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

Millet, H.; Belhumeur, P.; Hivonnait, A.; Bibault, F.; Dereli, M.; Tessier, A.; Erfanian, M.; Ribeiro, B.; Charpentier, F.; Croyal, M.; Montnach, J.; De Waard, M.

doi:10.1101/2025.09.17.676709

bioRxiv · 10.1101/2025.09.17.676709

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

Millet, H.·Belhumeur, P.·Hivonnait, A.·Bibault, F.·Dereli, M.·Tessier, A.·Erfanian, M.·Ribeiro, B.·Charpentier, F.·Croyal, M.·Montnach, J.·De Waard, M.

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Abstract

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.

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Millet, H., Belhumeur, P., Hivonnait, A., Bibault, F., Dereli, M., Tessier, A., Erfanian, M., Ribeiro, B., Charpentier, F., Croyal, M., Montnach, J., De Waard, M.. 2025-09-20. Late INa activation of cardiac TTX-sensitive sodium channels by AaH-II induces an arrhythmogenic phenotype. https://doi.org/10.1101/2025.09.17.676709

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