The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation

Abstract

Phosphorylation of the myosin regulatory light chain (RLC) in skeletal muscle has been proposed to act as a molecular memory of recent activation by increasing the rate of force development, ATPase activity, and isometric force at submaximal activation in fibers. It has been proposed that these effects stem from phosphorylation-induced movement of myosin heads away from the thick filament backbone. In this study, we examined the molecular effects of skeletal muscle myosin RLC phosphorylation using in vitro motility assays. We showed that, independently of the thick filament backbone, the velocity of skeletal muscle myosin is decreased upon phosphorylation due to an increase in the myosin duty cycle. Furthermore, we did not observe a phosphorylation-dependent shift in calcium sensitivity in the absence of the myosin thick filament. These data suggest that phosphorylation-induced movement of myosin heads away from the thick filament backbone explains only part of the observed phosphorylation-induced changes in myosin mechanics. Last, we showed that the duty cycle of skeletal muscle myosin is strain dependent, consistent with the notion that strain slows the rate of ADP release in striated muscle.

Fig. 1.

A: 8% polyacrylamide 8 M urea gel showing the reversibility of the phosphorylation reaction. Solid arrows denote dephosphorylated regulatory light chain (RLC); shaded arrows denote phosphorylated RLC. Lane a, endogenously dephosphorylated rabbit skeletal muscle myosin; lane b, Ca²⁺/CaM myosin light chain kinase (MLCK)-treated rabbit skeletal muscle (in vitro) dephosphorylated myosin; lane c, endogenously phosphorylated rabbit skeletal muscle myosin; lane d, calf intestinal alkaline phosphatase (CIAP)-treated rabbit skeletal muscle (in vitro) phosphorylated myosin. LC1–LC3, light chains 1–3. B: The unloaded sliding velocities of RLC-dephosphorylated (Dephos) and RLC-phosphorylated (Phos) myosins before and after treatment with either MLCK or CIAP were measured in the in vitro motility assay.

Fig. 2.

The duty cycles (f) of RLC-dephosphorylated and RLC-phosphorylated myosins were calculated by measuring the velocity of actin filament sliding vs. the number of myosin heads available to interact with the actin filament and fitting to Eq. 2. Each point represents the average sliding velocity of a single actin filament across 10 video frames; 176 actin filaments were followed for RLC-dephosphorylated myosin and 166 filaments were followed for RLC-phosphorylated myosin. The duty cycle for RLC-dephosphorylated myosin was 3.21 ± 0.45%, whereas the duty cycle for RLC-phosphorylated myosin was 4.64 ± 0.60%.

Fig. 3.

Comparison of the effect of applied strain on the duty cycle on RLC-dephosphorylated (A) and RLC-phosphorylated myosins (B). Each point represents the average sliding velocity of a single actin filament across 5–10 video frames; 145 actin filaments were followed for RLC-dephosphorylated myosin, and 204 actin filaments were followed for RLC-phosphorylated myosin. The duty cycle for strained dephosphorylated myosin was 6.8 ± 1.6%, and the duty cycle for strained phosphorylated myosin was 9.2 ± 2.0%. These measured duty cycles are ∼2-fold higher than the measured unstrained duty cycles.

Fig. 4.

A: regulated thin filament (reconstituted with tropomyosin and troponin) sliding velocity was measured as a function of calcium concentration at both 24 and 35°C. B: normalized plots of the regulated sliding velocities (V/V_max). Each point represents the average sliding velocity of 15–30 actin filaments averaged over 10 frames. The raw data were fit to Hill plots (Eq. 3). The best fit curves are indicated as follows: black solid line, RLC-dephosphorylated myosin at 35°C; shaded dotted line, RLC-phosphorylated myosin at 35°C; black dashed-dotted line; RLC-dephosphorylated myosin at 24°C; and shaded dashed line, RLC-phosphorylated myosin at 24°C. Each point represents the average velocity (mean ± SE).