Conformation of the myosin motor during force generation in skeletal muscle

Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide.     

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.     

Folding of the striated muscle myosin motor domain

We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1(795)GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25 degrees C and above the predominant product is an aggregate of S1(795)GFP or a complex with other lysate proteins. At 0 degrees C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1(795)GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg(2+)ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1(795)GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.     

Calcium sensitivity of vertebrate skeletal muscle myosin

The calcium sensitivity of vertebrate skeletal muscle myosin has been investigated. Adenosinetriphosphatase (ATPase) activity was assayed in a reconstituted system composed of either purified rabbit myosin plus actin or myosin plus actin, tropomyosin, and troponin. The calcium sensitivity of actomyosin Mg-ATPase activity was found to be directly affected by the ionic strength of the assay medium. Actomyosin assayed at approximately physiological ionic strength (120 mM KCl) demonstrated calcium sensitivity which varied between 6 and 52%, depending on the myosin preparation and the age of the myosin. Mg-ATPase activity was increased when calcium was present in the assay medium at physiological ionic strength. Conversely, actomyosin Mg-ATPase activity assayed at a lower ionic strength (15 mM KCl) was inhibited by addition of calcium. Addition of tropomyosin and troponin to the assay increased the calcium sensitivity of the system at the physiological ionic strength still further (up to 99% calcium sensitivity) and conferred calcium sensitivity on the system at the lower ionic strength (greater than 90% calcium sensitivity). A correlation also existed between myosin's calcium sensitivity and the phosphorylated state of light chain 2.     

A model for the active site of skeletal muscle myosin.

A model for a main element of the active site of skeletal muscle myosin is presented that relates directly to the 92 amino acid fragment (p₁₀) of myosin recently described by Elzinga &; Collins (1977). In this model, the substrate, an eight-membered cyclic complex of MgATP, fits tightly into a 16 amino acid segment of p₁₀ and interacts with seven of its amino acids. A main feature of the model is the important role played by the one molecule of N^τ-methylhistidine² that is present in each myosin heavy chain. At the site, it is postulated that this rare amino acid functions as a donor ligand to Mg²⁺. Once N^τ-methylhistidine is put in place next to the metal, the other amino acids that appear to form a pocket come easily into position around the MgATP. These amino acids with their postulated functions are: tyrosine 72, which through a Mg-bound water, or perhaps directly, is attached to the Mg; histidine 76, which donates a proton to the P_γ of ATP; lysine 78, which binds electrostatically to P_β of ATP; phenylalanines 80 and 81, which flank the purine ring of ATP; and aspartate 66, which forms a hydrogen bond to the 6-amino group of adenine. The Mg-coordination role ascribed to N^τ-methylhistidine 69 in skeletal muscle myosin could be taken by histidine 69 in cardiac myosin and in other muscle myosins that do not contain the methylated amino acid.The choice of p₁₀ to contain a main element of the active site is based on: (a) the presence in p₁₀ of the essential sulfhydryl groups, SH1 and SH2, whose modification affects the ATPase activity of myosin; (b) the presence in ρ₁₀ of N^τ-methylhistidine, an unusual amino acid whose methylation in skeletal muscle we take as an indicator for a special function at the active site; (c) the position of p₁₀ in the primary structure near the junction between subfragment 1 and subfragment 2 (the hinge region) where, we postulate, enzymatic events at the active site are coupled to movements of the hinge that occur during contraction; (d) indications that the DTNB light chain, probably involved in regulation, is also near the hinge; (e) the effects of MgATP at the active site on the chemical reactivity of three SH groups (SH1, SH2 and SH3) located near the hinge; and (f) the effect of hinge cleavage on the oxygen exchange reaction catalyzed at the active site. The correlation of all these observations forms the basis for our placement of part of the active site on p₁₀ near the subfragment 1-subfragment 2 hinge.     

Quantitative determination of calcium-activated myosin adenosine triphosphatase activity in rat skeletal muscle fibres

Summary A quantitative histochemical technique was developed for determining the kinetics of the calcium-activated myosin ATPase (Ca²⁺-myosin ATPase) reaction in rat skeletal muscle fibres. Using this technique, the maximum velocity (V_max) and the apparent Michaelis-Menten rate constant for ATP (K_app) of the Ca²⁺-myosin ATPase reaction were measured in type-identified fibres of the rat medial gastrocnemius (MG) muscle. The V_max and the K_app of the Ca²⁺-myosin ATPase reaction were lowest in type I fibres and highest (i.e., approx. two times greater) in type IIb fibres. The K_app in type IIa fibres was similar to that in type I. However, the V_max was 1.5 times greater in type IIa fibres, compared to type I fibres. Evidence is presented to suggest that the type IIb fibre population in the MG does not represent a single myosin isozyme. In addition, the broad range of V_max and K_app values indicates that there is marked heterogeneity in the myosin heavy chain and myosin light chain composition of myosin isozymes among individual fibres.     

