The long-term goal of our research is to document the molecular mechanisms of contraction in the striated muscle system, in particular to characterize the elementary molecular steps of the cross-bridge cycle in the cardiac muscle strips (myocardium). We selectively remove the thin filament from myocardium with a gelsolin treatment, and sequentially reconstitute the thin filament with G-actin, tropomyosin, and troponin complex. The degree of reconstitution is assessed by isometric tension, SDS-PAGE, electron microscopy, and confocal microscopy. At each stage of reconstitution, we assess the cross-bridge kinetics with the """"""""sinusoidal analysis"""""""" method. In this method, the length of the preparation is oscillated in sine waves of varying frequencies, and concomitant tension transients are analyzed in terms of three exponential processes. When the effects of ATP, ADP, and phosphate on the exponential processes are studied together with the ATP hydrolysis rate measurement, the 10 kinetic constants that characterize the elementary steps of the cross-bridge cycle can be deduced. To characterize the preparation at each stage of reconstitution, we also measure the ATP hydrolysis rate, the rate of tension redevelopment (ktr), maximum velocity of shortening (Vmax), and pCa-tension relationship. We propose to test the hypothesis that N-terminal negative charge of actin is essential in force generation in structured muscle system. We further propose to characterize the length of thin filament cooperativity experimentally, and test the hypothesis that the cooperativity is affected by the hydrophobic plug of actin. Our short-term goal is to document the thin filament activation mechanism by using thin-filament extracted and reconstituted models of myocardium, and determine the function of actin domains in the structured muscle system where force can be generated. In the long run, the method developed and the knowledge acquired will help to elucidate the mechanisms of cardiac dysfunction, including ischemia, hypertrophy, and familial hypertrophic cardiomyopathy.
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