Rabbit muscle phosphoglucomutase is a monomer

A procedure which avoids the use of heat or precipitation with salt at acid pH has been developed for the isolation of phosphoglucomutase from rabbit muscle. Contrary to earlier reports, the enzyme obtained by this procedure as well as by published techniques is a single polypeptide chain. This is true of phosphorylated as well as dephosphorylated proteins.     

Extraocular muscle in merosin-deficient muscular dystrophy: cation homeostasis is maintained but is not mechanistic in muscle sparing

Extraocular muscle is uniquely spared from damage in merosin-deficient congenital muscular dystrophy. Using a murine model, we have tested the hypothesis that the maintenance of calcium homeostasis is mechanistic in extraocular muscle protection. Atomic absorption spectroscopy has demonstrated a strong correlation between the perturbation of calcium homeostasis in hindlimb muscle that is severely damaged and the absence of changes in calcium in extraocular muscle. If, as in other skeletal muscles, extraocular muscle fibers are destabilized by merosin deficiency, we would expect an increase in total muscle calcium coupled with an adaptive response in the high capacity/speed of the sarcoplasmic reticulum of the eye muscle. However, we have not observed the expected increases in total muscle calcium content, Ca²⁺-ATPase activity, Na⁺/Ca²⁺ exchanger content, or smooth ER Ca²⁺-ATPase content that are predicted by this model. Instead, these results indicate that the increased membrane permeability that characterizes, and is potentially mechanistic in, myofiber degeneration in muscular dystrophy does not occur in merosin-deficient extraocular muscle. Thus, the high-capacity calcium-scavenging systems are not primarily responsible for extraocular muscle protection in muscular dystrophy.     

Estradiol's beneficial effect on murine muscle function is independent of muscle activity

Estradiol (E?) deficiency decreases muscle strength and wheel running in female mice. It is not known if the muscle weakness results directly from the loss of E? or indirectly from mice becoming relatively inactive with presumably diminished muscle activity. The first aim of this study was to determine if cage activities of ovariectomized mice with and without E? treatment differ. Ovariectomized mice were 19-46% less active than E?-replaced mice in terms of ambulation, jumping, and time spent being active (P ≤ 0.033). After E?-deficient mice were found to have low cage activities, the second aim was to determine if E? is beneficial to muscle contractility, independent of physical activities by the mouse or its hindlimb muscles. Adult, female mice were ovariectomized or sham-operated and randomized to receive E? or placebo and then subjected to conditions that should maintain physical and muscle activity at a constant low level. After 2 wk of hindlimb suspension or unilateral tibial nerve transection, muscle contractile function was assessed. Soleus muscles of hindlimb-suspended ovariectomized mice generated 31% lower normalized (relative to muscle contractile protein content) maximal isometric force than suspended mice with intact ovaries (P ≤ 0.049). Irrespective of whether the soleus muscle was innervated, muscles from ovariectomized mice generated ～20% lower absolute and normalized maximal isometric forces, as well as power, than E?-replaced mice (P ≤ 0.004). In conclusion, E? affects muscle force generation, even when muscle activity is equalized.     

Talin2 is induced during striated muscle differentiation and is targeted to stable adhesion complexes in mature muscle

The cytoskeletal protein talin serves as an essential link between integrins and the actin cytoskeleton in several similar, but functionally distinct, adhesion complexes, including focal adhesions, costameres, and intercalated disks. Vertebrates contain two talin genes, TLN1 and TLN2, but the different roles of Talin1 and Talin2 in cell adhesion are unclear. In this report we have analyzed Talin1 and Talin2 in striated muscle. Using isoform-specific antibodies, we found that Talin2 is highly expressed in mature striated muscle. Using mouse C2C12 cells and primary human skeletal muscle myoblasts as models of muscle differentiation, we show that Talin1 is expressed in undifferentiated myoblasts and that Talin2 expression is upregulated during muscle differentiation at both the mRNA and protein levels. We have also identified regulatory sequences that may be responsible for the differential expression of Talin1 and Talin2. Using GFP-tagged Talin1 and Talin2 constructs, we found that GFP-Talin1 targets to focal adhesions while GFP-Talin2 targets to abnormally large adhesions in myoblasts. We also found that ectopic expression of Talin2 in myoblasts, which do not contain appreciable levels of Talin2, dysregulates the actin cytoskeleton. Finally we demonstrate that Talin2, but not Talin1, localizes to costameres and intercalated disks, which are stable adhesions required for the assembly of mature striated muscle. Our results suggest that Talin1 is the primary link between integrins and actin in dynamic focal adhesions in undifferentiated, motile cells, but that Talin2 may serve as the link between integrins and the sarcomeric cytoskeletonin stable adhesion complexes in mature striated muscle.     

