Muscle force and stiffness during activation and relaxation: Implications for the actomyosin ATPase

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca²⁺]) and then relaxed (decreasing [Ca²⁺]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H₂PO₄⁻]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 µM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], crossbridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total P_i, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated P_i, P_i release from the cross-bridge is reversed, the state(s) before P_i release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before P_i release. In addition, if Ca²⁺ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.P_i state, it could do so before P_i release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 µM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca²⁺ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.