Myosin light chain phosphorylation during contraction of chicken fast and slow skeletal muscles

A modified automatic freezing apparatus (K. M. Kretzschmar and D. R. Wilkie, 1962, J. Physiol. (London), 202, 66–67) was used for studying light chain phosphorylation during the early phase of contraction of the fast, posterior latissimus dorsi, and slow, anterior latissimus dorsi, muscles of chicken at 37 °C. The frozen muscles were worked up under conditions which avoid artifacts in quantitating the level of light chain phosphorylation in contracting and resting muscles. The posterior latissimus dorsi muscle reached 80% of its maximal isometric tension at 0.1 s of tetanic stimulation. At the same time, light chain phosphorylation increased by 60% of its maximal extent. The peak tension of the posterior muscle at 0.2 s of stimulation was accompanied by maximal light chain phosphorylation. In case of the slow anterior latissimus dorsi muscle, maximal tetanic tension was developed in 2.5 – 5 s and light chain phosphorylation also proceeded at a much slower rate than in the fast posterior muscle. When contralateral posterior latissimus dorsi muscles were stimulated for 0.2 s and one muscle was frozen at the height of tetanus while the other muscle was allowed to relax and frozen 0.4 s after terminating the stimulation, both contracted and relaxed muscles exhibited maximal light chain phosphorylation. However, when the muscle was allowed to relax for 0.8 s before freezing, half of the phosphorylated light chain became dephosphorylated. The resting level of phosphate content of the light chain was restored in both the posterior and anterior muscles during a longer time after relaxation.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Myosin light chain phosphorylation and contractile performance of human skeletal muscle

Twitch tension and maximal unloaded velocity of human knee extensor muscles were studied under conditions of low phosphate content of the phosphorylatable light chains (P-light chains) of myosin and elevated phosphate content, following a 10-s maximal voluntary isometric contraction (MVC). After the MVC, twitch tension was significantly potentiated, with greater potentiation observed at a shorter muscle length (p less than 0.05). The MVC was associated with at least a twofold increase in phosphate content of the fast (LC2F) and two slow (LC2S and LC2S') P-light chains, but this increase was unrelated to muscle length. No significant differences in knee extension velocity were observed between conditions where P-light chains had low or elevated phosphate content. Positive but nonsignificant correlations were noted between the extent of twitch potentiation and phosphate content of individual P-light chains as well as the percentage of type II muscle fibres in vastus lateralis muscle. No significant relationships were determined for myosin light chain kinase activity and either P-light chain phosphorylation or type II fibre percentage. These data suggest that, unlike other mammalian fast muscles, P-light chain phosphorylation of mixed human muscles is not strongly associated with altered contractile performance.     

Phosphorylation in vivo of the P light chain of myosin in rabbit fast and slow skeletal muscles.

The P light chain of myosin is partially phosphorylated in resting slow and fast twitch skeletal muscles of the rabbit in vivo. The extent of P light-chain phosphorylation increases in both muscles on stimulation. Rabbit slow-twitch muscles contain two forms of the P light chain that migrate with the same electrophoretic mobilities as the two forms of P light chain in rabbit ventricular muscle. The rate of phosphorylation of the P light chain in slow-twitch muscle is slower than its rate of phosphorylation in fast-twitch muscles during tetanus. The rate of P light-chain dephosphorylation is slow after tetanic contraction of fast-twitch muscles in vivo. The time course of dephosphorylation does not correlate with the decline of post-tetanic potentiation of peak twitch tension in rabbit fast-twitch muscles. The frequency of stimulation is an important factor in determining the extent of P light-chain phosphorylation in fast- and slow-twitch muscles.     

Myosin from fast and slow skeletal and cardiac muscles of mammals of different size.

The ATPase activity of myosin and contraction time in extensor digitorum longus muscle, soleus muscle and cardiac muscle was compared in mammals differing in size. It was shown that the myosin ATPase activity of homologous muscles decreases and contraction time increases with increasing size of animals. The rate of tryptic digestion of myosin, the electrophoretic pattern of light chains of myosin and the effect of p-chloromercuribenzoate on ATPase activity of myosin were also studied. All these three myosin properties are very characteristic when the myosin from a fast muscle is compared with the myosin from a slow muscle of the same animal, but no relationship between these three myosin properties and ATPase activity of myosin was found, when homologous muscles of various mammals were compared.     

