Myotube Morphology Research Articles

Role of Trans‐signaling Molecules from Lewis Lung Carcinoma (LLC) Cells on C2C12 Myotube Atrophy and Protein Turnover Regulation

Cancer cachexia is a metabolic wasting syndrome observed in many cancer patients that is characterized by unintentional body weight loss. Imbalances in rates of protein synthesis and degradation are responsible for cancer‐induced skeletal muscle wasting. Lewis lung carcinoma (LLC) cell implantation is a widely used explant mouse model for examining cancer cachexia and muscle wasting. The purpose of this study is to investigate the influence of trans‐signals induced by LLC tumor cells on C2C12 myotube morphology and protein turnover regulation. C2C12 myotubes were treated with LLC cell culture medium (0%, 15% and 25%) for 4h, 24h and 72h. LLC medium treatment (25% for 72h) significantly decreased myotube diameter. The LLC medium induced both Stat3 and AMPK phosphorylation, while decreasing mTOR (S2448) and 4EBP1 (T37/46) phosphorylation in dose dependent manner. In addition, LLC treatment activated the FOXO3/Atrogin‐1 protein degradation signaling pathway. These results demonstrate that trans‐signaling molecules from LLC tumor cells can result in C2C12 myotube atrophy by both inhibiting mTOR regulation of protein synthesis pathway and activating protein degradation. (RO1CA121249)

Polyethylene glycol-based protein nanocapsules for functional delivery of a differentiation transcription factor

Transcription factors (TFs) can direct cell fate by binding to DNA and regulating gene transcription. Controlling the intracellular levels of specific TFs can therefore enable reprogramming of cellular function and differentiation. Direct delivery of recombinant TFs to target cells can thus have widespread therapeutic value, but has remained challenging due to structural fragility of TFs and inefficient membrane transduction. Here we describe the functional delivery of TFs using degradable polymeric nanocapsules to drive cellular differentiation. The nanocapsules were synthesized with poly(ethylene) glycol (PEG)-based monomers and intracellularly-degradable crosslinkers. Physical properties and release kinetics of the nanocapsules were optimized through tuning of monomer and crosslinker ratios to achieve enhanced delivery of cargo destined for the nuclei. The nanocapsules did not display cytotoxicity in primary cell lines up to concentrations of 5 μm. A recombinant myogenic transcription factor, MyoD, was delivered to the nuclei of myoblast cells using degradable nanocapsules to induce myogenic differentiation. MyoD was confirmed to be delivered to the nuclei of myoblasts using confocal microscopy and was demonstrated to be active in transcription through a luciferase-based reporter assay. More importantly, delivered MyoD was able to drive myoblast differentiation as evidenced by the hallmark elongated and multinuclear morphology of myotubes. The activation of downstream cascade was also confirmed through immunostaining of late myogenic markers myogenin and My-HC. The efficiency of differentiation achieved via nanocapsule delivery is significantly higher than that of native MyoD, and is comparable to that of plasmid transfection. The encapsulated MyoD can also withstand prolonged protease treatment and remain functional. The ease of preparation, biocompatibility and effective cargo delivery make the polymeric nanocapsule a useful tool to deliver a variety of recombinant TFs for therapeutic uses.

Caveolin isoform switch in differentiating C2C12 cells is not prevented by palmitate

Caveolae are membrane invaginations and important for cellular signaling. In skeletal muscle the expression of caveolin‐3 (cav3) is required for normal muscle fiber orientation and function, and caveolin‐1 (cav1) is constitutively expressed. Palmitate (palm) decreases membrane localization of cav3 in cardiomyocytes. Thus, we tested the hypothesis that palm impairs myotube formation in differentiating C2C12 cells. Two time points were tested: 1) After myotube formation: C2C12 myoblasts and myotubes were treated with 0.4 mM palm for 18 hours and 2) during differentiation: C2C12s were treated at 50–60% confluency. Western blot, IP and confocal microscopy determined expression and localization of cav1 and 3. In controls an isoform switch from cav1 in myoblasts to cav3 in myotubes occurred. Palm exposure after tube formation suppressed cav3 expression in confocal images. During differentiation, C2C12s form myotubes in the presence of palm, however the morphology was changed to long and spindly compared to controls. Palm increased the number of myotubes, but considering the changed morphology this suggests inhibition of fusion to larger myotubes. These data suggest that a caveolin isoform switch occurs in the differentiation of C2C12s from cav1 to cav3, and that cav3 expression is important for the correct morphology of myotubes formed.Support by AHA SDG 0930260N to IMP

