Myotube Nuclei Research Articles

Osteopontin, a chemotactic protein with cytokine-like properties, is up-regulated in muscle injury caused by Bothrops lanceolatus (fer-de-lance) snake venom

Osteopontin (OPN) is a chemotactic, adhesive protein whose receptors include some integrins and matrix proteins known to have role in inflammatory and repair processes. We examined the time course of OPN expression at acute and chronic stages after intramuscular injection of Bothrops lanceolatus venom in rats. Additionally, we examined the expression of CD68 (a marker for phagocytic macrophages) and the myogenic factors, myoD and myogenin. There was a biphasic upregulation of OPN (6–48 h and 3–14 days post-venom), i.e., during acute inflammation and myogenic cell proliferation and differentiation phases. OPN was detected in CD68 + macrophages, fibroblasts, normal and damaged myofibers, myoblasts and myotubes. Myogenin was expressed in the cytoplasm (atypical pattern) and nucleus of myoblasts and myotubes from 18 h to 7 days, after which it was expressed only in nuclei. Macrophage numbers, OPN and myogenin expression were still elevated at 7, 14 and 7 days. At 3 days, when OPN achieved the peak, some clusters of myoblasts were within regions of intense collagen deposition. Fibrosis may represent limitation for repairing processes and may explain the small diameter of regenerated fibers at 21 days post-venom. The expression of OPN in the course of venom-induced damage and regeneration suggests stages-specific mediation role along the whole process.

Chromatin modifications that support acetylcholine receptor gene activation are established during muscle cell determination and differentiation

Localization of acetylcholine receptors (AChRs) to the postsynaptic region of muscle is mediated in part by transcriptional mechanisms. An important way of regulating transcription is through targeting histone modifications on chromatin to distinct gene loci. Using chromatin immunoprecipitation, we examined the developmental regulation of certain histone modifications at the AChR epsilon subunit locus, including methylations at lysine residues K4 and K27 and acetylations at K9 and K14. We modeled various stages of muscle development in cell culture, including pre-determined cells, committed but undifferentiated myoblasts, and differentiated myotubes, and modeled synaptic myotube nuclei by stimulating myotubes with neuregulin (NRG) 1. We found that a pattern of histone modifications associated with transcriptional activation is targeted to the AChR epsilon subunit locus in myotubes prior to stimulation with NRG1 and does not change upon addition of NRG1. Instead, we found that during muscle cell determination and differentiation, specific histone modifications are targeted to the AChR epsilon subunit locus. Within the gene, at K4, dimethylation is induced during muscle cell determination, while trimethylation is induced during differentiation. At K27, loss of trimethylation and appearance of monomethylation occurs during determination and differentiation. In addition, in a region upstream of the gene, K4 di- and trimethylation, and K9/14 acetylation are induced in a distinct developmental pattern, which may reflect a functional regulatory element. These results suggest synaptic signaling does not directly target histone modifications but rather the histone modification pattern necessary for transcriptional activation is previously established in a series of steps during muscle development.

Down-regulation of MyoD by Calpain 3 Promotes Generation of Reserve Cells in C2C12 Myoblasts

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Myogenin expression is independent of agrin treatment in C2C12 cell culture

Skeletal muscle differentiation is influenced by myogenic regulatory factors, including MyoD and myogenin. Previous studies have reported that MyoD is expressed prior to myogenin. Our objective was to investigate this process using C2C12 cell culture. When C2C12 myoblasts proliferate to 80% density, growth medium (GM) is replaced with differentiation medium (DM). Subsequently, myoblasts fuse into myotubes within 72 hours in DM. During myotube formation, acetylcholine receptors (AChRs) cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. Using immunofluorescence, we observed MyoD in myoblast nuclei while myoblasts were in GM, with elimination of MyoD within 72 hours in DM. In contrast, we did not observe myogenin while myoblasts were in GM, with or without agrin treatment, but myogenin was observed in myotube nuclei within 24 hours in DM without agrin treatment. We conclude that myogenin expression is independent of agrin treatment in C2C12 cell culture.Wade A. Grow was supported by Midwestern University intramural funds.

