A goal of Dr. Kawai's research is to document the molecular mechanisms of contraction in cardiac muscle. Similar to an automobile engine, one power cycle of cardiac muscle consists of several steps: incorporate the fuel (called "ATP" in biology), burning the fuel (ATP hydrolysis to result in ADP and Pi), release the energy (force generation), and release the waste products (ADP and Pi). In muscle, this power cycle is performed by a molecular structure called "cross-bridges", so that the entire process is called "cross-bridge cycle". He is proposing to characterize elementary steps of the cross-bridge cycle and the mechanism of force generation. He uses skinned myocardium, because the chemical environment of contractile proteins can be modified and tension can be measured in this system. He removes one of its major components, the thin filament, and reconstitutes it with genetically altered mutant proteins. He then studies which step of the cross-bridge cycle is affected by a particular change in the amino acid sequence of a protein, and establishes the structure-function relationship. This procedure is equivalent of removing a critical part of the automobile engine, and replaces it with a modified part to examine engine's performance change. The proposed changes are made in the N-terminal of actin, and isoenzyme switch of a- and b- tropomyosin.
To determine the step modified by the specific substitution, he determines cross-bridge cycle with seven states. For this reason, he changes the length of the fibers sinusoidally at varying frequencies ranging four orders of magnitude, and observes the tension time course. The length change modifies the reaction rate constants, which in turn causes transient instability in the steady state, resulting in transitions between the states. These are observed as tension transients, from which four apparent rate constants are extracted. By studying the effects of MgATP, MgADP and phosphate (Pi), ten kinetic constants of the elementary steps that characterize the seven-state cross-bridge cycle can be deduced.
