

Inherited and Acquired Rhythm Disturbances in Sick Sinus Syndrome, Brugada Syndrome, and Atrial Fibrillation: Lessons from Preclinical Modeling

“…In particular, they systemically injected adeno-associated viral vectors carrying the human mutated SCN5A gene. Overexpression of R104W channels in vivo induced lowered heart rate, dramatic reduction of I Na current, prolonged RR intervals, and longer P-wave duration, indicative again of the dominant-negative effect observed previously in vitro [225].…”

“…Through these different models, it was possible to confirm the dominant-negative effect of this mutation, related to a defective N-terminal. [224,225] Several N-terminal variants, including R104W and R121W, and the mutated protein Y87C were overexpressed in human kidney TsA-201 and COS-7 cell lines. Mutant proteins showed binding to calmodulin (weak for R121W variant) and wild-type Nav1.5 channels.…”

Trafficking and Gating Cooperation Between Deficient Nav1.5-mutant Channels to Rescue INa

“…Unlike potassium channel genes, which encode one fourth of tetramers constituting functional channels, Na v 1.5 channels were thought to be structured as monomers, since the gene encodes the entire 4domain channel α-subunit. It was thus unexpected to report Na v 1.5 mutants with a dominant-negative effect on wildtype (WT) channels, i.e., a decrease of I Na exceeding the 50% of current density expected in case of haploinsufficiency observed when coexpressing some mutants with WT channels in a 1:1 ratio to mimic patient heterozygosity, as we and others did [2][3][4][5][6][7][8][9]. Furthermore, we demonstrated that Na v 1.5 α-subunits could interact with each other and that a trafficking-efficient mutant channel was able to drive a trafficking-deficient one to the surface membrane [3].…”

Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias

“…Some of these channelopathies are widely described in Section 4 of this review. Additionally, several SCN5A missenses can generate dominant-negative variants affecting Na v 1.5 trafficking or gating at the cell surface [39][40][41]. A recent study demonstrated that most of SCN5A missense that generate loss-of-function variants exert a dominant-negative effect that confers a high burden of BrS [42].…”