Albert M. Gordon - US grants

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 is 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 is to understand better how Ca activates muscles from the giant barnacle, Balanus nubilus, the muscle preparation we use extensively. Our fourth aim is 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.

This Program Project has as its focus the understanding of the regulation of contraction in cardiac muscle. Since contractile regulation in the heart is of such great significance to both normal and pathologic function, it is important to understand this mechanism. Although the regulatory architecture is common between cardiac and skeletal muscle, there are quantitative differences which give rise to differences in important physiological properties. These studies are designed to discover the protein isoforms and interactions which are responsible for these differences. In four projects, with three cores for support, we will answer a number of the crucial questions of contractile regulation in cardiac muscle. What are the interactions within and along the thin filament that occur during regulation? What role does strong cross- bridge attachment play in activating the thin filament along with Ca binding? What is the mode of regulation? Are the number of interacting sites regulated or the cross-bridge kinetics and how are cross-bridge kinetics regulated? How does sarcomere length have such a profound effect on cardiac contractile regulation? Is it through effects on Ca binding, strong cross-bridge attachment, or cross-bridge kinetics? With extensive collaboration and using preparations from isolated proteins in the in vitro motility assay, to skinned preparations, to intact preparations, we will use newly devised techniques to answer these questions. The results should have great significance to understanding contractile regulation in both the normal and pathologic heart. A more complete understanding will aid both in developing new drugs by providing an experimental model for the evaluation of the mechanism of action and in developing other approaches to therapy.

The objective of this project is to apply the techniques of the in vitro motility assay to investigate regulation of cardiac muscle contraction. Using the in vitro motility assay, one can test in a simplified system with well defined proteins whether Ca2+ regulates crossbridge (XBr) kinetics as suggested by some skinned and intact muscle studies and test which specific proteins are responsible for the observed differences in regulation between cardiac and skeletal muscle. It will also be possible to test aspects of regulation which might depend on the specific arrangement of filaments in the sarcomere and test whether XBr's can activate the thin filament, producing superactivation. Although the in vitro motility assay has been used for some time now to investigate the properties of molecular motors, this is the first direct application to the study of regulation in the actin-myosin system under more physiological conditions.

The long-term objective of this research is to understand the molecular events regulating contraction in cardiac and skeletal muscle. It is hypothesized that in the interaction of actin and myosin to produce contraction, regulation is achieved through interactions within the thin filament modulating the thin filament structure. Although there are some cases where modulation at the thick filament level is important (such as phosphorylation of the myosin light chains) most evidence supports the thin filament as the major site of control. Using probes on TnC to measure Ca2+ binding, we will investigate whether this binding is uniform along the thin filament and whether crossbridges affect this binding. By using probes of the thin filament structure and measurements of crossbridge (XBr) force and kinetics, we will test to what extent Ca binding to TnC per se and XBr attachment regulate thin filament activation leading to force and XBr kinetics. With major differences in regulation between cardiac and skeletal muscle, we will investigate a major mechanism by which Ca2+ binding to TnC initiates contraction, through the modification of the TnC-TnI and TnC-TnI-TnT interactions. Specifically we will investigate why the interaction between the cardiac isoforms of TnC-TnI are more electrostatic while that of the skeletal isoforms more hydrophobic. We will investigate whether this is maintained in the presence of TnT and which charged amino acids are responsible for this electrostatic interaction and the differing effects of phosphorylation. This will clarify the interactions between TnC-TnI- TnT that occur during the control of contraction. These aims will be achieved using skinned muscle preparations, fluorescent indicators on proteins exchanged into these preparations, protein binding studies using mutant regulatory proteins, and caged Ca to obtain rapid activation. Achieving these aims will help us understand how differences in molecular isoforms combined with a similar architecture of regulation can give rise to such large differences of regulation between cardiac and skeletal muscle.