Androgen receptor polyglutamine repeat length affects receptor activity and C2C12 cell development

Testosterone (T) has an anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. The effects of AR glutamine repeat length on skeletal muscle are unknown. In this study we examined the effect of AR CAG repeat length on AR function in C2C12 cells. AR expression vectors harboring 14, 24, and 33 CAG repeats were used to assess AR transcriptional activity. C2C12 cell proliferation, differentiation, gene expression, myotube formation, and myonuclear fusion index were assessed. Transcriptional activity increased with increasing repeat length and in response to testosterone (AR14 = 3.91 ± 0.26, AR24 = 25.21 ± 1.72, AR33 = 36.08 ± 3.22 relative light units; P < 0.001). Ligand activation was increased for AR33 (2.10 ± 0.04) compared with AR14 (1.54 ± 0.09) and AR24 (1.57 ± 0.05, P < 0.001). AR mRNA expression was elevated in each stably transfected line. AR33 cell proliferation (20,512.3 ± 1,024.0) was decreased vs. AR14 (27,604.17 ± 1,425.3; P < 0.001) after 72 h. Decreased CK activity in AR14 cells (54.9 ± 2.9 units/μg protein) in comparison to AR33 (70.8 ± 8.1) (P < 0.05) was noted. The myonuclear fusion index was lower for AR14 (15.21 ± 3.24%) and AR33 (9.97 ± 3.14%) in comparison to WT (35.07 ± 5.60%, P < 0.001). AR14 and AR33 cells also displayed atypical myotube morphology. RT-PCR revealed genotype differences in myostatin and myogenin expression. We conclude that AR polyglutamine repeat length is directly associated with transcriptional activity and alters the growth and development of C2C12 cells. This polymorphism may contribute to the heritability of muscle mass in humans.

Effect of a neonatal low-protein diet on the morphology of myotubes in culture and the expression of key proteins that regulate myogenesis in young and adult rats

To investigate the effects of a neonatal low-protein diet on the morphology of myotubes in culture and the expression of key proteins that regulate myogenesis in young and adult rats. Male Wistar rats (n=18) were suckled by mothers fed diets containing 17% protein (controls, C) or 8% protein (undernourished, UN). All rats were fed a normal protein diet after weaning. Muscles were removed from the legs of 42-, 60- and 90-day-old rats. Muscle cells were cultured to assess cell number, morphology and the expression of major proteins involved in myogenesis (Pax7, cadherins, β1 integrin, IL-4Rα and myogenin) by western blotting. IL-4 levels in culture supernatants were measured by ELISA. Offspring from mothers fed a low-protein diet showed a lower body weight gain. Cell number and myotube expansion were reduced in cultured muscle cells from UN, but the expression of myogenic marker proteins was unaltered. Dietary restriction during lactation had no impact on the synthesis of myogenic marker proteins, and myocyte differentiation occurred normally in the muscles of offspring aged 42, 60 or 90days. Nevertheless, the number and morphology of the myotubes are altered.

Androgen Receptor CAG Repeat Length Affects Receptor Activity and C2C12 Myogenic Potential

