Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers.

Abstract

Various functional roles for myosin light chain 2 (LC2) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC 2 on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca 2+. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC2, and (c) after recombination with LC 2. The extraction solution contained 20 mM EDTA, 20 or 50 mM KC1, and either imidazole or PO42-as a pH buffer (pH 7.0). The amount of LC2 extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC~ resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca 2+] by as much as 0.2 pCa units after LC~ extraction. At low concentrations of Ca 2+, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC~ to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC~. LCz extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC2 to the fiber segments. On the basis of these findings we conclude that LC2 may modulate the number of cross-bridges formed during Ca 2+ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC2 may modulate contraction via an influence upon the conformation of the S1-$2 hinge region of myosin.Address reprint requests to Dr.

C‐protein limits shortening velocity of rabbit skeletal muscle fibres at low levels of Ca2+ activation.

“…Margossian (1985) found that the stimulation of cardiac actin-activated myosin ATPase by Cprotein required LC2. We reported previously that partial extraction of LC2 also affects Vm.., but the effect was to slow velocity (Moss et al 1982 and was only observed in the high-velocity phase (Hofmann, Metzger, Greaser & Moss, 1990). If C-protein has its effects via an interaction with LC2, it may be necessary to postulate that LC2 or C-protein binds Ca2+ in order to account for the activation dependence of low-velocity Vmax.…”

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

“…The studies of Moss et al (30), Hofmann et al (13), Metzger and Moss (25), and Patel et al (31) have shown that partial extraction of RLC from skeletal muscle fibers increased the rate of tension redevelopment at submaximal [Ca 2ϩ ]. Our laboratory's studies (48) have revealed that removal of RLC decreased the rate of force development by a factor of two and that this could be restored by reincorporation of RLC in the fibers.…”

Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction

“…A graph of the force-pCa relationship was generated by plotting, for each pCa tested, the pooled relative force data from each of the fibers from the muscles of 6-, 24-, and 36-month-old rats. As described by Hofmann, et al, 54 , data were analyzed by least-squares regression using the Hill equation: $$logfalse[P_{\mathit{rel}}/false(1-P_{\mathit{rel}}false)false]=nfalse(logfalse[C{a}^{2+}false]+kfalse)$$ where P rel is force expressed as a fraction of maximal force, n is the Hill coefficient, and k is the intercept of the fitted line with the x axis, which corresponds to the [Ca 2+ ] at half-maximal force (pCa 50 ).…”