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 hyperpolarizatio n activated nonselective cation current (I-f) were recorded using whole-cel l voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at diffe rent 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 c ells 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) stimulate d 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 kina se (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. Intr acellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 mu M) completely inhibited the stimulation of th e 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 PD E2 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 earl y 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.