Testosterone (T) has a strong anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor activity in non-muscle tissue. The effects of AR glutamine repeat length on skeletal muscle are unknown. PURPOSE: To determine the effects of AR glutamine repeat length on AR function in skeletal muscle cells. METHODS: AR expression vectors carrying 14, 24, and 33 glutamine repeats, respectively, were constructed and AR transcriptional activity was determined in transfected C2C12 myoblasts using an AR sensor plasmid. Each vector was subsequently stably transfected into C2C12 cells to create 3 independent cell lines: C2C12AR14, C2C12AR24, and C2C12AR33. Cellular proliferation and creatine kinase (CK) activity were determined. Gene expression was assessed via RT-PCR. Myosin expression, myotube formation, and myonuclear fusion index were examined immunohistochemically. RESULTS: Transcriptional activity increased with increasing repeat length (3.91±0.26 vs. 25.21±1.72 vs. 36.08±3.22 relative light units in AR14, AR24, and AR33, respectively; p<0.001), in response to T. Significant AR protein expression was detected in AR14 line myoblasts only. In contrast, AR mRNA expression was elevated in each stable line in the myoblast stage and throughout differentiation. The proliferation of AR33 and AR24 cells was significantly decreased vs. AR14 (-25.7%, p<0.001, and -20.3%, p<0.05, respectively) after 72 hours. The CK activity of AR14 cells was decreased in comparison to AR24 and AR33 cells (54.9±2.9 vs. 68.3±2.2 and 70.8±8.1 units/ug protein, respectively; p<0.05) after 5 days of differentiation. The myonuclear fusion index was lower for both AR14 (15.21±3.24%, p<0.001) and AR33 (9.97±3.14%,<0.001)cells in comparison to C2C12 controls (35.07±5.60%). Both AR14 and AR33 cells displayed atypical myotube morphology. RT-PCR revealed differences in the expression of genes involved in differentiation, cell fusion, and cell cycle progression. CONCLUSIONS: AR polyglutamine repeat length affects androgen receptor function in skeletal muscle cells and induces morphological changes in developing myotubes. This study was Supported by NIH grants AG-022791 and AG-021500.

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling

Muscular dystrophy begins early in embryonic development deriving from stem cell loss and disrupted skeletal muscle formation

Examination of embryonic myogenesis of two distinct, but functionally related, skeletal muscle dystrophy mutants (mdx and cav-3(-/-)) establishes for the first time that key elements of the pathology of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy type 1C (LGMD-1c) originate in the disruption of the embryonic cardiac and skeletal muscle patterning processes. Disruption of myogenesis occurs earlier in mdx mutants, which lack a functional form of dystrophin, than in cav-3(-/-) mutants, which lack the Cav3 gene that encodes the protein caveolin-3; this finding is consistent with the milder phenotype of LGMD-1c, a condition caused by mutations in Cav3, and the earlier [embryonic day (E)9.5] expression of dystrophin. Myogenesis is severely disrupted in mdx embryos, which display developmental delays; myotube morphology and displacement defects; and aberrant stem cell behaviour. In addition, the caveolin-3 protein is elevated in mdx embryos. Both cav-3(-/-) and mdx mutants (from E15.5 and E11.5, respectively) exhibit hyperproliferation and apoptosis of Myf5-positive embryonic myoblasts; attrition of Pax7-positive myoblasts in situ; and depletion of total Pax7 protein in late gestation. Furthermore, both cav-3(-/-) and mdx mutants have cardiac defects. In cav-3(-/-) mutants, there is a more restricted phenotype comprising hypaxial muscle defects, an excess of malformed hypertrophic myotubes, a twofold increase in myonuclei, and reduced fast myosin heavy chain (FMyHC) content. Several mdx mutant embryo pathologies, including myotube hypotrophy, reduced myotube numbers and increased FMyHC, have reciprocity with cav-3(-/-) mutants. In double mutant (mdxcav-3(+/-)) embryos that are deficient in dystrophin (mdx) and heterozygous for caveolin-3 (cav-3(+/-)), whereby caveolin-3 is reduced to 50% of wild-type (WT) levels, these phenotypes are severely exacerbated: intercostal muscle fibre density is reduced by 71%, and Pax7-positive cells are depleted entirely from the lower limbs and severely attenuated elsewhere; these data suggest a compensatory rather than a contributory role for the elevated caveolin-3 levels that are found in mdx embryos. These data establish a key role for dystrophin in early muscle formation and demonstrate that caveolin-3 and dystrophin are essential for correct fibre-type specification and emergent stem cell function. These data plug a significant gap in the natural history of muscular dystrophy and will be invaluable in establishing an earlier diagnosis for DMD/LGMD and in designing earlier treatment protocols, leading to better clinical outcome for these patients.