Sequence Differences in the IQ Motifs of CaV1.1 and CaV1.2 Strongly Impact Calmodulin Binding and Calcium-dependent Inactivation

The proximal C terminus of the cardiac L-type calcium channel (Ca(V)1.2) contains structural elements important for the binding of calmodulin (CaM) and calcium-dependent inactivation, and exhibits extensive sequence conservation with the corresponding region of the skeletal L-type channel (Ca(V)1.1). However, there are several Ca(V)1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding Ca(V)1.2 residues, including three of the "IQ motif." To investigate the functional significance of these residue differences, we used native gel electrophoresis and expression in intact myotubes to compare the binding of CaM to extended regions (up to 300 residues) of the C termini of Ca(V)1.1 and Ca(V)1.2. We found that in the presence of Ca(2+) (either millimolar or that in resting myotubes), CaM bound strongly to C termini of Ca(V)1.2 but not of Ca(V)1.1. Furthermore, replacement of two residues (Tyr(1657) and Lys(1662)) within the IQ motif of a C-terminal Ca(V)1.2 construct with the divergent residues of Ca(V)1.1 (His(1532) and Met(1537)) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in Ca(V)1.1 caused a gain of CaM binding. In full-length Ca(V)1.2, substitution of these same two divergent residues with those of Ca(V)1.1 (Y1657H, K1662M) eliminated calcium-dependent inactivation of the heterologously expressed channel. Thus, our results reveal that a conserved difference between the IQ motifs of Ca(V)1.2 and Ca(V)1.1 has a profound effect on both CaM binding and calcium-dependent inactivation.

Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography-produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization.

Nuclear Exclusion of Forkhead Box O and Elk1 and Activation of Nuclear Factor-κB Are Required for C2C12-RasV12C40 Myoblast Differentiation

Activating ras point mutations are frequently found in skeletal muscle tumors such as rhabdomyosarcomas. In this study we investigated the impact of two different H-ras mutants in skeletal muscle differentiation: RasV12, a constitutively active form, and RasV12C40, a mutant deficient in Raf1 activation. Stably transfected C2C12-RasV12 myoblasts actively proliferated as indicated by the sustained expression of proliferating cell nuclear antigen and retinoblastoma at the hyperphosphorylated state and failed to express differentiation markers. This differentiation-defective phenotype was a consequence of the chronic p44/p42MAPK phosphorylation and the inability of the cells to activate AKT. Moreover, we observed that p44/p42MAPK activation in C2C12-RasV12 myoblasts phosphorylated the ETS-like transcription factor (ELK) 1, which translocates to the nuclei and seemed to be involved in maintaining myoblast proliferation. C2C12-RasV12C40 myoblasts cultured in low serum repressed phosphorylation of p44/p42MAPK and ELK1, resulting in cell cycle arrest and myogenic differentiation. Under this condition, activation of AKT, p70S6K, and p38MAPK was produced, leading to formation of myotubes in 3 d, 1 d earlier than in control C2C12-AU5 cells. Moreover, the expression of muscle-specific proteins, mainly the terminal differentiation markers caveolin-3 and myosin heavy chain, also occurred 1 d earlier than in control cells. Furthermore, AKT activation produced phosphorylation of Forkhead box O that led to nuclear exclusion and inactivation, allowing myogenesis. In addition, we found an induction of nuclear factor-kappaB activity in the nucleus in C2C12-RasV12C40 myotubes attributed to p38MAPK activation. Accordingly, muscle differentiation is associated with a pattern of transcription factors that involves nuclear exclusion ELK1 and Forkhead box O and the increase in nuclear factor-kappaB DNA binding.

