Undifferentiated Myoblasts Research Articles

Human Myosin-18B - A Versatile Actin Binding Protein

Class-18 myosins challenge our established view about myosins acting as molecular motors. No member of this class appears to have a significant ATPase activity, which is a prerequisite for motor activity. Humans express two myosin-18 isoforms, myosin-18A and myosin-18B. Whereas recent studies on myosin-18A shed some light on its cellular and biochemical mode of action, the molecular function of myosin-18B remains poorly understood. Class-18 myosins contain protein interaction domains outside their generic motor domain. In the case of myosin-18B this includes a large, N-terminal extension that shows no similarity to any known protein domain. Here, we show that the human myosin-18B motor domain binds to F-actin with an affinity of 4 µM. The isolated motor domain binds ATP but has no intrinsic ATPase activity. The large N-terminal extension is shown to directly bind to F-actin with an affinity of 7 µM. This interaction is nucleotide-independent but shows strong ionic strength dependence, which is indicative for a charge-mediated actin binding mechanism. We further analyzed the molecular function of the N-terminal extension by means of actin polymerization assays and found that the myosin-18B N-terminus inhibits F-actin assembly in vitro. Myosin-18B has previously been shown to be located in the cytoplasm of undifferentiated myoblasts. At later stages of differentiation it accumulates in myonuclei. Furthermore, it has been shown that cardiomyocytes display a partial sarcomeric pattern of myosin-18B alternating that of α-actinin-2. Based on our data, we propose a role for myosin-18B in the regulation of muscle sarcomere architecture during differentiation and the regulation of the nuclear actin pool.

Resveratrol promotes myogenesis and hypertrophy in murine myoblasts

BackgroundNutrigenomics elucidate the ability of bioactive food components to influence gene expression, protein synthesis, degradation and post-translational modifications.Resveratrol (RSV), natural polyphenol found in grapes and in other fruits, has a plethora of health benefits in a variety of human diseases: cardio- and neuroprotection, immune regulation, cancer chemoprevention, DNA repair, prevention of mitochondrial disorder, avoidance of obesity-related diseases. In skeletal muscle, RSV acts on protein catabolism and muscle function, conferring resistance against oxidative stress, injury and cell death, but its action mechanisms and protein targets in myogenesis process are not completely known. Myogenesis is a dynamic multistep process regulated by Myogenic Regulator Factors (MRFs), responsible of the commitment of myogenic cell into skeletal muscle: mononucleated undifferentiated myoblasts break free from cell cycle, elongate and fuse to form multinucleated myotubes. Skeletal muscle hypertrophy can be defined as a result of an increase in the size of pre-existing skeletal muscle fibers accompanied by increased protein synthesis, mainly regulated by Insulin Like Growth Factor 1 (IGF-1), PI3-K/AKT signaling pathways.Aim of this work was the study of RSV effects on proliferation, differentiation process and hypertrophy in C2C12 murine cells.MethodsTo study proliferative phase, cells were incubated in growth medium with/without RSV (0.1 or 25 μM) until reaching sub confluence condition (24, 48, 72 h). To examine differentiation, at 70% confluence, cells were transferred in differentiation medium both with/without RSV (0.1 or 25 μM) for 24, 48, 72, 96 hours. After 72 hours of differentiation, the genesis of hypertrophy in neo-formed myotubes was analyzed.ResultsData showed that RSV regulates cell cycle exit and induces C2C12 muscle differentiation. Furthermore, RSV might control MRFs and muscle-specific proteins synthesis. In late differentiation, RSV has positive effects on hypertrophy: RSV stimulates IGF-1 signaling pathway, in particular AKT and ERK 1/2 protein activation, AMPK protein level and induces hypertrophic morphological changes in neo-formed myotubes modulating cytoskeletal proteins expression.ConclusionsRSV might control cell cycle promoting myogenesis and hypertrophy in vitro, opening a novel field of application of RSV in clinical conditions characterized by chronic functional and morphological muscle impairment.

