The long-term objective of this research is to continue to study the process by which Ca2+ controls vertebrate striated muscle contraction. A significant gap exists in our knowledge of the activation mechanism with respect to its operation in both intact and skinned fibers. The health relatedness of the proposal is the potential for application of the results to cardiac muscle, where Ca2+ is also the key regulator of contraction and many inotropic agents act by influencing Ca2+ fluxes.
Our Specific Aims are directed at testing a current model for activation in which Ca2+ acts as a rate modulator governing the transition of weakly bound cross-bridges to the strongly bound state, which is not in accordance with the classical view that Ca2+ regulates the number of attached cross-bridges via a recruitment mechanism.
Our Aims are as follows. 1) Test whether, during the early phases of contraction, particularly at low levels of activation, the transition from detached to attached but low or non-tension generating states depends on Ca2+, thus indicating an activation mechanism more complex than simple regulation of a transition from weakly to strongly bound attached states. 2) Test whether activation level is influenced by mechanical perturbation, particularly at low levels of activation, thus testing whether changes in contractile properties following perturbations can simply be attributed to the dependence of a specific rate constant on an unchanged activation level. 3) Test whether the velocity of unloaded shortening, (Vu) and the curvature of the force-velocity curve are reduced by a reduction in activation level, thus testing the rate modulation theory prediction that the only influence of Ca2+ is on the forward rate constant governing the weakly-to-strongly bound state. A key feature of the Experimental Design is the intent to carry out a parallel study in both intact and skinned fibers. The Methodology exploits many techniques already developed, such as the dissection, mounting, stimulation/activation, microscopic observation of intact and skinned fibers and measurements of shortening velocities. Attached cross-bridge states will be characterized by techniques based on small amplitude sinusoidal length oscillations. The resulting tension oscillations will be separated into in-phase components (indicating attached cross-bridges) and quadrature components (indicating attached cross-bridges undergoing state transitions). Fluorescent dye technology will be used to report intracellular Ca2+.
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