title: Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Q-State Biosciences, Cambridge, MA, USA.
authors.forename: Graham T
authors.initials: GT
authors.lastname: Dempsey
authors.affiliation: GlaxoSmithKline-Safety Assessment, King of Prussia, PA, USA. Electronic address: khuram.w.chaudhary@gsk.com.
authors.forename: Khuram W
authors.initials: KW
authors.lastname: Chaudhary
authors.affiliation: Q-State Biosciences, Cambridge, MA, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Atwater
authors.affiliation: Q-State Biosciences, Cambridge, MA, USA.
authors.forename: Cuong
authors.initials: C
authors.lastname: Nguyen
authors.affiliation: GlaxoSmithKline-Safety Assessment, King of Prussia, PA, USA.
authors.forename: Barry S
authors.initials: BS
authors.lastname: Brown
authors.affiliation: GlaxoSmithKline-Alternative Discovery and Development, Cambridge, MA, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: McNeish
authors.affiliation: Department of Physics, Harvard University, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA.
authors.forename: Adam E
authors.initials: AE
authors.lastname: Cohen
authors.affiliation: Q-State Biosciences, Cambridge, MA, USA.
authors.forename: Joel M
authors.initials: JM
authors.lastname: Kralj
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D001145:Arrhythmias, Cardiac / Q000139:chemically induced / Q000503:physiopathology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002304:Cardiac Pacing, Artificial; D066126:Cardiotoxicity; D004353:Drug Evaluation, Preclinical / Q000379:methods; D055724:Electrophysiological Phenomena / Q000187:drug effects; D006801:Humans; D057054:Molecular Imaging / Q000379:methods*; D032383:Myocytes, Cardiac / Q000187:drug effects; D062308:Optogenetics / Q000379:methods*; D059629:Signal-To-Noise Ratio
chemical_list: D002121:Calcium Channel Blockers
keywords: Cardiomyocytes; Cardiotoxicity; Channelrhodopsin; Comprehensive in vitro Proarrhythmia Assay; Human induced pluripotent stem cells; Methods; Optogenetics; Pacing; Torsades de Pointes; Voltage indicator