High efficacy gold-KDEL peptide-siRNA nanoconstruct-mediated transfection in C2C12 myoblasts and myotubes

Gold nanoparticles (AuNP) were conjugated with cysteine terminated KDEL (Lys-Asp-Glu-Leu) peptide and siRNA directed against NADPH Oxidase 4 (Nox4). Fluorescence microscopy analysis provided evidence of cytocellular retrograde transport pathways and sub-cellular colocalization of AuNP nanoconstructs in both undifferentiated C2C12 myoblasts and differentiated C2C12 myotubes. The cellular trafficking of AuNP nanoconstructs in undifferentiated myoblasts suggests stable and efficient transfection of siRNA as demonstrated by colocalization of AuNP-delivered KDEL and siRNA. The cellular uptake of AuNP nanoconstructs was more efficient than Lipofectamine mediated transfection in differentiated myotubes (P<0.05) compared to undifferentiated myoblasts, suggesting that AuNP nanoconstructs provide an efficient platform for siRNA delivery to differentiated myotubes. The localization of these nanoconstructs in undifferentiated myoblasts suggests that most of the siRNA was localized in the endoplasmic reticulum (ER) with a minimal distribution in the Golgi bodies suggesting that the ER is a primary localization site for AuNP-KDEL mediated delivery of nanoconstructs. From the Clinical EditorThis team of investigators demonstrate that a delivery system composed of gold nanoparticle and KDEL based siRNA is superior to lipofectamine in delivering siRNA in differentiated myoblasts, paving the way to gene silencing methods in these cells for future therapeutic exploitation.

Highly efficient in vivo delivery of PMO into regenerating myotubes and rescue in laminin-α2 chain-null congenital muscular dystrophy mice

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches to the treatment of several neuromuscular disorders including Duchenne muscular dystrophy. The main weakness of this approach arises from the low efficiency and sporadic nature of the delivery of charge-neutral PMO into muscle fibers, the mechanism of which is unknown. In this study, to test our hypothesis that muscle fibers take up PMO more efficiently during myotube formation, we induced synchronous muscle regeneration by injection of cardiotoxin into the tibialis anterior muscle of Dmd exon 52-deficient mdx52 and wild-type mice. Interestingly, by in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. In addition, we showed that PMO or 2′-O-methyl phosphorothioate is taken up efficiently into C2C12 myotubes when transfected 24–72 h after the induction of differentiation but is poorly taken up into undifferentiated C2C12 myoblasts suggesting efficient uptake of PMO in the early stages of C2C12 myotube formation. Next, we tested the therapeutic potential of PMO for laminin-α2 chain-null dy3K/dy3K mice: a model of merosin-deficient congenital muscular dystrophy (MDC1A) with active muscle regeneration. We confirmed the recovery of laminin-α2 chain and slightly prolonged life span following skipping of the mutated exon 4 in dy3K/dy3K mice. These findings support the idea that PMO entry into fibers is dependent on a developmental stage in myogenesis rather than on dystrophinless muscle membranes and provide a platform for developing PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration.

Evidence for a Specific Uptake and Retention Mechanism for 25-Hydroxyvitamin D (25OHD) in Skeletal Muscle Cells

Little is known about the mechanism for the prolonged residence time of 25-hydroxyvitamin D (25OHD) in blood. Several lines of evidence led us to propose that skeletal muscle could function as the site of an extravascular pool of 25OHD. In vitro studies investigated the capacity of differentiated C2 murine muscle cells to take up and release 25OHD, in comparison with other cell types and the involvement of the membrane protein megalin in these mechanisms. When C2 cells are differentiated into myotubes, the time-dependent uptake of labeled 25OHD is 2-3 times higher than in undifferentiated myoblasts or nonmuscle osteoblastic MG63 cells (P < .001). During in vitro release experiments (after 25OHD uptake), myotubes released only 32% ± 6% stored 25OHD after 4 hours, whereas this figure was 60% ± 2% for osteoblasts (P < .01). Using immunofluorescence, C2 myotubes and primary rat muscle fibers were, for the first time, shown to express megalin and cubilin, endocytotic receptors for the vitamin D binding protein (DBP), which binds nearly all 25OHD in the blood. DBP has a high affinity for actin in skeletal muscle. A time-dependent uptake of Alexafluor-488-labeled DBP into mature muscle cells was observed by confocal microscopy. Incubation of C2 myotubes (for 24 hours) with receptor-associated protein, a megalin inhibitor, led to a 40% decrease in 25OHD uptake (P < .01). These data support the proposal that 25OHD, after uptake into mature muscle cells, is held there by DBP, which has been internalized via membrane megalin and is retained by binding to actin.

