The long term objective of this project is to develop techniques to investigate mechanical and energetic properties of isolated single skeletal muscle cells and to employ these techniques to gain further insight into the role of energetics in muscle design. Utilizing isolated single muscle cells from the frog, specific aims include: 1) develop techniques to measure simultaneously and quantitatively time course and amount of force and energy liberation during contraction, 2) determine relationship among maximum amplitude of force production per cross-sectional area, steady state rate of energy liberation, and concentration of solids in individual cells, 3) determine feasibility of estimating energy liberation attributed to Ca2+ cycling from steady rate of energy liberation in cells stretched to a resting sarcomere length of 3.8 Mum and delineate influence of agents known to alter Ca2+ cycling on this energy liberation, 4) determine relationship among tetanus force, energy liberation, and stiffness in the sarcomere length range of 1.7 to 2.2 Mum, 5) determine under conditions designed to alter cross-bridge cycling rate and/or maximum velocity of muscle shortening (Vo) relationships among Vo, steady rate of energy liberation and steady tetanus force, and 6) determine in three twitch fiber types relationships among Vo, steady rate of energy liberation, kinetics of force development and maintenance, concentration of solids, cell dimensions, and maximum force per cross-sectional area.
Specific Aim 7 is: perform preliminary studies designed to isolate viable, intact single cells from mammalian skeletal muscle. Energy liberation is measured using especially designed low heat capacity thermopiles. Concentration of solids in a living cell is calculated from a knowledge of the refractive index of the cell which will be determined by quantitative interference microscopy. Vo is measured by the slack step technique and stiffness is measured during ramp releases. These experiments will avoid difficulties of interpretation associated with investigations employing whole muscles. With single skeletal muscle cells, it eventually will be possible to relate energy cost of cross-bridge and Ca2+ cycling during contraction to mechanical characteristics such as rate of force development, fatigue resistance, and shortening velocity. These properties then can be related to structural and biochemical features of the same cell. This information will provide a clearer understanding of compromises involved in muscle design at a cellular level.
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