The aim of the proposed work is to investigate the process of chemomechanical transduction by the contractile apparatus of striated muscle. The economy of transduction (i.e. """"""""tension-cost"""""""", the ratio of ATPase/force) will be studied by simultaneously measuring isometric force and ATP hydrolysis in chemically skinned fibers from both fast- and slow- twitch skeletal muscle. ATPase will be estimated with a linked enzyme assay using a specially-designed microfluorometer. In addition, the maximal velocity of unloaded shortening (Vmax), stiffness, and the rate of redevelopment of force after a brief period of lightly-loaded isotonic shortening will be measured. In specific, the aim is to investigate whether the tension-cost varies with the level of activating Ca2+. Comparison of tension-cost of fasts-twitch and slow-twitch fibers will indicate likely differences between fiber types. The role of troponin C (TnC) and myosin phosphorylatable light chains (P-LC) in any fiber-type differences will be studied by exchange of TnC or P-LCs. The effect of phosphorylation of P-LCs will also be studied. Fiber-type and TnC or P-LC status will be verified using ultrasensitive polyacrylamide gel electrophoresis of single fibers. In addition to Ca2+, we will focus primarily on the effects of changes in pH, inorganic phosphate (Pi), and ADP because these variations are known to have significant effects on important steps of the cross-bridge cycle. Moreover, they are thought to play major roles in the decline of contractile force and Vmax observed with fatigue of intact muscle. In this regard, pH and Pi are especially important; therefore their interacting effects on the contractile apparatus will be examined using chemical analogs of Pi which we will synthesize. In conjunction with isometric force and ATPase, the other mechanical measures of cross-bridge number and behavior will provide detailed insight into the mechanisms by which fatigue or hypoxia adversely affect the contractile process.
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