Foxc2 induces Wnt4 and Bmp4 expression during muscle regeneration and osteogenesis

Proliferation and fusion of myoblasts is a well-orchestrated process occurring during muscle development and regeneration. Although myoblasts are known to originate from muscle satellite cells, the molecular mechanisms that coordinate their commitment toward differentiation are poorly understood. Here, we present a novel role for the transcription factor Forkhead box protein C2 (Foxc2) in regulating proliferation and preventing premature differentiation of activated muscle satellite cells. We demonstrate that Foxc2 expression is upregulated early in activated mouse muscle satellite cells and then diminishes during myogenesis. In undifferentiated C2C12 myoblasts, downregulation of endogenous Foxc2 expression leads to a decrease in proliferation, whereas forced expression of FOXC2 sustains proliferation and prevents differentiation into myotubes. We also show that FOXC2 induces Wnt signaling by direct interaction with the Wnt4 (wingless-type MMTV integration site family member-4) promoter region. The resulting elevated expression of bone morphogenetic protein-4 (Bmp4) and RhoA-GTP proteins inhibits the proper myoblast alignment and fusion required for myotube formation. Interestingly, continuous forced expression of FOXC2 alters the commitment of C2C12 myoblasts toward osteogenic differentiation, which is consistent with FOXC2 expression observed in patients with myositis ossificans, an abnormal bone growth within muscle tissue. In summary, our results suggest that (a) Foxc2 regulates the proliferation of multipotent muscle satellite cells; (b) downregulation of Foxc2 is critical for myogenesis to progress; and (c) sustained Foxc2 expression in myoblast cells suppresses myogenesis and alters their lineage commitment toward osteogenesis by inducing the Wnt4 and Bmp4 signaling pathways.

Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage

Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.

Abstract 2670: Metabolomic and molecular insights into pancreatic cancer-induced cachexia.

Abstract Pancreatic cancer is an aggressive and lethal neoplasm that induces cachexia and is typically detected at an advanced stage. Better understanding of the disease, early detection, and new therapeutic targets are urgently needed. Here we have investigated the metabolism of multiple pancreatic tumors, and their effect on mouse body weight. To understand the interactions between the tumor and normal tissue, we are developing a cell-based optical biosensor using genetically engineered myoblasts to detect the early onset of cachexia-inducing signals in muscle tissue. We have also characterized the expression of choline kinase (Chk), which is known to be overexpressed in aggressive cancer, and cyclooxygenase 2 (COX-2), a critically important inflammation mediator that significantly influences cancer angiogenesis, invasion, and metastasis. Human pancreatic adenocarcinoma cell lines Pa02C and Pa04C (Jones et al, Science 2008), as well as Panc1, and BxPC3 (obtained from ATCC), were inoculated subcutaneously in male SCID mice. Mouse bodyweight was followed for approximately 6 weeks. Tumors (500 mm3) were excised and freeze-clamped for immunoblot and high-resolution 1H MRS analyses. A loss of body weight was observed after inoculation of Pa04C and Panc1 cells but not BxPc3 and Pa02C cells. Panc1 tumors exhibited high expression levels of COX-2 and the highest levels of Chk. However, Pa04C tumors showed low levels of those two proteins. 1H MRS analysis revealed that Panc1 tumors contained the highest level of total choline, mainly due to a high level of phosphocholine, correlating with the high level of Chk. Panc1 tumors were also characterized by a high level of lactate. Pa04C tumors presented with the second highest levels of total choline and lactate. To determine the effect of pancreatic cancer cells on muscle, we transiently co-transfected primary human myoblasts with constitutive EF-1α driven eGFP expression and with either a control vector, triple-tandem repeat of a NFκB cis-element lacking a minimal promoter (mp) sequence, or with the same tandem repeat fused to a minimal promoter sequence (3xNFκB-mp) driving tdTomato expression. The transfected myoblasts were differentiated into myotubes and then treated for 24 h with conditioned medium from pancreatic tumor cells. We observed that the 3xNFκB-mp promoter was inducible in the presence of conditioned medium obtained from both Panc1 and Pa04C cells. No red fluorescence was induced in undifferentiated myoblasts or in confluent myoblasts. The acquisition of in vivo 1H MRSI is ongoing, and should further improve the characterization of those pancreatic cell lines, and the correlation between the metabolites measured in vivo and weight loss. Our data identify increased total choline and lactate as potential imaging biomarkers to detect pancreatic cancer, and Chk and COX-2 as potential therapeutic targets of this devastating disease. This work was supported by NIH P50CA103175. Citation Format: Marie-France Penet, Paul T. Winnard, Flonné Wildes, Yelena Mironchik, Tariq Shah, Anirban Maitra, Zaver M. Bhujwalla. Metabolomic and molecular insights into pancreatic cancer-induced cachexia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2670. doi:10.1158/1538-7445.AM2013-2670

