9318120 Kawai The molecular mechanisms of Ca2+ regulation of the contractile apparatus are investigated in mammalian cardiac muscles. The cross-bridge kinetics are assessed by the method called sinusoidal analysis, which changes the length of the muscle sinusoidally at varying frequencies, and measures concomitant amplitude and phase shift in tension. The length change perturbs the steady state distribution of the cross- bridges, and this perturbation results in the transient change in tension. From these data, 3 apparent rate constants are extracted. These are further studied as functions of the MgATP, MgADP, phosphate (Pi) concentrations, and 6 rate constants and 3 association constant (kinetic constants) that characterize a 7 state cross-bridge model are extracted. The rate limiting step is characterized by the ATP hydrolysis rate measurement. The strength of our approach is that the rate constants ranging 3 orders of magnitudes can be characterized by a single experiment. The role of cooperativity of the regulatory units of the thin filament in cross-bridge kinetics is investigated in this proposal. Skinned cardiac muscle is activated by varying concentrations of Ca2+, and the apparent rate constants are examined to see if the cross-bridge kinetics are affected by Ca2+. Troponin C (TnC) is partially extracted from skinned myocardium, the extracted preparation is activated in a solution containing high Ca2+ concentration (40 uM), and 10 kinetic constant that characterize elementary steps of the cross-bridge cycle are obtained as functions of the degree of TnC extraction. The preparation is also reconstituted with cardiac TnC in which Ca2+ specific binding site is destroyed by genetic engineering to examine if the binding of TnC alone affects the cross-bridge kinetics. These experiments will determine whether the cooperative activation of regulatory units on the thin filament affects elementary steps of the cross-bridge cycle. The Troponin complex is then placed into the turned on state regardless of Ca2+, either by extracting TnI or by using aTnC (turned on TnC), and cross-bridge kinetics. at varying Ca2+ concentrations are studied. This experiment will determine if there is an additional Ca2+ binding site, such as in the myosin light chain 2, and if Ca2+ binding into this site modifies the cross-bridge kinetics. The degree of extraction and reconstitution of troponin components is quantified by using SDS polyacrylamide gel electrophoresis, and phosphorylation by using isoelectric focusing gel. The proposed research uses biophysical, biochemical, engineering, and genetic engineering techniques, and it is aimed at understanding the basic mechanisms of contraction. ***
