Mechanism of Automaticity in Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells

J Mol Cell Cardiol. 2015 Apr;81:81-93. doi: 10.1016/j.yjmcc.2015.01.013. Epub 2015 Jan 30.

Abstract

Background and objectives: 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.

Keywords: Cell–cell coupling; Funny current; Human myocytes from stem cells; Optical mapping of calcium and action potentials; Spontaneous activity; Subcellular calcium waves.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Action Potentials / physiology
  • Aniline Compounds / pharmacology
  • Animals
  • Benzazepines / pharmacology
  • Caffeine / pharmacology
  • Calcium / metabolism*
  • Cardiovascular Agents / pharmacology
  • Cell Differentiation
  • Cell Line
  • Cellular Reprogramming
  • Connexin 43 / metabolism
  • Dependovirus / genetics
  • Genetic Vectors
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / drug effects
  • Induced Pluripotent Stem Cells / metabolism*
  • Isoproterenol / pharmacology
  • Ivabradine
  • Microtubules / drug effects
  • Microtubules / metabolism
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • Patch-Clamp Techniques
  • Phenyl Ethers / pharmacology
  • Sarcoplasmic Reticulum / drug effects
  • Sarcoplasmic Reticulum / metabolism*
  • Sodium-Calcium Exchanger / antagonists & inhibitors
  • Sodium-Calcium Exchanger / metabolism*
  • Tetracaine / pharmacology
  • Transfection

Substances

  • Aniline Compounds
  • Benzazepines
  • Cardiovascular Agents
  • Connexin 43
  • GJA1 protein, human
  • Phenyl Ethers
  • SEA 0400
  • Sodium-Calcium Exchanger
  • Tetracaine
  • Caffeine
  • Ivabradine
  • Isoproterenol
  • Calcium