Inhibition Of Myogenic Differentiation Research Articles

SnRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders and has no cure. Due to an unknown molecular mechanism, FSHD displays overlapping manifestations with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FSHD is caused by aberrant gain of expression of the transcription factor double homeobox 4 (DUX4), which triggers a pro-apoptotic transcriptional program resulting in inhibition of myogenic differentiation and muscle wasting. Regulation of DUX4 activity is poorly known. We identify Matrin 3 (MATR3), whose mutation causes ALS and dominant distal myopathy, as a cellular factor controlling DUX4 expression and activity. MATR3 binds to the DUX4 DNA-binding domain and blocks DUX4-mediated gene expression, rescuing cell viability and myogenic differentiation of FSHD muscle cells, without affecting healthy muscle cells. Finally, we characterize a shorter MATR3 fragment that is necessary and sufficient to directly block DUX4-induced toxicity to the same extent as the full-length protein. Collectively, our data suggest MATR3 as a candidate for developing a treatment for FSHD.

Tetrandrine Inhibits Skeletal Muscle Differentiation by Blocking Autophagic Flux.

Tetrandrine is well known to act as a calcium channel blocker. It is a potential candidate for a tumor chemotherapy drug without toxicity. Tetrandrine inhibits cancer cell proliferation and induces cell death through apoptosis and autophagy. As cancer patients usually experience complications with sarcopenia or muscle injury, we thus assessed the effects of tetrandrine on skeletal muscle cells. We report in this study that a low dose of tetrandrine (less than 5 μM) does not affect the proliferation of C2C12 myoblasts, but significantly inhibits myogenic differentiation. Consistently, tetrandrine inhibited muscle regeneration after BaCl2-induced injury. Mechanistic experiments showed that tetrandrine decreased the p-mTOR level and increased the levels of LC3 and SQSTM1/p62 during differentiation. Ad-mRFP-GFP-LC3B transfection experiments revealed that the lysosomal quenching of GFP signals was suppressed by tetrandrine. Furthermore, the levels of DNM1L/Drp1, PPARGA1 and cytochrome C (Cyto C), as well as caspase 3 activation and ROS production, were decreased following tetrandrine administration, indicating that the mitochondrial network signaling was inhibited. Our results indicate that tetrandrine has dual effects on autophagic flux in myoblasts during differentiation, activation in the early stage and blockade in the late stage. The ultimate blocking of autophagic flux by tetrandrine led to the disruption of mitochondria remodeling and inhibition of myogenic differentiation. The inhibitory effects of tetrandrine on skeletal muscle differentiation may limit its application in advanced cancer patients. Thus, great attention should be paid to the clinical use of tetrandrine for cancer therapy.

Mitochondrial calcium uptake regulates tumour progression in embryonal rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is characterised by a failure of cells to complete skeletal muscle differentiation. Although ERMS cells are vulnerable to oxidative stress, the relevance of mitochondrial calcium homoeostasis in oncogenesis is unclear. Here, we show that ERMS cell lines as well as primary tumours exhibit elevated expression of the mitochondrial calcium uniporter (MCU). MCU knockdown resulted in impaired mitochondrial calcium uptake and a reduction in mitochondrial reactive oxygen species (mROS) levels. Phenotypically, MCU knockdown cells exhibited reduced cellular proliferation and motility, with an increased propensity to differentiate in vitro and in vivo. RNA-sequencing of MCU knockdown cells revealed a significant reduction in genes involved in TGFβ signalling that play prominent roles in oncogenesis and inhibition of myogenic differentiation. Interestingly, modulation of mROS production impacted TGFβ signalling. Our study elucidates mechanisms by which mitochondrial calcium dysregulation promotes tumour progression and suggests that targeting the MCU complex to restore mitochondrial calcium homoeostasis could be a therapeutic avenue in ERMS.

Trans-cinnamaldehyde protects C2C12 myoblasts from DNA damage, mitochondrial dysfunction and apoptosis caused by oxidative stress through inhibiting ROS production.

