The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation

Abstract

(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.in vitro motility assay; mechanics ALL KNOWN MUSCLE SYSTEMS share the characteristic that the regulation of actomyosin interaction is mediated by the gated release of calcium. The specific mechanism by which calcium regulates this interaction depends on the muscle type. In molluscan muscle, calcium binds directly to the myosin essential light chain (ELC) that, together with the regulatory light chain (RLC) and part of the myosin heavy chain (MHC), constitutes the myosin regulatory domain that switches on the myosin motor in response to calcium binding (86). In vertebrate cardiac and skeletal muscle, calcium binds to troponin C, causing a shift in the position of tropomyosin along actin and allowing myosin cross bridges to bind strongly to the thin filament and shorten the sarcomere (for review, see Ref. 22). Smooth muscle has yet another pathway for activation in which calcium binding to calmodulin activates myosin light chain kinase (MLCK), phosphorylating the RLC and activating smooth muscle contraction (for review, see Ref. 70).It was shown by Perrie et al. (50) that vertebrate striated muscle RLC could also be phosphorylated by activated MLCK, and although this interaction is not necessary for activation of contraction, it appears to modulate skeletal and cardiac muscle contractility (40). Phosphorylation of the RLC in striated muscle fibers has been shown to enhance both the magnitude (13,52,66,74,79) and rate of tension development (42, 73) as well as to increase the ATPase rate (79) at submaximal calcium levels (for review, see Ref. 71). Furthermore, it has been shown that myosin phosphorylation can cause potentiation of posttetanic twitch (39, 40) and the rate of cross-bridge attachment (14,42,48,73,79). These effects have also been shown to be removed by knocking out skeletal muscle MLCK (89). The extent of RLC phosphorylation correlates with the frequency of act...

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry

“…In this scheme, straining the myosin traps ADP from ATP hydrolysis in the myosin active site by increasing the affinity of myosin for ADP so that the ADP from ATP hydrolysis becomes unexchangeable with the medium, making the myosin less sensitive to exogenously added ADP. This is consistent with observations of skeletal muscle myosin under strain (24,27), which also shows a strain-induced reduction in the affinity for exogenously added ADP. This is also consistent with a kinetic scheme in which there is a strain-dependent isomerization in the ADP-bound state (28).…”

“…Thus, the reductions in force seen in these mutants could be due to changes in any of these parameters. It is unlikely that the mutations change the number of heads bound to the surface because neither phosphorylation of the RLC nor depletion of the RLC, both more drastic changes in the myosin structure than these one-amino-acid changes, alters the number of heads on the surface (6,23,24). Furthermore, the unloaded velocity and the unloaded duty cycle are statistically indistinguishable, suggesting that the step size of the myosin is unchanged by these mutations.…”

“…Our previous research with skeletal muscle myosin suggests that myosin with phosphorylated RLC exhibits a greater strain sensitivity than dephosphorylated myosin (23,24). It has been shown that phosphorylation of the mutant light chains can restore calcium binding to the RLC and restore mutation-induced changes in the RLC structure (12).…”

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

“…This value is in the middle of the wide range reported by previous studies on heart muscle preparations that used noncardiac isoforms of MLCK (25,(31)(32)(33) and similar to the value (30-40%) reported for β-cardiac myosin in vitro (45). Enhancement of maximum isometric force by cRLC phosphorylation is also observed for skeletal muscle myosin (25), and the effects of RLC phosphorylation on the motor function of a range of muscle myosin isoforms have been attributed to an increase in the myosin lever arm stiffness (46,47) or step size (48).…”

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

“…Perry suggested that myosin light chains could exist as charged variants (Perrie et al 1972(Perrie et al , 1973, shortly after which phosphorylation was established as a significant mechanism in cardiac and skeletal muscle (Frearson and Perry 1975). Myosin RLC phosphorylationinduced enhancement of isometric force and increased rates of muscle contraction have been reported in a wealth of studies from different laboratories (Colson et al 2010;Davis et al 2001;Greenberg et al 2009;Scruggs and Solaro 2011;Sheikh et al 2012;Sweeney et al 1993;Warren et al 2012). Specifically, RLC phosphorylation was shown to accelerate the rate of cross-bridge entry into the force generating state by regulating the proximity as well as the interaction of myosin with actin, thereby increasing Ca 2+ sensitivity and amplitude of force at all activation levels (Colson et al 2010;Szczesna et al 2002).…”