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Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

Inhibition of the mammalian target of rapamycin (mTOR) with the macrolide rapamycin or pharmacological suppression of KATP channel opening translated to scar expansion of the myocardial infarcted (MI) adult female rodent heart. The present study tested the hypotheses that rapamycin‐mediated scar expansion was sex‐specific and that mTOR signaling directly influenced KATP channel subunit expression/activity. Scar size was significantly larger in post‐MI male rats as compared to the previous data reported in post‐MI female rats. The reported scar expansion of rapamycin‐treated post‐MI female rats was not observed following the administration of the macrolide to post‐MI male rats. Protein levels of the KATP channel subunits Kir6.2 and SUR2A and phosphorylation of the serine2448 residue of mTOR were similar in the normal heart of adult male and female rats. By contrast, greater tuberin inactivation characterized by the increased phosphorylation of the threonine1462 residue and reduced raptor protein levels were identified in the normal heart of adult female rats. Rapamycin pretreatment of phorbol 12,13‐dibutyrate (PDBu)‐treated neonatal rat ventricular cardiomyocytes (NNVMs) suppressed hypertrophy, inhibited p70S6K phosphorylation, and attenuated SUR2A protein upregulation. In the presence of low ATP levels, KATP channel activity detected in untreated NNVMs was significantly attenuated in PDBu‐induced hypertrophied NNVMs via a rapamycin‐independent pathway. Thus, rapamycin administration to post‐MI rats unmasked a sex‐specific pattern of scar expansion and mTOR signaling in PDBu‐induced hypertrophied NNVMs significantly increased SUR2A protein levels. However, the biological advantage associated with SUR2A protein upregulation was partially offset by an mTOR‐independent pathway that attenuated KATP channel activity in PDBu‐induced hypertrophied NNVMs. [ABSTRACT FROM AUTHOR]

Phosphorylation of the cardiac Na V 1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of Na V 1.5 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 V 1.5, 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 V 1.5, and suggest that the regulation of Na V 1.5 by FHF2 phosphorylation is highly complex., Etoc Summary: Lesage et al . identify the phosphorylation sites of FHF2 from mouse left ventricular Na V 1.5 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 Na V 1.5 channels.