Abstract
BACKGROUND: SCN5A encodes the cardiac NaV1.5 (voltage-gated Na(+) channel), classically known for initiating action potentials and recently implicated in cardiomyocyte metabolism via mitochondrial Na(+)/Ca(2+) exchange. SCN5A variants are linked to arrhythmias and heart failure, but mechanisms controlling SCN5A/NaV1.5 expression and its metabolic interface remain understudied. METHODS: We used bioinformatic approaches to identify novel SCN5A regulatory features and discovered an alternative polyadenylation (APA) signal downstream of exon 2, which is conserved in humans and several other species but not mice. To test its function, we generated knock-in mice harboring the human APA signal. Western blotting, cell fractionation, and fluorescence microscopy were used to characterize the resulting truncated protein isoform that localizes to mitochondria. Mitochondrial functions and metabolites were assessed in neonatal rat cardiomyocytes, human-induced pluripotent stem cell-derived cardiomyocytes, and mouse hearts overexpressing the novel isoform. RESULTS: We identified a well-conserved APA signal downstream of SCN5A exon 2, yielding a truncated transcript isoform (SCN5A-short). Reanalysis of cardiac APA-seq and mRNA-seq data reveals reduced SCN5A-short expression in failing human hearts. Knock-in of the human APA signal into mice enables expression of SCN5A-short while decreasing full-length SCN5A mRNA. SCN5A-short encodes a novel NaV1.5-NT (N-terminal fragment of NaV1.5) that localizes to the mitochondrial matrix in cardiomyocytes and mouse hearts. Exogenous expression of NaV1.5-NT in cultured cardiomyocytes enhances mitochondrial respiration, ATP production, and mitochondrial ROS while depleting NADH. Native polyacrylamide gel electrophoresis analyses indicate that this coincides with enhanced CI (complex I) activities, as well as context-dependent alterations of CV (complex V) assembly. Moreover, moderate cardiomyocyte-targeted NaV1.5-NT expression in mice was sufficient to rewire the cardiac metabolome, with suggestive evidence of increased fatty acid oxidation. CONCLUSIONS: APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT, a novel mitochondrial-targeted peptide that supports cardiomyocyte metabolism. While the precise molecular mechanisms remain unresolved, these findings highlight an unforeseen alternative pathway for expanding SCN5A-mitochondrial crosstalk, with potential implications for metabolic changes in heart failure and arrhythmias.