Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging.

INTRODUCTION The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative seeks an in vitro test to accurately predict clinical Torsades de Pointes (TdP). We developed a cardiotoxicity assay incorporating simultaneous measurement of the action potential (AP) waveform and Ca(2+) transient (CT) in human iPSC-derived cardiomyocytes (CMs). Concurrent optogenetic pacing provided a well-controlled electrophysiological background. METHODS We used the Optopatch platform for all-optical electrophysiology (Hochbaum et al., 2014). In a monolayer culture, a subset of cells expressed a genetically encoded, calcium and voltage reporter, CaViar (Hou, Kralj, Douglass, Engert, & Cohen, 2014), while others expressed a channelrhodopsin variant, CheRiff. Optical pacing of CheRiff-expressing cells synchronized the syncytium. We screened 12 compounds (11 acute, 1 chronic) to identify electrophysiological (AP rise time, AP50, AP90, beat rate) and CT effects in spontaneously beating and paced cultures (1Hz, 2Hz). RESULTS CaViar reported spontaneous and paced APs and CTs with high signal-to-noise ratio and low phototoxicity. Quinidine, flecainide, E-4031, digoxin and cisapride prolonged APs, while verapamil and nifedipine shortened APs. Early after depolarizations (EADs) were elicited by quinidine, flecainide and cisapride. All but four compounds (amiodarone, chromanol, nifedipine, verapamil) prolonged AP rise time. Nifedipine and verapamil decreased CT amplitude, while digoxin increased CT amplitude. Pentamidine prolonged APs after chronic exposure. DISCUSSION The Optopatch platform provides a robust assay to measure APs and CTs in hiPSC-CMs. This addresses the CiPA mandate and will facilitate comparisons of cell-based assays to human clinical data.

[1]  M. Sanguinetti,et al.  A mechanistic link between an inherited and an acquird cardiac arrthytmia: HERG encodes the IKr potassium channel , 1995, Cell.

[2]  Gary Gintant,et al.  Rechanneling the cardiac proarrhythmia safety paradigm: a meeting report from the Cardiac Safety Research Consortium. , 2014, American heart journal.

[3]  Praveen Shukla,et al.  Chemically defined generation of human cardiomyocytes , 2014, Nature Methods.

[4]  Robert Passier,et al.  Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro--correlation between contraction force and electrophysiology. , 2015, Biomaterials.

[5]  Calum A. MacRae,et al.  Wnt11 patterns a myocardial electrical gradient via regulation of the L-type Ca2+ channel , 2010, Nature.

[6]  P. Lipp,et al.  Genetically encoded Ca2+ indicators in cardiac myocytes. , 2014, Circulation research.

[7]  Maurizio Recanatini,et al.  Safety of Non-Antiarrhythmic Drugs that Prolong the QT Interval or Induce Torsade de Pointes , 2002, Drug safety.

[8]  Rene Spijker,et al.  Differentiation of Human Embryonic Stem Cells to Cardiomyocytes: Role of Coculture With Visceral Endoderm-Like Cells , 2003, Circulation.

[9]  Thomas Weiser,et al.  The Electrophysiological Effects of Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and in Guinea Pig Isolated Hearts , 2011, Cellular Physiology and Biochemistry.

[10]  F. Guengerich,et al.  Mechanisms of drug toxicity and relevance to pharmaceutical development. , 2011, Drug metabolism and pharmacokinetics.

[11]  James A Thomson,et al.  Human Embryonic Stem Cells Develop Into Multiple Types of Cardiac Myocytes: Action Potential Characterization , 2003, Circulation research.

[12]  D. T. Yue,et al.  Optical mapping of optogenetically shaped cardiac action potentials , 2014, Scientific Reports.

[13]  T. Baird,et al.  Preclinical QT safety assessment: cross-species comparisons and human translation from an industry consortium. , 2014, Journal of pharmacological and toxicological methods.

[14]  C Antzelevitch,et al.  The potential for QT prolongation and proarrhythmia by non-antiarrhythmic drugs: clinical and regulatory implications. Report on a policy conference of the European Society of Cardiology. , 2000, European heart journal.

[15]  N. Hellen,et al.  Action potential morphology of human induced pluripotent stem cell-derived cardiomyocytes does not predict cardiac chamber specificity and is dependent on cell density. , 2015, Biophysical journal.

[16]  B. Fermini,et al.  Comparative gene expression profiling in human-induced pluripotent stem cell--derived cardiocytes and human and cynomolgus heart tissue. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[17]  R Lazzara,et al.  Multiple mechanisms in the long-QT syndrome. Current knowledge, gaps, and future directions. The SADS Foundation Task Force on LQTS. , 1996, Circulation.

[18]  Nathaniel Huebsch,et al.  Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales. , 2015, Tissue engineering. Part C, Methods.

[19]  Charles E. Leonard,et al.  Cisapride and ventricular arrhythmia. , 2008, British journal of clinical pharmacology.

[20]  S. Yamanaka,et al.  The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells. , 2009, Biochemical and biophysical research communications.