The Effects Of Quercetin On Ros Generation And Myotube Area In C2C12 Muscle Cells

PURPOSE: The relationship between intracellular ROS generation and skeletal muscle proteolysis remains unclear. This research tested the hypotheses that elevated intracellular ROS generation induces measurable loss of myonuclei and myotube area in cultured skeletal muscle and these effects are attenuated with quercetin, a natural antioxidant. METHODS: C2C12 mouse muscle cells were grown to 70% confluence then supplemented with 2% horse serum to initiate differentiation into myotubes. At 4 days post-differentiation, myotubes were treated with 5μm quercetin for 18hrs. At 5 days post-differentiation, myotubes were loaded with 10μM 2',7'-dichlorodihydrofluorescein, an intracellular ROS probe. Baseline fluorescence was measured on a microplate reader, then culture media was supplemented with 200μM H2O2. Fluorescence measurements were taken at 6, 12, and 24 hours post- H2O2 administration to examine ROS generation. Immunocytochemistry (antibody staining of myonuclei (DAPI) and myosin heavy chain (MF-20)) and fluorescence microscopy were performed at baseline, 6, 12, and 24hrs to examine myotube morphology. Metamorph™ imaging software was used to quantify myonuclei and myotube area under each experimental condition. RESULTS: Myotubes treated with 200μM H2O2 displayed higher DCFH fluorescence at 6 (42%), 12 (32%), and 24hrs (26%) relative to untreated controls (p<0.001). Conversely, myotubes treated with 5μM quercetin prior to H2O2 exposure displayed lower DCFH fluorescence than did either myotubes treated with H2O2 alone (p<0.001) or untreated controls (p<0.001). Morphological analysis revealed a 21% loss of myotube area at 24hrs in cells treated with H2O2 alone as compared with untreated controls (p = 0.025). Myotubes pre-treated with quercetin displayed only a 13% loss of area at 24hrs; however, this was not different from myotubes treated with H2O2 alone. The ratio of myonuclei/myotube area (1.25±0.3 SEM) was not different between any of the treatment groups at baseline, 6, 12, and 24hrs, which suggests a proportional decrease in myonuclei and myotube area at 24hrs. CONCLUSIONS: Findings demonstrate that H2O2 stimulates increased ROS generation and leads to a significant loss of myotube area. Pre-treatment with quercetin abrogates ROS generation but does not attenuate loss of myotube area.

Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the alpha-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease.

W01.111 A critical role for the phosphoinositide-3-kinase signal transduction pathway in the activation of apolipoprotein E gene expression

In previous studies, inhibition of cholesterol synthesis by HMG CoA reductase inhibitors (HMGRI) was associated with myotoxicity in cultures of neonatal rat skeletal myotubes, and rhabdomyolysis in rats, rabbits, and humansin vivo. In vitromyotoxicity was directly related to HMGRI-induced depletion of mevalonate, farnesol, and geranylgeraniol, since supplementation with these intermediate metabolites abrogated the toxicity. Both farnesol and geranylgeraniol are required for the posttranslational modification, or isoprenylation, of essential regulatory proteins in mammalian cells. The objective of the present study was to measure changes in protein isoprenylation in cultured neonatal rat skeletal muscle cells exposed for 24 hr to increasing concentrations of pravastatin or lovastatin. Proteins were labeled with [3H]mevalonate, [3H]farnesyl pyrophosphate (FPP), or [3H]geranylgeranyl pyrophosphate (GGPP), and then separated by SDS–PAGE and quantitated by scintillation counting and densitometry of autoradiographs. Mevalonate and FPP labeling of the majority of proteins increased in a concentration-dependent manner, even at concentrations greater than 2 μmlovastatin and 25 μmpravastatin that completely inhibited cholesterol synthesis. In contrast, mevalonate and FPP labeling of three protein bands with molecular weights of 26.6, 27.7, and 28.9 kDa was markedly inhibited at concentrations higher than 1 μmlovastatin and 400 μmpravastatin, which inhibited protein synthesis and disrupted myotube morphology after longer exposures in a previous study. In contrast, these proteins were equally well labeled by GGPP at all HMGRI concentrations tested, suggesting that isoprenylation of the 26.9-, 27.8-, and 28.9-kDa proteins requires geranylgeraniol. The results of this study indicate that HMGRI-induced myotoxicity is most likely related to reduced posttranslational modification of specific regulatory proteins by geranylgeraniol.

