Primary Mouse Myoblasts Research Articles

Role of H2 Calponin in Myoblast Differentiation, Fusion and Myogenesis

Actin cytoskeleton plays a pivotal role in myoblast motility, adhesion, fusion and differentiation into myotubes. However, how the actin cytoskeleton is regulated during myogenesis is largely unknown. H2-calponin is a regulatory protein that stabilizes the actin cytoskeleton and regulates cell motility and adhesion. In the present study, we investigated the role of h2 calponin in the regulation of myoblast migration, differentiation and fusion. The results showed that h2-calponin is expressed at significant levels in undifferentiated mouse primary satellite cells and C2C12 myoblasts. In migrating satellite cells and C2C12 myoblasts, h2-calponin co-localizes with actin stress fibers at both the leading edge and the tail regions. In newly fused myotubes derived from primary satellite cells, h2-calponin is mainly present at the ends of the myotubes. The level of h2-calponin significantly decreases during satellite cell and myoblast differentiation and fusion, and becomes undetectable in mature myocytes. Primary satellite cells isolated from h2-calponin gene knockout mice had significantly decreased cell spreading area indicating reduced substrate adhesion and increased rate of cell proliferation as compared with wild type controls. Biochemical and histological studies revealed that skeletal muscles of h2-calponin knockout mice retain normal muscle weight and myofilament protein compositions. Myotubes derived from h2-calponin-null satellite cells showed apparently normal sarcomere striations and expression of myofilament proteins. Therefore, deletion of h2-calponin does not abolish the maximum capacity of myoblast function and myogenesis. However, in vitro differentiation studies revealed that h2-calponin-hull myoblasts had higher efficiency of fusion, as indicated by increased fusion index, than wild type controls. The data suggest that the function of h2-calponin provides a novel mechanism in regulating actin cytoskeleton activity during myoblast differentiation, fusion and myogenesis.

Abstract 13433: Angiotensin II Type 2 Receptor Regulates Skeletal Myoblast Differentiation: Implications for treatment of Cachexia and Skeletal Muscle Wasting

Patients with advanced congestive heart failure (CHF) or chronic kidney disease (CKD) often have increased angiotensin II (Ang II) levels and cachexia. We previously demonstrated that Ang II infusion in rodents causes skeletal muscle wasting and decreases muscle regenerative potential via Ang II type 1 receptor (AT1R) signaling, likely contributing to cachexia in CHF and CKD. However, the potential role of Ang II type 2 receptor (AT2R) signaling in skeletal muscle physiology remains unknown. We found that AT2R expression was robustly increased in mouse skeletal myoblasts during differentiation, suggesting that the AT2R plays an important role in skeletal muscle regeneration. To test this hypothesis, we infused mice with AT2R antagonist PD123319 (PD, 30 mg/kg/d) or agonist CGP123319 (CGP, 1 μg/kg/min) during cardiotoxin (CTX)-induced muscle injury and regeneration. PD reduced the size of regenerating myofibers (727.5±54.6 and 516.0±37.0 μm2 in sham and PD, respectively, p<0.05) and expression of the myoblast differentiation markers myogenin and eMyHC (56.9% and 40.2% decrease in PD, respectively. p<0.01), whereas CGP had the opposite effects. siRNA mediated AT2R knockdown in mouse primary myoblasts suppressed the increase of myogenin and desmin, resulting in lowered differentiation. We analyzed changes in phosphoprotein levels in myoblasts after AT2R knockdown by phosphoprotein array and identified multiple changes, including increased phospho-ERK1/2 levels. Importantly, inhibition of ERK1/2 restored normal myoblast differentiation in the setting of AT2R knockdown, suggesting the AT2R positively regulates myoblast differentiation by reducing ERK1/2 activity. Furthermore, we found that skeletal muscle regeneration was reduced (decreased regenerating myofiber size and myogenin/desmin expression) in a mouse myocardial infarction model of CHF, concomitantly with markedly blunted increase of AT2R expression, strongly suggesting that the AT2R plays an important role in the reduction of skeletal muscle function in CHF. These data indicate that AT2R signaling positively regulates myoblast differentiation and potentiates skeletal muscle regeneration, providing a new therapeutic target in wasting disorders such as CHF and CKD.

