The general objective of this research is to investigate the mechanism of control of contraction of striated muscle in both normal and diseased states at the cellular and molecular level. There are several specific aims in this research. The first is to estimate the Ca bound to the filaments and determine whether this binding depends on cross-bridge interaction. These studies utilize aequorin luminescence to measure Ca in barnacle single muscle fibers under controlled stimulation and mechanical conditions.
The second aim i s to investigate the mechanism of hystersis in Ca sensitivity in muscles whereby muscles are more sensitive to calcium when the Ca concentration is being decreased as in relaxation than when it is being increased as at the beginning of contraction. Experiments will be done to test the hypothesis that hysteresis is caused by a cross-bridge dependent increase in Ca binding to the activating sites, and not by phosphorylation. Skinned barnacle frog, rabbit, and rat muscles will be used in this study.
Our third aim i s to understand better how Ca activates muscles from the giant barnacle, Balanus nubilus, the muscle preparation we use extensively.
Our fourth aim i s to develop a technique of measuring Ca bound to various sites of Tn-C in skinned mammalian skeletal muscle using selective extraction, fluorescent labeling and reintroduction of labeled Tn-C into skinned muscle fibers and measuring the fluorescence that accompanies Ca activation. This technique will be used to study Ca binding to the Ca specific and Ca-Mg sites on Tn-C to correlate this binding with contraction and with factors which change cross-bridge interaction such as sarcomere length, MgATP, hysteresis, rigor, and step changes in length. Using this same technique, we will attach a fluorescent label to measure thin filament activation to test its relationship to cross-bridge interaction. Finally, we will measure the relative time courses of the sequential events in activation from Ca binding, to thin filament activation, to cross-bridge interaction, to force production during Ca activation of contraction and relaxation.
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