During a normal contractile cycle of a skeletal muscle cell, calcium ions (Ca) are released from the sarcoplasmic reticulum (SR) into the myoplasm under electrical control of the transverse-tubular membranes; Ca then moves by diffusion and binds to troponin on the myofilaments to activate contraction. On a slower time scale, the SR Ca pump re-sequesters Ca within the SR to restore the fiber's resting state. This application proposes to study the function of Ca signaling molecules in their native environment, including ryanodine receptors (RyRs, the Ca release channels of the SR), the myoplasmic Ca buffers (including ATP, troponin, and parvalbumin), and the SR Ca pump. Experiments will be carried out on intact (normally- functioning) fast-twitch and slow-twitch fibers from adult mice. Studies will also be carried out on fibers from mice with specific genetic abnormalities, including mdx mice (an animal model of Duchenne muscular dystrophy). Individual muscle cells will be micro-injected with a fluorescent Ca indicator (e.g., fluo-3 or furaptra), and changes in fluorescence, reflective of changes in myoplasmic free [Ca]; will be recorded on either a home-built confocal microscope (for spatially-resolved recordings) or an optical bench apparatus (for spatially-averaged recordings). The signals to be measured include local Ca signals ( Ca sparks and Ca flames ), which are thought to be produced by activity of a small number of RyRs, and global Ca signals (e.g., those evoked by action potentials), which reflect the synchronous activation of many RyRs. The mechanism of action of dantrolene, a pharmacological agent used to treat malignant hyperthermia, will be investigated in frog intact fibers. The results will be interpreted with the aid of computer models that quantify the SR Ca release flux, the binding of Ca by its myoplasmic buffers, the diffusion of Ca and its mobile buffers, and the transport of Ca by the Ca pump. The results will yield new knowledge regarding (i) the mechanisms that control the opening and closing of RYRS in their native environment, (ii) the kinetics of the reactions between Ca and its buffers, and (iii) the kinetics of Ca pumping. Overall, the knowledge obtained will contribute to understanding Ca signaling in normal muscle cells and alterations in the signaling pathways by diseases and drugs. Treatments of muscle conditions involving alterations in Ca signaling will likely improve with increased understanding of the underlying Ca signaling mechanisms. The rise and fall in the concentration of calcium (Ca) within a voluntary/skeletal muscle cell provide the immediate signals for muscle contraction and relaxation, respectively. This project will measure changes in Ca concentration in fast-twitch and slow-twitch fibers of mouse muscle and will interpret the changes with quantitative computer models. The knowledge obtained will contribute to understanding Ca signaling in normal muscle cells and how this signaling is altered by specific diseases and drugs. Treatments of muscle conditions involving alterations in Ca signaling will likely improve with increased understanding of the underlying Ca movements. ? ? ? ?
