The goals of the proposed research are to understand the sequential states in calcium activation of muscle contraction leading to the biochemical and mechanical activity of the myofilaments and to know whether, how, and when this process is modified physiologically by the interaction of cross- bridges with the thin filament. Experiments relating to regulation are aimed at determining whether calcium binding to the regulatory protein TnC of skinned muscle fibers in influenced by cross-bridges, and the state of the cross-bridge leading to a further interaction between calcium and cross-bridges. Additionally, experiments are aimed at determining whether cross-bridge activation through calcium binding to the regulatory sites is affected by calcium binding to the calcium-magnesium sites of TnC. In the case of studies on TnI, the aims are to determine whether the interaction between TnC and TnI differs when calcium is bound to TnC activating sites, cross-bridges are bound to actin, and when both calcium and cross-bridges are bound. Experiments relating to the flexibility of the thin filament are aimed at determining whether these motions play a regulatory role through the binding of calcium to the activating sites leading to a structural change in the thin filament or increased flexibility. Studies toward accomplishing these objectives will involve a careful but sensitive approach using the information present in the emitted fluorescence from labeled regulatory proteins incorporated into a physiological relevant system, skinned-single skeletal muscle fibers. The approach involves monitoring the fluorescence using a micro-spectrofluorimeter to follow absorption and emission spectra, excited stage lifetimes, and fluorescence polarization anisotropy as a means of evaluating the processes influencing the fluorescent emission. That the skinned muscle fiber preparation is capable of generating tension, provides additional and important correlations of the fluorescent properties with such physiological parameters as tension, stiffness, and shortening. Methods also include the use of ATP analogs and other compounds as a means of estimating the influence of different cross-bridge states on the regulatory proteins. The proposed experiments bear directly on current views of calcium activation mechanisms, and ultimately the understanding of basic muscle contractile mechanisms. That either cardiac or skeletal muscle performance, whether by disease or under drug action, often involve modification of calcium sensitivity of regulation further justifies the examination of feedback relations between cross-bridge formation and activation of the thin filament by calcium.
| Yates, L D; Coby, R L; Luo, Z et al. (1993) Filament overlap affects TnC extraction from skinned muscle fibres. J Muscle Res Cell Motil 14:392-400 |
