Phosphate Release and Force Generation in Skeletal Muscle Fibers

Abstract

Rapid laser pulse-induced photolysis of an adenosine triphosphate precursor in muscle fibers abruptly initiated cycling of the cross-bridges. The accompanying changes in tension and stiffness were related to elementary mechanochemical events of the energy-transducing mechanism. When inorganic phosphate was present at millimolar concentrations during liberation of adenosine triphosphate in the absence of calcium, relaxation was accelerated. Steady active tension in the presence of calcium was decreased but the approach to final tension was more rapid. These results suggest that, during energy transduction, formation of the dominant force-generating cross-bridge state is coupled to release of inorganic phosphate in a reaction that is readily reversible.

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

“…Kinetic parameters of force changes upon rapid increases in [P i ] were obtained by fitting transients by the biphasic function described for relaxation (25). The function consists of a linear term to obtain the rate constant k þPi(1) and the duration t þPi(1) of the initial slow force decline (phase 1) and an exponential term with the rate constant k þPi (2) for the subsequent rapid exponential force decay (phase 2).…”

“…3 C), and linear regression analysis (not illustrated) yields a strong correlation of average amplitude of ''give'' and force reduction (r ¼ 0.983, p ¼ 0.0026). Also, the propagation speed of ''give'' and the force kinetics of phase 2 (k þPi (2) ) changed in parallel with the [P i ] (Fig. 3 D) with a very strong correlation of propagation speed and k þPi(2) (r ¼ 0.999, p ¼ 3.8 Â 10 À5 ).…”

“…Relaxing/activating standard solutions without added P i contained either 3 mM K 4 Cl 2 CaEGTA (activating) or 3 mM K 4 Cl 2 EGTA (relaxing solution), 10 mM imidazole, 1 mM K 2 Cl 2 Na 2 MgATP, 3 mM MgCl 2 , 47.7 mM Na 2 CrP, 2 mM DTT, pH 7.0 at 10 C, ionic strength (m) ¼ 0.17 M. The standard solutions contained 0.16 5 0.04 mM (mean 5 SD) contaminant P i as determined by a phosphate assay kit (E-6646; Molecular Probes, Eugene, OR). To keep a constant m for all solutions, in solutions with additional P i for each mM of added P i , the [Na 2 CrP] was reduced by 0.65 mM.…”

“…At same final [P i ], k þPi(1) is identical to k TR and k ACT and to the rate constant k ÀPi of the single-exponential force rise in response to a rapid drop in [P i ]. k þPi (2) but not k þPi(1) nor k ÀPi is altered by the initial [P i ], thus by the prehistory of contraction. These results suggest that the major force decay upon rapid increase in [P i ] does not reflect the kinetics of the ''force-generating step'' but is determined by sarcomere dynamics whereas in the absence of ''give'', P i -induced force kinetics simply report the kinetics of rate-limiting transitions in the cross-bridge cycle.…”

“…To understand how cross-bridges convert chemical into mechanical energy, the force-generating transitions in this ATPase cycle, i.e., the force-generating steps, have to be clarified. 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.…”