Kinetics of force generation and phosphate release in skinned rabbit soleus muscle fibers

Abstract

The kinetics of the force generating and Pi release steps of the actomyosin-adenosinetriphosphatase (ATPase) cycle have been compared in Ca(2+)-activated skinned fibers of rabbit soleus (slow twitch) and psoas (fast twitch) muscle. Pi was rapidly photogenerated within the fiber lattice by laser flash photolysis of caged Pi [1-(2-nitro)phenylethyl phosphate]. Pi reduces isometric tension in the steady state but is less effective in slow-twitch muscle than in fast-twitch muscle (e.g., 14 mM Pi reduces tension by 29 +/- 4.6% in slow and by 47 +/- 5.3% in fast). The tension response to a sudden increase in Pi concentration in slow-twitch muscle has four phases, but as in fast-twitch muscle, only phase II (an exponential decline in force) appears to be caused by Pi binding to cross bridges, whereas the other three phases are probably indirect effects caused by caged Pi photolysis. The amplitude of phase II is consistent with the steady-state reduction in force by Pi. The rate of phase II (kappa Pi) is 3.9 +/- 0.33 s-1 at 20 degrees C and 0.28 +/- 0.02 s-1 at 10 degrees C (1 mM Pi). kappa Pi is thus 33 times slower in slow-twitch muscle than in fast at 20 degrees C and 84 times slower at 10 degrees C. In contrast to fast-twitch muscle, in slow muscle kappa Pi is sufficiently slow to partially limit the ATPase turnover rate.

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi]

“…Thus, having to start from higher initial [P i ] to determine k þPi (2) values at high final [P i ] would change a linear toward a hyperbolic-shaped relation. In line with this idea, an exploration of the k þPi -[P i ] relation over a small [P i ]-range (1-3 mM), by inducing P i -jumps from same initial [P i ] with the caged-P i technique, also yielded a linear relation (17). In summary, when initiated from the same [P i ], P i -induced force kinetics depend linearly on the [P i ].…”

“…There is a tendency for the k þPi -[P i ] relation to become more hyperbolic with increasing temperature (13). In contrast, P i -jump studies on slow skeletal muscle fibers (17) and skinned cardiac myocyte bundles (16) revealed linear relations. Similarly, here with cardiac myofibrils, k þPi(2) increased linearly with the final [P i ].…”

“…It is generally accepted that the first and major force-generating step is not coupled to the cleavage of ATP into ADP and inorganic phosphate (P i ) but rather to the subsequent, reversible release of P i , e.g., through the backdoor of the myosin molecule (1)(2)(3)(4). To explore the chemomechanical coupling, force changes were induced in muscle fibers by rapid length changes (5-10), temperature jumps (5,11), pressure jumps (12), or photolytic release of caged-P i (13)(14)(15)(16)(17), and the kinetics of these force changes were interpreted in terms of cross-bridge models. The rapid change in [P i ] provides a well-defined approach because it specifically perturbs the reversible equilibrium of the P i -release.…”

“…Force kinetics after rapid change of [P i ] were first explored in skinned skeletal muscle fibers (14,17) and cardiac myocyte bundles (16). Flash photolysis of caged-P i in these preparations enabled the investigators in these studies to study the kinetics of the force decay induced by rapid increase of [P i ].…”

“…In contrast to k TR and k ACT, which were interpreted to reflect rate-limiting transitions in the cross-bridge cycle, k Pi was interpreted to report the kinetics of the force-generating isomerization. Forward and reverse rate constants of this isomerization were determined from the hyperbolic relation of k Pi versus the [P i ] (13)(14)(15)17) and from the hyperbolic [P i ]-dependence of the force kinetics induced by sinusoidal length changes (6,7). Former cross-bridge models (3,18) were revised by adding a force-generating ADP.P i -binding state to the cross-bridge model.…”