A mechanistic evaluation of sodium channel modulators in hiPSC-derived cardiomyocytes using fluorescence-based sodium flux imaging.

Drug-induced cardiotoxicity remains a significant challenge in pharmaceutical development. While patch-clamp remains the gold standard for evaluating ion channel effects, its low throughput limits early-phase screening. Multi-electrode array (MEA) systems offer higher throughput but often fail to mechanistically distinguish between various sodium channel modulators, such as agonists versus antagonists. The purpose of this study was to develop a cardiac safety assessment method capable of mechanistically distinguishing sodium channel modulators. In this study, we developed a cardiac safety assessment method using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with fluorescence-based intracellular Na+ flux imaging. We characterized hiPSC-CM batches by verifying the expression of key ion channel genes, including SCN5A, ATP1A1, and SLC8A1. Functional responses to sodium channel modulators were evaluated using MEA analysis. Intracellular Na+ intensity changes were quantified using the sodium sensitive dye CoroNa Green. MEA analysis confirmed functional responses to various Nav1.5 inhibitors and activators, though distinguishing specific modes of action was limited. By utilizing the sodium-sensitive dye CoroNa Green, we successfully quantified real-time Na+ intensity changes. Our results demonstrate that Nav1.5 activators (e.g., Aconitine) and Na+/K+-ATPase inhibitors (e.g., Ouabain, Digoxin) significantly increase intracellular Na+ levels at both 1-h and 24-h intervals. This imaging-based approach provides a mechanistic tool for distinguishing sodium channel modulators, offering an alternative for next-generation cardiovascular safety pharmacology.