Dissecting Actomyosin Mechanochemistry Using Blebbistatin As A Pharmacological Tool

Muscle contraction results from cyclic interactions between actin and myosin under turnover of ATP. Here, we use the myosin II-specific inhibitor blebbistatin as a pharmacological tool to elucidate this interaction. Particularly we consider the hypotheses that: 1. the effects of blebbistatin depend on the level of myosin regulatory light chain phosphorylation1 and 2. blebbistatin stabilizes the start of the actomyosin power-stroke state, characterized by an absence of inorganic phosphate (Pi) in the active site2. In vitro motility assays (IVMAs) and skinned muscle fiber studies were performed using preparations from fast rabbit muscle. Blebbistatin (1µM) inhibited sliding velocity in the IVMA (∼50%) independently of the RLC phosphorylation status, suggesting that the phosphorylation dependence in muscle cells is fully attributed to myosin head-backbone interactions. The inhibitory effect was approximately half in conditions of reduced ionic strength from 130 to 60 mM, whereas the effect was similar at different MgATP concentrations (0.01 −1 mM). Muscle fiber data showed a reduced isometric force development by 36 ± 11% and 89 ± 5% at 2µM and 10µM blebbistatin, respectively. The force enhancement during an imposed stretch to the fiber during activation (10% of fiber length; 1 length/s) was unaltered. An interpretation of the blebbistatin effects linked to a mecahano-kinetic model of muscle contraction is consistent with an increased population of a start-of-power stroke actomyosin state without Pi in the active site, and it is necessary for the assumption that blebbistatin stabilizes actomyosin states with bound Pi.