Toward animal-free cardiac safety testing: Early detection of cardiotoxic reactions via multimodal in vitro readouts

INTRODUCTION: As regulatory agencies such as the FDA move toward phasing out animal testing in preclinical studies, there is a growing demand for human-relevant, cell-based models for cardiac safety assessment. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a powerful tool for evaluating genotype-specific cardiotoxicity, particularly in individuals with inherited arrhythmia syndromes such as long QT syndrome (LQTS). However, the predictive accuracy of these models remains limited by variability across different assay platforms. OBJECTIVES: This study aimed to systematically characterize quinidine-induced cardiotoxicity in control and LQTS hiPSC-CMs across three complementary assay platforms and to determine whether label-free mechanical phenotyping provides earlier and more sensitive markers of toxicity than conventional electrophysiology alone. METHODS: We systematically evaluated quinidine, a well-known QT-prolonging agent, using three complementary techniques-multielectrode array (MEA), patch clamp, and digital holographic imaging (DHI)-in control and LQTS hiPSC-CMs. MEA and patch clamp were used to assess field potentials and action potentials, whereas DHI was employed to quantify contractile dynamics, including relaxation and resting periods, under matched experimental conditions. RESULTS: MEA and patch clamp detected concentration-dependent electrical abnormalities, including action potential prolongation and early afterdepolarizations, whereas DHI quantified contractile dysfunction in LQTS samples at concentrations as low as 1 µM, significantly prolonging the relaxation period (+32.0 ± 4.6%) and shortening the resting period (-67.0 ± 1.2%) versus baseline (p < 0.001). These mechanical changes remained detectable when MEA signals were lost or patch-clamp traces became unstable, indicating that DHI yields an earlier, more sensitive marker of quinidine-induced cardiotoxicity. CONCLUSION: Integrating label-free mechanical phenotyping with conventional electrophysiology improves early detection of drug-induced cardiotoxicity. As the field advances toward more ethical and predictive approaches, multimodal hiPSC-CM platforms will be essential for advancing in vitro cardiac safety testing.