Mouse muscle phosphofructokinase is partially phosphorylated.

Phosphofructokinase isolated from mouse skeletal muscle 18 hours after intraperitoneal injection of [${}^{32}\text{P]-}\text{PO}{}_4{}^{\mathrm{3?}}$ contained 0.12 to 0.15 moles of covalently bound phosphate per protomer on the basis of the specific activity of radiolabel in the γ-position of ATP. Under identical conditions, muscle pyruvate kinase and aldolase had no covalently bound phosphate.     

Myonase is localized in skeletal muscle myofibrils

A novel chymotrypsin-like proteinase termed myonase was previously purified from MDX-mouse skeletal muscle [Hori et al. (1998) J. Biochem. 123, 650-658]. Western blots and immunohistochemical analyses showed that myonase was present within myocytes of both MDX-mouse and control mouse, and subcellular fractionation showed that it was associated with myofibrils. No significant difference was observed on Western blots between the amounts of myonase in myofibrils of MDX-mouse and control mouse, but the amount of myonase recoverable as a pure protein was 5-10-fold more when MDX-mouse was the source of the skeletal muscle. Myofibrils also possessed an endogenous inhibitor of myonase, whose inhibitory activity at physiological pH (pH 7.4) depended on salt concentration, stronger inhibition being observed at a low salt concentration. Inhibition at alkaline pH (pH 9) was weak and independent of salt concentration. Myonase in myofibrils was partially released at neutral pH by a high salt concentration (>0.6 M NaCl). However, even at 4 M NaCl, more than 80% of myonase remained within the myofibrils. Under alkaline conditions, release of myonase from myofibril was more extensive. At pH 12, myonase was almost completely present in the soluble fraction. Release of myonase under these conditions coincided with the solubilization of other myofibrillar proteins.     

PDGF-receptor concentration is elevated in regenerative muscle fibers in dystrophin-deficient muscle.

Dystrophin-deficient muscle undergoes sudden, postnatal onset of muscle necrosis that is either progressive, as in Duchenne muscular dystrophy, or successfully arrested and followed by regeneration, as in most muscles of mdx mice. The mechanisms regulating regeneration in mdx muscle are unknown, although the possibility that there is renewed expression of genes regulating embryonic muscle cell proliferation and differentiation may provide testable hypotheses. Here, we examine the possibility that necrotic and regenerating mdx muscles exhibit renewed or increased expression of PDGF-receptors. PDGF-binding to receptors on muscle has been shown previously to be associated with myogenic cell proliferation and delay of muscle differentiation. We find that PDGF-receptors are present in 4-week-old mdx mice in muscles that undergo brief, reversible necrosis (hindlimb muscles) or progressive necrosis (diaphragm), as well as in 4-week-old control mouse muscles. Immunoblots indicate that the concentrations of PDGF-receptors in 4-week-old dystrophic (necrotic) and control muscles are similar. Prenecrotic, dystrophic fibers and control fibers possess some cell surface labeling of fibers treated with anti-PDGF-receptor and viewed by indirect immunofluorescence. Necrotic fibers in dystrophic muscle show cytoplasmic labeling for PDGF-receptors and labeling of perinuclear regions at the muscle cell surface. Adult dystrophic muscle displays higher concentrations of PDGF-receptor in both regenerated muscle (hindlimb) and progressively necrotic muscle (diaphragm) than found in controls. Anti-PDGF-receptor labeling of regenerated, dystrophic muscle is observed primarily in granules surrounding central nuclei or surrounding nuclei located at the surface of regenerated fibers. No labeling of perinuclear regions of control muscle or prenecrotic fibers was observed. Myonuclei fractionated from adult mdx hindlimb muscles contained no PDGF-receptor, indicating that PDGF-receptor-positive structures are not tightly associated with nuclei or within nuclei. L6 myoblasts show PDGF-receptor distributed diffusely on the cell surface. Stimulation of L6 myoblasts with 10 ng/ml of PDGF-BB causes receptor internalization and concentration in granules at perinuclear regions. Thus, PDGF stimulation of myoblasts causes a redistribution of PDGF-receptors to resemble receptor localization observed during muscle regeneration. These findings implicate PDGF-mediated mechanisms in regeneration of dystrophic muscle.     

