We plan to extend methods that have been developed for the dispersion of single cardiac myocytes in rat, guinea pig and frog to larger species such as dog, rabbit and cat. In addition some properties of human cardiac myocytes will be examined. Cardiac myocytes will be dispersed acutely using enzymatic methods of dissociation. A rigorous program for evaluting the success of the method has been established and includes measurements of ultrastructure, biochemical behaviour and electrophysiological properties. The usefulness of a wide species representation arises from the fact that each species has special advantages. For example, dog is a favorite preparation for cardiac sarcolemma, rabbit is useful for pacemaker studies, rat provides many genetic models of cardiac disease, frog has no t-tubule system, etc. In addition, the generalizibility of results is enhanced using our approach. Cellular parameters that will be evaluated include Ca tolerance, myocyte ultrastructure, electrophysiological properties including cable properties, resting potential, membrane voltage and ligan-gated current, contractile activity, ion content, ion fluxes, mitochondrial function, sarcolemmal binding and cyclic nucleotide production. Reconstitution experiments will be done in which single channels from lipid-enriched SL vesicles are isolated by patch clamp. Calcium channels will be studied particularly from the point of view of the dihydropyridines, a new important class of Ca channel modulators. These agents have agonist and antagonist effects, are highly potent and very specific. The binding of these agents to sarcolemmal Ca channels will be compared to the functional effects measured from single channel and whole cell currents. Particular attention will be paid to state-dependent binding. The currents underlying the pacemaker potentials in rabbit pacemaker and subsidiary pacemaker tissue will be examined and a synthesis of the pacemaking potential in the light of these findings will be made. The effects of autonomic transmitters, adenosine, and ATP on pacemaking will also be examined.
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