The aim of the proposed work is to investigate the economy of chemomechanical transduction (i.e. the ratio of chemical to mechanical activity) by the contractile apparatus of striated muscle under a variety of experimental conditions. This will be done by simultaneously measuring isometric force and ATP hydrolysis in skinned fibers from thin-filament regulated muscle (from frog and rabbit) as well as from thick-filament regulated muscle (from scallop). ATPase will be estimated using a linked-enzyme assay, by observing the decrease of NADH fluorescence using a microfluorometer which has been constructed in this laboratory. In specific, the aim is to investigate whether the tension-cost (i.e. the ratio of ATPase/force) varies with the level of activating calcium. Comparison of tension-cost of fast and slow twitch fibers from rabbit will indicate likely differences between fiber types, while comparison of tension-cost of fast twitch fibers from rabbit and frog will investigate possible species differences. Further comparison of tension-cost of the aforementioned thin-filament regulated muscles with that of a thick-filament regulated scallop muscle will assess the influence of regulatory mode on contractile economy. Further experiments will investigate tension-cost under conditions which are known to influence calcium sensitivity and/or force production of the contractile machinery, to wit: variations of solution pH, substrate (MgATP), ionic strength, Mg2+, temperature, and substrate/product ratios (i.e. free energy), as well as isotopic substitution of solvent water by deuterium oxide (D20). A mechanical length control apparatus which can rapidly perturb the length of skinned fiber segments will be used to monitor fiber stiffness as a measure of the number of attached crossbridges. After a quick release, the multiphasic process of force recovery will be studied to indicate changes in the rate of crossbridge rotation and detachment. Lastly, the maximal velocity of shortening will be measured using a slack-test technique. In conjunction with force and ATPase, these measures of crossbridge number and behavior will provide insight into the mechanisms by which these various experimental conditions affect the contractile apparatus. This work will be of clinical interest, as well, in that the negative ionotrophy observed with fatigue and hypoxia of skeletal and cardiac muscles is associated with changes in intracellular pH, substrate, and free energy. At present, however, it is not clear how these changes influence the contractile process. It is hoped that the present study might shed some light on this problem.
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