Establishment of beta-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development

beta-Adrenergic modulation of the L-type Ca2+ current (I-CaL) was character ized for different developmental stages in murine embryonic stem cell-deriv ed cardiomyocytes using the whole-cell patch-clamp technique at 37 degrees C, Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bo dies grown for 7 days (7d). I-CaL was insensitive to isoproterenol, forskol in, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocyte s (from 7+1d to 7+2d) but highly stimulated by these substances in late dev elopmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during develo pment. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the st imulatory response to forskolin and 8-bromo-cAMP was relatively weak. The f orskolin effect was strongly augmented by ATP-gamma-S. At this stage, basal I-CaL was stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, a nd IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimu lation of I-CaL by the catalytic subunit of the cAMP-dependent protein kina se A (PKA) was found to be similar (about 3 times) throughout development a nd in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca2+ channel related to phosphorylation occurred during dev elopment, I-CaL was stimulated by isoproterenol in the presence of a PKA in hibitor and GTP-gamma-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through th e stimulatory GTP binding protein, G(s). We conclude that uncoupling and/or low expression of G(s) protein accounted for the I-CaL insensitivity to be ta-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced beta-adrenergic response is due, at least i n part, to high intrinsic PDE and phosphatase activities.