Cardiac conduction is an important function of the heart. To date, accurate measurement of conduction velocity (CV) in vitro is hindered by the low spatial resolution and poor signal-to-noise ratio of microelectrode arrays (MEAs), or the cytotoxicity and end-point analysis of fluorescence optical imaging. Here, we have developed a new label-free method based on defocused brightfield imaging to quantify CV by analyzing centroid displacements and contraction trajectories of each cardiomyocyte in a monolayer of human stem cell-derived cardiomyocytes (iPSC-CMs). Our data revealed that the time delay between intracellular calcium release and the initiation of cell contraction is highly consistent across cardiomyocytes; however, the duration a cell takes to reach its maximum beating magnitude varies significantly, proving that the time delay in excitation-contraction coupling is largely constant in iPSC-CMs. Standard calcium imaging of the same iPSC-CM populations (~106 cells) was conducted for comparison with our label-free method. The results confirmed that our label-free method was capable of achieving highly accurate CV mapping (17.64 ± 0.89 cm/s vs. 17.95 ± 2.29 cm/s, p-value>0.1). Additionally, our method effectively revealed various shapes in cell beating pattern. We also performed label-free CV mapping on disease-specific iPSC-CM monolayers with plakophilin-2 (PKP2) knockdown, which effectively quantified their low CV values and further validated the arrhythmogenic role of PKP2 mutation in arrhythmogenic right ventricular cardiomyopathy (ARVC) through the disruption of cardiac conduction. The label-free method offers a cytotoxic-free technique for long-term measurement of dynamic beating trajectories, beating propagation and conduction velocities of cardiomyocyte monolayers.
Keywords: Arrhythmia; Beating trajectories; Conduction velocity; Excitation-contraction coupling; Label-free mapping; iPSC-cardiomyocyte.
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