0019 A human cardiomyocyte-based platform for the profiling of drug- induced effects on cardiac contractility: Predicting inotropic mechanisms of action Najah Abi-Gerges, Tim Indersmitten, Ky Truong, William Nguyen, Ismael Tapia, Nathalie Nguyen, Paul E. MIller, Andrea Ghetti AnaBios Corporation, San Diego, CA, United States of America Drug-induced effects on cardiac contractility can lead to serious adverse events including heart failure and therefore limit the utility of innovative treatments. We sought to develop a human cardiomyocyte contractility assay that has the potential to simultaneously predict drug-induced inotropic risk and generate multi-parameter data to profile different inotropic mechanisms of action. Adult human primary cardiomyocytes from ethically consented organ donors were used to measure contractility transients using an imaging-based platform (IonOptix). We tracked changes in contractility parameters to infer both drug-induced inotropic effect (sarcomere shortening) as well as the mechanisms of action based on cluster analysis of a set of 11 contractility parameters. We addressed the clinical relevance of this approach using a panel of 26 inotropes (17 positive, 9 negative) covering diverse mechanisms of action. Each inotrope was tested separately at multiple concentrations. All 17 positive and 9 negative inotropes exerted concentration-dependent increases and decreases in sarcomere shortening, respectively. For example, a 316% increase and 93% decrease in sarcomere shortening occurred with isoprotere- nol (non-selective β-adrenoceptor agonist) and ryanodine (ryanodine receptor inhibitor, IC 50 = 0.16 μM) at top test concentrations of 0.03 μM and 10 μM, respectively. Very distinct changes to the kinetics of the contractility transient were found to be associated with positive and negative inotropes. Interestingly, the multi-parametric readout allowed for the differentiation of drugs operating via distinct mechanisms. Hierarchical clustering of contractility transient param- eters, coupled with principal component analysis, enabled the classification of subsets of both positives as well as negative inotropes, in a mechanism-related mode. This approach enables the identifica- tion of the inotropic potential of novel molecules and facilitates informed mechanistic-based decision making and risk management at the preclinical stage. doi:10.1016/j.vascn.2019.05.019 0020 Development of a new CiPA-compliant MOA in vitro assay on stem cell-derived cardiomyocytes including automated data analysis and model parameter optimization Stefan Mann a , Juliane Heide a , Razvan Airini b,c , Florin Epureanu b,c , Alexandru Deftu b,c , Antonia Teona Deftu b,c , Beatrice Mihaela Radu b,c , Thomas Knott d , Bogdan Amuzescu b,c a Cytocentrics Bioscience GmbH, Cologne, Germany b Department of Biophysics & Physiology, Faculty of Biology, University of Bucharest, Bucharest, Romania c Life, Environmental and Earth Sciences Division, Research Institute of the University of Bucharest (ICUB), Bucharest, Romania d CytoBioScience Inc., San Antonio, TX, United States of America The Cardiac in vitro Proarrhythmia Assay (CiPA) aims to improve proarrhythmogenic risk prediction. The elements (1) ion channel inhibition, (2) action potential (AP) modeling, and (3) human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) surrogate AP must be calibrated before first-in-human (FIH) trials. Missing link is a patch-clamp mechanism-of-action (MOA) assay to calibrate ion channel data with hiPSC-CM surrogate AP. We aim to develop a new complex automated patch-clamp assay on hiPSC-CM combining voltage and current-clamp protocols, fast parameter optimization of a modified O'Hara-Rudy 2011 (ORd) model, and generation of a waveform representing the sum of ion currents inhibited by a drug during externally paced AP to be used as stimulus file in a dynamic-clamp-type protocol in order to restore the original AP shape distorted by that drug. Experiments were performed with CytoPatch™2 automated patch-clamp equipment on Cor.4 U® cardiomyocytes (Ncardia), using physiological solutions and a well- defined combination of voltage-clamp and current-clamp protocols. We analyzed voltage-clamp data with an automated script and obtained similar current density distribution histograms of fast voltage-dependent Na + current (I Na ), L-type Ca 2+ current (I CaL ), delayed rectifier K + current (I K ), transient outward K + current (I to ) and “funny” current (I f ) for n = 42 Cor.4 U® cells approached via classical (ruptured) whole-cell patch-clamp and n = 28 Cor.4 U® hiPSC-CM approached via β-escin perforated patch-clamp. The percentage of change in time was reduced by up to one order of magnitude for I CaL , I K and I to , and AP shape was more stable for escin- perforated experiments. Via Na + substitution with choline we proved the identity of I Na , and with specific blockers nifedipine, ZD7288, cisapride and chromanol 293B we suppressed the corre- sponding current components. We developed a fast parameter optimization algorithm for a modified ORd model including temper- ature dependence of gating for multiple currents (from own experiments) and a 4-state circular allosteric model of I f (Männikkö et al. 2005) and applied it in sequential or parallel variants, obtaining accurate fits of experimentally recorded AP. Our results prove that β- escin perforated patch-clamp experiments on Cor.4 U® hiPSC-CM offer sufficient stability of ion currents and AP shape for CiPA- compliant pharmacology experiments including dynamic-clamp protocols. doi:10.1016/j.vascn.2019.05.020 0021 Physiologic I Kr augmentation in human-derived stem cell cardiomyocytes for improved cardiotoxicity drug screening Mark W. Nowak a , Brian K. Panama b , Sanjot Singh a , Qiuming Gong c , Randall L. Rasmusson b , Zhengfeng Zhou c , Glenna C.L. Bett b a Cytocybernetics, Pendelton, NY, United States of America b Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY, United States of America c Ohio Health and Science University, Portland, OR, United States of America Identifying block of I Kr by candidate drugs is an important step in drug safety screening. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) allow I Kr to be studied in a cardiomyo- cyte-specific environment, but the system presents experimental challenges. Our objective was to develop a stable and physiologically- relevant assay for determining I Kr drug block in hiPSC-CMs and the effect of the drug on the action potential (AP). We examined I Kr in hiPSC-CMs (iCell, Cellular Dynamics), using voltage clamp and stimulated APs. We used the Cybercyte dynamic clamp system to record stable hiPSC-CM APs with physiological resting potentials. We Abstracts 8