A Comparative Review of Canine and Human Rhabdomyosarcoma With Emphasis on Classification and Pathogenesis

Rhabdomyosarcomas are a diverse group of malignant mesenchymal neoplasms exhibiting variable levels of differentiation toward skeletal myocytes. Neoplastic cells may resemble relatively undifferentiated myoblasts, satellite cells, or more differentiated elongated spindle cells and multicellular myotubes. In veterinary medicine, classification into subtypes and variants is based on an outdated system derived from human pathology and is solely based on histologic characteristics. In contrast, classification of human rhabdomyosarcoma is based on histologic, immunohistochemical, and molecular diagnostic techniques, and subclassification has clinical and prognostic relevance. Relevance of tumor subtyping has not been established in veterinary medicine. Recent discoveries of components of the molecular pathogenesis and genomes of human rhabdomyosarcomas have led to new diagnostic techniques and revisions of the human classification system. The current classification system in veterinary medicine is reviewed in light of these changes. Diagnosis of rhabdomyosarcoma using histopathology, electron microscopy, and the clinical aspects of human and canine rhabdomyosarcomas is compared. The clinical features and biologic behavior of canine rhabdomyosarcomas are compared with canine soft tissue sarcomas.

Lamins in development, tissue maintenance and stress

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Automated Gas-Phase Purification for Accurate, Multiplexed Quantification on a Stand-Alone Ion-Trap Mass Spectrometer

Isobaric tagging enables the acquisition of highly multiplexed proteome quantification, but it is hindered by the pervasive problem of precursor interference. The elimination of coisolated contaminants prior to reporter tag generation can be achieved through the use of gas-phase purification via proton transfer ion/ion reactions (QuantMode); however, the original QuantMode technique was implemented on the high-resolution linear ion-trap-Orbitrap hybrid mass spectrometer enabled with electron transfer dissociation (ETD). Here we extend this technology to stand-alone linear ion-trap systems (trapQuantMode, trapQM). Facilitated by the use of inlet beam-type activation (i.e., trapHCD) for production and observation of the low mass-to-charge reporter region, this scan sequence comprises three separate events to maximize peptide identifications, minimize duty cycle requirements, and increase quantitative accuracy, precision, and dynamic range. Significant improvements in quantitative accuracy were attained over standard methods when using trapQM to analyze an interference model system comprising tryptic peptides of yeast that we contaminated with human peptides. Finally, we demonstrate practical benefits of this method by analysis of the proteomic changes that occur during mouse skeletal muscle myoblast differentiation. While the reduced duty cycle of trapQM led to the identification of fewer proteins than conventional operation (4050 vs 2964), trapQM identified more significant differences (>1.5 fold, 1362 vs 1132, respectively; p < 0.05) between the proteomes of undifferentiated myoblasts and differentiated myotubes and nearly 10-fold more differences with changes greater than 5-fold (96 vs 12). We further show that our trapQM dataset is superior for identifying changes in protein abundance that are consistent with the metabolic and structural changes known to accompany myotube formation.

Skeletal muscle secreted factors prevent glucocorticoid-induced osteocyte apoptosis through activation of β-catenin

It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect osteocytes from cell death through activation of the Wnt/β-catenin pathway, as MT-CM induces β-catenin nuclear translocation and β-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve osteocyte viability.

Biomechanical characterization of a desminopathy in primary human myoblasts

Heterozygous mutations of the human desmin gene on chromosome 2q35 cause hereditary and sporadic myopathies and cardiomyopathies. The expression of mutant desmin brings about partial disruption of the extra sarcomeric desmin cytoskeleton and abnormal protein aggregation in the sarcoplasm of striated muscle cells. The precise molecular pathways and sequential steps that lead from a desmin gene defect to progressive muscle damage are still unclear. We tested whether mutant desmin changes the biomechanical properties and the intrinsic mechanical stress response of primary cultured myoblasts derived from a patient carrying a heterozygous R350P desmin mutation. Compared to wildtype controls, undifferentiated mutant desmin myoblasts revealed increased cell death and substrate detachment in response to cyclic stretch on flexible membranes. Moreover, magnetic tweezer microrheometry of myoblasts using fibronectin-coated beads showed increased stiffness of diseased cells. Our findings provide the first evidence that altered mechanical properties may contribute to the progressive striated muscle pathology in desminopathies. We postulate that the expression of mutant desmin leads to increased mechanical stiffness, which results in excessive mechanical stress in response to strain and consecutively to increased mechanical vulnerability and damage of muscle cells.

