Control Of Muscle Differentiation Research Articles

Mesenchymal stem cell transplant as an intervention to ameliorate disuse-induced muscle atrophy in a mouse model of simulated microgravity

BackgroundThe hindlimb unloaded (HU) mouse model exhibits disuse-induced muscle atrophy. However, effective interventions remain elusive. We investigated the therapeutic potential of mesenchymal stem cells (MSC) transplant on muscle decline in HU mice. MethodsWe divided 4-month-old male c57BL/6j mice into controls and HU mice treated with PBS as placebo (HU-PBS) or MSCs (HU-MSC; one million cells/100 μl PBS into gastrocnemius muscles once a week) for three weeks. We measured muscle mass, grip strength, and an unbiased transcriptome analysis of gastrocnemius muscles. ResultsMSC treatment prevented muscle atrophy and improved grip strength in HU mice. Transcriptome analysis revealed MSC-induced unique (557 genes) and differential (1214 genes) expressions of several genes compared to the HU-PBS group. GO and KEGG term analysis revealed an HU-induced downregulation of pathways associated with the regulation of contractile apparatus, neuromuscular junction, and satellite cell function, which were partly reversed with MSC treatment. Lastly, MSC treatment also upregulated the pathways controlling muscle differentiation and growth in the HU mice. ConclusionAltogether, we report the therapeutic potential of MSCs in treating disuse-induced muscle atrophy and weakness. Our study may help unravel novel molecular mechanisms involved in MSCs-induced muscle restoration.

Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.

LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo, such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics. Graphic abstract: Graphical representation of the single myofiber isolation method. Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.

MyoD-induced circular RNA CDR1as promotes myogenic differentiation of skeletal muscle satellite cells

Many protein coding and non-coding genes interplay in governing skeletal muscle formation. Nevertheless, comparing with the linear transcripts, functions of covalently closed circular RNAs (circRNAs), the new frontier of regulatory non-coding RNA (ncRNAs) molecules, remain largely unknown. Here, we identify CDR1as (antisense to the cerebellar degeneration-related protein 1 transcript, also termed as ciRS-7), a well-known cancer and neuron circRNA, plays a significant role in virtually controlling muscle differentiation. CDR1as is highly expressed in muscles of the mid-embryonic goat foetus, and activated at the initiation of myogenic differentiation in vitro. MyoD (myogenic differentiation protein 1), a driven transcription factor for myogenesis, promotes CDR1as by binding on its 5′ flank region (−646 to −634 bp, neighbouring the predicted transcription start site at −580 bp). Overexpression or knockdown of CDR1as dramatically induces or impedes muscle differentiation program, respectively. By competitively binding to miR-7 (microRNA 7), CDR1as relieves the downregulation of IGF1R (insulin like growth factor 1 receptor) caused by miR-7 and consequently activates muscle differentiation. These results unveil that CDR1as plays critical roles in myogenic differentiation, which extends the versatile functions of CDR1as in mammal development and disease.

Study of the Chronology of Expression of Ten Extracellular Matrix Molecules during the Myogenesis in Cattle to Better Understand Sensory Properties of Meat.

The sensory properties of beef are known to depend on muscle fiber and intramuscular connective tissue composition (IMCT). IMCT is composed of collagens, proteoglycans and glycoproteins. The differentiation of muscle fibers has been extensively studied but there is scarcity in the data concerning IMCT differentiation. In order to be able to control muscle differentiation to improve beef quality, it is essential to understand the ontogenesis of IMCT molecules. Therefore, in this study, we investigated the chronology of appearance of 10 IMCT molecules in bovine Semitendinosus muscle using immunohistology technique at five key stages of myogenesis. Since 60 days post-conception (dpc), the whole molecules were present, but did not have their final location. It seems that they reach it at around 210 dpc. Then, the findings emphasized that since 210 dpc, the stage at which the differentiation of muscle fibers is almost complete, the differentiation of IMCT is almost completed. These data suggested that for the best controlling of the muscular differentiation to improve beef sensory quality, it would be necessary to intervene very early (before the IMCT constituents have acquired their definitive localization and the muscle fibers have finished differentiating), i.e., at the beginning of the first third of gestation.

