Hyperpolarization-activated Cyclic Nucleotide-Modulated 'HCN' Channels Confer Regular and Faster Rhythmicity to Beating Mouse Embryonic Stem Cells

J Physiol. 2008 Feb 1;586(3):701-16. doi: 10.1113/jphysiol.2007.144329. Epub 2007 Nov 22.

Abstract

The hyperpolarization-activated cation current (I(f)), and the hyperpolarization-activated cyclic nucleotide-modulated 'HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. I(f) and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing I(f), and the density of I(f) in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The I(f) blocker ZD7288 (0.3 microm) reduced I(f) and the beating rate of embryoid bodies. Taken together, the activation kinetics of I(f) and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of I(f) to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / pharmacology
  • Action Potentials / physiology*
  • Animals
  • Cardiac Electrophysiology
  • Cardiotonic Agents / pharmacology
  • Cell Line
  • Cholinergic Agents / pharmacology
  • Cyclic Nucleotide-Gated Cation Channels / metabolism*
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / drug effects
  • Embryonic Stem Cells / metabolism*
  • Heart / embryology*
  • Heart / physiology
  • Heart Rate / physiology*
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
  • Ion Channels / metabolism*
  • Isoproterenol / pharmacology
  • Mice
  • Myocardial Contraction / physiology
  • Myocardium / cytology
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism
  • Patch-Clamp Techniques
  • Potassium Channels
  • Protein Isoforms / metabolism

Substances

  • Cardiotonic Agents
  • Cholinergic Agents
  • Cyclic Nucleotide-Gated Cation Channels
  • HCN3 protein, mouse
  • Hcn2 protein, mouse
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
  • Ion Channels
  • Potassium Channels
  • Protein Isoforms
  • Isoproterenol
  • Acetylcholine