The Spatial Mapping of the Metabolic Cofactor NADH within Live Progenitor Stem Cells

NADH is a naturally occurring bi-product and regulatory metabolite associated with cellular respiration. The quantification using the difference lifetime of autofluorescence of free and bound NADH has the potential to enhance the understanding of a range of cellular processes including apoptosis, cancer pathology and enzyme kinetics. Fluorescence lifetime imaging microscopy (FLIM) enables not only examination of the spatial location of the cofactor within live cells but also of its state.Here we describe the use of phasor FLIM to spatially map the fluorescence lifetimes of NADH in both free and bound form within live undifferentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. The phasor representation enables the possibility for a direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells to enable a global analysis.A comparison of myoblast cells induced to differentiate through serum starvation and undifferentiated cells show differing spatial distribution of the different forms of NADH. Cells due to undergo differentiation displayed a short lifetime representing free NADH situated around the cytoplasmic periphery and a longer lifetime attributed to the presence of bound NADH just outside of the nucleus. Differentiated cells displayed redirection of the distribution of free NADH located mainly within the nucleus while the bound form remained directly comparable to that of the other cells. Furthermore, there appears to be a spatial shift in the distribution of lifetimes at a pixel level within the phasor plot. We show that the states of differentiation of myocytes may be determined through the phasor FLIM analysis of the autofluorescent properties of NADH.

Stau1 regulates Dvl2 expression during myoblast differentiation

Post-transcriptional regulation of gene expression by RNA-binding proteins has pivotal roles in many biological processes. We have shown that Stau1, a conserved RNA-binding protein, negatively regulates myogenesis in C2C12 myoblasts. However, its target mRNAs in regulation of myogenesis remain unknown. Here we describe that Stau1 positively regulates expression of Dvl2 gene encoding a central mediator of Wnt pathway in undifferentiated C2C12 myoblasts. Stau1 binds to 3′ untranslated region (UTR) of Dvl2 mRNA and Stau1 knockdown shortened a half-life of the mRNA containing Dvl2 3′ UTR. After induction of myogenic differentiation, association of Stau1 with 3′ UTR of Dvl2 mRNA was decreased. Correlated with the decrease in the association, the Dvl2 mRNA level was reduced during myogenesis. A forced expression of Dvl2 markedly inhibited progression of myogenic differentiation. Our results suggest that Dvl2 has an inhibitory role in myogenesis and Stau1 coordinates myogenesis through the regulation of Dvl2 mRNA.

The SCF-Fbxo40 Complex Induces IRS1 Ubiquitination in Skeletal Muscle, Limiting IGF1 Signaling

Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/IRS1/PI3K/Akt pathway. However the effect of IGF1 in differentiated muscle is limited by IRS1 ubiquitination and proteasome-mediated breakdown. In skeletal muscle, IGF1R activation sensitizes IRS1 to degradation, and a screen for the responsible E3 ligase identified Fbxo40 as mediating this rapid turnover of IRS1, since IRS1 loss can be rescued by knockdown of Fbxo40. In biochemical assays, an SCF E3 ligase complex containing Fbxo40 directly ubiquitinates IRS1, and this activity is enhanced by increased tyrosine phosphorylation of IRS1. Fbxo40 is muscle specific in expression and is upregulated during differentiation. Knockdown of Fbxo40 induces dramatic hypertrophy of myofibers. Mice null for Fbxo40 have increased levels of IRS1 and demonstrate enhanced body and muscle size during the growth phase associated with elevated IGF1 levels. These findings establish an important means of restraining IGF1's effects on skeletal muscle.

Gene expression during normal and FSHD myogenesis

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how.MethodsUsing exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods.ResultsMany of the ~17,000 examined genes were differentially expressed (> 2-fold, p < 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types.ConclusionsDUX4's pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4's inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.

