The major event leading to the activation of a skeletal muscle is: an electrical depolarization of the sarcolemma that spreads along the T- system to trigger the release of Ca2+ from the terminal sacs (TC) of the sarcoplasmic reticulum (SR), binding of Ca2+ to the troponin leading to actin-myosin interaction. Subsequently, Ca2+ is re-accumulated by a SR calcium pump producing relaxation. This is an outline of events, and there are further details that are important to know if one is to understand this system fully. One of the details not well understood is the mechanism by which direct electric stimulation of vertebrate striated muscles induces a state (hitherto referred to as fatigue), during which the contractile force first declines and subsequently the muscle becomes mechanically refractory to further stimulation. We have found that fatigue in frog fibers is associated with a lack of activation of groups of myofibrils and with a localized lack of Ca release in the muscle cross-sectional area. We want to investigate further the mechanisms responsible for the failure of Ca release and myofibril activation. In fatigued fibers tension can be recovered with K-depolarization and all the myofibrils can be activated and tension can be recovered by releasing Ca with caffeine plus post-fatigue contractures are larger that pre-fatigue ones. Therefore we want to test the hypothesis that the primary cause of failure probably lies in one of the earlier steps of e-c coupling: either the tubular action potential fails to propagate all along the T- system or the T-system-TC-signal that brings about the Ca release from the SR is altered in some myofibrils. We want to test this hypothesis by 1) recording images of topological Ca release changes during fatigue induced with different patterns of stimulation; 2) measuring internal pH changes to see if, in fact, they correlate or not in real time with the development of fatigue; 3) measure whether fatigue is the result of a lack of activation of groups of myofibrils within the muscle cell which would lead to an increase in internal load or frictional load against which the muscle cell contracts, and thereby to a decrease in maximal velocity of shortening (V omicron) observed in fatigued muscle; 4) measure if the tubular action potential is altered during development of fatigue; 5) see whether the vacuolation observed during fatigue is associated with measurable changes in fiber electrical capacitance, and to observe if there is a correlation between recovery from fatigue and disappearance of vacuoles and swelling. This will be done by a combination of optical and mechanical techniques which include images of Ca release, intracellular pH and potentiometric measurements in isolated single muscle cells.
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