In the intact heart and in isolated preparations of heart, force generators may vary in the amount of force and the maximum velocity of shortening they develop under optimal calcium-activation. In this study of the regulation of cardiac contractile performance, the overall objectives are to: 1) define specifically the changes in function of the cardiac contractile proteins that can be produced by neuroendocrine activity and alteration of resting tension; 2) identify the changes in the contractile proteins themselves associated with the modified function; and 3) determine how these mechanisms are integrated into the overall function of individual cardiac cells.
The specific aims for this period of support are to: 1) determine the steps in the cross-bridge cycle that are regulated in order to produce different levels of contractility of the contractile proteins; 2) define the changes in cross-bridge function that occur in terms of development of force, velocity of shortening, ATPase activity, and in some cases, position of the cross-bridge mass; 3) identify the changes, in particular phosphorylation, in the contractile proteins that are responsible for the modification of the rates of steps within the cross- bridge cycle. The studies will be carried out on isolated cardiac tissue, in which the properties will be altered by changes in resting tension, mechanical activity, superfusion or perfusion medium temperature, duration of superfusion and stimulation by catecholamines. Isometric force will be measured before and maximum Ca-activated force and maximum velocity of shortening will be measured after chemical skinning. Position of the cross-mass will be measured by X diffraction. The slack length method will be used to measure maximum velocity, and Ca- and actin-activated ATPase myosin will be characterized in cryostatic sections by quantitative histocytochemistry. The remaining tissue will be analyzed by 2-dimensional gel electrophoresis. Particular attention will be focused on myosin light chain. In some studies 32P will be used to radioactively label phosphorylated proteins, and proteolysis followed by H.P.L.C. will be used to determine the specific amino acids that have been phosphorylated. This will show what proteins are changed in the regulation of the contractile proteins. Since the tension in the different contractile states can vary from almost zero to maximum, and the economy of energy use can vary, this type of regulation is crucial in the adaption of the normal heart to different hemodynamic demands and metabolic conditions, either normal or pathological. Knowledge of this type of regulation may even provide a basis for the development of new drugs for treatment of impaired cardiac function.
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