Simultaneous Widefield Voltage and Dye-Free Optical Mapping Quantifies Electromechanical Waves in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

[Image: see text] Coupled electromechanical waves define a heart’s function in health and diseases. Optical mapping of electrical waves using fluorescent labels offers mechanistic insights into cardiac conduction abnormalities. Dye-free/label-free mapping of mechanical waves presents an attractive non-invasive alternative. In this study, we developed a simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used as follows: (1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-cardiomyocytes (CMs); (2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature industrial CMOS cameras; (3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence-imaged) and mechanical (dye-free-imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wavefront tortuosity. During regular pacing, the dye-free-imaged conduction velocity and electrical wave velocity are correlated; both modalities are sensitive to pharmacological uncoupling and dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover the strong frequency dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CMs inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.