Troponin I (TnI) is a thin filament inhibitory protein that shuttles between actin and troponin C as a function of Ca2+. It acts as a molecular switch controlling the reaction of cross-bridges with actin in striated myofilaments. There is also evidence for TnI isoform switching in developing heart. Our objective is to determine the structural basis for the unique function of the cardiac TnI isoform in myofilament Ca2+ regulation. We think that these unique to heart by which the myofilament response to Ca2+ is modulated by length, protein phosphorylation and pH. To meet our objectives we use recombinant TnI that has been altered by site directed and deletion mutations. The specific aims are: (1) To express and isolate wild type recombinant cardiac TnI (cTnI) and mutants and to characterize and compare the recombinant proteins with native cTnI; (2) To identify the regions of cTnI that are important in unique aspects of the activation of the cardiac thin filament by Ca2+; (3) To determine the significance of TnI in the feedback effect of cross-bridges on TnC Ca2+-binding and length dependence of activation of cardiac myofilaments; (4) To identify the regions of cTnI that are important in inducing high pH sensitivity to the Ca2+-regulation of the cardiac actin- myosin reaction as compared to that of slow and fast skeletal muscle; and (5) To determine the relative significance of the multiple and unique phosphorylation sites on cTnI that are substrates for protein kinase A and for protein kinase C. The approach to these aims includes measurements of TnC Ca2+ -binding, force and ATPase activity in reconstituted preparations and skinned fibers functioning under different conditions of pCa, pH, protein phosphorylation and extent of cross-bridge reaction with the thin filaments. Interactions between recombinant TnI and TnC are measured using affinity chromatography insights into the regulation of cardiac myofilaments contraction by TnI and its potential significance in cardiac physiology, development, pathology and pharmacology.