Novel Storm-Based Quantitative Assessment of Relative Protein Localization Reveals New Role for Sodium Channel β1 Subunit in Cardiac Conduction

Abstract

We recently demonstrated that cardiac sodium channels (Nav1.5) localized within gap junction (GJ) -adjacent microdomains within the intercalated disc (ID) may enable ephaptic coupling between cardiac myocytes. Further we demonstrated conduction slowing and elevated arrhythmia risk secondary to disruption of close membrane apposition (<10nm) within these microdomains. Confocal micrographs of guinea pig ventricular myocardium revealed expression of β1 (SCN1b), a sodium channel auxiliary subunit and cell adhesion molecule, throughout the interplicate regions of the ID. Therefore, we hypothesized that β1-mediated adhesion may be key to close membrane apposition within Nav1.5-rich ID microdomains and thereby, to ephaptic coupling. In order to assess β1-localization within the ID, we analyzed STORM images of guinea pig ventricular sections immunolabeled for β1 along with Cx43 and N-cadherin (N-Cad) using custom algorithms. Overall, 16±2% of Cx43 clusters overlapped with a β1 cluster while 30±3% had a β1 cluster <200nm away, with a median distance of 55±6nm between Cx43 and β1. In contrast, only 7±3% of N-Cad overlapped with β1 while 16±2% had β1 <200nm away (p<0.05 vs. Cx43-β1), with a median distance of 154±98nm between N-Cad and β1. These data suggest preferential β1 localization adjacent Cx43 GJ relative (interplicate ID regions) to N-cadherin (plicate ID regions). Next we examined β1-mediated adhesion using electric cell-substrate impedance spectroscopy (ECIS): 1610 cells heterologously overexpressing β1 revealed 3-fold higher barrier function relative to native 1610 cells. Further, βadp1, a peptide mimetic of the β1 adhesion domain, inhibited barrier function in dose dependent fashion in β1-overexpressing 1610 cells but not in native 1610 cells. Taken together our results support a role for β1-mediated adhesion in modulating ephaptic coupling in the heart and highlight it as a potential target for novel anti-arrhythmic therapy.