Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Abstract

Late Na+ current ( INaL) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na+ channel 1.5 (Nav1.5), resulting in enhanced INaL. Conversely, an increase of INaL would be expected to cause elevation of intracellular Ca2+ and activation of CaMKII. However, a relationship between enhancement of INaL and activation of CaMKII has yet to be demonstrated. We investigated whether Na+ influx via Nav1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the INaL activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Nav1.5 (but not Nav1.1 or Nav1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Nav1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Nav1.5 mutant (N1325S). The effects of both ATX-II and the N1325S mutation were reversed by either INaL inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Nav1.5 coimmunoprecipitation. The same association between CaMKII and Nav1.5 was also found in N1325S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or INaL also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Nav1.5-dependent increase in Na+ influx leads to activation of CaMKII, which in turn phosphorylates Nav1.5, further promoting Na+ influx. Pharmacological inhibition of either CaMKII or Nav1.5 can ameliorate cardiac dysfunction caused by excessive Na+ influx.