Selective activation of tetrodotoxin-sensitive sodium channels by AaH-II drives arrhythmogenic late Na⁺ current in the heart

Increased late sodium current (I_NaL) is a central mechanism underlying both inherited and acquired cardiac arrhythmias. Although Na_v1.5 is the dominant cardiac sodium channel, multiple tetrodotoxin-sensitive (TTX-S) Na_v isoforms are also expressed in cardiomyocytes, particularly within transverse tubules, where they influence excitation-contraction coupling. Whether these channels contribute directly to pathological I_NaL and arrhythmogenesis remains unresolved, in part because available pharmacological tools lack isoform selectivity. Herein, our objective was to determine the contribution of TTX-S Na_v channels to pathological cardiac I_NaL and arrhythmia and to establish selective pharmacological tools to dissect their role. Using automated patch-clamp and human Na_v isoform profiling, we show that the reference I_NaL inducer ATX-II predominantly activates TTX-resistant Na_v1.5 Na_v channels. However, ATX-II lacks Na_v isoform selectivity when inappropriately used, questioning the conclusions reached by numerous cardiac I_NaL studies. In contrast, AaH-II, a peptide from Androctonus australis hector scorpion venom, more selectively and potently enhances I_NaL through TTX-S Na_v isoforms. In human iPS-derived cardiomyocytes, TTX-S I_NaL leads to action potential prolongation. In adult ventricular cardiomyocytes, AaH-II induces abnormal Ca²⁺ handling and spontaneous Ca²⁺ release events. In isolated hearts and in vivo, selective TTX-S I_NaL activation produces conduction abnormalities, QT prolongation, and ventricular proarrhythmic events, which are prevented by nanomolar tetrodotoxin concentrations that spare Na_v1.5. These findings demonstrate that TTX-S sodium channels are sufficient to generate arrhythmogenic late Na⁺ current in the heart, independently of Na_v1.5. AaH-II provides a powerful new tool to selectively probe TTX-S I_NaL and reveals these channels as previously underappreciated contributors to cardiac electrical instability. Targeting TTX-S Na_v channels may therefore represent a novel and potentially safer strategy for antiarrhythmic therapy.