The adoption of human-relevant New Approach Methodologies for non-clinical safety assessment has accelerated following recent regulatory changes such as the FDA Modernisation Act 2.0. In cardiac safety evaluation, assessing only individual ion channels like hERG can overlook integrated electrophysiological mechanisms that drive proarrhythmic risk. Parameters derived from action potential (AP) waveform analysis, including triangulation and engagement of repolarisation reserve, provide additional insight into these effects. This study evaluated whether optical voltage imaging of human iPSC-derived cardiomyocytes (hiPSC-CMs) can detect compound-induced electrophysiological changes beyond hERG inhibition and link waveform alterations to underlying ion channel pharmacology.
Using fluorescence-based voltage recordings from synchronously beating hiPSC-CM monolayers, we measured AP waveform parameters following exposure to compounds with selective or multichannel cardiac activity. Triangulation showed clear compound- and concentration-dependent modulation consistent with known pharmacology. Selective hERG blockade by dofetilide caused marked increases in triangulation, while multichannel compounds such as quinidine exhibited broader effects consistent with reduced repolarisation reserve. Calcium channel blockers produced minimal changes except at high concentrations. Lower-risk hERG inhibitors, including moxifloxacin, induced limited triangulation compared with dofetilide. Overall, AP waveform analysis in hiPSC-CMs enabled mechanistic differentiation of multichannel electrophysiological effects and offers a translational tool for human-relevant cardiac safety testing.