Myosin light chain patterns of individual fast and slow-twitch fibres of rabbit muscles

Summary Single muscle fibres were isolated by microdissection from freeze-dried samples of rabbit psoas and soleus muscles. The individual fibres were typed according to qualitative histochemical reactions for succinate dehydrogenase or NADH-tetrazolium reductase and for alkaline Ca²⁺-activated myofibrillar myosin ATPase after acid or alkaline preincubation. Methods are described for electrophoretic analysis by means of polyacrylamide disc electrophoresis in the presence of SDS of total myofibrillar proteins in single fibres after pre-extraction of soluble proteins. Fast-twitch white fibres revealed a myosin light chain pattern characteristic of fast-type myosin with three light chains of apparent molecular weights of 22,300 (LC₁), 18,400 (LC₂) and 16,000 (LC₃). Fast-twitch red fibres were indistinguishable in this respect from fast-twitch white fibres and showed an identical pattern of myosin light chains. Slow-twitch fibres could be characterized by a myosin light chain pattern typical of myosin of slow-twitch muscles with peptides of the apparent molecular weights of 23,500 (LC_1Sa), 23,000 (LC_1Sb) and 18,500 (LS_2S). Slow-twitch fibres isolated from soleus as well as from psoas muscle were indistinguishable with regard to their myosin light chain patterns, thus suggesting that fibres of the same histochemical type correspond in their myosin light chain patterns irrespective of their origin from different muscles.Dedicated to the memory of Ernest Gutmann who has contributed so much to our knowledge on differentiation of muscle and who died on August 6, 1977     

Myosin light chain phosphorylation in intact human muscle

The phosphate content of the fast (LC2F) and two slow (LC2S and LC2S1) phosphorylatable light chains (P-light chains) in myosin isolated from biopsy samples of rested human vastus lateralis muscle averaged 0.21, 0.28 and 0.25 mol of phosphate per mol of P-light chain, respectively. Following a 10 s maximal contraction, phosphate content was increased by almost 2-fold in the fast and two slow P-light chains. After prolonged, moderate cycling activity phosphate content was only slightly increased in the three P-light chains. These data suggest that, unlike animal skeletal muscle, myosin light chain kinase and phosphatase activities are similar in human fast and slow muscle fibres.     

Myosin light chain phosphorylation and growth cone motility

According to the treadmill hypothesis, the rate of growth cone advance depends upon the difference between the rates of protrusion (powered by actin polymerization at the leading edge) and retrograde F-actin flow, powered by activated myosin. Myosin II, a strong candidate for powering the retrograde flow, is activated by myosin light chain (MLC) phosphorylation. Earlier results showing that pharmacological inhibition of myosin light chain kinase (MLCK) causes growth cone collapse with loss of F-actin-based structures are seemingly inconsistent with the treadmill hypothesis, which predicts faster growth cone advance. These experiments re-examine this issue using an inhibitory pseudosubstrate peptide taken from the MLCK sequence and coupled to the fatty acid stearate to allow it to cross the membrane. At 5-25 microM, the peptide completely collapsed growth cones from goldfish retina with a progressive loss of lamellipodia and then filopodia, as seen with pharmacological inhibitors, but fully reversible. Lower concentrations (2.5 microM) both simplified the growth cone (fewer filopodia) and caused faster advance, doubling growth rates for many axons (51-102 microm/h; p <.025). Rhodamine-phalloidin staining showed reduced F-actin content in the faster growing growth cones, and marked reductions in collapsed ones. At higher concentrations, there was a transient advance of individual filopodia before collapse (also seen with the general myosin inhibitor, butanedione monoxime, which did not accelerate growth). The rho/rho kinase pathway modulates MLC dephosphorylation by myosin-bound protein phosphatase 1 (MPP1), and manipulations of MPP1 also altered motility. Lysophosphatidic acid (10 microM), which causes inhibition of MPP1 to accumulate activated myosin II, caused a contracted collapse (vs. that due to loss of F-actin) but was ineffective after treatment with low doses of peptide, demonstrating that the peptide acts via MLC phosphorylation. Inhibiting rho kinase with Y27632 (100 microM) to disinhibit the phosphatase increased the growth rate like the MLCK peptide, as expected. These results suggest that: varying the level of MLCK activity inversely affects the rate of growth cone advance, consistent with the treadmill hypothesis and myosin II powering of retrograde F-actin flow; MLCK activity in growth cones, as in fibroblasts, contributes strongly to controlling the amount of F-actin; and the phosphatase is already highly active in these cultures, because rho kinase inhibition produces much smaller effects on growth than does MLCK inhibition.     

