Calcium‐dependent Nedd4‐2 upregulation mediates degradation of the cardiac sodium channel Nav1.5: implications for heart failure

Aim Reductions in voltage‐gated sodium channel (Nav1.5) function/expression provide a slowed‐conduction substrate for cardiac arrhythmias. Nedd4‐2, which is activated by calcium, post‐translationally modulates Nav1.5. We aim to investigate whether elevated intracellular calcium ([Ca2+]i) reduces Nav1.5 through Nedd4‐2 and its role in heart failure (HF). Methods Using a combination of biochemical, electrophysiological, cellular andin vivomethods, we tested the effect and mechanism of calcium on Nedd4‐2 and in turn Nav1.5. Results Increased [Ca2+]i, following 24‐h ionomycin treatment, decreased sodium current (INa) density and Nav1.5 protein without altering itsmRNAin both neonatal rat cardiomyocytes (NRCMs) andHEK293 cells stably expressing Nav1.5. The calcium chelatorBAPTA‐AMrestored the reduced Nav1.5 andINainNRCMs pre‐treated by ionomycin. Nav1.5 was decreased by Nedd4‐2 transfection and further decreased by 6‐h ionomycin treatment. These effects were not observed in cells transfected with the catalytically inactive mutant, Nedd4‐2 C801S, or with Y1977A‐Nav1.5 mutant containing the impaired Nedd4‐2 binding motif. Furthermore, elevated [Ca2+]iincreased Nedd4‐2, the interaction between Nedd4‐2 and Nav1.5, and Nav1.5 ubiquitination. Nav1.5 protein is decreased, whereas Nedd4‐2 is increased in volume‐overloadHFrat hearts, with increased co‐localization of Nav1.5 with ubiquitin or Nedd4‐2 as indicated by immunofluorescence staining.BAPTA‐AMrescued the reduced Nav1.5 protein,INaand increased Nedd4‐2 in hypertrophiedNRCMs induced by isoproterenol or angiotensinII. Conclusion Calcium‐mediated increases in Nedd4‐2 downregulate Nav1.5 by ubiquitination. Nav1.5 is downregulated and co‐localizes with Nedd4‐2 and ubiquitin in failing rat heart. These data suggest a role of Nedd4‐2 in Nav1.5 downregulation inHF.