Functional expression and regulation of the hyperpolarization activated non-selective cation current in embryonic stem cell-derived cardiomyocytes

The biophysical and pharmacological characteristics of the hyperpolarization activated nonselective cation current (I-f) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. The cation current was detected in a large percentage (65%) of early stage (EDX, 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 I-f decreased (45 %), but I-f densities (15.5 + 0.9 pA pF(-1), n = 20) were increased. The muscarinic agonist carbachol (CCh, 1 mu M) depressed basal I-f 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 mu M) stimulated I-f in LDS cells by 33 +/- 5.2% (n = 6) but not in EDS cells (n = 5). Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PRA, 7 mu M) stimulated I-f in EDS cells by 37.0 +/- 2.9%, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 mu M) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 mu M) completely inhibited the stimulation of the L-type Ca2+ current (I-Ca,I-L) as well as of I-f by ISO (1 mu M). 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 I-Ca,I-L and I-f in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated I-Ca,I-L but not I-f. The present work demonstrates that I, is functionally expressed during early cardiomyogenesis. Similar to I-Ca,I-L, I-f is regulated during embryonic development by phosphorylation via PKA. In contrast to I-Ca,I-L, I-f is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.