Translational level of acetylcholine receptor α mRNA in mouse skeletal muscle is regulated by YB-1 in response to neural activity

Y-box-binding protein 1 (YB-1) binds to mRNAs and affects translation. In this study, we focused on skeletal muscle, in which YB-1 expression is restricted to the early postnatal period, and found that YB-1 binds to acetylcholine receptor α-subunit (AChR α) mRNA. Although transcription of the AChR α gene is known to be regulated by myogenic transcription factors, translational control of the mRNA in response to neuromuscular transmission has not been examined. In undifferentiated C2C12 myoblasts, expression of AChR α remained at a low level. However, translation of the mRNA was increased by knockdown of YB-1. Continued overexpression of YB-1 prevented the cells from differentiating. In myotubes, which show clustering of AChRs, translation of the mRNA was induced within 3h after treatment with nicotine. The effect of nicotine was inhibited by α-bungarotoxin, and in the presence of cycloheximide the level of AChR α was reduced, even after nicotine treatment. Sucrose gradient analysis revealed that in nicotine-treated myotubes, YB-1-containing polysomes were shifted to the heavier-sedimenting fractions, and showed an apparent decrease in the amount of YB-1 bound to AChR α mRNA. These results suggest that in skeletal muscle cells, neural activity reduces the molar ratio of YB-1 relative to its binding AChR α mRNA, leading to an increase of ribosome binding to the mRNA, and thus activating translation. Furthermore, in postnatal growing mice, as has already been shown, the level of AChR α mRNA declined during the early period with maturation of neuromuscular synapses, but the translation level was found to increase transiently at postnatal day 10, when the level of YB-1 was markedly reduced. It is suggested that although the level of AChR α mRNA is reduced, the translation can be induced by alteration of the ratio of YB-1 protein to the mRNA.

P5.77 Sarcomeric gene and protein expression during human myogenesis in vitro

In vertebrates, skeletal muscle is found throughout the body and forms during the entire life span. Adult skeletal muscle displays a high regenerative capacity, with satellite cells as the primary source of this remarkable ability. Satellite cells are quiescent muscle progenitors located between myofibers and their surrounding basal lamina. The formation of skeletal muscle cells, myogenesis, is a complex process, which takes place during embryonic development, postnatal growth and adult muscle regeneration. Myogenesis is marked by specific changes in muscle cell morphology and cytoarchitecture through the fusion of mononucleated myoblasts into multinucleated myotubes and the assembly of contractile sarcomeric myofibrils. The formation of contractile myofibrils involves the transition of myoblast cytoarchitecture, which requires a change in the expression of proteins from non-muscle-specific to muscle-specific isoforms. In this study, combined molecular and immunocytochemistry methods were used to investigate the expression profile of sarcomeric genes and proteins during myoblast proliferation and differentiation in human muscle cell line. We observed the expression of a panel of sarcomeric gene transcripts together with the early expression of a number of the investigated sarcomeric proteins at the proliferated myoblasts. In addition, after 6 days of differentiation, the majority of cells expressed all the investigated sarcomeric proteins. The results presented here provide novel insights into the sequential onset of the sarcomeric genes and proteins and support the previous notion that non-committed stem cells exist in the adult muscle satellite cells during myogenesis in vitro. In addition, our study demonstrates the effectiveness of a differentiation system, which transforms undifferentiated and star-shaped myoblasts, into long multinucleated myotubes, displaying the features of contractile muscle cells.

Expression and association of TRPC1 with TRPC3 during skeletal myogenesis

TRPC1 and TRPC3 proteins are widely expressed in skeletal muscles in forming calcium-permeable channels. Herein we characterize the expression pattern of TRPC transcripts during skeletal myogenesis in C2C12 myoblasts. We used polymerase chain reaction and Western blotting to detect expression levels, immunohistochemistry for subcellular localization, and co-immunoprecipitation techniques to assess interaction. TRPC1 localizes to the cytoplasm and is enriched in the perinuclear region in undifferentiated myoblasts. Expression of TRPC1 increases significantly during myogenesis and resides mainly in differentiated myocytes and myotubes. TRPC3 is absent in undifferentiated myoblasts, is dramatically upregulated in differentiated culture, and is preferentially expressed in myotubes. Physical interaction of TRPC1-TRPC3 was observed, suggesting the possible existence of heteromers. Expression of TRPC1 and TRPC3 is tightly regulated during myogensis. Evidence of TRPC1-TRPC3 interaction was first demonstrated in a muscle cell line. The functional consequences of this interaction remain to be established.