Abstract 11138: Superresolution Studies of Sodium Channels Within Intercalated Disk Microdomains Suggest Novel Arrhythmia Mechanism

Abstract

Pore-forming (Nav1.5) and auxiliary (β1; SCN1b) subunits of cardiac sodium channels are enriched at the cardiomyocyte intercalated disk (ID). Mathematical models suggest that this may facilitate conduction via ephaptic mechanisms. We recently demonstrated Nav1.5 enrichment (gSTED superresolution microscopy) and close membrane apposition (<10 nm; electron microscopy) within the perinexus, a microdomain surrounding connexin43 (Cx43) gap junctions (GJ). These data identified the perinexus as a candidate structure for the cardiac ephapse. Further studies using gSTED and STORM superresolution microscopy revealed Nav1.5 and β1 enrichment within ID regions not containing dense clusters of Cx43 and N-Cadherin. Notably, both were identified within the perinexus: Overall, 22% of Nav1.5 & β1 were located within perinexal regions while only 2 and 5% respectively overlapped with Cx43 clusters. Importantly, acute interstitial edema (AIE) increased intermembrane distance at perinexal, but not at non-perinexal sites in adult guinea pig myocardium. Functionally, this correlated with decreased transverse conduction velocity (CV-T; 15.2±0.3 vs. 19.6±0.1cm/s) and increased anisotropic ratio (AR; 3.0±0.2 vs. 2.8±0.1) relative to control, in perfused guinea pig ventricles. Nav1.5 blockade (0.5 μM flecainide) by itself decreased CV (18%) without changing AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T (13.0±0.6cm/s), increased AR (3.3±0.2) and increased spontaneous arrhythmias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model including ephaptic coupling and the ID localization of Nav1.5 could recapitulate these results. Next we investigated the role of β1 in ephaptic coupling: Electrical cell-substrate impedance spectroscopy of 1610 cells heterologously overexpressing β1 revealed 3-fold higher paracellular resistance relative to native 1610 cells. These data along with the known cell adhesion function of β1 in neural tissue suggest that β1-mediated adhesion may facilitate close membrane apposition within the perinexus. Taken together, our results identify β1-mediated adhesion as a novel determinant of anisotropic conduction and potential antiarrhythmic target.