Developmental Changes in β-Adrenergic Modulation of L-Type Ca ²⁺ Channels in Embryonic Mouse Heart

Abstract

In the adult mammalian myocardium, cellular Ca2+ entry is regulated by the sympathetic nervous system. L-type Ca2+ channel currents are markedly increased by beta-adrenergic (beta-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the developing mammalian heart, the regulation of Ca2+ entry by this enzyme cascade has not been clearly established, because changes in receptor density and coupling to downstream elements of the signaling cascade are known to occur during embryogenesis. In this study, we systematically investigated the regulation of L-type Ca2+ channel currents during development of the murine embryonic heart. We used conventional whole-cell and perforated-patch-clamp procedures to study modulation of L- type Ca2+ channel currents and to assay functional activity of distinct steps in the beta-A signaling cascade in murine embryonic myocytes at different stages of gestation. Our data indicate that the L-type Ca2+ channels in early-stage (day-11 to -13) myocytes are unresponsive to either isoproterenol or cAMP. L-type Ca2+ channels in late-stage (day-17 to -19) murine myocytes, however, exhibit responses to isoproterenol and cAMP similar to responses in adult cells, providing evidence that the beta-A cascade becomes functionally active during this period of embryonic development. We found that L-type Ca2+ channel activity in early-stage cells is increased by cell dialysis with the catalytic subunit of cAMP-dependent protein kinase (cA-PK) and that dialysis of early-stage cells with the holoenzyme of cA-PK restores functional responses to forskolin and cAMP, but not to isoproterenol. Our results provide strong evidence that a key factor in the early-stage insensitivity of L-type Ca2+ channels to cAMP is the absence, or low expression level, of the holoenzyme of cA-PK but that in addition, another element in the signaling cascade upstream from adenylate cyclase is expressed at a nonfunctional level or is uncoupled from the cascade and thus contributes to L-type Ca2+ channel insensitivity to beta-A agonists in early stages of the developing murine heart.

Establishment of Ionic Channels and Signalling Cascades in the Embryonic Stem Cell-Derived Primitive Endoderm and Cardiovascular System

“…As reported for early murine cardiomyocytes early stage ES cell-derived cardiomyocytes did not express functionally coupled ß-adrenoceptors [Slotkin et al, 1994;An et al, 1996], whereas intact muscarinic signalling was present at this stage . The most relevant finding was, however, the prominent role of nitric oxide (NO) as a key signalling molecule during early embryonic development .…”

Differentiation of Human Embryonic Stem Cells to Cardiomyocytes

“…Here, the lack of cAMP-dependent protein kinase appeared to be the limiting factor. 12,19 Thus, although hES-derived and early human fetal cardiomyocytes show some features in common with early mouse cardiomyocytes, their calcium channel modulation resembles that in the adult mouse. hES cells may thus represent an excellent system for studying changes in calcium channel function during early human development, which appears to differ significantly from that in mice.…”

TNF-.ALPHA. Rapidly Antagonizes the .BETA.-Adrenergic Responses of the Chloride Current in Guinea-Pig Ventricular Myocytes.

“…During development, modulatory responses to catecholamines can be affected by changes in the relative expression levels of the -adrenergic signaling cascade component. 35 Enhancement of ICl can be caused by a -adrenergic receptor-mediated cAMP-dependent phosphorylation of the Cl -channel. To elucidate whether the inhibitory action of TNF-was exerted by a modulation of the cAMP-dependent phosphorylation pathway, pharmacological tools such as FK, 8-bromo-cAMP and IBMX, were used.…”