The goal of this research is to understand the mechanisms by which transient tears at the sarcolemma during exercise might lead to long-term damage within a muscle cell. Muscles in Duchenne muscular dystrophy, in which the initial lesion is greater levels of transient sarcolemmal tearing, exhibit similar ultrastructural damage as eccentric exercise. Duchenne dystrophic muscles develop greater activity of calcium-specific leak channels which in turn are responsible for elevated resting intracellular free calcium ([Ca2+]i) and higher rates of calcium- dependent proteolysis. We hypothesize that, during exercise, local calcium influx through transient tears leads to local activation of calcium leak channels via proteolysis. Eventually, accumulation of activated leak channels will increase calcium influx, resting [Ca2+]i, and activation of calcium-dependent degradative pathways. Transient sarcolemmal tears during contractile activity, especially in dystrophic muscle, may represent the initial step in the activation of processes which eventually mediate muscle cell death. This study will examine this hypothesis through the following questions: (1) What are the spatial and temporal changes in [Ca2+]i near a rigorously defined, reproducible wounding event? (2) Does wounding lead to local activation of leak channels, and, if so, what changes in (Ca2+]i are required to activate channels? Is wound-induced leak channel activation mediated by proteolysis and by effects on the cytoskeleton? (3) How is activity of the leak channel modulated by proteolysis or manipulation of the cytoskeleton in excised patches? (4) Is activation of calcium-dependent degradative processes after contractile activity dependent on calcium influx through more active leak channels? (Question 1) Precise wounds on cultured mouse skeletal myotubes will be followed by measurement of spatial and temporal changes in [Ca2+]i. (Question 2) Knowledge of [Ca2+]i changes will then be employed to determine the relationship between calcium influx and channel activation using patch clamp methods. (Question 3) The regulation of channel activity by proteolysis and modulation of the cytoskeleton will be examined using the excised inside-out patch clamp configuration. (Question 4) Myotubes will be subject to long periods of contractile activity or quiescence and dihydropyridine inhibitors of the leak channel to correlate inhibition of leak channels with decreased rates of calcium-dependent enzymatic processes.
