RT Journal Article SR Electronic T1 FHF2 phosphorylation and regulation of native myocardial NaV1.5 channels JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.01.31.526475 DO 10.1101/2023.01.31.526475 A1 Adrien Lesage A1 Maxime Lorenzini A1 Sophie Burel A1 Marine Sarlandie A1 Floriane Bibault A1 Dan Maloney A1 Jonathan R. Silva A1 R. Reid Townsend A1 Jeanne M. Nerbonne A1 Céline Marionneau YR 2023 UL http://biorxiv.org/content/early/2023/02/03/2023.01.31.526475.abstract AB Phosphorylation of the cardiac NaV1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of NaV1.5 by phosphorylation of its accessory proteins remains elusive. Using a phosphoproteomic analysis of NaV 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 NaV1.5, 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 NaV 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 NaV 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 NaV1.5, and suggest that the regulation of NaV1.5 by FHF2 phosphorylation is highly complex.eTOC Summary Lesage et al. identify the phosphorylation sites of FHF2 from mouse left ventricular NaV1.5 channel complexes. While no roles for FHF2 phosphosites could be recognized yet, the findings demonstrate differential FHF2-dependent regulation of neonatal and adult mouse ventricular NaV1.5 channels.Competing Interest StatementThe authors have declared no competing interest.AalanineEglutamateFHF2Fibroblast growth factor Homologous Factor 2FHF2-VYisoform VY of Fibroblast growth factor Homologous Factor 2INapeak Na+ currentINaLlate Na+ currentIPimmunoprecipitationmαNaVPANanti-NaV channel subunit mouse monoclonal antibodyMSMass SpectrometryMS1mass spectrum of peptide precursorsMS2 or MS/MSfragmentation mass spectrum of peptides selected in narrow mass range (2 Da) from MS1 scanNaVvoltage-gated Na+ channelpSphosphoserinepTphosphothreonineSserineTthreonineWTWild-Type.