Abstracts

This is a Late-Breaking abstract.

Rationale: Dravet syndrome (DS) is an inherited epileptic condition resulting from a loss-of-function in Na_v1.1. DS patients have a disproportionately high risk of SUDEP, though the mechanism remains elusive. Previous studies have suggested that global reduction in Na_v1.6 decreases incidence of seizures and thereby reduces SUDEP. Intriguingly, DS cardiomyocytes exhibit a paradoxical increase in Na⁺ influx. Here we hypothesize that the Dravet associated Na_V1.6 remodeling in cardiac nanodomains results in Na⁺ loading near Na⁺/Ca²⁺ handling machinery, which underlies Ca²⁺ mishandling and arrhythmias that contribute to SUDEP.

Methods: Cardiac-specific NaV1.6 knockout mice (cNaV1.6KO) were obtained by crossing C57BL/6 mice with loxP sites flanking Exon 1 of the Scn8a gene (custom generated by Transgenic and Gene Targeting Core and Mutation Generation and Detection Core at the University of Utah) with transgenic mice harboring Cre under the cardiac specific β-myosin-heavy chain (Myh7) promoter (Tg(Myh7-cre)1Jmk), a generous gift from Dr. Federica Accornero, OSU. These mice were bred into DS mice (129S-Scn1atm1Kea/Mmjax; Cat. #037107-JAX), purchased from Jackson Laboratories (Bar Harbor, ME, USA), to create the DSxcNav1.6KO line.

Results: DS mice (Scn1a^+/-) exhibited similar expression levels of the predominant NaV isoform in the heart, Na_V1.5 relative to wild type (WT); however, expression of Na_V1.6 was increased in DS hearts. To determine localization of Na_v1.6 expression we used stochastic optical reconstruction microscopy (STORM; < 20 nm lateral resolution). STORM demonstrated an increase in fraction of Na_V1.6 clusters near (< 100 nm) Ca²⁺release channels, ryanodine receptor 2 (RyR2) and Na⁺/Ca²⁺ exchange (NCX) within DS transverse (T)-tubules of cardiomyocytes relative to WT. Next, we examined the functional consequences of increased Na_V1.6 clusters near Ca²⁺ handling machinery using scanning ion conductance microscopy (SICM)-guided patch clamp and confocal Ca²⁺ imaging. These approaches revealed an increase in persistent Na⁺ activity within T-tubules in the DS relative to WT, which was coupled to enhanced Ca²⁺ release in the form of Ca²⁺ sparks. On a whole-cell level, DS cardiomyocytes displayed enhanced persistent Na⁺ current coupled with increased frequency of Ca²⁺ waves (Figure 1A). This abnormal Ca²⁺ handling, in turn, translated into an increased arrhythmia burden (Figure 1B) and mortality. Consistent with pathological increase Na_V1.6 in DS, cardiac-specific Na_V1.6 knockout (cNa_V1.6-KO) resulted in a reduction in persistent Na_V activity, arrhythmogenic Ca²⁺ release, and mortality in DS.

Conclusions: Na_V1.6 may play a critical role in sudden cardiac death associated with DS that contributes to SUDEP. Our results suggest, for the first time, that cardiac Na_V1.6 may serve as a druggable target in the heart for arrhythmia and SUDEP prevention.

Funding: This work was supported by NIH grants R01HL155378 and R01NS121234 (Radwański), R01HL148736 (Veeraraghavan), and T32HL149637 (King). National Science Foundation Graduate Research Fellowship, NSF Fellow ID: 2019259354 (Struckman). American Heart Association 19TPA34910191 (Radwański), 20TPA35460040 (Veeraraghavan), 22POST915300 (Tarasov).