Experiments proposed here test the hypothesis that an important element in intrinsic and extrinsic regulation of cardiac function is modulation of the process by which Ca2+ - binding to TNC controls the actin-myosin reaction. A general objective is to know how and when the Ca-signalling mechanism is altered by i) the chemical and mechanical state of the myofilaments and ii) the population of isoforms of myofilament proteins.
The aims are: (1) To know whether shifts in isoform population of myofilament proteins other than TNC influence the length dependence of myofibrillar Ca2+ -activation; (2) To determine if shifts in isoforms of myofilament proteins influence the effect of actin-cross-bridge interactions on TNC Ca2+ -binding and conformation; (3) To determine the influence of shifts in myofilament isoform population on the effect of chemical mileau (pH, Pi) on myofilament response to Ca2+; (4) To know how changes in phosphorylation of isoforms of TNT and TNI affect myofilament activity and regulation; (5) To measure the altered myofilament response to Ca2+ in intact heart preparations with known isoform population of myofilament proteins. The approach to these aims involves the use of perfused hearts, of myofilaments at various levels of organization containing isoforms of TNT and TNI. Isoforms are identified by PAGE and immunoblots. Ca-binding is measured directly to TNC in skinned fibers and reconstituted preparations. Fluorescent probes attached to TNC and TNT are used as reporters of Ca2+ -binding and conformational changes. Actin-cross-bridge reactions are controlled by changes in length, by changes in concentrations of heavy meromyosin in reconstituted preparations, and by conditions altering weak and strong binding states. TNT is phosphorylated i) in skinned fibers and reconstituted preparations using TNT kinase and protein kinase C and ii) in intact muscle by stimulation of the protein kinase C pathway in preparations treated with 32P. Myofilament Ca2+ -response in intact preparations is measured with the aequorin technique by plotting steady light against steady force in preparations stimulated tetanically in different Ca2+ concentrations. These studies provide insight into the mechanism of altered myofilament response to Ca2+ and its potential significance in myocardial physiology, pathology and pharmacology.
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