Skeletal muscle HIF-1alpha expression is dependent on muscle fiber type

Oxygen homeostasis is an essential regulation system for cell energy production and survival. The oxygen-sensitive subunit alpha of the hypoxia inducible factor-1 (HIF-1) complex is a key protein of this system. In this work, we analyzed mouse and rat HIF-1alpha protein and mRNA expression in parallel to energetic metabolism variations within skeletal muscle. Two physiological situations were studied using HIF-1alpha-specific Western blotting and semiquantitative RT-PCR. First, we compared HIF-1alpha expression between the predominantly oxidative soleus muscle and three predominantly glycolytic muscles. Second, HIF-1alpha expression was assessed in an energy metabolism switch model that was based on muscle disuse. These two in vivo situations were compared with the in vitro HIF-1alpha induction by CoCl(2) treatment on C(2)C(12) mouse muscle cells. HIF-1alpha mRNA and protein levels were found to be constitutively higher in the more glycolytic muscles compared with the more oxidative muscles. Our results gave rise to the hypothesis that the oxygen homeostasis regulation system depends on the fiber type.     

Glycogenin activity in human skeletal muscle is proportional to muscle glycogen concentration

The de novo biosynthesis of glycogen is catalyzed by glycogenin, a self-glucosylating protein primer. To date, the role of glycogenin in regulating glycogen metabolism and the attainment of maximal glycogen levels in skeletal muscle are unknown. We measured glycogenin activity after enzymatic removal of glucose by alpha-amylase, an indirect measure of glycogenin amount. Seven male subjects performed an exercise and dietary protocol that resulted in one high-carbohydrate leg (HL) and one low-carbohydrate leg (LL) before testing. Resting muscle biopsies were obtained and analyzed for total glycogen, proglycogen (PG), macroglycogen (MG), and glycogenin activity. Results showed differences (P < 0.05) between HL and LL for total glycogen (438.0 +/- 69.5 vs. 305.7 +/- 57.4 mmol glucosyl units/kg dry wt) and PG (311.4 +/- 38.1 vs. 227.3 +/- 33.1 mmol glucosyl units/kg dry wt). A positive correlation between total muscle glycogen content and glycogenin activity (r = 0.84, P < 0.001) was observed. Similar positive correlations (P < 0.05) were also evident between both PG and MG concentration and glycogenin activity (PG, r = 0.82; MG, r = 0.84). It can be concluded that glycogenin does display activity in human skeletal muscle and is proportional to glycogen concentration. Thus it must be considered as a potential regulator of glycogen synthesis in human skeletal muscle.     

TNF-alpha is a mitogen in skeletal muscle

Emerging evidence suggests that tumor necrosis factor (TNF)- plays a role in muscle repair. To determine whether TNF- modulates satellite cell proliferation, the current study evaluated TNF- effects on DNA synthesis in primary myoblasts and on satellite cell activation in adult mouse muscle. Exposure to recombinant TNF- increased total DNA content in rat primary myoblasts dose-dependently over a 24-h period and increased the number of primary myoblasts incorporating 5-bromo-2'-deoxyuridine (BrdU) during a 30-min pulse labeling. Systemic injection of TNF- stimulated BrdU incorporation by satellite cells in muscles of adult mice, whereas no BrdU was incorporated by satellite cells in control mice. TNF- stimulated serum response factor (SRF) binding to the serum response element (SRE) present in the c-fos gene promoter and stimulated reporter gene expression controlled by the same element. Our data suggest that TNF- activates satellite cells to enter the cell cycle and accelerates G₁-to-S phase transition, and these actions may involve activation of early response genes via SRF. cytokine; cell cycle; satellite cells; serum response factor; c-fos     

VAMP2 is expressed in muscle satellite cells and up-regulated during muscle regeneration

