Rapid ionic current phenotyping (RICP) identifies mechanistic underpinnings of iPSC-CM AP heterogeneity

As a renewable, easily accessible, human-derived in vitro model, human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) are a promising tool for studying arrhythmia-related factors, including cardiotoxicity and congenital proarrhythmia risks. An oft-mentioned limitation of iPSC-CMs is the abundant cell-to-cell variability in recordings of their electrical activity. Here, we develop a new method, rapid ionic current phenotyping (RICP), that utilizes a short (10 s) voltage clamp protocol to quantify cell-to-cell heterogeneity in key ionic currents. We correlate these ionic current dynamics to action potential recordings from the same cells and produce mechanistic insights into cellular heterogeneity. We present evidence that the L-type calcium current is the main determinant of upstroke velocity, rapid delayed rectifier K+ current is the main determinant of the maximal diastolic potential, and an outward current in the excitable range of slow delayed rectifier K+ is the main determinant of action potential duration. We measure an unidentified outward current in several cells at 6 mV that is not recapitulated by iPSC-CM mathematical models but contributes to determining action potential duration. In this way, our study both quantifies cell-to-cell variability in membrane potential and ionic currents, and demonstrates how the ionic current variability gives rise to action potential heterogeneity. Based on these results, we argue that iPSC-CM heterogeneity should not be viewed simply as a problem to be solved but as a model system to understand the mechanistic underpinnings of cellular variability. New & Noteworthy We present rapid ionic current phenotyping (RICP), a current quantification approach based on an optimized voltage clamp protocol. The method captures a rich snapshot of ionic currents that provides quantitative information about multiple currents (e.g., ICaL, IKr) in the same cell. The protocol helped to identify key ionic determinants of cellular action potential heterogeneity in iPSC-CMs. This included unexpected results, such as the critical role of IKr in establishing the maximum diastolic potential. ### Competing Interest Statement The authors have declared no competing interest.