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

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 Ca²⁺-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 Ca²⁺-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.