Membrane trafficking is one of the most important mechanisms involved in the establishment and maintenance of the forms and functions of the cell. However, it is poorly understood in skeletal muscle cells. In this study, we have focused on vesicle-associated membrane proteins (VAMPs), which are components of the vesicle docking and fusion complex, and have performed immunostaining to investigate the expression of VAMPs in rat skeletal muscle tissue. We have found that VAMP2, but not VAMP1 or VAMP3, is expressed in satellite cells. VAMP2 is also expressed in myofibers in the soleus muscle and nerve endings. This is consistent with previous studies in which VAMP2 has been shown to regulate GLUT4 trafficking in slow-twitch myofibers in soleus muscle and neurotransmitter release in nerve endings. As satellite cells are quiescent myogenic cells, the expression of VAMP2 has further been examined in regenerating muscles after injury by the snake venom, cardiotoxin; we have observed enhanced expression of VAMP2 in immature myotubes with a peak at 3 days after injury. Our findings suggest that VAMP2 plays roles in quiescent satellite cells and is involved in muscle regeneration. The nature of the material transported in the VAMP2-bearing vesicles in satellite cells and myotubes is still under investigation. This work was supported by a research grant (17A-10) for nervous and mental disorders from the Ministry of Health, Labor, and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

The tumor suppressor adenomatous polyposis coli (APC) is a crucial regulator of many stem cell types. In constantly cycling stem cells of fast turnover tissues, APC loss results in the constitutive activation of a Wnt target gene program that massively increases proliferation and leads to malignant transformation. However, APC function in skeletal muscle, a tissue with a low turnover rate, has never been investigated. Here we show that conditional genetic disruption of APC in adult muscle stem cells results in the abrogation of adult muscle regenerative potential. We demonstrate that APC removal in adult muscle stem cells abolishes cell cycle entry and leads to cell death. By using double knockout strategies, we further prove that this phenotype is attributable to overactivation of β-catenin signaling. Our results demonstrate that in muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression and radically diverge from previous observations concerning stem cells in actively self-renewing tissues.     

Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function

Adiponectin (Ad) is linked to various disease states and mediates antidiabetic and anti-inflammatory effects. While it was originally thought that Ad expression was limited to adipocytes, we demonstrate here that Ad is expressed in mouse skeletal muscles and within differentiated L6 myotubes, as assessed by RT-PCR, Western blot, and immunohistochemical analyses. Serial muscle sections stained for fiber type, lipid content, and Ad revealed that muscle fibers with elevated intramyocellular Ad expression were consistently type IIA and IID fibers with detectably higher intramyocellular lipid (IMCL) content. To determine the effect of Ad on muscle phenotype and function, we used an Ad-null [knockout (KO)] mouse model. Body mass increased significantly in 24-wk-old KO mice [+5.5 +/- 3% relative to wild-type mice (WT)], with no change in muscle mass observed. IMCL content was significantly increased (+75.1 +/- 25%), whereas epididymal fat mass, although elevated, was not different in the KO mice compared with WT (+35.1 +/- 23%; P = 0.16). Fiber-type composition was unaltered, although type IIB fiber area was increased in KO mice (+25.5 +/- 6%). In situ muscle stimulation revealed lower peak tetanic forces in KO mice relative to WT (-47.5 +/- 6%), with no change in low-frequency fatigue rates. These data demonstrate that the absence of Ad expression causes contractile dysfunction and phenotypical changes in skeletal muscle. Furthermore, we demonstrate that Ad is expressed in skeletal muscle and that its intramyocellular localization is associated with elevated IMCL, particularly in type IIA/D fibers.     

Delta-sarcoglycan is required for early zebrafish muscle organization

Mutations in sarcoglycans (alpha-, beta-, gamma-, and delta-) have been linked with limb girdle muscular dystrophy (LGMD) types 2C-F in humans. We have cloned the zebrafish orthologue encoding delta-sarcoglycan and mapped the gene to linkage group 21. The predicted zebrafish delta-sarcoglycan protein is highly homologous with its human orthologue including conservation of two of the three predicted glycosylation sites. Like other members of the dystrophin-associated protein complex (DAPC), delta-sarcoglycan localizes to the sarcolemmal membrane of the myofiber in adult zebrafish, but is more apparent at the myosepta in developing embryos. Zebrafish embryos injected with morpholinos against delta-sarcoglycan were relatively inactive at 5 dpf, their myofibers were disorganized, and swim bladders uninflated. Immunohistochemical and immunoblotting experiments show that delta-, beta-, and gamma-sarcoglycans were all downregulated in the morphants, whereas dystrophin expression was unaffected. Whereas humans lacking delta-sarcoglycan primarily show adult phenotypes, our results suggest that delta-sarcoglycan plays a role in early zebrafish muscle development.