The Myogenic Factor Myf5 Supports Efficient Skeletal Muscle Regeneration by Enabling Transient Myoblast Amplification

The myogenic factor Myf5 defines the onset of myogenesis in mammals during development. Mice lacking both Myf5 and MyoD fail to form myoblasts and are characterized by a complete absence of skeletal muscle at birth. To investigate the function of Myf5 in adult skeletal muscle, we generated Myf5 and mdx compound mutants, which are characterized by constant regeneration. Double mutant mice show an increase of dystrophic changes in the musculature, although these mice were viable and the degree of myopathy was modest. Myf5 mutant muscles show a small decrease in the number of muscle satellite cells, which was within the range of physiological variations. We also observed a significant delay in the regeneration of Myf5 deficient skeletal muscles after injury. Interestingly, Myf5 deficient skeletal muscles were able to even out this flaw during the course of regeneration, generating intact muscles 4 weeks after injury. Although we did not detect a striking reduction of MyoD positive activated myoblasts or of Myf5-LacZ positive cells in regenerating muscles, a clear decrease in the proliferation rate of satellite cell-derived myoblasts was apparent in satellite cell-derived cultures. The reduction of the proliferation rate of Myf5 mutant myoblasts was also reflected by a delayed transition from proliferation to differentiation, resulting in a reduced number of myotube nuclei after 6 and 7 days of culture. We reason that Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification. Disclosure of potential conflicts of interest is found at the end of this article.

Cytotoxicity induced in myotubes by a Lys49 phospholipase A 2 homologue from the venom of the snake Bothrops asper: Evidence of rapid plasma membrane damage and a dual role for extracellular calcium

Acute muscle tissue damage, myonecrosis, is a typical consequence of envenomations by snakes of the family Viperidae. Catalytically-inactive Lys49 phospholipase A 2 homologues are abundant myotoxic components in viperid venoms, causing plasma membrane damage by a mechanism independent of phospholipid hydrolysis. However, the precise mode of action of these myotoxins remains unsolved. In this work, a cell culture model of C2C12 myotubes was used to assess the action of Bothrops asper myotoxin II (Mt-II), a Lys49 phospholipase A 2 homologue. Mt-II induced a dose- and time-dependent cytotoxic effect associated with plasma membrane disruption, evidenced by the release of the cytosolic enzyme lactate dehydrogenase and the penetration of propidium iodide. A rapid increment in cytosolic Ca 2+ occurred after addition of Mt-II. Such elevation was associated with hypercontraction of myotubes and blebbing of plasma membrane. An increment in the Ca 2+ signal was observed in myotube nuclei. Elimination of extracellular Ca 2+ resulted in increased cytotoxicity upon incubation with Mt-II, suggesting a membrane-protective role for extracellular Ca 2+. Chelation of cytosolic Ca 2+ with BAPTA-AM did not modify the cytotoxic effect, probably due to the large increment induced by Mt-II in cytosolic Ca 2+ which overrides the chelating capacity of BAPTA-AM. It is concluded that Mt-II induces rapid and drastic plasma membrane lesion and a prominent Ca 2+ influx in myotubes. Extracellular Ca 2+ plays a dual role in this model: it protects the membrane from the cytolytic action of the toxin; at the same time, the Ca 2+ influx that occurs after membrane disruption is likely to play a key role in the intracellular degenerative events associated with Mt-II-induced myotube damage.

Enhancer of Polycomb1, a Novel Homeodomain Only Protein-binding Partner, Induces Skeletal Muscle Differentiation

Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.

Arginine supplementation induces myoblast fusion via augmentation of nitric oxide production

The semi-essential amino acid, L-arginine (L-Arg), is the substrate for endogenous synthesis of nitric oxide, a molecule that is involved in myoblast proliferation and fusion. Since L-Arg supply may limit nitric oxide synthase (NOS) activity in endothelial cells, we examined L-Arg supplementation in differentiating mouse myoblasts and tested the hypothesis that L-Arg exerts direct effects on myoblast fusion via augmentation of endogenous nitric oxide production. C(2)C(12) myoblasts in differentiation media received one of the following treatments for 120 h: 1 mM L-Arg, 0.1 mM N-nitro-L-arginine methyl ester (L-NAME), L-Arg + L-NAME, 10 mM L-Lysine, or no supplement (Control). Cultures were fixed and stained with hematoxylin and eosin for microphotometric image analysis of myotube density, nuclear density, and fusion index (% of total nuclei in myotubes). Endogenous production of nitric oxide during the treatment period peaked between 24 and 48 h. L-Arg amplified nitric oxide production between 0 and 24 h and increased myotube density, total nuclei number, and nuclear fusion index. These L-Arg effects were prevented by the NOS inhibitor, L-NAME. Further, L-Lysine, a competitive inhibitor of L-Arg uptake, repressed nitric oxide production and reduced myotube density and fusion index. In summary, L-Arg augments myotube formation and increases nitric oxide production in a process limited by cellular L-Arg uptake.

