Upstream alternative polyadenylation inSCN5Aproduces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration

Abstract

SUMMARYSCN5Aencodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials.SCN5Agene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the firstSCN5Acoding exon. This yields aSCN5A-shorttranscript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-lengthSCN5AmRNA ratios in failing hearts. Knock-in of the humanSCN5AAPA sequence into mice is sufficient to enable expression ofSCN5A-short transcript, while significantly decreasing expression of full-lengthSCN5AmRNA. Notably,SCN5A-short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation ofSCN5Aproduces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.