DEVELOPMENTAL-CHANGES IN BETA-ADRENERGIC MODULATION OF L-TYPE CA2+ CHANNELS IN EMBRYONIC MOUSE HEART

In the adult mammalian myocardium, cellular Ca2+ entry is regulated by the sympathetic nervous system. L-type Ca2+ channel currents are mark edly increased by beta-adrenergic (P-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the develo ping mammalian heart, the regulation of Ca2+ entry by this enzyme casc ade has not been clearly established, because changes in receptor dens ity and coupling to downstream elements of the signaling cascade are k nown to occur during embryogenesis. In this study, we systematically i nvestigated the regulation of L-type Ca2+ channel currents during deve lopment of the murine embryonic heart. We used conventional whole-cell and perforated-patch-clamp procedures to study modulation of L-type C a2+ channel currents and to assay functional activity of distinct step s in the P-A signaling cascade in murine embryonic myocytes at differe nt stages of gestation. Our data indicate that L-type Ca2+ channels in early-stage (day-11 to -13) myocytes are unresponsive to either isopr oterenol or cAMP. L-type Ca2+ channels in late-stage (day-17 to -19) m urine myocytes, however, exhibit responses to isoproterenol and cAMP s imilar to responses in adult cells, providing evidence that the beta-A cascade becomes functionally active during this period of embryonic d evelopment. We found that L-type Ca2+ channel activity ih 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 cel ls with the holoenzyme of cA-PK restores functional responses to forsk olin and cAMP, but not to isoproterenol. Our results provide strong ev idence that a key factor in the early-stage insensitivity of L-type Ca 2+ channels to cAMP is the absence, or low expression level, of the ho loenzyme of cA-PK but that in addition, another element in the signali ng cascade upstream from adenylate cyclase is expressed at a nonfuncti onal 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.