Multiparametric cardiomyocyte profiling with single‐LED excitation reveals subtype‐specific electromechanical coupling in LQTS in vitro

Cardiac excitation-contraction coupling (ECC) is an essential and tightly regulated process that links action potentials to intracellular calcium release and mechanical contraction. At the whole-heart level, ECC is captured by integrative evaluation of electrical and mechanical components of the cardiac cycle through parameters termed electromechanical delay (EMD) and electromechanical window (EMW). Alterations in the EMD or EMW reflect impaired electromechanical coupling and have been associated with increased arrhythmic risk across multiple cardiac diseases. However, it is currently unclear whether and how equivalent metrics can be derived and interpreted in vitro or meaningfully applied to patient-specific human disease models. Here, we implement ECC profiling in vitro using a single-LED optical imaging system that enables simultaneous measurements of membrane voltage, intracellular calcium and contractility. This approach allows quantification of individual transients and their temporal relationships. To investigate distinct perturbations of ECC, we applied this platform to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) representing three mechanistically distinct ECC perturbations: a repolarization defect (LQT2, KCNH2 p.A561V), a calmodulin-associated calcium-handling defect (CALM-LQTS, CALM1 p.F142L) and pharmacological contractile uncoupling by myosin ATPase inhibition (blebbistatin). Cellular analogues of the clinical EMD and EMW were robustly quantifiable and provided mechanistically informative ECC signatures that were not entirely captured by conventional metrics focused on asynchronous individual transient analysis. Pharmacological targeting of the main cardiac ion currents further validated these findings and confirmed the robustness of the ECC quantification. This novel technique provides a basis for more sensitive assessment of disease phenotypes, arrhythmogenic risk and pharmacological effects in human cellular models, supporting the development of new approach methodologies. KEY POINTS: A single-LED optical platform enables fully synchronized, simultaneous measurement of voltage, calcium and contraction in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) at a high spatial and temporal resolution. In vitro analogues of the clinical electromechanical delay and electromechanical window can be robustly quantified in hiPSC-CM monolayers and behave consistently with their in vivo counterparts under genetic and pharmacological perturbations. Despite comparable APD80 prolongation, LQT2 and CALM-LQTS hiPSC-CMs display divergent electromechanical signatures, demonstrating that integrative ECC metrics resolve mechanistic differences not captured by conventional single-transient analyses. Pharmacological recapitulation of the disease phenotypes further confirmed these observations. In vitro ECC profiling offers a sensitive approach for distinguishing mechanistic perturbations of cardiac function and improving the characterization of arrhythmogenic risk.