title: Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells.
abstract: OBJECTIVE
  The creation of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) has spawned broad excitement borne out of the prospects to diagnose and treat cardiovascular diseases based on personalized medicine. A common feature of hiPS-CMs is their spontaneous contractions but the mechanism(s) remain uncertain.
  
  
  METHODS
  Intrinsic activity was investigated by the voltage-clamp technique, optical mapping of action potentials (APs) and intracellular Ca(2+) (Cai) transients (CaiT) at subcellular-resolution and pharmacological interventions.
  
  
  RESULTS
  The frequency of spontaneous CaiT (sCaiT) in monolayers of hiPS-CMs was not altered by ivabradine, an inhibitor of the pacemaker current, If despite high levels of HCN transcripts (1-4). HiPS-CMs had negligible If and IK1 (inwardly-rectifying K(+)-current) and a minimum diastolic potential of -59.1±3.3mV (n=18). APs upstrokes were preceded by a depolarizing-foot coincident with a rise of Cai. Subcellular Cai wavelets varied in amplitude, propagated and died-off; larger Cai-waves triggered cellular sCaTs and APs. SCaiTs increased in frequency with [Ca(2+)]out (0.05-to-1.8mM), isoproterenol (1μM) or caffeine (100μM) (n≥5, p<0.05). HiPS-CMs became quiescent with ryanodine receptor stabilizers (K201=2μM); tetracaine; Na-Ca exchange (NCX) inhibition (SEA0400=2μM); higher [K(+)]out (5→8mM), and thiol-reducing agents but could still be electrically stimulated to elicit CaiTs. Cell-cell coupling of hiPS-CM in monolayers was evident from connexin-43 expression and CaiT propagation. SCaiTs from an ensemble of dispersed hiPS-CMs were out-of-phase but became synchronous through the outgrowth of inter-connecting microtubules.
  
  
  CONCLUSIONS
  Automaticity in hiPS-CMs originates from a Ca(2+)-clock mechanism involving Ca(2+) cycling across the sarcoplasmic reticulum linked to NCX to trigger APs.
authors: Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Jong J
authors: JJ
authors: Kim
authors: Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Lei
authors: L
authors: Yang
authors: Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Bo
authors: B
authors: Lin
authors: University of Iowa, Carver College of Medicine, Division of Cardiovascular Medicine, Iowa City, IA 52242, USA.
authors: Xiaodong
authors: X
authors: Zhu
authors: Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Bin
authors: B
authors: Sun
authors: Center for Cellular and Systems Electrophysiology, University at Buffalo, State University of New York, Buffalo, NY 14214, USA.
authors: Aaron D
authors: AD
authors: Kaplan
authors: Center for Cellular and Systems Electrophysiology, University at Buffalo, State University of New York, Buffalo, NY 14214, USA; Departments of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, NY 14214, USA; Departments of Gynecology-Obstetrics, University at Buffalo, State University of New York, Buffalo, NY 14214, USA.
authors: Glenna C L
authors: GC
authors: Bett
authors: Center for Cellular and Systems Electrophysiology, University at Buffalo, State University of New York, Buffalo, NY 14214, USA; Departments of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, NY 14214, USA.
authors: Randall L
authors: RL
authors: Rasmusson
authors: University of Iowa, Carver College of Medicine, Division of Cardiovascular Medicine, Iowa City, IA 52242, USA.
authors: Barry
authors: B
authors: London
authors: Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address: gsalama@pitt.edu.
authors: Guy
authors: G
authors: Salama
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001552:Benzazepines / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002317:Cardiovascular Agents / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D065150:Cellular Reprogramming; D018031:Connexin 43 / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D000077550:Ivabradine; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010647:Phenyl Ethers / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013748:Tetracaine / Q000494:pharmacology; D014162:Transfection
chemical_list: D000814:Aniline Compounds; D001552:Benzazepines; D002317:Cardiovascular Agents; D018031:Connexin 43; C500216:GJA1 protein, human; D010647:Phenyl Ethers; C430918:SEA 0400; D019831:Sodium-Calcium Exchanger; D013748:Tetracaine; D002110:Caffeine; D000077550:Ivabradine; D007545:Isoproterenol; D002118:Calcium
keywords: Cell–cell coupling; Funny current; Human myocytes from stem cells; Optical mapping of calcium and action potentials; Spontaneous activity; Subcellular calcium waves
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