The myosin co-chaperone UNC-45 is required for skeletal and cardiac muscle function in zebrafish

The assembly of myosin into higher order structures is dependent upon accessory factors that are often tissue-specific. UNC-45 acts as such a molecular chaperone for myosin in the nematode Caenorhabditis elegans, in both muscle and non-muscle contexts. Although vertebrates contain homologues of UNC-45, their requirement for muscle function has not been assayed. We identified a zebrafish gene, unc45b, similar to a mammalian unc-45 homologue, expressed exclusively in striated muscle tissue, including the somites, heart and craniofacial muscle. Morpholino-oligonucleotide-mediated knockdown of unc45b results in paralysis and cardiac dysfunction. This paralysis is correlated with a loss of myosin filaments in the sarcomeres of the trunk muscle. Morphants lack circulation, heart looping and display severe cardiac and yolk-sac edema and also demonstrate ventral displacement of several jaw cartilages. Overall, this confirms a role for unc45b in zebrafish motility consistent with a function in myosin thick filament assembly and stability and uncovers novel roles for this gene in the function and morphogenesis of the developing heart and jaw. These results suggest that Unc45b acts as a chaperone that aids in the folding of myosin isoforms required for skeletal, cranial and cardiac muscle contraction.     

Slow myosin in developing rat skeletal muscle

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of kinetic properties of the ATPase reaction of arterial smooth muscle myosin with skeletal muscle myosin

Myosin was prepared from arterial smooth muscle, and a hybrid actomyosin was formed from arterial myosin and rabbit skeletal muscle F-actin. We performed kinetics on the ATPase reaction [EC 3.6.1.3] of arterial myosin and the hybrid actomyosin at high ionic strength, and compared the kinetic properties of arterial myosin ATPase with those of skeletal muscle myosin ATPase. No significant difference was found between these two myosins in the size of the initial Pi burst, the amount of bound nucleotides, and the rates of various elementary steps in the ATPase reaction. On the other hand, two important differences were observed between the hybrid actomyosin and skeletal muscle actomyosin: (i) The amounts of ATP necessary for complete dissociation of the hybrid and skeletal muscle actomyosins were 2 and 1 mol/mol of myosin, respectively. (ii) The rate of dissociation of the hybrid actomyosin induced by ATP was much lower than that of skeletal muscle actomyosin and also was lower than that of fluorescence enhancement.     

Phosphorylation of rabbit skeletal muscle myosin in situ

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.     

Dissociation and reassociation of rabbit skeletal muscle myosin

Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25°) and in the obsence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4°C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5–10 per cent release of alkali light chains, LC₁ and LC₃, and a 50 per cent dissociation of the Ca²⁺ binding light chain, LC₂, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15–20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca²⁺ binding sites.     

Polymorphism of myosin among skeletal muscle fiber types

An immunocytochemical approach was used to localize myosin with respect to individual fibers in rat skeletal muscle. Transverse cryostat sections of rat diaphragm, a fast-twitch muscle, were exposed to fluorescein-labeled immunoglobulin against purified chicken pectoralis myosin. Fluorescence microscopy revealed a differential response among fiber types, identified on the basis of mitochondrial content. All white and intermediate fiber but only about half of the red fiber reacted with his antimyosin. In addition, an alkali-stable ATPase had the same pattern of distribution among fibers, which is consistent with the existence of two categories of red fibers. The positive response of certain red fibers indicates either that their myosin has antigenic determinants in common with "white" myosin, or that the immunogen contained a "red" myosin. Myosin, extracted from a small region of the pectorlis which consists entirely of white fibers, was used to prepare an immunoadsorbent column to isolate antibodies specific for white myosin. This purified anti-white myosin reacted with the same fibers of the rat diaphragm that had reacted with the white, intermediate, and some red fibers are sufficiently homologous to share antigenic determinants. In a slow-twitch muscle, the soleus, only a minority of the fiber reacted with antipectoralis myosin. The majority failed to respond; hence, they are not equivalent to intermediate fibers of the diaphragm; despite their intermediate mitochondrial content. Immunocytochemical analysis of two different musles of the rat has demonstrated that more than one isoenzyme of myosin can exist in a single muscle, and that individual fiber types can be recognized by immunological differences in their myosin. We conclude that, in the rat diaphragm, there are at least two immunochemically distinct types of myosin and four types of muscle fibers: white, intermediate, and two red. We suggest that these fibers correspond to the four types of motor units described by Burke et al. (Burke, R. E., D. N. Levine, P. Tsairis, and F. E. Zajac, III 1973. J. Physiol. (Lond) 234:723-748.)in the cat gastrocnemius.`     

Autophosphorylation of untrained and well-trained skeletal muscle myosin

Earlier the autophosphorylation of myosin and the labile phosphate (P) content of rabbit skeletal muscle was reported [6, 7, 9]. The present paper describes that the endogeneous preformed P level in fresh preparation of exercised muscle is higher than that of untrained control one. It was revealed that the presence of a significant amount of mitochondrial myosin (with much higher P content) in the well-trained human muscle preparations falsified the appreciation of myofibrillar myosin. Therefore, a reliable myofibrillar preparation with correct P content from exercised subjects was obtained only after the separation of mitochondrial fraction. The P content of fresh preparations can be increased by phosphorylation even in the exercised muscle myosins up to the most higher level in human samples. The phosphoryl group incorporation from [gamma-32P]ATP into the rabbit and hare myosins was checked by radioactive tracer technique, and confirmed by total P content determination performed parallel with molybdate test. It was stated that under present circumstances the labelled 32P incorporation was lower even at an optimal substrate concentration than that of P value obtained directly with molybdate method; because the total P content of preparations had not exchanged during 2 min incubation. So it has been concluded from [gamma-23P] phosphoryl group assayments that much higher amount of P was incorporated into P-Arg, N pi-P-His and fraction 2 as compared with unappreciated labelled P level of the inorganic P (P-Ser, P-Thr), P-Lys, N tau-P-His and minor fractions. From these observations it has been considered that the P-Arg, N pi-P-His and fraction 2 take part in the contraction mechanism and in the course of physical training.