Phosphorylation of the regulatory light chains of myosin affects Ca²⁺ sensitivity of skeletal muscle contraction

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca²⁺ sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca²⁺ sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in -log of the Ca²⁺ concentration producing half-maximal activation = ∼0.25). The same was true for the Ca²⁺ sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca²⁺ sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca²⁺, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca²⁺ binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.