The phosphate (P(i)) dissociation step of the cross-bridge cycle was investigated in skinned rat ventricular myocytes to examine its role in force generation and Ca 2+ regulation in cardiac muscle. Pulse photolysis of caged P(i) (α-carboxyl-2-nitrobenzyl phosphate) produced up to 3 mM P(i) within the filament lattice, resulting in an approximately exponential decline in steady-state tension. The apparent rate constant, k(Pi), increased linearly with total P(i) concentration (initial plus photoreleased), giving an apparent second-order rate constant for P(i) binding of 3100 M -1 s -1 , which is intermediate in value between fast and slow skeletal muscles. A decrease in the level of Ca 2+ activation to 20% of maximum tension reduced k(Pi) by twofold and increased the relative amplitude by threefold, consistent with modulation of P(i) release by Ca 2+ . A three-state model, with separate but coupled transitions for force generation and P(i) dissociation, and a Ca 2+ sensitive forward rate constant for force generation, was compatible with the data. There was no evidence for a slow phase of tension decline observed previously in fast skeletal fibers at low Ca 2+ , suggesting differences in cooperative mechanisms in cardiac and skeletal muscle. In separate experiments, tension development was initiated from a relaxed state by photolysis of caged Ca 2+ . The apparent rate constant, k(Ca), was accelerated in the presence of high P(i), consistent with close coupling between force generation and P(i) dissociation, even when force development was initiated from a relaxed state. k(Ca) was also dependent on the level of Ca 2+ activation. However, significant quantitative differences between k(Pi) and k(Ca), including different sensitivities to Ca 2+ and P(i), indicate that caged Ca 2+ tension transients are influenced by additional Ca 2+ dependent but P(i)-independent steps that occur before P(i) release. Data from both types of measurements suggest that kinetic transitions associated with P(i) dissociation are modulated by the Ca 2+ regulatory system and partially limit the physiological rate of tension development in cardiac muscle.
Araujo, A., & Walker, J. W. (1996). Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium. Biophysical Journal, 70(5), 2316–2326. https://doi.org/10.1016/S0006-3495(96)79797-7