Abstract P1092: Voltage Gated Sodium Channel Subunit Mimetic Peptide βadp1 Increases Regulated Intramembrane Proteolysis Of Scn1b/β1

Abstract

Clinical treatment for cardiac arrhythmias based on targeting ion channels is limited and can have deadly side effects. We previously reported that acute administration of βadp1 resulted in decreased action potential conduction velocity and increased arrhythmias in isolated guinea pig hearts. These changes were associated with disruption of trans-homophilic adhesion of the β1 subunit within gap junction-adjacent perinexal domains in intercalated discs. Using Electric Cell-Substrate Impedance Sensing (ECIS), we found that βadp1 shows opposing acute and long-term effects on β1 mediated adhesion. βadp1 decreases β1 mediated adhesion in 1610 cells stably expressing SCN1B/β1 (1610β1) over the first 24 hours, but after 48 hours is associated with increased adhesion. Regulated Intramembrane Proteolysis (RIP) of β1 sequentially by BACE-1 and γ-secretase results in a cleaved intracellular fragment that translocates to the nucleus and alters transcription of various genes, including VGSC subunits. Therefore, we hypothesized the long-term gain-of-adhesion-function with βadp1 treatment resulted from increased RIP of β1, with downstream VGSC gene expression effects. Inhibition of γ-secretase with DAPT accumulates a C-terminal fragment (CTF) of β1 during RIP, enabling quantification of changes to RIP. Thus, 1610β1 cells were treated with 50μM βadp1, 1μM DAPT, 50μM βadp1+1μM DAPT or 0.1% DMSO control. Cells were fixed for β1 immunofluorescence (IF) or lysate was collected at 6-, 24-, and 48-hours post treatments for Western blotting. DAPT treatment significantly increased CTF levels compared to control at all timepoints (p<.0001). However, βadp1+DAPT treatment resulted in greater accumulation of β1 CTF levels over DAPT alone at each timepoint. (n=6, 6 hr: p<.01, 24 hr: p<.01, 48 hr: p<.05). βadp1+DAPT treatment caused a significant increase in full-length β1 at 6 hours, but no difference at 24 or 48 hours. The data suggest depletion of β1 by increased RIP of β1 after βadp1 treatment. IF showed an increase in abundance of β1 48 hours after βadp1 treatment compared to DMSO. In sum, our data indicates that βadp1 may upregulate RIP of β1 with potential downstream effects on gene transcription of VGSC subunits, suggesting a path to a novel class of antiarrhythmics.