Our hypothesis is that modulation of Ca control of cardiac myofibrils is a regulatory device by which heart cells adjust their activity during beat-to-beat regulation. Our objectives are: 1) to understand the nature of Ca regulation of cardiac myofibrillar activity; 2) to show that the Ca regulation mechanism is modulated physiologically by covalent (protein phosphorylation) and non-covalent modifications (changes in H ion and Mg 2 ion) of the myofibrils; and 3) to understand the mechanism of these modifications. Our approaches to these objectives involve: 1) studies with myofibrils, thick and thin filaments and their constituent proteins, and """"""""reconstituted myofibrils"""""""" in which we systematically study Ca binding properties and ATPase as functions of myofibrillar phosphorylation, pH and free Mg; 2) studies with chemically """"""""skinned"""""""" heart muscle fibers in which we measure tension cost (force/ATPase), unloaded shortening velocity and tension transients during quick length changes as functions of free Ca in varying conditions of myofibrillar phosphorylation, pH and free Mg; and 3) studies with isolated perfused work performing hearts in which we relate time dependent functional changes to levels of troponin I and P-light chain phosphorylation. We have also proposed experiments aimed at preparing and characterizing the P-light chain kinase and phosphatase so that we can use this information and the preparations themselves to adjust in vitro levels of myofibrillar phosphorylation. Our methods involve newly developed 1) techniques for preparing functional, non-denatured myofibrils which retain in vivo levels of light chain and TnI phosphorylation, 2) techniques for determining the levels of phosphorylation in these proteins, 3) techniques for varying free Ca 2 ions in our incubation media without using EGTA, and 4) techniques for measuring tension cost in the fiber preparations.
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