Abstract PR02: Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is composed of skeletal myoblast-like cells that have lost the capacity to terminally differentiate. This suggests that RMS cells may contain a factor that blocks normal muscle differentiation. Because cell cycle arrest is coupled to muscle differentiation, identifying putative negative regulators of differentiation could lead to novel therapeutic approaches aimed at fostering terminal differentiation. To gain insight into the events that normally trigger the initial phase of muscle differentiation, we carried out a high content cell-based screen using detection of Myogenin, an early marker of muscle differentiation, as a readout that we measured by automated fluorescence microscopy. We focused on identifying kinases that hinder this developmental transition because of the potential to develop pharmacological inhibitors of “hit” kinases. Using an siRNA library targeting over 600 kinases, we identified 56 as putative negative regulators of myogenic differentiation: 43 with and 19 without PD332991, a Cdk4/6 inhibitor included as a sensitizer. Network analysis showed that 47% of the hits identified without the sensitizer, including SRC family kinases Src and Fyn, were just one interaction node away from MyoD- or Mef2-family myogenic regulatory factors. Although some of the hit kinases were previously implicated in myogenesis, many others were not. Further, 9 (16%) of the hits are causally linked with cancer, based on KEGG and COSMIC databases. Pathway analyses highlighted certain cancer-associated pathways, like EPHA/Ephrin B signaling, the MAPK kinase pathway, de novo pyrimidine synthesis, and mTOR signaling. Among the hits relevant to RMS, Mtor was particularly interesting because this gene encodes a kinase regarded to positively regulate skeletal muscle differentiation. We confirmed our screen findings that Mtor blocks muscle differentiation by showing that its knockdown increases the transcription of a panel of muscle-specific genes in an established myoblast cell line and primary mouse myoblasts. Induction of muscle genes by Mtor knockdown correlated with G0/G1 cell cycle arrest that normally accompanies differentiation. Rapamycin mimicked the effects of Mtor knockdown on muscle gene expression and cell proliferation, implying a role for mTorc1. Finally, preliminary analysis of RMS gene expression data demonstrated that higher expression of muscle differentiation genes correlates with improved survival. These data highlight the potential for mTOR inhibitors to foster the expression of muscle specific genes, pushing the myoblast-like tumor cells to a more differentiated state. Altering their biology in this way may improve outcome. On-going efforts are exploring the mechanisms acting downstream of Mtor to impede muscle differentiation and directly evaluating pro-differentiation effects of mTOR inhibition in RMS models. This abstract is also presented as Poster A1. Citation Format: Raphael A. Wilson, Jing Liu, Lin Xu, Yanbin Zheng, Stephen X. Skapek. Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR02.

Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP

Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1KD cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1KD cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1KD cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1KD cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell–cell fusion. Toca-1KD cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASPKO MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1KD cells restored myotube formation of Toca-1KD cells. Thus, our results suggest that Toca-1KD cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.

Angiopoietin 1 Enhances the Proliferation and Differentiation of Skeletal Myoblasts

Angiopoietin 1 (Ang1) plays an important role in various endothelial functions, such as vascular integrity and angiogenesis; however, less is known about its function outside of the endothelium. In this study, we examined whether Ang1 has direct effects on skeletal muscle cells. We found that Ang1 exhibited myogenic potential, as it promoted the proliferation, migration, and differentiation of mouse primary skeletal myoblasts. The positive effect of Ang1 on myoblast proliferation could have been mediated by the a7 and b1 integrins. We also found that Ang1 potentiated cellular Ca2+ movements in differentiated myotubes in response to stimuli, possibly through the increased expression of two Ca2+-related proteins, namely, Orai1 and calmodulin. Ang1 also increased Orai1 and calmodulin expression in mouse hearts in vivo. These results provide an insight into the molecular mechanisms by which Ang1 directly affects the myogenesis of striated muscle.

Surface modification of polypyrrole/biopolymer composites for controlled protein and cellular adhesion

The ability to control the interaction between proteins and cells with biomaterials is critical for the effective application of materials for a variety of biomedical applications. Herein, the surface modification of the biological dopant dextran sulphate-doped polypyrrole (PPy-DS) with poly(ethylene glycol) to generate a biomaterial interface that is highly resistant to protein and cellular adhesion is described. Thiolated poly(ethylene glycol) (PEG-thiol) was covalently bound to PPy-DS backbone via a thiol-ene reaction. The surface resistance to an extracellular matrix protein fibronectin increased with increasing molecular weight and concentration of PEG-thiol, and was further optimised via increasing the reaction temperature and the pH of the reactant aqueous solution. Optimised surface modification conditions substantially reduced interfacial protein adsorption, with the complete inhibition of adhesion and colonisation by primary mouse myoblasts. PEG-thiol-modified inherently conducting polymers are highly protein resistant multifunctional materials that are promising compounds for a range of biomedical and aquatic applications.

