The objective of this project is to map the contribution of Ca2+ sequestration, Ca2+ dissociation from troponin (TN) and cross-bridge detachment to the multiple phases of relaxation rate in skeletal muscle during normal and fatigue conditions. We will do this by selectively altering each of these biochemical events and determining the effect on relaxation rate and/or [Ca2+]. Intact and mechanically skinned frog muscle fibers with functionally intact SR will be studied at 10 degrees C. Normal rates of contraction and relaxation have been achieved in skinned fibers by rapidly and transiently increasing sarcoplasmic [Ca2+] by use of a photolabile caged Ca2+ (Specific Aim #1) and rapid relaxation has been induced by chelating Ca2+ with a photolabile chelator (diazo-2). We will determine (Specific Aims #2 and #3) how relaxation rate is influenced by modification of: 1) rate of Ca2+ sequestration, 2) rate of Ca2+ dissociation from TN, 3) rate of cross-bridge detachment and 4) number of attached cross-bridges at the time of relaxation. Rate of Ca2+ sequestration will be modified by altering SR Ca2+ ATPase activity with thapsigargin, by manipulating [Mg/PA], or by rapid Ca2+ chelation with diazo-2. Biologically active mutant TNCs which exhibit a 10-fold difference in the rate of Ca2+ dissociation from their Ca2+ specific sites will be reconstituted into skinned fibers and tested for their effect on relaxation rate. The number of attached cross-bridges at the time of relaxation will be modified by altering [Ca2+] and [vanadate]. Cross-bridge detachment rate will be estimated from the final rate of relaxation from rigor induced by caged ATP in the absence of Ca2+. Cross-bridge detachment rate will be decreased by increasing [ADP] and increased by increasing [P/i]. We will determine (Specific Aim #4) which aspect of relaxation, SR Ca2+ uptake, Ca2+ off rate from TN or cross-bridge detachment, is most influenced by fatigue. Experiments will concentrate on the consequences of decreases in pH and in the chemical free energy change of ATP hydrolysis and their mechanisms of action in producing fatigue. We will develop (Specific Aim #5) fluorescent techniques which allow us to monitor Ca2+ transients, Ca2+ association-dissociation from TN and force during contraction-relaxation in skinned fibers. In summary, these studies will define how Ca2+ sequestration rate, Ca2+ off rate from TN and cross-bridge detachment rate contribute or map to the multiple phases of relaxation observed in skeletal muscle.
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