Molecular composition and functional properties of f-channels in murine embryonic stem cell-derived pacemaker cells

• Published in: Journal of molecular and cellular cardiology 2009-03, Vol.46 (3), p.343-351
• Main Authors: Barbuti, Andrea, Crespi, Alessia, Capilupo, Daniela, Mazzocchi, Nausicaa, Baruscotti, Mirko, DiFrancesco, Dario
• Format: Article
• Language: English
• Subjects: Acetylcholine - pharmacology; Action Potentials - drug effects; Action Potentials - physiology; Adrenergic beta-Agonists - pharmacology; Animals; Benzazepines - pharmacology; Biological Clocks - drug effects; Biological Clocks - physiology; Cardiovascular; Cell Line; Cholinergic Agents - pharmacology; Cyclic Nucleotide-Gated Cation Channels - antagonists & inhibitors; Cyclic Nucleotide-Gated Cation Channels - metabolism; Embryonic stem cells; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; f-channels; Heart Conduction System; Hyperpolarization-activated cation channel; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Isoproterenol - pharmacology; Kinetics; Membrane Potentials - drug effects; Membrane Potentials - physiology; Mice; Muscarinic receptors; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Pacemaker channels; Potassium Channels - metabolism; Protein Isoforms - antagonists & inhibitors; Protein Isoforms - metabolism; Receptors, Neurotransmitter - agonists; Receptors, Neurotransmitter - antagonists & inhibitors; Receptors, Neurotransmitter - metabolism; β-adrenergic receptors
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2008.12.001

Abstract Mouse embryonic stem cells (mESCs) differentiate into all cardiac phenotypes, and thus represent an important potential source for cardiac regenerative therapies. Here we characterize the molecular composition and functional properties of “funny” (f-) channels in mESC-derived pacemaker cells. Following differentiation, a fraction of mESC-derived myocytes exhibited action potentials characterized by a slow diastolic depolarization and expressed the If current. If plays an important role in the pacemaking mechanism of these cells since ivabradine (3 μM), a specific f-channel inhibitor, inhibited If by about 50% and slowed rate by about 25%. Analysis of If kinetics revealed the presence of two populations of cells, one expressing a fast- and one a slow-activating If ; the two components are present both at early and late stages of differentiation and had also distinct activation curves. Immunofluorescence analysis revealed that HCN1 and HCN4 are the only isoforms of the pacemaker channel expressed in these cells. Rhythmic cells responded to β-adrenergic and muscarinic agonists: isoproterenol (1 μM) accelerated and acetylcholine (0.1 μM) slowed spontaneous rate by about 50 and 12%, respectively. The same agonists caused quantitatively different effects on If : isoproterenol shifted activation curves by about 5.9 and 2.7 mV and acetylcholine by − 4.0 and − 2.0 mV in slow and fast If -activating cells, respectively. Accordingly, β1- and β2-adrenergic, and M2-muscarinic receptors were detected in mESC-derived myocytes. Our data show that mESC-derived pacemaker cells functionally express proteins which underlie generation and modulation of heart rhythm, and can therefore represent a potential cell substrate for the generation of biological pacemakers.