Myosin transitions in developing fast and slow muscles of the rat hindlimb

Abstract. Myosin isozymes from the slow soleus and fast EDL muscles of the rat hindlimb were analyzed by pyrophosphate gel electrophoresis, by peptide mapping of heavy chains, and by antibody staining. At the earliest stage examined, 20 days gestation, distinctions between the developing fast and slow muscles were seen by all these criteria; all fibers in the distal hindlimb reacted strongly with antibody to adult fast myosin. Some fibers also reacted with antibody to adult slow myosin; these fibers had a precise, axial distribution in the hindlimb. This pattern of staining which includes the entire soleus, foreshadows the adult distribution of slow fibers and may indicate that the specific pattern of innervation of the limb is already determined. In the early developing soleus there are four fetal and neonatal isozymes plus two isozymes present in equal proportions in the 'slow' area of the pyrophosphate gel. The mobility of these two slow isozymes decreases with maturity and the slowest moving isozyme gradually becomes the dominant species. Thus early diversity between the soleus and EDL is expressed by myosins which are distinct from the mature isozymes. The relative proportion of slow isozymes significantly increases with development and as this occurs the fetal and neonatal isozymes are progressively eliminated. Transiently at least one mature fast isozyme appears in the soleus. This is present at 15 days postpartum and probably correlates with the population of fast, type II fibers, which comprise 50% of this muscle cell population at 15 days. The EDL contained three fetal and neonatal isozymes and only one slow isozyme which does not change in mobility with age. Slow isozymes in the soleus and EDL are thus not identical. Each muscle underwent a unique series of changes until the adult pattern of isozymes and heavy chains was reached about one month postpartum.     

Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments.

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.     

Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy

Hyperphosphorylation of myosin regulatory light chain (RLC) in cardiac muscle is proposed to cause compensatory hypertrophy. We therefore investigated potential mechanisms in genetically modified mice. Transgenic (TG) mice were generated to overexpress Ca2+/calmodulin-dependent myosin light chain kinase specifically in cardiomyocytes. Phosphorylation of sarcomeric cardiac RLC and cytoplasmic nonmuscle RLC increased markedly in hearts from TG mice compared with hearts from wild-type (WT) mice. Quantitative measures of RLC phosphorylation revealed no spatial gradients. No significant hypertrophy or structural abnormalities were observed up to 6 months of age in hearts of TG mice compared with WT animals. Hearts and cardiomyocytes from WT animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses, but the responses for TG mice were attenuated. Additional biochemical measurements indicated that overexpression of the Ca2+/calmodulin-binding kinase did not perturb other Ca2+/calmodulin-dependent processes involving Ca2+/calmodulin-dependent protein kinase II or the protein phosphatase calcineurin. Thus, increased myosin RLC phosphorylation per se does not cause cardiac hypertrophy and probably inhibits physiological and pathophysiological hypertrophy by contributing to enhanced contractile performance and efficiency.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.     

Regulation of glucose uptake in rat slow and fast skeletal muscles

1. Regulation of glucose uptake was compared between extensor digitorum longus (EDL) and soleus (Sol) muscles in rats. 2. Insulin stimulated glucose uptake more in EDL than in Sol. 3. Under high concentrations of insulin, the glucose uptake was higher in EDL than Sol. 4. Inhibition of oxidative phosphorylation by anoxia or an uncoupler stimulated glucose uptake more in EDL than in Sol. 5. Anoxia abolished the effect of insulin on glucose uptake in both EDL and Sol. 6. The blocker to glucose transport system reduced glucose uptake more in Sol than in EDL.     

Power outputs of slow and fast skeletal muscles of mice

The purpose of this study was to contrast the frequency-power relationship of slow soleus and fast extensor digitorum longus (EDL) muscles to their frequency-force relationships and to investigate factors involved in the development of maximum power during a single contraction. Stimulation frequency-force and stimulation frequency-power relationships were determined for soleus and EDL muscles of the mouse for single contractions in situ at 35 degrees C. Power was measured during isovelocity shortening contractions with displacement through 10% of fiber length at the optimum velocity. Optimum velocity was defined as the shortening velocity for the generation of maximum power for a given stimulation frequency. Both force (N/cm2) and power (watts/kg) increased with stimulation frequency until a plateau was reached. For the frequency-force relationship, the curve for soleus muscles was merely shifted to the left of that for EDL muscles. In contrast, the power developed by EDL muscles was greater than that of soleus muscles (P less than 0.05) at each stimulation frequency. The higher power was a direct consequence of higher optimum velocities for EDL muscles compared with soleus muscles.