(1054-C) Multiplexed Cardiomyopathy and Proarrhythmia (Cardiotoxicity) KIC Assay of Human iPSC-Cardiomyocytes
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: We report the first single-cell kinetic (live-cell) functional high throughput screen (HTS) of combined cardiomyopathy of proarrhythmia (cardiotoxicity) on hiPSC-cardiomyocytes with which we correctly identified 30/31 adverse drug reactions (ADRs) during assay development. Cardiotoxicity, which includes arrhythmias and structural cardiomyocyte damage, is a leading cause of drug failures clinically and post-FDA approval. Reports of clinical predictivity of animal studies, which are laborious and expensive, include 84.7% (Valentin, et al., 2022) for arrhythmia; 50% (Ewart, et al., 2014), and 78.5% (Bhatt, et al., 2019) for myopathy and blood pressure; and 57.8% for all cardiac disorders from >70 years of adverse event reports for all FDA approved drugs (Clark, et al., 2018). Human stem cell models derived from induced pluripotent stem cells (hiPSCs) are more species-relevant but have largely failed to achieve both high predictivity of human clinical outcomes and HTS scalability. Most hiPSC-model screens rely on whole-well or whole-field averaged data (e.g., FLIPR, MEA, and Impedance), which average out details such as early and delayed afterdepolarizations (EADs and DADs) and produce more artifacts than single-cell kinetic image cytometry® (KIC®). Having previously published single-cell Kinetic Image Cytometry (KIC) with prediction arrhythmogenic liability accuracy of ~90% using Ca2+ kinetics as a readout (Pfeiffer, et al., 2016), which improved to 95% with deep learning (Serrano, et al., 2022), we added myopathy by multiplexing readouts of Ca2+ (Cal-520), plasma membrane action potentials (V, BeRST 1, AKA, PhoS-VSD-1), contraction (from V and Ca2+ videos), and mitochondrial membrane potential (TMRM), with Hoechst (for nuclei to seed single-cell image segmentation) of metabolically matured hiPSC-CMs. V and Ca2+ were acquired in a single interleaved video at 60 Hz (30 Hz per channel). Also, for the first time, we implemented fully automated single-cell analyses/detection of contraction, early- and delayed afterdepolarizations (EADs and DADs), waveform alternans, tachycardia, and fibrillation. For assay validation, we report results for 31 drugs (effector classes: 6 sarcoplasmic reticulum, 10 plasma membrane ion channel, 8 mitochondrial/myosin, 7 negative reference) screened on combinations of six sources of hiPSC-cardiomyocytes. The data are more complex than we expected, but largely predict clinical outcomes. For example, for omecamtiv mecarbil (which showed no improvement in exercise tolerance and failed FDA approval to treat HF), for which we previously reported substiantial inotropy on isolated canine cardiomyocytes, we first saw (0.625-0.95 μM) prolongation with no change in contraction amplitude, and then decreased amplitude at 2.5 µM (18.6X free effective therapeutic plasma concentration, ETPCf, of Cmax) and arrest at 10 µM (74.3X ETPCf of Cmax). We present multiplexed single-cell cardiomyopathy and proarrhythmia screening as a potentially powerfully predictive tool for reducing animal use and developing drugs with higher margins of safety that are safer and more effective.