Identification of MyoD-Responsive Transcripts Reveals a Novel Long Non-coding RNA (lncRNA-AK143003) that Negatively Regulates Myoblast Differentiation

Myogenic differentiation factor (MyoD) is a master transcription factor in muscle development and differentiation. Although several long non-coding RNAs (lncRNAs) linked to MyoD have been found to influence muscle development, the functions of many lncRNAs have not been explored. Here we utilized lncRNA and mRNA microarray analysis to identify potential lncRNAs regulated by MyoD in muscle cells. A total of 997 differentially expressed lncRNAs (335 up-regulated and 662 down-regulated) and 1,817 differentially expressed mRNAs (148 up-regulated and 1,669 down-regulated) were identified after MyoD knockdown in C2C12 cells. Functional predictions suggested that most lncRNAs are involved in the biological pathways related to muscle differentiation and cell cycle with co-expressed genes. To gain further insight into the MyoD-mediated lncRNA expression in muscle differentiation, tissue expression profiles and MyoD overexpression were performed, and we found one of the candidate lncRNAs-AK143003 was significantly regulated by MyoD. Further analyses showed its noncoding ability and cytoplasmic localisation. Silencing of AK143003 stimulated the accumulation of myogenic marker genes, whereas AK143003 overexpression led to their decreased synthesis. This study identified a multitude of MyoD-mediated lncRNAs for further investigation and identified a novel lncRNA, lnc-AK143003, which plays a role in controlling muscle differentiation.

Abstract 150: Identification of SYNPO2, SYNM, LMOD1, PDLIM7 and PLN as Novel Markers of Smooth Muscle Cells in Atherosclerosis

Smooth muscle cell (SMC) activation is a hallmark of vascular remodeling associated with downregulation of SMC specific genes and altered cell function. Our aim was to identify a novel set of genes silenced following SMC activation in vascular disease. We compared microarrays from carotid plaques (n=177) vs. undiseased arteries and symptomatic vs. asymptomatic patients and found that ‘muscle contraction’, ‘muscle differentiation’ and ‘cytoskeleton based migration’ were the most significantly downregulated pathways (p=6.06e-23; 5.22e-11; 4.62e-11 respectively). Genes clustered in these categories were known SMC markers together with a novel set of genes functionally coupled to actin cytoskeleton: SYNPO2, SYNM, LMOD1, PDLIM7 and PLN. Transcription factors regulating the expression of these genes showed enrichment of SRF, MYOD1 and MYOGENIN, known to control muscle differentiation. Downregulation of these genes was validated in independent microarrays from carotid (n=21) and coronary plaques (n=38). Their expression was positively correlated to typical SMC markers both in human plaques (Pearson r>0.8, p<0.0001) and rat carotid intimal hyperplasia (r>0.9, p<0.0001). In microarrays from rat carotid balloon injury, these genes were downregulated in the early phases of tissue remodeling but gradually reappeared in the mature intima. By immunohistochemistry, the proteins localized to SMCs in normal human vessels, but were mostly absent in plaques and restenosis tissues. In vitro, SYNPO2 and PDLIM7 localized to actin filaments, SYNM to cortical cytoskeleton, PLN to nucleus while LMOD1 was rapidly lost in cultured human and primary rat SMCs. Polymorphisms in some of these genes associated with carotid intima-media thickness in high-risk coronary artery disease subjects (n=3400). By eQTL analyses, rs11746443 influenced PDLIM7 expression in plaques. Interestingly, this polymorphism constitutes the binding site for HEY1 transcription factor implicated in vascular development. We identified a panel of novel SMC proteins, which are lost in vascular disease and reflect the altered phenotype of SMCs in vascular remodeling. Our results indicate that these could be early and sensitive markers of the SMCs dedifferentiation in atherosclerosis.

The nucleoporin gp210/Nup210 controls muscle differentiation by regulating nuclear envelope/ER homeostasis.

Previously, we identified the nucleoporin gp210/Nup210 as a critical regulator of muscle and neuronal differentiation, but how this nucleoporin exerts its function and whether it modulates nuclear pore complex (NPC) activity remain unknown. Here, we show that gp210/Nup210 mediates muscle cell differentiation in vitro via its conserved N-terminal domain that extends into the perinuclear space. Removal of the C-terminal domain, which partially mislocalizes gp210/Nup210 away from NPCs, efficiently rescues the differentiation defect caused by the knockdown of endogenous gp210/Nup210. Unexpectedly, a gp210/Nup210 mutant lacking the NPC-targeting transmembrane and C-terminal domains is sufficient for C2C12 myoblast differentiation. We demonstrate that the endoplasmic reticulum (ER) stress-specific caspase cascade is exacerbated during Nup210 depletion and that blocking ER stress-mediated apoptosis rescues differentiation of Nup210-deficient cells. Our results suggest that the role of gp210/Nup210 in cell differentiation is mediated by its large luminal domain, which can act independently of NPC association and appears to play a pivotal role in the maintenance of nuclear envelope/ER homeostasis.