E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

The E2A proteins are basic helix-loop-helix transcription factors that regulate proliferation and differentiation in many cell types. In muscle cells, the E2A proteins form heterodimers with muscle regulatory factors such as MyoD, which then bind to DNA and regulate the transcription of target genes essential for muscle differentiation. We now demonstrate that E2A proteins are primarily localized in the nucleus in both C2C12 myoblasts and myotubes, and are degraded by the ubiquitin proteasome system evidenced by stabilization following treatment with the proteasome inhibitor, MG132. During the differentiation from myoblast to myotube, the cellular abundance of E2A proteins is relatively unaltered, despite significant changes (each approximately 5-fold) in the relative rates of protein synthesis and protein degradation via the ubiquitin-proteasome system. The rate of ubiquitin-proteasome-mediated E2A protein degradation depends on the myogenic differentiation state (t 1/2 approximately 2 h in proliferating myoblasts versus t 1/2 > 10 h in differentiated myotubes), and is also associated with cell cycle in non-muscle cells. Our findings reveal an important role for both translational and post-translational regulatory mechanisms in mediating the complex program of muscle differentiation determined by the E2A proteins.

Organization of Calcium Channel β1a Subunits in Triad Junctions in Skeletal Muscle

In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle alpha1S subunit, the beta1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of beta1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (alpha1S-null) myotubes, YFP-beta1a-CFP and CFP-beta1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta1a does not appear to bind to other cellular proteins in the absence of alpha1S. FRET efficiencies for these cytoplasmic beta1a subunits were approximately 6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled alpha1S (in dysgenic or beta1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta1-null myotubes, FRET efficiencies of doubly labeled beta1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled beta1a (CFP or YFP at the N or C terminus) were expressed in beta1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta1a subunits are located >10 nm from one another.

Fgfr4 Is Required for Effective Muscle Regeneration in Vivo: DELINEATION OF A MyoD-Tead2-Fgfr4 TRANSCRIPTIONAL PATHWAY

Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds; however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites and tested candidate regulators in a 27-time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5'-CATTCCT-3'), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose-dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoD-Tead2-Fgfr4 pathway important for effective muscle regeneration.

FoxO1 Stimulates Fatty Acid Uptake and Oxidation in Muscle Cells through CD36-dependent and -independent Mechanisms

Emerging evidence documents a key function for the forkhead transcription factor FoxO1 in cellular metabolism. Here, we investigate the role of FoxO1 in the regulation of fatty acid (FA) metabolism in muscle cells. C2C12 cells expressing an inducible construct with either wild type FoxO1 or a mutant form (FoxO1/TSS) refractory to the protein kinase B inhibitory effects were generated. FoxO1 activation after myotube formation altered the expression of several genes of FA metabolism. Acyl-CoA oxidase and peroxisome proliferator-activated receptor delta mRNA levels increased 2.2-fold and 1.4-fold, respectively, whereas mRNA for acetyl-CoA carboxylase decreased by 50%. Membrane uptake of oleate increased 3-fold, and oleate oxidation increased 2-fold. Cellular triglyceride content was also increased. The enhanced FA utilization induced by FoxO1 was mediated by a severalfold increase in plasma membrane level of the fatty acid translocase FAT/CD36 and eliminated by cell treatment with the CD36 inhibitor sulfo-N-succinimidyl-oleate. We conclude that FoxO1 activation induces coordinate increases in FA uptake and oxidation and that these effects are mediated, at least in part, by membrane enrichment in CD36. The data suggest that FoxO1 contributes to preparing the muscle cell for the increased reliance on FA metabolism that is characteristic of fasting. Dysregulation of FoxO1 in muscle could contribute to intramuscular lipid accumulation and insulin resistance by maintaining activation of FA uptake.