Flt3L is a novel regulator of skeletal myogenesis

Various cues initiate multiple signaling pathways to regulate the highly coordinated process of skeletal myogenesis. Myoblast differentiation comprises a series of ordered events starting with cell cycle withdrawal and ending with myocyte fusion, with each step probably controlled by multiple extracellular signals and intracellular signaling pathways. Here we report the identification of Fms-like tyrokine kinase 3 ligand (Flt3L) signaling as a novel regulator of skeletal myogenesis. Flt3L is a multifunctional cytokine in immune cells, but its involvement in skeletal muscle formation has not been reported. We found that Flt3L is expressed in C2C12 myoblasts, with levels increasing throughout differentiation. Knockdown of Flt3L, or its receptor Flt3, suppresses myoblast differentiation, which is rescued by recombinant Flt3L or Flt3, respectively. Differentiation is not rescued, however, by recombinant ligand when the receptor is knocked down, or vice versa, suggesting that Flt3L and Flt3 function together. Flt3L knockdown also inhibits differentiation in mouse primary myoblasts. Both Flt3L and Flt3 are highly expressed in nascent myofibers during muscle regeneration in vivo, and Flt3L siRNA impairs muscle regeneration, validating the physiological significance of Flt3L function in myogenesis. We have identified a cellular mechanism for the myogenic function of Flt3L, as we show that Flt3L promotes cell cycle exit that is necessary for myogenic differentiation. Furthermore, we identify Erk as a relevant target of Flt3L signaling during myogenesis, and demonstrate that Flt3L suppresses Erk signaling through p120RasGAP. In summary, our work reveals an unexpected role for an immunoregulatory cytokine in skeletal myogenesis and a new myogenic pathway.

STIM1 negatively regulates Ca2+ release from the sarcoplasmic reticulum in skeletal myotubes

STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca²⁺ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca²⁺ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca²⁺-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca²⁺ releases from the SR in response to KCl [evoking ECC (excitation-contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca²⁺-independent manner. These results suggest that STIM1 negatively regulates Ca²⁺ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

Skeletal Muscle Atrophy in Bioengineered Skeletal Muscle: A New Model System

Skeletal muscle atrophy has been well characterized in various animal models, and while certain pathways that lead to disuse atrophy and its associated functional deficits have been well studied, available drugs to counteract these deficiencies are limited. An ex vivo tissue-engineered skeletal muscle offers a unique opportunity to study skeletal muscle physiology in a controlled in vitro setting. Primary mouse myoblasts isolated from adult muscle were tissue engineered into bioartificial muscles (BAMs) containing hundreds of aligned postmitotic muscle fibers expressing sarcomeric proteins. When electrically stimulated, BAMs generated measureable active forces within 2-3 days of formation. The maximum isometric tetanic force (Po) increased for ∼3 weeks to 2587±502 μN/BAM and was maintained at this level for greater than 80 days. When BAMs were reduced in length by 25% to 50%, muscle atrophy occurred in as little as 6 days. Length reduction resulted in significant decreases in Po (50.4%), mean myofiber cross-sectional area (21.7%), total protein synthesis rate (22.0%), and noncollagenous protein content (6.9%). No significant changes occurred in either the total metabolic activity or protein degradation rates. This study is the first in vitro demonstration that length reduction alone can induce skeletal muscle atrophy, and establishes a novel in vitro model for the study of skeletal muscle atrophy.

191st ENMC International Workshop: Recent advances in oculopharyngeal muscular dystrophy research: From bench to bedside 8-10 June 2012, Naarden, The Netherlands

This workshop hosted a group of 19 clinicians and basic scientist from 5 countries (France, USA, Canada, Denmark and The Netherlands). Recent research developments and possible collaboration in advancing therapies for oculopharyngeal muscular dystrophy (OPMD), a late onset, rare and progressive neurodegeneration disorder were discussed in this meeting. The disease is caused by alanine expansion mutations in the gene encoding for poly(A)-binding protein nuclear 1 (PABPN1), resulting in accumulation of mutant PABPN1 in the cell nucleus and the formation of insoluble aggregates. Therefore, OPMD is categorized together with neurodegenerative disorders that are caused by single amino acid repeats. While the genetic cause for those disorders is often recognized, the molecular basis for symptoms is still obscure. PABPN1 is ubiquitously expressed and it is not known why symptoms develop only after midlife and initially only in subsets of skeletal muscles [1,2]. While the genetic cause for OPMD was reported in 1998 and good surgical treatments exist for major early symptoms, there are currently no medical options for the more general skeletal muscle involvement, no validated biomarkers and no clinical severity scale.