A Novel Isoform of Met Receptor Tyrosine Kinase Blocks Hepatocyte Growth Factor/Met Signaling and Stimulates Skeletal Muscle Cell Differentiation

Hepatocyte growth factor (HGF) and its receptor, Met, regulate skeletal muscle differentiation. In the present study, we identified a novel alternatively spliced isoform of Met lacking exon 13 (designated Δ13Met), which is expressed mainly in human skeletal muscle. Alternative splicing yielded a truncated Met having extracellular domain only, suggesting an inhibitory role. Indeed, Δ13Met expression led to a decrease in HGF-induced tyrosine phosphorylation of Met and ERK phosphorylation, as well as cell proliferation and migration via sequestration of HGF. Interestingly, in human primary myoblasts undergoing differentiation, Δ13Met mRNA and protein levels were rapidly increased, concomitantly with a decrease in wild type Met mRNA and protein. Inhibition of Δ13Met with siRNA led to a decreased differentiation, whereas its overexpression potentiated differentiation of human primary myoblasts. Furthermore, in notexin-induced mouse injury model, exogenous Δ13Met expression enhanced regeneration of skeletal muscle, further confirming a stimulatory role of the isoform in muscle cell differentiation. In summary, we identified a novel alternatively spliced inhibitory isoform of Met that stimulates muscle cell differentiation, which confers a new means to control muscle differentiation and/or regeneration.

TM4SF5 suppression disturbs integrin α5-related signalling and muscle development in zebrafish.

TM4SF5 (transmembrane 4L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5 mRNA was expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day post-fertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5-related signalling.

ZEB1 Imposes a Temporary Stage-Dependent Inhibition of Muscle Gene Expression and Differentiation via CtBP-Mediated Transcriptional Repression

Skeletal muscle development is orchestrated by the myogenic regulatory factor MyoD, whose activity is blocked in myoblasts by proteins preventing its nuclear translocation and/or binding to G/C-centered E-boxes in target genes. Recent evidence indicates that muscle gene expression is also regulated at the cis level by differential affinity for DNA between MyoD and other E-box binding proteins during myogenesis. MyoD binds to G/C-centered E-boxes, enriched in muscle differentiation genes, in myotubes but not in myoblasts. Here, we used cell-based and in vivo Drosophila, Xenopus laevis, and mouse models to show that ZEB1, a G/C-centered E-box binding transcriptional repressor, imposes a temporary stage-dependent inhibition of muscle gene expression and differentiation via CtBP-mediated transcriptional repression. We found that, contrary to MyoD, ZEB1 binds to G/C-centered E-boxes in muscle differentiation genes at the myoblast stage but not in myotubes. Its knockdown results in precocious expression of muscle differentiation genes and acceleration of myotube formation. Inhibition of muscle genes by ZEB1 occurs via transcriptional repression and involves recruitment of the CtBP corepressor. Lastly, we show that the pattern of gene expression associated with muscle differentiation is accelerated in ZEB1(-/-) mouse embryos. These results set ZEB1 as an important regulator of the temporal pattern of gene expression controlling muscle differentiation.

BAF60 A, B, and Cs of muscle determination and renewal

Developmental biologists have defined many of the diffusible and transcription factors that control muscle differentiation, yet we still have only rudimentary knowledge of the mechanisms that dictate whether a myogenic progenitor cell forms muscle versus alternate lineages, including those that can be pathological in a state of disease or degeneration. Clues about the molecular basis for lineage determination in muscle progenitors are only now emerging from studies of chromatin modifications that avail myogenic genes for transcription, together with analysis of the composition and activities of the chromatin-modifying complexes themselves. Here we review recent progress on muscle determination and explore a unifying theme that environmental cues from the stem or progenitor niche control the selection of specific subunit variants of the switch/sucrose nonfermentable (SWI/SNF) chromatin-modifying complex, creating a combinatorial code that dictates whether cells adopt myogenic versus nonmyogenic cell fates. A key component of the code appears to be the mutually exclusive usage of the a, b, and c variants of the 60-kD structural subunit BAF60 (BRG1/BRM-associated factor 60), of which BAF60c is essential to activate both skeletal and cardiac muscle programs. Since chromatin remodeling governs myogenic fate, the combinatorial assembly of the SWI/SNF complex might be targeted to develop drugs aimed at the therapeutic reduction of compensatory fibrosis and fatty deposition in chronic muscular disorders.

