FHF2 phosphorylation and regulation of native myocardial Na_V1.5 channels

Abstract

Phosphorylation of the cardiac Na_V1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of Na_V1.5 by phosphorylation of its accessory proteins remains elusive. Using a phosphoproteomic analysis of Na_V channel complexes purified from mouse left ventricles, we identified nine phosphorylation sites on Fibroblast growth factor Homologous Factor 2 (FHF2). To determine the roles of phosphosites in regulating Na_V1.5, we developed two models from neonatal and adult mouse ventricular cardiomyocytes in which FHF2 expression is knockdown and rescued by WT, phosphosilent or phosphomimetic FHF2-VY. While the increased rates of closed-state and open-state inactivation of Na_V channels induced by the FHF2 knockdown are completely restored by the FHF2-VY isoform in adult cardiomyocytes, sole a partial rescue is obtained in neonatal cardiomyocytes. The FHF2 knockdown also shifts the voltage-dependence of activation towards hyperpolarized potentials in neonatal cardiomyocytes, which is not rescued by FHF2-VY. Parallel investigations showed that the FHF2-VY isoform is predominant in adult cardiomyocytes, while expression of FHF2-VY and FHF2-A is comparable in neonatal cardiomyocytes. Similar to WT FHF2-VY, however, each FHF2-VY phosphomutant restores the Na_V channel inactivation properties in both models, preventing identification of FHF2 phosphosite roles. FHF2 knockdown also increases the late Na⁺ current in adult cardiomyocytes, which is restored similarly by WT and phosphosilent FHF2-VY. Together, our results demonstrate that ventricular FHF2 is highly phosphorylated, implicate differential roles for FHF2 in regulating neonatal and adult mouse ventricular Na_V1.5, and suggest that the regulation of Na_V1.5 by FHF2 phosphorylation is highly complex.