Abstract
The second phase of the biphasic force decay upon release of phosphate from caged phosphate was previously interpreted as a signature of kinetics of the force-generating step in the cross-bridge cycle. To test this hypothesis without using caged compounds, force responses and individual sarcomere dynamics upon rapid increases or decreases in concentration of inorganic phosphate [P(i)] were investigated in calcium-activated cardiac myofibrils. Rapid increases in [P(i)] induced a biphasic force decay with an initial slow decline (phase 1) and a subsequent 3-5-fold faster major decay (phase 2). Phase 2 started with the distinct elongation of a single sarcomere, the so-called sarcomere "give". "Give" then propagated from sarcomere to sarcomere along the myofibril. Propagation speed and rate constant of phase 2 (k(+Pi(2))) had a similar [P(i)]-dependence, indicating that the kinetics of the major force decay (phase 2) upon rapid increase in [P(i)] is determined by sarcomere dynamics. In contrast, no "give" was observed during phase 1 after rapid [P(i)]-increase (rate constant k(+Pi(1))) and during the single-exponential force rise (rate constant k(-Pi)) after rapid [P(i)]-decrease. The values of k(+Pi(1)) and k(-Pi) were similar to the rate constant of mechanically induced force redevelopment (k(TR)) and Ca(2+)-induced force development (k(ACT)) measured at same [P(i)]. These results indicate that the major phase 2 of force decay upon a P(i)-jump does not reflect kinetics of the force-generating step but results from sarcomere "give". The other phases of P(i)-induced force kinetics that occur in the absence of "give" yield the same information as mechanically and Ca(2+)-induced force kinetics (k(+Pi(1)) ∼ k(-Pi) ∼ k(TR) ∼ k(ACT)). Model simulations indicate that P(i)-induced force kinetics neither enable the separation of P(i)-release from the rate-limiting transition f into force states nor differentiate whether the "force-generating step" occurs before, along, or after the P(i)-release.