Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.     

Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle

The presence and potential physiological role of the erythropoietin receptor (Epo-R) were examined in human skeletal muscle. In this study we demonstrate that Epo-R is present in the endothelium, smooth muscle cells, and in fractions of the sarcolemma of skeletal muscle fibers. To study the potential effects of Epo in human skeletal muscle, two separate studies were conducted: one to study the acute effects of a single Epo injection on skeletal muscle gene expression and plasma hormones and another to study the effects of long-term (14 wk) Epo treatment on skeletal muscle structure. Subjects (n = 11) received a single Epo injection of 15,000 IU (double blinded, cross over, placebo). A single Epo injection reduced myoglobin and increased transferrin receptor and MRF-4 mRNA content within 10 h after injection. Plasma hormones remained unaltered. Capillarization and fiber hypertrophy was studied in subjects (n = 8) who received long-term Epo administration, and muscle biopsies were obtained before and after. Epo treatment did not alter mean fiber area (0.84 +/- 0.2 vs. 0.72 +/- 0.3 mm(2)), capillaries per fiber (4.3 +/- 0.5 vs. 4.4 +/- 1.3), or number of proliferating endothelial cells. In conclusion, the Epo-R is present in the vasculature and myocytes in human skeletal muscle, suggesting a role in both cell types. In accordance, a single injection of Epo regulates myoglobin, MRF-4, and transferrin receptor mRNA levels. However, in contrast to our hypothesis, prolonged Epo administration had no apparent effect on capillarization or muscle fiber hypertrophy.     

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.     

Differential Expression of PTP1D, a Protein Tyrosine Phosphatase with Two SH2 Domains, in Slow and Fast Skeletal Muscle Fibers

We show that PTP1D, a protein tyrosine phosphatase that contains two SH2 domains, is preferentially expressed in slow skeletal muscle fibers. Immunohistochemical staining using polyclonal antibodies against PTP1D demonstrated that PTP1D was expressed in a subpopulation of rodent muscle fibers. These fibers were identified as slow Type I fibers based on histochemical ATPase assays and slow myosin heavy chain expression. Northern and Western analyses showed that PTP1D levels were higher in predominantly slow muscles than in predominantly fast muscles. This differential expression of PTP1D in slow muscle fibers appeared by birth. In cultures of mouse myogenic cells, PTP1D was expressed after MyoD and myogenin and appeared in myotubes derived from embryonic, fetal, and postnatal myoblasts. Remarkably, PTP1D was organized into sarcomeres in a pattern coincident with myosin heavy chain, suggesting that PTP1D associates with a component of the thick filament. These results show that PTP1D is preferentially expressed in slow muscle fibers. We speculate that PTP1D may play a role in slow muscle fiber function and differentiation.     

Quantifying the temporospatial expression of postnatal porcine skeletal myosin heavy chain genes.

Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber type-restricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed.     

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

Ephrin-A3 promotes and maintains slow muscle fiber identity during postnatal development and reinnervation

Each adult mammalian skeletal muscle has a unique complement of fast and slow myofibers, reflecting patterns established during development and reinforced via their innervation by fast and slow motor neurons. Existing data support a model of postnatal "matching" whereby predetermined myofiber type identity promotes pruning of inappropriate motor axons, but no molecular mechanism has yet been identified. We present evidence that fiber type–specific repulsive interactions inhibit innervation of slow myofibers by fast motor axons during both postnatal maturation of the neuromuscular junction and myofiber reinnervation after injury. The repulsive guidance ligand ephrin-A3 is expressed only on slow myofibers, whereas its candidate receptor, EphA8, localizes exclusively to fast motor endplates. Adult mice lacking ephrin-A3 have dramatically fewer slow myofibers in fast and mixed muscles, and misexpression of ephrin-A3 on fast myofibers followed by denervation/reinnervation promotes their respecification to a slow phenotype. We therefore conclude that Eph/ephrin interactions guide the fiber type specificity of neuromuscular interactions during development and adult life.     

The vitamin C transporter SVCT2 is down-regulated during postnatal development of slow skeletal muscles

Vitamin C plays key roles in cell homeostasis, acting as a potent antioxidant as well as a positive modulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. Besides, SVCT2 is up-regulated upon the early fusion of primary myoblasts. However, our knowledge of the postnatal expression profile of SVCT2 remains scarce. Here we have analyzed the expression of SVCT2 during postnatal development of the chicken slow anterior and fast posterior latissimus dorsi muscles, ranging from day 7 to adulthood. SVCT2 expression is consistently higher in the slow than in the fast muscle at all stages. After hatching, SVCT2 expression is significantly down-regulated in the anterior latissimus dorsi, which nevertheless maintains a robust slow phenotype. Taking advantage of the C2C12 cell line to recapitulate myogenesis, we confirmed that SVCT2 is expressed in a biphasic fashion, reaching maximal levels upon early myoblasts fusion and decreasing during myotube growth. Together, these findings suggest that the dynamic expression levels of SVCT2 could be relevant for different features of skeletal muscle physiology, such as muscle cell formation, growth and activity.     

Differential expression of calcineurin and SR Ca2+ handling proteins in equine muscle fibers during early postnatal growth.

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.     

Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol- anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.     

Muscle LIM protein plays both structural and functional roles in skeletal muscle

Muscle LIM protein (MLP) has been suggested to be an important mediator of mechanical stress in cardiac tissue, but the role that it plays in skeletal muscle remains unclear. Previous studies have shown that it is dramatically upregulated in fast-to-slow fiber-type transformation and also after eccentric contraction (EC)-induced muscle injury. The functional consequences of this upregulation, if any, are unclear. In the present study, we have examined the skeletal muscle phenotype of MLP-knockout (MLPKO) mice in terms of their response to EC-induced muscle injuries. The data suggest that while the MLPKO mice recover completely after EC-induced injury, their torque production lags behind that of heterozygous littermates in the early stages of the recovery process. This lag is accompanied by decreased expression of the muscle regulatory factor MyoD, suggesting that MLP may influence gene expression. In addition, there is evidence of type I fiber atrophy and a shorter resting sarcomere length in the MLPKO mice, but no significant differences in fiber type distribution. In summary, MLP appears to play a subtle role in the maintenance of normal muscle characteristics and in the early events of the recovery process of skeletal muscle to injury, serving both structural and gene-regulatory roles. eccentric contractions; passive tension