Coupling of USP10 de-ubiquitination and chaperone-mediated autophagy causes cardiac sodium channel degradation and cardiac arrhythmias

SCN5A encodes cardiac sodium channel Na_v1.5 that maintains normal electrophysiological functions of hearts. Loss-of-function variants of Na_v1.5 reduce sodium current densities (I_Na) and cause arrhythmias such as cardiac conduction block or Brugada syndrome. The regulatory mechanisms of Na_v1.5 functions are not fully understood. The aim of this study was to identify novel proteins that interact with Na_v1.5 and characterize their regulatory mechanisms on Na_v1.5 and arrhythmias.

GST pull-down coupled with mass spectrometry, co-immunoprecipitation, and mutational analysis were used to identify de-ubiquitinating enzyme USP10 as a novel Na_v1.5-interacting protein, and showed that USP10 reduces Na_v1.5 protein expression and I_Na densities in vitro. AAV9-mediated cardiac overexpression of USP10 in mice reduced Na_v1.5 protein expression, I_Na and I_Ca-L densities, shortened APD, and caused delayed ventricular activation, spontaneous atrioventricular conduction block, sinus pause, and ventricular tachycardia induced with electrical pacing. Cardiac knockdown of USP10 in Scn5a^+/− mice restored Na_v1.5, I_Na, and I_Ca-L to levels comparable to wild-type mice, and alleviated the conduction delay and premature ventricular contractions. Mechanistically, USP10 increased Na_v1.5 protein degradation through chaperone-mediated autophagy (CMA) as the effect was blocked by lysosome inhibitor CQ and inhibition of CMA using siRNA targeting LAMP2A or HSC70, but not by proteasomal inhibitor MG132. Mutational analysis identified the key CMA degradation motif of Na_v1.5 as EKRFQ^431–435. USP10 decreased Na_v1.5 ubiquitination and increased binding of Na_v1.5 to HSC70. Mutational analysis identified K⁴³⁰ of Na_v1.5 as the USP10 de-ubiquitination site, and K430R mutation blocked regulation of Na_v1.5 by USP10.

We identified a novel CMA-mediated pathway regulating degradation of Na_v1.5 by coupling with USP10-mediated de-ubiquitination at K⁴³⁰ of Na_v1.5, which resulted in reduced I_Na densities and cardiac conduction defects. Knockdown of USP10 alleviated arrhythmias in Scn5a^+/− mice, providing a novel therapeutic strategy for treating arrhythmias with reduced I_Na.