The general goals of the proposed research has been to understand the process by which Ca2+ activates the mechanical and biochemical activity of cardiac myofilaments, and to known whether, how, and when this process is modified physiologically by intrinsic mechanisms and by mechanisms extrinsic to the myofilaments involving protein phosphorylation. Experiments relating to intrinsic regulation are aimed at determination of whether Ca2+ binding to regulatory sites in the myofilament lattice are influenced by the sarcomere length and/or by force-generating cross-bridge complexes with the thin filament. Experiments relating to extrinsic regulation focus on phosphorylation of troponin I (TnI), the P-light chain of myosin, and C-protein. The aims are to determine the effect of TnI phosphorylation on the binding of cross-bridges to the thin filament, and also to determine whether there are isoforms of TnI and how variations in the population of the TnI isoforms affect myofilament activity and regulation. In the case of studies on C-protein and the P-light chain of myosin, the aims are to determine whether these proteins affect myofilament activation by Ca2+ and whether their activity is influenced by the level of protein phosphorylation. The approach to these aims involves measurement of levels of covalent phosphorylation of myofilament proteins in hearts freeze-clamped during the response to inotropic perturbations. The approach also involves preparation of each of the main myofilament proteins, analysis of the isoforms of TnI and myosin heavy chain, reconstitution with variations in the levels of covalent phosphorylation and measurement of cross-bridge binding to actin and actomyosin ATPase activity. Mechanical studies using chemically skinned fibers include measurements of tension, stiffness, Vmax, and kinetics of the rise and fall of force during "jumps" and "dives" in the free Ca2+ surrounding the myofilaments. Methods also include newly developed procedures for making multiple measurements of Ca2+ binding to chemically skinned preparations of heart muscle, characterized for TnC content, sarcomere length and extent of actin-cross-bridge interaction. The proposed experiments bear directly on current perceptions of the regulation of cardiac output by the Starling mechanism and by the autonomic nervous system, and whether extrinsic and intrinsic regulation of the Ca2+ activation of the myofilaments might represent a locus of lesions in heart failure.