The Sodium Channel Auxiliary Subunit β1 is Structurally Critical for Cardiac Conduction: Evidence from the Single Molecule Scale to the Whole Organ

Abstract

Electrical impulse propagation in the heart's ventricles is thought to occur by means of direct flow of electric current from cell to cell via gap junctions (GJs) composed of connexin43 (Cx43). We recently demonstrated that cardiac sodium channels (Nav1.5) localized within the perinexus, an intercalated disk (ID) nanodomain adjacent to Cx43 GJ, may enable ephaptic coupling between cardiac myocytes. We hypothesized that the cell adhesion function of β1 may contribute to close intermembrane apposition within these nanodomains and thereby facilitate ephaptic coupling. Super-resolution STochastic Optical Reconstruction Microscopy-based Relative Localization Analysis (STORM-RLA) identified two populations of NaV1.5 within the ID in guinea pig ventricles: One, localized to the perinexus, within 200nm of Cx43 GJs, accounted for 47% of ID-localized NaV1.5 and the other, which co-distributed with N-Cadherin to plicate ID regions, accounted for 29% of ID-localized NaV1.5. These findings were confirmed by immuno-electron microscopy. Confocal microscopy revealed β1 enrichment at the ID, and suggested preferential localization to GJ-enriched interplicate ID regions. STORM-RLA confirmed the preferential localization of β1 to interplicate ID regions: Perinexal nanodomains accounted for 48% of ID-localized β1, whereas N-Cadherin-rich plicate regions accounted for only 8%. In order to test the role of β1 adhesion in the heart, we developed a novel peptide inhibitor of β1 adhesion - βadp1. In electric cell-substrate impedance spectroscopy studies, it inhibited the barrier function in β1-overexpressing 1610 cells but not in native 1610 cells. In guinea pig ventricles, βadp1 (100 µM) compromised the diffusion-resistance of the ID as assessed by perfusion of fixable fluorescent dyes: A 0.5kD molecule preferentially accumulated within IDs in control hearts but not in βadp1-treated hearts. Perinexal intermembrane spacing measured by transmission electron microscopy was increased in βadp1-treated hearts (48 ± 4 µm) compared to untreated hearts (17± 1µm) and those treated with a scrambled control peptide, βadp1-scr (22 ± 1 µm). Notably, neither peptide affected membrane spacing at other ID locations. In optical mapping studies, βadp1 but not βadp1-scr preferentially decreased transverse conduction velocity, increased anisotropy and precipitated spontaneous tachyarrhythmias in a dose-dependent manner. In conclusion, these results suggest a critical role of β1-mediated adhesion in generating close apposition between NaV1.5-rich perinexal membranes and thereby, facilitating ephaptic conduction in the heart. Further, these data highlight β1-mediated adhesion as a target for novel, mechanistically-driven antiarrhythmic therapy.