Oxidative stress-induced myoblast damage is one of the major causes of skeletal muscle loss associated with inhibition of myogenic differentiation and muscle dysfunction. Trans-cinnamaldehyde (tCA), the most common essential oil constituent in cinnamon, is known to possess strong anti-oxidant activity. However, it has not been determined whether tCA can protect myoblasts from oxidative damage. The aim of this study was to investigate the protective effect of tCA against oxidative stress-induced damage in mouse myoblast C2C12 cells. To examine the efficacy of tCA to protect against oxidative damage, cell viability, morphological changes, DNA damage, mitochondrial membrane potential (MMP) analysis, reactive oxygen species (ROS) generation, and Western blotting were applied. tCA suppressed hydrogen peroxide (H2O2)-induced growth inhibition and DNA damage by blocking abnormal ROS accumulation. In addition, tCA attenuated apoptosis by suppressing loss of MMP and cytosolic release of cytochrome c, increasing the rate of Bcl-2/Bax expression and reducing the activity of caspase-3 in H2O2-stimulated cells, suggesting that tCA protected C2C12 cells from mitochondria-mediated apoptosis caused by oxidative stress. The results showed that tCA may be useful as a potential treatment for the prevention and treatment of various oxidative stress-related muscle disorders in the future.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on acetylcholinesterase during myogenic differentiation of contractile rat primary skeletal muscle cells

Emerging data indicate that prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could interfere with myogenic differentiation in vivo. Acetylcholinesterase (EC3.1.1.7; AChE), an enzyme critical for cholinergic neurotransmission, is abundantly expressed in neurons and mature myotubes, and we recently found that muscle AChE expression was suppressed in parallel with the inhibition of myogenic differentiation upon TCDD treatment in mouse C2C12 cells. This TCDD-induced suppression of muscle AChE was proposed to involve an aryl hydrocarbon receptor (AhR)-independent mechanism, but the precise underlying mechanism remains unclear. Considering the widely recognized role of muscular activity in AChE expression and its potential crosstalk with the AhR signaling pathway, we sought to investigate the effect of TCDD on muscle AChE expression in the presence of muscular activity. Therefore, we employed a highly contractile rat primary skeletal muscle culture system in which AChE activity and the expression of genes related to it (AChE T subunit and collagen Q (ColQ)) were increased during the myogenic differentiation process. Although TCDD treatment successfully induced the expression of genes regulated by AhR activation, the treatment exerted no notable effects on myogenic differentiation. Moreover, muscle AChE enzymatic activity and mRNA level remained unchanged following TCDD treatment, and only ColQ mRNA expression was slightly increased after 4-day treatment with TCDD (10−10 M). The compensatory role of muscle-contraction-related signaling pathways in this newly identified unresponsiveness of muscle AChE to TCDD warrants further investigation.

CCAAT/Enhancer Binding Protein \u03b2 inhibits myogenic differentiation via ID3

Myogenesis is regulated by the coordinated expression of muscle regulatory factors, a family of transcription factors that includes MYOD, MYF5, myogenin and MRF4. Muscle regulatory factors are basic helix-loop-helix transcription factors that heterodimerize with E proteins to bind the regulatory regions of target genes. Their activity can be inhibited by members of the Inhibitor of DNA binding and differentiation (ID) family, which bind E-proteins with high affinity, thereby preventing muscle regulatory factor-dependent transcriptional responses. CCAAT/Enhancer Binding protein beta (C/EBPβ) is a transcription factor expressed in myogenic precursor cells that acts to inhibit myogenic differentiation, though the mechanism remains poorly understood. We identify Id3 as a novel C/EBPβ target gene that inhibits myogenic differentiation. Overexpression of C/EBPβ stimulates Id3 mRNA and protein expression, and is required for C/EBPβ-mediated inhibition of myogenic differentiation. Misexpression of C/EBPβ in myogenic precursors, such as in models of cancer cachexia, prevents the differentiation of myogenic precursors and we show that loss of Id3 rescues differentiation under these conditions, suggesting that the stimulation of Id3 expression by C/EBPβ is an important mechanism by which C/EBPβ inhibits myogenic differentiation.

Effect of sodium 4-phenylbutyrate on Clenbuterol-mediated muscle growth.