Satellite cells regulate the skeletal muscle environment by inhibiting fibroblast function

Interaction between satellite cells and the skeletal muscle extracellular matrix (ECM) has been demonstrated, but a role for satellite cells in regulating the ECM during hypertrophy has not been defined. We utilized mouse primary myoblasts and fibroblasts in co‐culture in addition to a genetically modified mouse model (Pax7‐DTA) to investigate the role of satellite cells in the regulation of muscle ECM. The Pax7‐DTA mouse allows for the conditional depletion of satellite cells in adult muscle following tamoxifen administration. Vehicle/tamoxifen treated animals were randomized to sham or synergist ablation surgery, leading to overload of the plantaris muscle for 1 and 8 weeks. Microarray analysis after 1 week of surgery showed the loss of satellite cells was associated with increased expression of numerous collagens and ECM structural genes. Depletion of satellite cells also led to attenuated hypertrophy and fibrosis of the plantaris with increases in both ECM deposition and fibroblast number following 8 weeks of overload. Co‐culturing mouse myoblasts with fibroblasts led to decreased mRNA expression of Col Ia2, IIIa1, VIa2 and Fibronectin compared to fibroblasts cocultured with fibroblasts, suggesting a paracrine role of myoblasts that inhibits fibroblast ECM production. We conclude that a novel function of satellite cells both at rest and during hypertrophy is to regulate muscle fibroblasts and the ECM.Funding: NIAMS R01AR060701, R21AG34453 and the Jeane B. Kempner Postdoctoral Award.

Multiple Phytoestrogens Inhibit Cell Growth and Confer Cytoprotection By Inducing Manganese Superoxide Dismutase Expression

Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. As data on phytoestrogens continues to accumulate, it is clear that there is significant overlap in the cellular effects elicited by these various compounds. Here, we show that one mechanism by which a number of phytoestrogens achieve their growth inhibitory and cytoprotective effects is via induction of the mitochondrial manganese superoxide dismutase (MnSOD). Eight phytoestrogens, including resveratrol, coumestrol, kaempferol, genistein, daidzein, apigenin, isoliquirtigenin and glycitin, were tested for their ability to induce MnSOD expression in mouse C2C12 and primary myoblasts. Five of these, resveratrol, coumestrol, kaempferol, genistein and daidzein, significantly increased MnSOD expression, slowed proliferative growth and enhanced stress resistance (hydrogen peroxide LD50) . When siRNA was used to prevent the MnSOD induction by genistein, coumestrol or daidzein, none of these compounds exerted any effect on proliferative growth, and only the effect of coumestrol on stress resistance persisted. The estrogen antagonist ICI182780 prevented the increased MnSOD expression and also the changes in cell growth and stress resistance, indicating that these effects are mediated by estrogen receptors (ER). The absence of effects of resveratrol or coumestrol, but not genistein, in ERβ-null cells further indicated that this ER in particular is important in mediating these effects. Thus, an ER-mediated induction of MnSOD expression appears to underlie the growth inhibitory and cytoprotective activities of multiple phytoestrogens.

Angiopoietin 1 enhances the proliferation and differentiation of skeletal myoblasts

Angiopoietin 1 (Ang1) plays an important role in various endothelial functions, such as vascular integrity and angiogenesis; however, less is known about its function outside of the endothelium. In this study, we examined whether Ang1 has direct effects on skeletal muscle cells. We found that Ang1 exhibited myogenic potential, as it promoted the proliferation, migration, and differentiation of mouse primary skeletal myoblasts. The positive effect of Ang1 on myoblast proliferation could have been mediated by the α7 and β1 integrins. We also found that Ang1 potentiated cellular Ca(2+) movements in differentiated myotubes in response to stimuli, possibly through the increased expression of two Ca(2+) -related proteins, namely, Orai1 and calmodulin. Ang1 also increased Orai1 and calmodulin expression in mouse hearts in vivo. These results provide an insight into the molecular mechanisms by which Ang1 directly affects the myogenesis of striated muscle.

Flt3L is a novel regulator of skeletal myogenesis

Various cues initiate multiple signaling pathways to regulate the highly coordinated process of skeletal myogenesis. Myoblast differentiation comprises a series of ordered events starting with cell cycle withdrawal and ending with myocyte fusion, with each step probably controlled by multiple extracellular signals and intracellular signaling pathways. Here we report the identification of Fms-like tyrokine kinase 3 ligand (Flt3L) signaling as a novel regulator of skeletal myogenesis. Flt3L is a multifunctional cytokine in immune cells, but its involvement in skeletal muscle formation has not been reported. We found that Flt3L is expressed in C2C12 myoblasts, with levels increasing throughout differentiation. Knockdown of Flt3L, or its receptor Flt3, suppresses myoblast differentiation, which is rescued by recombinant Flt3L or Flt3, respectively. Differentiation is not rescued, however, by recombinant ligand when the receptor is knocked down, or vice versa, suggesting that Flt3L and Flt3 function together. Flt3L knockdown also inhibits differentiation in mouse primary myoblasts. Both Flt3L and Flt3 are highly expressed in nascent myofibers during muscle regeneration in vivo, and Flt3L siRNA impairs muscle regeneration, validating the physiological significance of Flt3L function in myogenesis. We have identified a cellular mechanism for the myogenic function of Flt3L, as we show that Flt3L promotes cell cycle exit that is necessary for myogenic differentiation. Furthermore, we identify Erk as a relevant target of Flt3L signaling during myogenesis, and demonstrate that Flt3L suppresses Erk signaling through p120RasGAP. In summary, our work reveals an unexpected role for an immunoregulatory cytokine in skeletal myogenesis and a new myogenic pathway.

Ex-Vivo Gene Therapy Approach for Continuous Delivery of BDNF and Its Potential Use for Neurological Disorders (P02.013)

Objective: Evaluating persistent delivery of BDNF by gene therapy. Background Brain-derived neurotrophic factor (BDNF) potently prevents neuronal death and stimulate neuronal function. BDNF gene delivery to the entorhinal cortex ameliorated neuronal death, improved hippocampus-dependent memory AD. Similarly it supports the survival of nigral dopaminergic neurons in Parkinson9s disease. BDNF is a neuroprotective agent when administered within hours of ischaemia, enhancing axonal sprouting and synaptic function. Unfortunately its use is hampered by short-half life, limited biodistribution and limited bioavailability. Thus, the ideal system for BDNF administration must achieve effective concentrations at sites of degenerating neurons and delivery must be sustained. Design/Methods: G8 primary mouse myoblasts were transfected with a plasmid containing the cDNA for human bdnf. In order to prevent allograft rejections, genetically engineered myoblasts expressing human BDNF cells were encapsulated within alginate-PLL microcapsules with a molecular cut-off size of 300KDa, thus permeable to bdnf (27KDa). C57BL/6 mice (n=3) were implanted intraperitoneally, each mouse with approximately 9 x 106 recombinant BDNF-secreting myoblasts. Levels of BDNF expression in vivo and in vitro were measured by ELISA. Clinical evaluation and histological analysis were performed to evaluate side effects. Results: Transgene expression of BDNF by engineered myoblasts was verified by ELISA. Engineered myoblasts produced human BDNF at a rate of 430pg/106 cells/day. Mice were bled on day 0, 3 and 7. There were low but detectable levels of bdnf in plasma of treated mice suggesting its persistent bioavailability BDNF is known to cross the blood-brain barrier. Histopathological analysis disclosed no side effects. Conclusions: The remarkable potency of growth factors in preventing cell death and stimulating cell function in CNS disease provides incentive for this research. Encapsulated recombinant myoblast expressing BDNF overcome problems of dosing, administration and sustained bioavailability that might have a substantial impact in neurological diseases. This therapeutic approach will be evaluated in degenerative conditions like AD. Disclosure: Dr. Gomez-Vargas has nothing to disclose. Dr. Hortelano has nothing to disclose. Dr. Rathbone has received research support from Sanofi-Aventis.

Skeletal muscle fibroblast collagen expression is negatively regulated by satellite cells

Interaction between satellite cells and components of the extracellular matrix (ECM) of skeletal muscle has been demonstrated, and recent evidence suggests that satellite cells may regulate ECM remodeling following overload in muscle. We utilized mouse primary myoblasts and fibroblasts in co‐culture in addition to a genetically modified mouse model (Pax7‐DTA) to investigate the role of satellite cells in regulation of skeletal muscle ECM. The Pax7‐DTA mouse allows for the conditional ablation of >90% of satellite cells in adult mice following the administration of tamoxifen. Control/tamoxifen treated animals were randomized to sham or synergist ablation surgery, leading to overload of the plantaris muscle for 2 weeks. Microarray analysis was performed to determine if the loss of satellite cells altered plantaris gene expression. The loss of satellite cells under rest and overload conditions was associated with increased expression of a number of different collagens, as well as ECM structural and remodeling genes. Co‐culturing mouse myoblasts with fibroblasts led to a 40% decrease in the mRNA expression of Col VIa2 and Fibronectin compared to fibroblasts co‐cultured with fibroblasts. These results suggest that satellite cells are involved in the regulation and maintenance of the ECM both under rest and during muscle hypertrophy.Funding: NIAMS R01AR060701 and the Jeane B. Kempner Postdoctoral Fellowship.

Insulin Receptor Substrate protein 53 kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell–extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.

The Cavβ1 Subunit Regulates Gene Expression in Muscle Progenitor Cells

CaVβ subunits are traditionally considered constituents of CaV complexes (CaV1or2, CaVβ, and CaV α2/δ), where they localize at the plasma membrane and serve to regulate channel expression and gating properties. Recent publications also show CaVβ subunit localization in the nucleus. This phenomenon has been observed under a variety of conditions (different cell types, β subunit isoforms, co-expressed proteins, etc). However, the mechanisms responsible for CaVβ subunit nuclear shuttling, as well as a physiological role for this nuclear localization, remain major questions. Ongoing work in our laboratory has shown that muscle progenitor cells (myoblasts) express CaVβ1 protein (but not CaV1 subunits) in both the cytoplasm and nucleus and that the loss of CaVβ1 expression impairs proliferation in these cells. To better understand the mechanisms that link CaVβ1 nuclear localization with control of proliferation, we have conducted large-scale screening experiments designed to identify which genes are directly regulated by CaVβ1, as well as its protein binding partners. To test if CaVβ1 may regulate gene expression, we conducted microarray experiments on RNA extracted from wild type, heterozygous, and CaVβ1 -null mouse primary myoblasts. A number of gene transcripts were found to be differentially regulated based on the relative amount of CaVβ1 expression. To identify specific CaVβ1 target genes, we performed chromatin immunoprecipitation -on-a-chip experiments to locate which promoter regions CaVβ1 bound to across the entire mouse genome. Nuclear binding partners of CaVβ1 were screened using affinity purification of CaVβ1a-YFP from myoblast nuclear fractions coupled with mass spectrometry. Finally, the importance of CaVβ1 in embryonic myogenesis was explored in CaVβ1 -null mice. Our results support the idea of CaVβ subunits acting as transcription factors and regulating gene expression independently from CaV's, and suggest these functions may be particularly important to progenitor cell growth.

PAX3-FOXO1 Induces Cannabinoid Receptor 1 to Enhance Cell Invasion and Metastasis

Alveolar rhabdomyosarcoma (ARMS) is a muscle-derived childhood tumor characterized by production of oncogenic PAX3/7-FOXO1 chimeric transcription factors. While downstream targets of the PAX3-FOXO1 oncoprotein in ARMS have been defined, the functional relevance of these targets is unclear. Here, we show that upregulation of the cannabinoid receptor 1 (Cnr1/Cb1) by PAX3-FOXO1 in mouse primary myoblasts and ARMS cell lines, contributes to PAX3-FOXO1 phenotypes, both in vivo and in vitro. In primary myoblasts, Cnr1 was dispensable for PAX3-FOXO1 to mediate cell proliferation, differentiation, or transformation; however, Cnr1 function was essential to increase the invasive capacity conferred by PAX3-FOXO1 overexpression in these cells. Genetic or pharmacologic abrogation of Cnr1 inhibited the enhanced basement membrane invasion induced by PAX3-FOXO1. Cnr1 loss by either route also dramatically reduced lung metastasis formation. Taken together, our findings strongly implicate Cnr1 as a novel tractable target to inhibit ARMS invasion and metastasis.

Regulation of myotube formation by the actin-binding factor drebrin

BackgroundMyogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.MethodsRNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells.ResultsIn a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated.ConclusionsOur findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.