P18 INK4c and p27 KIP1 are required for cell cycle arrest of differentiated myotubes

Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis. These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27. In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals. Under differentiation conditions, only p27 −/− or p18 −/− p27 −/− myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency. Escape from cell cycle arrest was significantly greater in p18 −/− p27 −/− myotubes than in p27 −/− myotubes. Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18 −/− p27 −/− myotube proliferation to encompass approximately half of the nuclei. p18 −/− p21 −/− and p21 −/− p27 −/− myotubes remain terminally arrested. Nuclei within individual restimulated p18 −/− p27 −/− myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects. Increasing the time of differentiation or serum stimulation of p18 −/− p27 −/− myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases. During the first 24 h of serum stimulation, the p18 −/− p27 −/− myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication. Apoptosis is increased in restimulated p18 −/− p27 −/− myotube nuclei, but is not specific for any cell cycle phase. These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation. While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest. We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest.

Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages.

Adult skeletal muscle contains populations of satellite cells and muscle-derived stem cells that are capable of forming multinucleate myotubes. The purpose of this study was to determine the phenotype of cells isolated from a common satellite cell isolation and passaging procedure from whole skeletal muscle. To ascertain the characteristics of the cellular phenotype, the myogenic markers MyoD and desmin, the satellite-cell-specific marker Pax7, and the haemopoietic stem cell markers CD34 and CD45 were examined by immunohistochemical analysis. Immediately after isolation, > 90% myogenic marker-positive cells were positive for desmin, MyoD and Pax7. In contrast, approximately 10% of the isolated cells expressed only CD34 or CD45. After three passages, the percentage of cells that were positive for the myogenic markers desmin, MyoD and Pax7 was reduced to approximately 55%, while the population of CD34- or CD45-positive cells increased to approximately 30% after the third passage. Immunohistochemical detection of bromodeoxyuridine demonstrated that the number of proliferating cells decreased progressively after each passaging. Finally, after the third passage the percentage of nuclei in myotubes decreased from 46.7% to 12.5%. Since passaging of muscle progenitor cells is common practice, the results of the current report suggest that characterization of cell heterogeneity needs to be made frequently.

Heat stress-induced localization of small heat shock proteins in mouse myoblasts: intranuclear lamin A/C speckles as target for αB-crystallin and Hsp25

We examined the effect of heat stress on localization of two sHsps, αB-crystallin and Hsp25, and of Hsc70, a member of a different class of heat shock proteins (Hsps), in both undifferentiated and differentiated mouse C2C12 cells. Under normal conditions, αB-crystallin and Hsp25 are found in the cytoplasm; only αB-crystallin is also found in the nucleus, distributed in a speckled pattern. Hsc70 is found to be homogeneously distributed throughout the cell. On heat stress, all these proteins translocate almost entirely into the nucleus and upon recovery relocate to the cytoplasm. Dual staining experiments using C2C12 myoblasts show that αB-crystallin and Hsp25, but not Hsc70, colocalize with the intranuclear lamin A/C and the splicing factor SC-35, suggesting interactions of sHsps and intranuclear lamin A/C. Interestingly, none of these proteins are found in the myotube nuclei. Upon heat stress, only Hsc70 translocates into the myotube nuclei. This differential entry of αB-crystallin and Hsp25 into the nuclei of myoblasts and myotubes upon heat stress may have functional role in the development and/or in the maintenance of muscle cells. Our study therefore suggests that these sHsps may be a part of the intranuclear lamin A/C network or stabilizing this specific network.