A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA

SummaryRecently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 “sponges” miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

Eric Olson

In his classic fable, The Gnat and the Bull , Aesop (620 BC–560 BC)—that astute observer of human nature—teaches us the sad reality that the smaller the mind, the greater the conceit. One of the things I like about Eric Olson is that he embodies the notion that a truly great mind is free of conceit. Although he is one of the most successful scientists of our time and despite his extraordinary accomplishments and recognitions, Eric has managed to keep a simple, warm, genuine, down-to-earth style. I find this to be overwhelmingly refreshing, and almost as admirable as his scientific prowess. I hope the readers will enjoy this foray into the human dimension of Eric’s rich and well-rounded personality—his life-long passion for science, his work ethics, his love of music, his views on science and faith, his concern for those who entrust their career to him, and his total lack of arrogance. Eric is one of the best role models for young investigators to emulate, not only from a scientific but also from a human standpoint. Although now he is a veritable icon in cardiovascular research, he tells us that it was not easy to get to that point. This should be a good lesson for others. Indeed, it is never easy in science, for anybody. I hope that those who are at the inception of their careers will be inspired by reading how Eric overcame initial challenges and developed a stunning upward trajectory using the old-fashioned (and irreplaceable) virtues of hard work and perseverance. — —Roberto Bolli Eric Olson, Professor of Molecular Biology at the University of Texas Southwestern, Dallas, Tex, knows how to build muscle. Indeed, he has been studying muscle development since he landed his first faculty position in the mid 1980s. Since that time, Olson and …

Control of Muscle Differentiation by a Mitochondria-Targeted Fluorophore

During muscle differentiation, mitochondria undergo dramatic changes in their morphology and distribution to prepare for the higher rate of energy consumption. By applying a mitochondria-targeted rosamine library in C2C12 myogenesis, we discovered one compound that controls muscle differentiation. When treated to undifferentiated myoblasts, our selected compound, B25, inhibited myotube formation, and when treated to fully differentiated myotubes, it induced fission of multinucleated myotubes into mononucleated fragments. Compared to myoseverin, which is known for inducing myotube fission by destabilizing microtubules, B25 affects neither microtubule stability nor cell cycle. Further investigation identified that B25 induces myotube fission through the activation of NF-kappaB, which is one of the important signaling pathways linked to skeletal muscle differentiation. So far, the use of small-molecule fluorophores is limited in the discovery of labeling agents or sensors. In addition to their potential as a sensor, here we show the application of fluorescent small molecules in the discovery of a bioactive probe that induces a specific cellular response.

The Him Gene Reveals a Balance of Inputs Controlling Muscle Differentiation in Drosophila

SummaryTissue development requires the controlled regulation of cell-differentiation programs. In muscle, the Mef2 transcription factor binds to and activates the expression of many genes and has a major positive role in the orchestration of differentiation [1–4]. However, little is known about how Mef2 activity is regulated in vivo during development. Here, we characterize a gene, Holes in muscle (Him), which our results indicate is part of this control in Drosophila. Him expression rapidly declines as embryonic muscle differentiates, and consistent with this, Him overexpression inhibits muscle differentiation. This inhibitory effect is suppressed by mef2, implicating Him in the mef2 pathway. We then found that Him downregulates the transcriptional activity of Mef2 in both cell culture and in vivo. Furthermore, Him protein binds Groucho, a conserved, transcriptional corepressor, through a WRPW motif and requires this motif and groucho function to inhibit both muscle differentiation and Mef2 activity during development. Together, our results identify a mechanism that can inhibit muscle differentiation in vivo. We conclude that a balance of positive and negative inputs, including Mef2, Him, and Groucho, controls muscle differentiation during Drosophila development and suggest that one outcome is to hold developing muscle cells in a state with differentiation genes poised to be expressed.