Myoblast structure affects subsequent skeletal myotube morphology and sarcomere assembly

Skeletal myogenesis is a precise procedure marked by specific changes in muscle cell morphology and cytoarchitecture. Cessation of proliferation by skeletal muscle precursor cells (myoblasts) coincides with the induction of fusion to form multinucleated myotubes and the initiation of differentiation, the process through which sarcomeres are formed. Concurrently, there is a distinct upregulation in expression of muscle-specific isoforms and an extreme downregulation of non-muscle-specific cytoskeletal isoforms. The sarcomere is the contractile unit of the cell and is comprised of a number of different proteins aggregated and aligned in very ordered arrays along the myotube. It is this rigorously controlled alignment that gives striated muscle its characteristic “striped” appearance. Previous studies, conducted predominantly in cardiac muscle, propose models for the development of the sarcomere that attribute little of the differentiative process to the myoblast morphology and cytoskeletal arrangement. In this study, perturbation of myoblast morphology and cytoskeletal arrangement by transfection with nonmuscle actin genes in the mouse skeletal muscle cell line C2 resulted in myotubes of both varied morphology and sarcomeric structure. The results presented herein not only provide novel insights into the formation of the sarcomere in skeletal muscle, but also suggest a role for myoblast morphology and cytoskeletal structure in the subsequent differentiation of the myotube.

Overexpression of Interleukin-15 Induces Skeletal Muscle Hypertrophy in Vitro: Implications for Treatment of Muscle Wasting Disorders

Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL-15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and α-actin accumulation in differentiated myotubes. Secreted factors from IL-15-overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF-I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions.

Myotube morphology, and expression and distribution of collagen type I during normal and low score normal avian satellite cell myogenesis

Myogenic satellite cells are essential for postnatal muscle growth and the regeneration of muscle in response to injury. An understanding of how the extracellular matrix affects satellite cell activity, and the temporal and spatial expression of extracellular matrix macromolecules is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), satellite cell proliferation and differentiation rates are significantly lower than that observed in normal chicken satellite cells, which may be attributed to a late embryonic increase in the expression of decorin. Satellite cell-derived morphological properties, collagen type I expression, and the spatial distribution of collagen type I were investigated during normal and LSN satellite cell proliferation and differentiation. These studies showed a decrease in LSN myotube length and the number of nuclei per myotube. Collagen type I expression was similar between the LSN and normal satellite cell cultures during the course of proliferation and differentiation. However, the spatial distribution of collagen type I was altered in the LSN cultures 48 h after the initiation of fusion. The LSN cultures exhibited a premature extracellular distribution of collagen type I compared to the normal satellite cells.

HMG CoA Reductase Inhibitor-Induced Myotoxicity: Pravastatin and Lovastatin Inhibit the Geranylgeranylation of Low-Molecular-Weight Proteins in Neonatal Rat Muscle Cell Culture

In previous studies, inhibition of cholesterol synthesis by HMG CoA reductase inhibitors (HMGRI) was associated with myotoxicity in cultures of neonatal rat skeletal myotubes, and rhabdomyolysis in rats, rabbits, and humansin vivo. In vitromyotoxicity was directly related to HMGRI-induced depletion of mevalonate, farnesol, and geranylgeraniol, since supplementation with these intermediate metabolites abrogated the toxicity. Both farnesol and geranylgeraniol are required for the posttranslational modification, or isoprenylation, of essential regulatory proteins in mammalian cells. The objective of the present study was to measure changes in protein isoprenylation in cultured neonatal rat skeletal muscle cells exposed for 24 hr to increasing concentrations of pravastatin or lovastatin. Proteins were labeled with [3H]mevalonate, [3H]farnesyl pyrophosphate (FPP), or [3H]geranylgeranyl pyrophosphate (GGPP), and then separated by SDS–PAGE and quantitated by scintillation counting and densitometry of autoradiographs. Mevalonate and FPP labeling of the majority of proteins increased in a concentration-dependent manner, even at concentrations greater than 2 μmlovastatin and 25 μmpravastatin that completely inhibited cholesterol synthesis. In contrast, mevalonate and FPP labeling of three protein bands with molecular weights of 26.6, 27.7, and 28.9 kDa was markedly inhibited at concentrations higher than 1 μmlovastatin and 400 μmpravastatin, which inhibited protein synthesis and disrupted myotube morphology after longer exposures in a previous study. In contrast, these proteins were equally well labeled by GGPP at all HMGRI concentrations tested, suggesting that isoprenylation of the 26.9-, 27.8-, and 28.9-kDa proteins requires geranylgeraniol. The results of this study indicate that HMGRI-induced myotoxicity is most likely related to reduced posttranslational modification of specific regulatory proteins by geranylgeraniol.

