Maltsev V A, Ji G J, Wobus A M, Fleischmann B K, Hescheler J
Division of Cardiovascular Medicine, Henry Ford Heart and Vascular Institute, Detroit, MI, USA.
Circ Res. 1999 Feb 5;84(2):136-45. doi: 10.1161/01.res.84.2.136.
beta-Adrenergic modulation of the L-type Ca2+ current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37 degreesC. 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 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-gamma-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 Ca2+ channel related to phosphorylation occurred during development. ICaL was stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-gamma-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 beta-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced beta-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities.
利用全细胞膜片钳技术，在37℃条件下对小鼠胚胎干细胞来源的心肌细胞不同发育阶段的L型钙电流（ICaL）的β-肾上腺素能调节特性进行了研究。心肌细胞首次出现在培养7天（7d）的胚胎干细胞来源的胚状体中。在非常早期发育阶段（VEDS）的心肌细胞（从7 + 1d到7 + 2d）中，ICaL对异丙肾上腺素、福斯可林和8-溴-cAMP不敏感，但在晚期发育阶段（LDS）的心肌细胞（从7 + 9d到7 + 12d）中受到这些物质的高度刺激，这表明所有信号级联成分在发育过程中功能上变得耦合。在早期发育阶段（EDS）的细胞（从7 + 3d到7 + 5d）中，对福斯可林和8-溴-cAMP的刺激反应相对较弱。ATP-γ-S可强烈增强福斯可林的作用。在此阶段，基础ICaL受到非选择性磷酸二酯酶（PDE）抑制剂异丁基甲基黄嘌呤、对PDE II、III和IV亚型具有选择性的PDE抑制剂以及磷酸酶抑制剂冈田酸的刺激。在整个发育过程以及成年小鼠心室心肌细胞中，发现cAMP依赖性蛋白激酶A（PKA）催化亚基对ICaL的刺激作用相似（约3倍），这表明在发育过程中与磷酸化相关的钙通道没有发生结构变化。在LDS心肌细胞而非VEDS心肌细胞中，在存在PKA抑制剂和GTP-γ-S的情况下，异丙肾上腺素可刺激ICaL，这表明通过刺激性GTP结合蛋白Gs介导的膜限定刺激途径的发育。我们得出结论，Gs蛋白的解偶联和/或低表达导致了VEDS心肌细胞中ICaL对β-肾上腺素能刺激不敏感。此外，在7 + 4d阶段的EDS细胞中，β-肾上腺素能反应降低至少部分是由于高内在PDE和磷酸酶活性。
