Simultaneous Optical Imaging of Action Potentials and Calcium Transients in Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Abstract Cardiovascular diseases have emerged as one of the leading causes of human mortality, but the discovery of new drugs has been hindered by the absence of suitable in vitro platforms. In recent decades, continuously refined protocols for differentiating human induced pluripotent stem cells (hiPSCs) into hiPSC‐derived cardiomyocytes (hiPSC‐CMs) have significantly advanced disease modeling and drug screening; however, this has led to an increasing need to monitor the function of hiPSC‐CMs. The precise regulation of action potentials (APs) and intracellular calcium (Ca 2+ ) transients is critical for proper excitation‐contraction coupling and cardiomyocyte function. These important parameters are usually adversely affected in cardiovascular diseases or under cardiotoxic conditions and can be measured using optical imaging–based techniques. However, this procedure is complex and technologically challenging. We have adapted the IonOptix system to simultaneously measure APs and Ca 2+ transients in hiPSC‐CMs loaded with the fluorescent dyes FluoVolt and Rhod 2, respectively. This system serves as a powerful high‐throughput platform to facilitate the discovery of new compounds to treat cardiovascular diseases with the cellular phenotypes of abnormal APs and Ca 2+ handling. Here, we present a comprehensive protocol for hiPSC‐CM preparation, device setup, optical imaging, and data analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Maintenance and seeding of hiPSC‐CMs Basic Protocol 2 : Simultaneous detection of action potentials and Ca 2+ transients in hiPSC‐CMs