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

EURO-Laminopathies is a European Commissionfunded research project, including 13 scientific group leaders in the fields of human genetics, clinical research, structural biology, molecular cell biology, and pharmaceutical industry, and one administrative manager from eight countries (Austria, France, Germany, Italy Israel, Spain, Switzerland and the United Kingdom). The consortium held its kick-off meeting as an International ENMC Workshop in Naarden on the weekend of March 10–12, 2006. The EURO-Laminopathies project aims at understanding the molecular mechanisms of laminopathies, which are rare human diseases linked tomutations in genes encoding nuclear envelope proteins, such as A-type lamins (LMNA), proteins involved in the post-translational processing of A-type lamins (ZMPSTE24), and lamin-binding proteins (EMD, LBR, LAP2). Laminopathies are clinically manifested after birth, can affect different tissues andprogressively develop during childhood or adolescence, often leading to early death. Efficient therapies have been hampered by the lack

Acheron, a novel member of the Lupus Antigen family, is induced during the programmed cell death of skeletal muscles in the moth Manduca sexta

In order to identify novel genes associated with the initiation of programmed cell death during development, we employed a differential screening protocol to isolate cDNAs that were induced when the intersegmental muscles (ISM) of the moth Manduca sexta become committed to die at the end of metamorphosis. In this report we provide the first description of Acheron (Achn), a novel protein that was isolated in this screen. Acheron contains three Lupus antigen (La) repeats, nuclear localization and export (NLS and NES) signals, and an RNA recognition motif. Achn defines a new subfamily of La proteins that appears to have branched from authentic La protein relatively late in metazoan evolution. Achn is widely expressed in various insect, mouse and human tissues. Consistent with its expression during ISM death, Achn has been shown in separate studies to control muscle differentiation and apoptosis in both mice and zebrafish. These data define Achn as a newly discovered regulatory molecule that presumably mediates a variety of developmental and homeostatic processes in animals.

Identification of gene networks involved in retinoic acid control of muscle differentiation

Identification of gene networks involved in retinoic acid control of muscle differentiation

Foxd3 mediates zebrafish myf5 expression during early somitogenesis

Myf5, one of the basic helix–loop–helix transcription factors, controls muscle differentiation and is expressed in somites during early embryogenesis. However, the transcription factors bound to the cis-elements of myf5 are poorly understood. In this study, we used the yeast one-hybrid assay and found that Forkhead box d3 (Foxd3) interacted specifically with the −82/−62 cassette, a key element directing somite-specific expression of myf5. The dual-luciferase assay revealed that the expression of Foxd3 potently transactivated the myf5 promoter. Knocking down foxd3 with morpholino oligonucleotide (MO) resulted in a dramatic down-regulation of myf5 in somites and adaxial cells but not in the presomitic mesoderm. On the other hand, myod expression remained unchanged in foxd3 morphants. Foxd3 mediation of myf5 expression is stage-dependent, maintaining myf5 expression in the somites and adaxial cells during the 7- to 18-somite stage. Furthermore, in the pax3 morphant, the expression of foxd3 was down-regulated greatly and the expression of myf5 was similar to that of the foxd3 morphant. Co-injection of foxd3 mRNA and pax3-MO1 greatly restored the expression of myf5 in the somites and adaxial cells, suggesting that pax3 induces foxd3 expression, which then induces the expression of myf5. This report is the first study to show that Foxd3, a well-known regulator in neural crest development, is also involved in myf5 regulation.

Myostatin and NF-κB Regulate Skeletal Myogenesis Through Distinct Signaling Pathways

Myostatin (Mstn) is a potent negative regulator of skeletal development shown to inhibit myoblast proliferation by impinging on cell cycle and suppressing the synthesis of MyoD. Moreover, Mstn causes muscle wasting and its expression is linked with several conditions of muscle loss, mainly dystrophy and cachexia. NF-κB is a transcription factor that is constitutively active in proliferating myoblasts and also plays a role in cell growth control and skeletal muscle differentiation. NF-κB inhibits myogenesis by promoting myoblast growth and inducing loss of MyoD, and NF-κB activity is required in states of muscle wasting. However, the extracellular factors that regulate NF-κB activity to modulate myogenesis are currently not known. Given the similarities in Mstn and NF-κB activities in muscle cells, we investigated the possibility that Mstn-induced regulation of myogenesis may signal via NF-κB. Using a variety of assays to monitor for NF-κB activity, we found that Mstn signaling does not activate NF-κB in differentiating C2C12 myoblasts, nor is the constitutive activity of NF-κB required for Mstn-mediated inhibition of myogenesis. Likewise, in pre-differentiated myotubes, Mstn signaling induces only a modest activation of NF-κB DNA binding activity. We also investigated whether NF-κB inhibition of myogenesis may occur through the regulation of Mstn. However, activation of NF-κB by TNFα or IL-1β failed to induce Mstn expression. These results thus highlight the distinctive differences by which Mstn and NF-κB signal to regulate myogenesis, a finding which broadens our understanding of how these pathways function in both development and disease.