[21]  A. Bruening-Wright,et al.  The action potential and comparative pharmacology of stem cell-derived human cardiomyocytes. , 2010, Journal of pharmacological and toxicological methods.

[22]  C. January,et al.  Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. , 1998, Biophysical journal.

[23]  Gary Gintant,et al.  ILSI-HESI cardiovascular safety subcommittee initiative: evaluation of three non-clinical models of QT prolongation. , 2006, Journal of pharmacological and toxicological methods.

[24]  H. R. Lu,et al.  Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICHS7B Guidelines , 2008, British journal of pharmacology.

[25]  Harold Bien,et al.  Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution. , 2006, Progress in biophysics and molecular biology.

[26]  H. Himmel Drug-induced functional cardiotoxicity screening in stem cell-derived human and mouse cardiomyocytes: effects of reference compounds. , 2013, Journal of pharmacological and toxicological methods.

[27]  Icilio Cavero,et al.  Comprehensive in vitro Proarrhythmia Assay, a novel in vitro/in silico paradigm to detect ventricular proarrhythmic liability: a visionary 21st century initiative , 2014, Expert opinion on drug safety.

[28]  Robert W. Mills,et al.  Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor , 2014, Stem cell reports.

[29]  H. Ahammer,et al.  Optical multisite monitoring of cell excitation phenomena in isolated cardiomyocytes , 1995, Pflügers Archiv.

[30]  S. Nattel,et al.  Ranolazine: Ion‐channel‐blocking actions and in vivo electrophysiological effects , 2004, British journal of pharmacology.

[31]  International Conference on Harmonisation; guidance on S7B Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals; availability. Notice. , 2005, Federal register.

[32]  Dougal Maclaurin,et al.  Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin , 2013, Proceedings of the National Academy of Sciences.

[33]  Tomoaki Inoue,et al.  Electrophysiological characterization of cardiomyocytes derived from human induced pluripotent stem cells. , 2011, Journal of pharmacological sciences.

[34]  Samouil L. Farhi,et al.  All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins , 2014, Nature Methods.

[35]  Ofer Binah,et al.  Human Embryonic and Induced Pluripotent Stem Cell–Derived Cardiomyocytes Exhibit Beat Rate Variability and Power-Law Behavior , 2012, Circulation.

[36]  Jonathan A. Bernstein,et al.  Using iPS cells to investigate cardiac phenotypes in patients with Timothy Syndrome , 2011, Nature.

[37]  J. Shryock,et al.  Antagonism by Ranolazine of the Pro-Arrhythmic Effects of Increasing Late INa in Guinea Pig Ventricular Myocytes , 2004, Journal of cardiovascular pharmacology.

[38]  I. Judson,et al.  HDAC Inhibitors and Cardiac Safety , 2007, Clinical Cancer Research.

[39]  A. Camm,et al.  The potential for QT prolongation and pro-arrhythmia by non-anti-arrhythmic drugs: clinical and regulatory implications. Report on a Policy Conference of the European Society of Cardiology. , 2000, Cardiovascular research.

[40]  A. Bril,et al.  Electrophysiological characterization of BRL-32872 in canine Purkinje fiber and ventricular muscle. Effect on early after-depolarizations and repolarization dispersion. , 1999, European journal of pharmacology.

[41]  A. Camm,et al.  Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. , 2003, Cardiovascular research.

[42]  Van V. Brantner,et al.  Spending on new drug development1. , 2010, Health economics.

[43]  George A. Truskey,et al.  Modeling the mitochondrial cardiomyopathy of Barth syndrome with iPSC and heart-on-chip technologies , 2014, Nature Medicine.

[44]  J. Gibson,et al.  Human stem cell-derived cardiomyocytes detect drug-mediated changes in action potentials and ion currents. , 2014, Journal of pharmacological and toxicological methods.

[45]  Y. Kuryshev,et al.  Pentamidine-Induced Long QT Syndrome and Block of hERG Trafficking , 2005, Journal of Pharmacology and Experimental Therapeutics.

[46]  Rachel D. Vanderlaan,et al.  Electrophysiological profiling of cardiomyocytes in embryonic bodies derived from human embryonic stem cells: therapeutic implications. , 2003, Circulation research.

[47]  Lior Gepstein,et al.  In vitro electrophysiological drug testing using human embryonic stem cell derived cardiomyocytes. , 2009, Stem cells and development.

[48]  N. McMahon,et al.  Comparison of electrophysiological data from human-induced pluripotent stem cell-derived cardiomyocytes to functional preclinical safety assays. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[49]  James A Thomson,et al.  High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents. , 2011, American journal of physiology. Heart and circulatory physiology.

[50]  Gregory F. Lewis,et al.  High-throughput cardiac safety evaluation and multi-parameter arrhythmia profiling of cardiomyocytes using microelectrode arrays. , 2015, Toxicology and applied pharmacology.

[51]  Jun Zhou,et al.  Pentamidine reduces hERG expression to prolong the QT interval , 2005, British journal of pharmacology.

