Abstract 10687: Structural Remodeling of Na V 16 Channel Clusters Contributes to Arrhythmogenic Late Sodium Current in the Presence of D96V Mutant Calmodulin

Abstract

Calmodulin facilitates normal sodium channel (Na V ) inactivation, necessary to prevent proarrhythmic late sodium current (I Na ). Previous studies have implicated Na V 1.6 dysfunction in late I Na -mediated arrhythmias. Therefore we investigated Na V dysregulation by arrhythmogenic CaM mutant D96V. Chinese hamster ovary cells expressing Na V 1.6 were transfected with either WT- or D96V-CaM. STED microscopy revealed enlarged Na V 1.6 clusters in cells receiving D96V-CaM relative to WT. Therefore we explored Na V 1.6 remodeling in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V). Confocal microscopy confirmed expression of Na V 1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RyR2 in cD96V hearts, consistent with T-tubular localization. STORM microscopy enabled the quantitative assessment of Na V 1.6 clustering. In both WT and cD96V hearts, ~50% of Na V 1.6 clusters localized <100 nm from RyR2. However, Na V 1.6 density within these regions increased 67% in cD96V relative to WT. We probed the functional impact of this surprising remodeling using SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings. The number of NaVs as well as frequency of late Na V 1.6 burst openings were significantly increased in cD96V myocytes relative to WT (Fig 1). Previous work implicates aberrant late Na V 1.6 activity in proarrhythmic Ca2+ mishandling. Indeed, STORM revealed significant enrichment of NCX near Na V 1.6 in both WT and cD96V. Consistent with these data cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. This effect was reversed by cardiac-specific Na V 1.6 knockout. To our knowledge, this is the first report of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, which may shed mechanistic insight into calmodulinopathy.