Functional expression and regulation of the hyperpolarization activated non-selective cation current in embryonic stem cell-derived cardiomyocytes.
1. The biophysical and pharmacological characteristics of the hyperpolarization activated non- selective cation current (If) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. 2. The cation current was detected in a large percentage (65 %) of early stage (EDS, differentiated for 7 + 3-4 days) cells at a current density of 11.4 +/- 0.6 pA pF-1 (n = 47). In late stage (LDS, differentiated for 7 + 9-12 days) cells the percentage of cells expressing If decreased (45 %), but If densities (15.5 +/- 0.9 pA pF-1, n = 20) were increased. 3. The muscarinic agonist carbachol (CCh, 1 microM) depressed basal If in EDS cells by 45.7 +/- 6.5 %, n = 5) and was without effect in LDS cardiomyocytes (n = 4). The beta-adrenoceptor agonist isoprenaline (ISO, 1 microM) stimulated If in LDS cells by 33 +/- 5.2 % (n = 6) but not in EDS cells (n = 5). 4. Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PKA, 7 microM) stimulated If in EDS cells by 37.0 +/- 2.9 %, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 microM) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 microM) completely inhibited the stimulation of the L-type Ca2+ current (ICa,L) as well as of If by ISO (1 microM). 5. Extracellular superfusion with phosphodiesterase (PDE) inhibitors - IBMX, a non-selective antagonist, Erythro-9-(2-hydoxy-3-nonyl)adenine (EHNA), a PDE2 antagonist and rolipram, a PDE4 antagonist - resulted in stimulation of ICa,L and If in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated ICa,L, but not If. 6. The present work demonstrates that If is functionally expressed during early cardiomyogenesis. Similar to ICa,L, If is regulated during embryonic development by phosphorylation via PKA. In contrast to ICa,L, If is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.
Abi-Gerges
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Institute of Neurophysiology, University of Cologne, D-50931 Cologne, Germany and Laboratoire de Cardiologie Cellulaire & Moleculaire, INSERM U-446, Faculty of Pharmacy, University of Paris-Sud, F-92296 Châtenay-Malabry, France.
Ji
G J
GJ
Lu
Z J
ZJ
Fischmeister
R
R
Hescheler
J
J
Fleischmann
B K
BK
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism; D002412:Cations / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D006321:Heart / Q000196:embryology*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D015221:Potassium Channels; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D020746:Calcium Channels, L-Type; D002412:Cations; D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D018721:Muscarinic Agonists; D009419:Nerve Tissue Proteins; D010726:Phosphodiesterase Inhibitors; D015221:Potassium Channels; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases