The specific goal for this project is to understand the developmental differences in properties and adrenergic regulation of Ca2+ current density (I(Ca), pA/pF) in the rabbit heart. Differences between adult and newborn cells may be due to differences at the level of the number and types of receptors, the amount and type of G proteins, the amount and type of adenylyl cyclase, the amounts and types of phosphodiesterases, phosphatases, kinases or other aspects of the Ca2+ current modulatory pathway. We will test specific hypotheses related to developmental differences in competitive interaction of kinases and phosphatase enzymes on adrenergic regulation of Ca2+ current. Our first hypothesis is that: Inhibition of PPase in newborn has greater effect due to a greater PPase activity and different PPase types in NB cells. We will evaluate the developmental changes in the activity of specific kinases and PPases both in the basal state and with stimulation via the cAMP pathway. Specific tests will be done concerning the role of protein kinase A and other kinases in regulating the basal current magnitude, in specific types of PPases involved in dephosphorylation of Ca2+ channels, and in the dual action of cAMP in regards to enhancing kinase activity and inhibiting PPase activity as a means of increasing the availability of Ca2+ channels. Our second hypothesis is that: The multiple sites of phosphorylation that regulate availability and gating mode of Ca2+ channels are differentially phosphorylated in newborn and adult cells. Single channel studies with both kinase and PPase inhibitors will be used to specifically test hypotheses relating to the mode changes of Ca2+ channel opening as determined by the actions of kinases and PPases. Our third hypothesis is that: differences in the distribution of specific isoforms of inhibitory G proteins and differences in specific isoforms of adenylyl cyclase provide a mechanism for the greater inhibitory effects of carbachol in NB than in AD cells and also the differential effects of adenosine compared to carbachol in the inhibition of the stimulation of I(Ca) by isoproterenol and forskolin. We will expand the time frame over which we study these developmental differences by studies of I(Ca) amplitude and modulation to fetal heart cells. These studies will be important in clarifying the biochemical basis of the developmental changes we observe, with specific emphasis on developmental changes in the expression and function of phosphatase enzymes. Our proposal is the first comprehensive evaluation of developmental changes in mammalian cardiac ionic current magnitude and adrenergic regulation. Ca2+ currents play major roles in controlling the action potential duration as well as the supply of Ca2+ to control contractility. The proposed studies will contribute toward our goal of improving the treatment of cardiac dysfunction in infants and children.
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