Establishment of b-Adrenergic Modulation of L-Type Ca Current in the Early Stages of Cardiomyocyte Development

b-Adrenergic modulation of the L-type Ca current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37°C. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d). ICaL was insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 711d to 712d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 719d to 7112d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 713d to 715d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-g-S. At this stage, basal ICaL was stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, and IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimulation of ICaL by the catalytic subunit of the cAMP-dependent protein kinase A (PKA) was found to be similar (about 3 times) throughout development and in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca channel related to phosphorylation occurred during development. ICaL was stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-g-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through the stimulatory GTP binding protein, Gs. We conclude that uncoupling and/or low expression of Gs protein accounted for the ICaL insensitivity to b-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 714d stage, the reduced b-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities. (Circ Res. 1999;84:136-145.)