Bibault, F. | bioRxiv Science

bioRxiv2025

Late INa activation of cardiac TTX-sensitive sodium channels by AaH-II induces an arrhythmogenic phenotype

AimsLate sodium current (INaL) is a key contributor to cardiac arrhythmias, but its precise origin and arrhythmogenic potential from tetrodotoxin-sensitive (TTX-S) sodium (Nav) channels remain unclear. While the FDA-endorsed toxin ATX-II has been widely used to model INaL-associated arrhythmogenesis, it lacks selectivity, limiting its utility in dissecting the roles of individual Nav channel subtypes. This study investigates the proarrhythmic impact of TTX-S Nav channel activation using AaH-II*, a scorpion venom-derived peptide with selective efficacy for TTX-S channels. Methods and ResultsUsing automated patch-clamp recordings, we characterized AaH-II* selectivity across human Nav isoforms and demonstrated potent, preferential activation of INaL in hNav1.1, 1.2, 1.3, and 1.6 over the TTX-resistant cardiac isoform hNav1.5. Calcium imaging in isolated adult rat cardiomyocytes showed that low nanomolar concentrations of AaH-II* induced spontaneous calcium release events and arrhythmogenic calcium transients, even in the absence of Nav1.5 activation. Ex vivo multielectrode array recordings in Langendorff-perfused rat hearts confirmed dose-dependent ventricular conduction slowing, prolonged repolarization, and increased arrhythmia burden, all mitigated by TTX. In vivo, intravenous AaH-II* administration in rats elicited QTc prolongation, atrioventricular block, and ventricular tachyarrhythmias, which were significantly suppressed by TTX pretreatment. ConclusionWe identify AaH-II* as a powerful and selective tool to study INaL from TTX-S Nav channels in cardiac tissue. Our findings reveal that TTX-S channel-mediated INaL alone is sufficient to induce arrhythmias and that pharmacological inhibition of these channels offers a promising antiarrhythmic strategy. These results advocate for broader consideration of TTX-S Nav channels as targets in arrhythmia research and drug safety screening.

physiology↗

bioRxiv2023

FHF2 phosphorylation and regulation of native myocardial NaV1.5 channels

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 SummaryLesage 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.

physiology↗