| Regulation of contraction in muscle is a cooperative process involving the activation effects of ionized calcium (Ca2+) and actomyosin crossbridge formation. General myocardial EC coupling pathway involves a Ca 2+ influx during the plateau phase of the action potential, triggering more Ca2+ from the sarcoplasmic reticulum (SR). Binding of this Ca2+ to the contractile machinery within the sarcomere induces myofilament force development, energy for Ca2+ handling, and mechanical work that requires adenosine triphosphate (ATP). Hydrolysis of ATP inducts myosin globular head attachment to exposed myosin binding sites on actin, forming a crossbridge. To develop a detailed understanding of these molecular events underlying muscle contraction and its regulation, a characterization of its mechanical properties, chemical identity, and structural features are needed, as well as an evaluation of the kinetics of all relevant steps in the crossbridge cycle. Integrating information from such studies would provide meaningful quantitative models of crossbridge actions and their regulation that would elucidate the contractile machinery turned on by Ca 2+ in the intact sarcomere and help combat the progression of cardiac diseases. Addressing this problem calls for studies that rely on recent advances in contractile protein mutagenesis that allow for the precise placement of fluorescent probes upon the contractile proteins. In the intact sarcomere, measuring mechanical force and probing for fluorescence simultaneously is a major engineering problem due to the techniques and small sizes involved. We present an apparatus designed to allow for the kinetic measurement of mechanical force in the intact sarcomere, with myofibril preparation designed to bypass the SR, while probing for molecular events directly and simultaneously. This provides a quantitative model that could describe in a testable manner the roles of direct Ca2+ activation, ATP turnover rates, contractile protein phosphorylation, and their relationships and effects on force production, and would further help untangle some of the complex cooperative relationships in the intact sarcomere. |