[52]  G. Salama,et al.  Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart , 1976, Science.

[53]  Guy Salama,et al.  Simultaneous maps of optical action potentials and calcium transients in guinea‐pig hearts: mechanisms underlying concordant alternans , 2000, The Journal of physiology.

[54]  Yasunari Kanda,et al.  Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an iPS cell-derived cardiomyocyte sheet: multi-site validation study. , 2014, Journal of pharmacological sciences.

[55]  Philip Wong,et al.  Pharmacoelectrophysiology of viral-free induced pluripotent stem cell-derived human cardiomyocytes. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[56]  D. Zipes,et al.  New antiarrhythmic agents: amiodarone, aprindine, disopyramide, ethmozin, mexiletine, tocainide, verapamil. , 1978, The American journal of cardiology.

[57]  P. Lipp,et al.  Screening Action Potentials: The Power of Light , 2011, Front. Pharmacol..

[58]  José Jalife,et al.  Optical Imaging of Voltage and Calcium in Cardiac Cells & Tissues , 2012, Circulation research.

[59]  James L Stevens,et al.  The future of drug safety testing: expanding the view and narrowing the focus. , 2009, Drug discovery today.

[60]  Gary Gintant,et al.  An evaluation of hERG current assay performance: Translating preclinical safety studies to clinical QT prolongation. , 2011, Pharmacology & therapeutics.

[61]  Hung-Fat Tse,et al.  Calcium Homeostasis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes , 2011, Stem Cell Reviews and Reports.

[62]  D. Walker,et al.  Pharmacokinetic/pharmacodynamic assessment of the effects of E4031, cisapride, terfenadine and terodiline on monophasic action potential duration in dog , 2001, Xenobiotica; the fate of foreign compounds in biological systems.

[63]  K. Summers,et al.  Molecular genetics of long QT syndrome. , 2010, Molecular genetics and metabolism.

[64]  Stefan R. Pulver,et al.  Ultra-sensitive fluorescent proteins for imaging neuronal activity , 2013, Nature.

[65]  Ronald A. Li,et al.  Functional sarcoplasmic reticulum for calcium-handling of human embryonic stem cell-derived cardiomyocytes: Insights for driven maturation , 2008, Cell Research.

[66]  Euan A Ashley,et al.  Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. , 2013, Cell stem cell.

[67]  Peter Kohl,et al.  Simultaneous Voltage and Calcium Mapping of Genetically Purified Human Induced Pluripotent Stem Cell–Derived Cardiac Myocyte Monolayers , 2012, Circulation research.

[68]  J. Verducci,et al.  MICE Models: Superior to the HERG Model in Predicting Torsade de Pointes , 2013, Scientific Reports.

[69]  V. Fast,et al.  Microscopic conduction in cultured strands of neonatal rat heart cells measured with voltage-sensitive dyes. , 1993, Circulation research.

[70]  J. Cinca,et al.  Sarcoplasmic reticulum and L‐type Ca2+ channel activity regulate the beat‐to‐beat stability of calcium handling in human atrial myocytes , 2011, The Journal of physiology.

[71]  U. Ravens,et al.  Atrial selectivity of antiarrhythmic drugs , 2013, The Journal of physiology.

[72]  Liang Guo,et al.  Refining the human iPSC-cardiomyocyte arrhythmic risk assessment model. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[73]  M. Lazdunski,et al.  HERG and KvLQT1/IsK, the cardiac K+ channels involved in long QT syndromes, are targets for calcium channel blockers. , 1998, Molecular pharmacology.

[74]  Richard T. Lee,et al.  Stem-cell therapy for cardiac disease , 2008, Nature.

[75]  Roger Y. Tsien,et al.  Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires , 2012, Proceedings of the National Academy of Sciences.

[76]  C. Lawrence,et al.  Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes? , 2006, Journal of pharmacological and toxicological methods.

[77]  Lior Gepstein,et al.  Modelling the long QT syndrome with induced pluripotent stem cells , 2011, Nature.

[78]  Florian Engert,et al.  Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents , 2014, Front. Physiol..

[79]  J. Paavola,et al.  Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells. , 2011, Journal of molecular and cellular cardiology.

[80]  P Smith,et al.  Concordance of the toxicity of pharmaceuticals in humans and in animals. , 2000, Regulatory toxicology and pharmacology : RTP.

[81]  P. Doevendans,et al.  Differentiation of cardiomyocytes in floating embryoid bodies is comparable to fetal cardiomyocytes. , 2000, Journal of molecular and cellular cardiology.

[82]  M. Cannell,et al.  Imaging Ca2+ Nanosparks in Heart With a New Targeted Biosensor , 2014, Circulation research.

[83]  J F Leclercq,et al.  Possible mechanisms of the arrhythmias in the long QT syndrome. , 1985, European heart journal.

[84]  D. Fisher Recent insights into the regulation of cardiac Ca2+ flux during perinatal development and in cardiac failure. , 1995, Current opinion in cardiology.