LOW DOSE ULTRASOUND EFFECTS ON SKELETAL MUSCLE REGENERATION IN RAT TIBIALIS ANTERIOR 366

Therapeutic ultrasound (US) is commonly used after skeletal muscle injury to increase circulation, decrease pain, and accelerate tissue healing. The objective of this study was to test the hypothesis that thermal or radiant energy increases the number of replicating satellite cells and myotubes seen after a muscle crush injury. Focal muscle damage was produced by forceps crush to the anterior tibialis muscles of male, adult Sprague-Dawley rats. Animals were divided into treatment (n=3) and sham (n=3) groups. The treatment group received three 5 minute applications of 3 MHz, pulsed US at 0.5 w/cm2. Treatments were given every other day. The sham group received the sound head application for the same time period but no sonation. All animals received injections of BrdU for localizing mitotically active cells. At 6 days after injury (3 US treatments), myotubes and young myofibers that stained positive for embryonic myosin were apparent in both groups. Myotube morphology was similar between the two groups. BrdU positive nuclei were noted both deep to(satellite cells) and external to (fibroblasts and inflammatory cells) the basal laminae of the myotubes. Preliminary quantification of the BrdU positive nuclei deep to the myotube basal laminae demonstrated no significant difference between the groups (p >.05). These results suggest that low dose US may not have a significant effect on satellite cell proliferation as seen at 6 days post injury.

Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation.

The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5-transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF-alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.

Neonatal Centronuclear Myopathy with N-Cam Decorated Myotubes

Muscle specimens from two baby males with fatal X-linked recessive (XLR) form of central nuclear myopathy (CNM) were studied by immunochemistry using cell surface molecules (N-CAM) and by electron microscopy. In both neonates muscle fibres with morphology of myotubes strongly expressed N-CAM. The expression of N-CAM in non regenerating postnatal human muscle suggests that neonatal X-linked fatal form of CNM is characterized by arrest or delay of maturation of muscle fibres.

Thymopoietin, a thymic polypeptide, prevents nicotinic agonist-induced morphological changes in neonatal muscle cells in culture

Thymopoietin, a polypeptide hormone isolated from thymus and involved in immune function, potently inhibited [ 125I]α-bungarotoxin binding to neonatal muscle cells in culture ( IC 50 = 3.8nM) and blocked carbachol-stimulated 22Na uptake with an IC 50 of 1.9 ± 0.2nM and 23 ± 7nM at a half-maximal and maximal concentration of carbachol, respectively. Studies were subsequently done to evaluate potential long-term functional consequences of this interaction of thymopoietin at the nicotinic receptor. Exposure (1–3 days) of neonatal muscle cells in culture to nicotine (3 × 10 −6 M) or carbachol (1 × 10 −6 M) resulted in a decline in myotube branching and a decrease in myotube length. Thymopoietin did not appreciably alter myotube morphology on its own; however, it prevented the effects of nicotine and carbachol on muscle cell morphology at concentrations (1–10 nM) which corresponded well to those with which thymopoietin interacted at the receptor. The action of α-bungarotoxin on the myotubes was very similar to that of thymopoietin. These studies suggest that the endogenously occurring polypeptide, thymopoietin, has the potential to modulate muscle cell morphology through an interaction at the nicotinic receptor.

Studies on the role of microtubules in myofibrillogenesis

Go-localization of microtubnle (MT) and muscle myosin (MHC) myofibril immunofluoreseoneo in developing myotubes of chicken skeletal muscle cultures was observed by using double staining of tubulin and MHC indirect immunofluorescence. 12-o-tetradecanoyl-phorbol-13-acetate (TPA) selectively and reversibly blocks myofibrillogenesis and alters the morphology of myotubes into myosacs where MTs are present in radiating pattern. When the arrested myogenic cells recover and start myofibrillogenesis after released from TPA, prior to the emergence of myofibrils, the pro-existing MTs become bipolarly aligned eoineidontly with the tubular restoration of cell shape. Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips. That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested. Taxol and eoleomid disturbed MT polymerization and disposition, and interfered with the normal spatial assembly of myofibrils in developing myotubos.