0235 : In vivo overexpression of a cardiac sodium channel mutant in mice

Loss-of-function mutations in the cardiac Na+ channel α-subunit gene, SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by ventricular fibrillation and sudden cardiac death. We previously evidenced, in HEK cells, the dominant-negative effect of the R104W BrS mutation in Nav1.5, inducing the retention of the wild-type (WT) channel and the proteasomal degradation of the mutant protein. To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAV).We used a dual AAV vector strategy combining viral DNA recombination and trans-splicing. One-week old mice were injected with two AAV serotypes capsid 9: one, packaging the cardiac specific troponin-T promoter, the 5’ half of hSCN5A, the 5’ donor site of a synthetic intron and a highly recombinogenic sequence; and another, packaging the same recombinogenic sequence, the 3’ acceptor site of the synthetic intron, the 3’ half of hSCN5A, the gfp gene as a reporter, and the SV40 polyA signal. Six weeks after injection, the hSCN5A full-gene expression and the percentage of transduced cardiac cells were assessed by qPCR, western blot (WB) analysis and immunohistochemistry on transduced heart tissues. The Na+ current was recorded by the patchclamp technique in isolated cardiomyocytes.Both WT and mutant human Nav1.5 transcripts and proteins were observed by RT-qPCR, WB and immunohistochemistry on injected-mice heart tissues. Patch-clamp recordings in WT-channel injected mice evidenced a two-fold increase of the Na+ current. In contrast, the cardiac Na+ current of R104Winjected mice was impaired (i.e. the current density was decreased by 45% and the activation was shifted by -4mV).Our data suggest that the trans-splicing and viral DNA recombination strategy using AAV9 serotype and a cardiac-specific promoter is successful to overexpress WT or mutant Na+ channels in mouse hearts. This approach allowed us to modulate the cardiac Na+ current in adult mice