Ventricular arrhythmias are the major cause of heart failure (HF) -associated deaths. Previous studies demonstrated that HF is associated with raised diastolic Ca2+ (1) and decreased expression and dysfunction of the cardiac voltage-gated sodium channel Nav1.5 (2). These changes may provide both triggers and a slowed-conduction substrate for cardiac arrhythmias (3). However, the mechanisms of Nav1.5 down-regulation in HF have not been fully elucidated. The ubiquitin ligase Nedd4-2, which is well expressed in the heart and activated by elevated calcium concentration, is a major factor for Nav1.5 degradation (4). Thus, we hypothesized that elevated diastolic calcium in HF increases Nedd4-2 expression, leading to reduced membrane density of Nav1.5. Male Sprague-Dawley rats (140-160g, n=12) were used to establish a volume-overload HF model by producing abdominal arterio-venous fistula (5). Western blot and immunofluorescence assay were used to detect the expression and co-localization of Nav1.5 and Nedd4-2 in heart tissues and isolated cardiomyocytes. Cell biology and patch clamp were further performed to investigate the mechanisms of Nav1.5 downregulation in HEK293 cells stably expressing Nav1.5 (Nav1.5-HEK). Values are presented as means ± S.E.M., and one-way ANOVA was used for multiple comparisons. Nav1.5 expression is decreased whereas Nedd4-2 expression is increased in volume-overload HF hearts. These changes are accompanied by increased co-localization of Nav1.5 with ubiquitin or Nedd4-2 in cardiomyocytes. The intracellular calcium concentration ([Ca2+]i) was elevated by ionomycin (IM) treatment. In Nav1.5-HEK cells, 24-hour IM treatment decreased Nav1.5 expression in whole-cell lysates and membrane fractions, and reduced the peak sodium current (INa density: IM 24h -153.30±13.76 vs. Ctrl -318.06±36.49 pA/pF, P<0.01). However, 6-hour IM treatment did not significantly alter total Nav1.5 expression but decreased INa (INa density: IM 6h -134.05±16.74 vs. Ctrl -318.06±36.49 pA/pF, P<0.01). The gating properties of Nav1.5 were not significantly altered by 24-hour or 6-hour IM treatment. Nav1.5 expression and current were decreased by Nedd4-2 transfection and further decreased under 6-hour IM treatment. These effects were not observed in cells transfected with the catalytically inactive mutant, Nedd4-2-C801S. Furthermore, elevated [Ca2+]i increased Nedd4-2 expression and enhanced the interaction between Nav1.5 and Nedd4-2 indicated by co-immunoprecipitation in Nav1.5-HEK cells. In conclusion, Nav1.5 is downregulated and co-localizes with Nedd4-2 and ubiquitin in a volume-over load HF model. Calcium-mediated expression and activation of Nedd4-2 reduces Nav1.5 expression and currents in Nav1.5-HEK cells. These data suggest a role of Nedd4-2 in Nav1.5 downregulation in HF.