The central objective of these studies is to examine developmental and ischemic modulation of mechanisms by which Ca enters cardiac cells. Ischemia-induced dysfunction of the Ca channel or Na/Ca exchange may contribute to abnormalities of myocardial contraction and relaxation displayed by some immature hearts after ischemic arrest associated with cardiac surgery. The hypothesis that calcium channel is more a victim than an instigator of ischemia and reperfusion injury will be examined in cardiac membranes and intact cultured ventricular cells of various ages. It will be determined whether elements of ischemica and reperfusion including substrate deprivation, hypoxia, acidosis and hyperkalemia alter ligand binding, contractile response to Ca agonists and antagonists, and Ca45 influx. Calcium channel ligand binding properties change during cardiac development. As the first approach to understanding age-related alterations in myocardial sensitivity to ischemia, binding properties, stoichiometry, and allosteric interactions for the 3 classes of ligands will be examined in membranes of ventricular tissue from immature, neonatal and mature chick heart and will also be examined in intact cultured ventricular cells from chick tissue of comparable stages of development. Then, ontogeny of calcium channel function will be examined because development of ligand binding sites and of the ability of the Ca channel to actually gate Ca influx may be noncoordinate. Calcium channel function will be examined by measuring the contractile response of ventricular strips and of monolayers of cultured cells to specific Ca channel agonists and antagonists. Using monolayer cultures, Ca channel function in relationship to developmental age will also be examined by measuring the Ca45 uptake that is sensitive to calcium channel agonists and antagonists. Na/Ca exchanbe will also be assessed. For ligand binding and functional experiments intact, spontaneously contracting ventricular cells will be utilized because they offer several distinct advantages: 1) ligand binding is modulated by use and membrane potential; this cannot be examined in membranes, 2) unidirectional Ca flux can be cleanly resolved and related to ligand binding under identical conditions, 3) elements of ischemia can be precisely defined and controlled. These studies will provide fundamental new insights into the control of susceptibility to ischemic injury of immature and neontal myocardium.
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