Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium

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 Ca2+ regulation in cardiac muscle. Pulse photolysis of caged Pi (alpha-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 s(-1), which is (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 Ca2+ 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 Ca2+. A three-state model, with separate but coupled transitions for force generation and P-i dissociation, and a Ca2+-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 Ca2+, 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 Ca2+, 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 Ca2+ activation, However, significant quantitative differences between k(Pi) and k(Ca), including different sensitivities to Ca2+ and P-i, indicate that caged Ca2+ tension transients are influenced by additional Ca2+-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 Ca2+ regulatory system and partially limit the physiological rate of tension development in cardiac muscle.