L-NAME Prevents Stretch-induced Increases in Myotube Size and Nuclear Number

1037 The myotube stretch model allows for isolation and study of cell signaling pathways intrinsic to stretch-induced myocyte growth in the absence of humoral responses inherent to in vivo studies. PURPOSE: To determine if inhibition of nitric oxide synthase (NOS) activity via L-NAME attenuates stretch-induced hypertrophy of myotubes. METHODS: C2C12 myoblasts were cultured on silastic membranes suspended between concave metal clamps attached to a computer-controlled linear stepper motor. Cultures were maintained in growth medium (DMEM supplemented with 10% fetal bovine serum) in a humidified incubator at 37°C, 5% CO2 until 80% confluency was reached, then switched to differentiation media (DM; DMEM supplemented with 2% horse serum). Hypertrophy was induced in some cultures by an intermittent linear stretch protocol. The protocol involved three 60-min bouts of stretch each consisting of 12 sets of 10 stretch cycles (± 10% length change applied at a rate of 20%/s). One set was completed every 5 min for the 60 min period. Stretch bouts were completed at 12h intervals beginning at 96h post serum withdrawal. DM was refreshed for all groups (stretched and non-stretched) before each stretching bout. Cultures were divided into four treatment groups (n = 3 cultures/group): 1) Control, 2) L-NAME (100μM in DM), 3) Stretch only, and 4) L-NAME + Stretch. Four hours after the last stretching bout (124 hrs), cultures were fixed with 10% methanol and stained with hematoxylin and eosin (H&E). Microphotometric analysis was performed on digital light microscopy images calibrated with a stage micrometer. Myotube width (calculated as the average of ten transverse measurements taken along the length of each myotube), and the number of myonuclei/myotube were recorded in three separate fields of view for each culture. RESULTS: The stretch protocol increased average myotube width (+17%, P < 0.05) and number of myonuclei/myotube (+28%, P < 0.05). L-NAME treatment alone reduced myotube area 18% compared to control cultures (P < 0.05). Further, L-NAME treatment during the stretch protocol eliminated stretchinduced myotube growth (i.e no difference between L-NAME+Stretch and L-NAME only; P > 0.05). Myonuclei content in cultures treated with L-NAME did not differ (P > 0.05) from controls with or without stretch. CONCLUSIONS: Stretch-induced hypertrophy and myonuclear accretion in myotubes was attenuated with L-NAME treatment. Additionally, L-NAME treatment alone caused a reduction in myotube size and nuclei number. Thus, myotube growth and stretch-induced hypertrophy is dependent upon intrinsic NOS activity.

IL-4: A Fusion Signal

One function of the cytokine interleukin 4 (IL-4) when released by T cells is to stimulate macrophage fusion. Horsley et al. show that IL-4 is also a fusion-stimulating signal for nascent myotubes and promotes myotube growth by enhancing fusion of myoblasts to existing myotubes. Calcium signaling is essential for muscle differentiation and one family of transcription factors activated by calcium is the nuclear factor of activated T cells (NFAT). Several NFAT isoforms are temporally activated during myogenesis, but NFAT2c is the isoform specifically activated in nascent myotubes just after fusion. Using NFAT2c -/- myoblasts, the authors showed that myotube size and nuclei number could be restored to wild-type amounts by application of IL-4. Furthermore, IL-4 expression and secretion was increased during the period following initial myotube formation and was absent in the NFAT2c -/- myotube cultures resulting in smaller (narrower) myotubes with fewer nuclei. The importance of IL-4 and the IL-4 receptor α (IL-4Rα) was confirmed in that IL-4 -/- or IL-4Rα -/- mouse muscles showed decreased myofiber size and myotube nuclei number compared with wild-type muscle. Using a coculture fusion assay, the authors showed that the myotube-released IL-4 stimulated the IL-4Rα-expressing myoblasts to fuse with existing myotubes. Thus, these experiments not only address fundamental questions in muscle development and recovery from injury, but also provide a role for IL-4 outside of the immune system. V. Horsley, K. M. Jansen, S. T. Mills, G. K. Pavlath, IL-4 acts as a myoblast recruitment factor during mammalian muscle growth. Cell 113 , 483-494 (2003). [Online Journal]