Previously, we highlighted induction of an integrated stress response (ISR) gene program in skeletal muscle of pigs treated with a beta-adrenergic agonist. Hence we tested the hypothesis that the ER-stress inhibitor, sodium 4-phenylbutyrate (PBA), would inhibit Clenbuterol-mediated muscle growth and reduce expression of genes that are known indicators of an ISR in mice. Clenbuterol (1mg/kg/day) administered to C57BL6/J mice for 21 days increased body weight (p<0.001), muscle weights (p<0.01), and muscle fibre diameters (p<0.05). Co-administration of PBA (100mg/kg/day) did not alter the Clenbuterol-mediated phenotype, nor did PBA alone have any effects compared to that of the vehicle treated mice. Clenbuterol increased skeletal muscle mRNA expression of phosphoserine amino transferase 1 (PSAT1, p<0.001) and cyclophillin A (p<0.01) at day 3, but not day 7. Clenbuterol decreased mRNA expression of activating transcription factor (ATF) 4 and ATF5 at day 3 (p<0.05) and day 7 (p<0.01), X-box binding protein 1 (XBP1) variant 2 mRNA at day 3 only (p<0.01) and DNA damage inducible transcript 3 (DDIT3/CHOP) mRNA at day 7 only (p<0.05). Co-administration of PBA had no effect on Clenbuterol-induced changes in skeletal muscle gene expression. In contrast, treatment of C2C12 myotubes with 5mM PBA (8hr) attenuated the thapsigargin-induced ISR gene program. Prolonged (24-48hr) treatment with PBA caused atrophy (p<0.01), reduced neoprotein synthesis (p<0.0001) and decreased expression of myogenin and fast myosin heavy chain genes (p<0.01), indicating an inhibition of myogenic differentiation. In summary, Clenbuterol did not induce an ISR gene program in mouse muscle. On the contrary, it reduced expression of a number of ISR genes, but it increased expression of PSAT1 mRNA. Co-administration of PBA had no effect on Clenbuterol-mediated muscle growth or gene expression in mice, whereas PBA did inhibit thapsigargin-induced ISR gene expression in cultured C2C12 cells and appeared to inhibit myogenic differentiation, independent of altering ISR gene expression.

Smad4 restricts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration.

Skeletal muscle regenerative potential declines with age, in part due to deficiencies in resident stem cells (satellite cells, SCs) and derived myogenic progenitors (MPs); however, the factors responsible for this decline remain obscure. TGFβ superfamily signaling is an inhibitor of myogenic differentiation, with elevated activity in aged skeletal muscle. Surprisingly, we find reduced expression of Smad4, the downstream cofactor for canonical TGFβ superfamily signaling, and the target Id1 in aged SCs and MPs during regeneration. Specific deletion of Smad4 in adult mouse SCs led to increased propensity for terminal myogenic commitment connected to impaired proliferative potential. Furthermore, SC-specific Smad4 disruption compromised adult skeletal muscle regeneration. Finally, loss of Smad4 in aged SCs did not promote aged skeletal muscle regeneration. Therefore, SC-specific reduction of Smad4 is a feature of aged regenerating skeletal muscle and Smad4 is a critical regulator of SC and MP amplification during skeletal muscle regeneration.

Nickel affects gill and muscle development in oriental fire-bellied toad (Bombina orientalis) embryos

The developmental toxicity of nickel was examined in the embryos of Bombina orientalis, a common amphibian in Korea. Based on a standard frog embryo teratogenesis assay, the LC50 and EC50 for malformation of nickel after 168h of treatment were 33.8μM and 5.4μM, respectively. At a lethal concentration (100μM), nickel treatment decreased the space between gill filaments and caused epithelial swelling and abnormal fusion of gill filaments. These findings suggest that nickel affects the functional development of gills, leading to embryonic death. At sublethal concentrations (1–10μM), nickel produced multiple embryonic abnormalities, including bent tail and tail dysplasia. At 10μM, nickel significantly decreased tail length and tail muscle fiber density in tadpoles, indicating inhibition of myogenic differentiation. Before hatching, the pre-muscular response to muscular response stages (stages 26–31) were the most sensitive period to nickel with respect to tail muscle development. During these stages, MyoD mRNA was upregulated, whereas myogenic regulatory factor 4 mRNA was downregulated by 0.1μM nickel. Calcium-dependent kinase activities in muscular response stage embryos were significantly decreased by nickel, whereas these activities were restored by exogenous calcium. In tadpoles, 10μM nickel significantly decreased the expression of the myosin heavy chain and the 12/101 muscle marker protein in the tail. Expression was restored by exogenous calcium. Our results indicate that nickel affects muscle development by disrupting calcium-dependent myogenesis in developing B. orientalis embryos.

Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation.

Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics.

Changes in Serum Levels of Myokines and Wnt-Antagonists after an Ultramarathon Race.

BackgroundRegular physical activities have a positive effect on the muscular skeletal system but overstrenuous exercise may be different. Transiently suppressed bone formation and increased bone resorption after participation in a 246-km ultradistance race has been demonstrated.PurposeThe aim of this study was to analyze effects of the Spartathlon race on novel musculoskeletal markers.MethodsVenous blood samples were obtained before and immediately after the race from 19 participants of the Spartathlon. From 9 runners who were available 3 days after the start blood was drawn for a third time. Serum levels of myostatin, an inhibitor of myogenic differentiation, and its opponent follistatin as well as sclerostin and dickkopf-1, both of them inhibitors of the wnt signaling pathway, and markers of bone turnover were determined.ResultsSerum levels of myostatin were significantly higher after the race. Serum follistatin only showed a transient increase. Sclerostin levels did not significantly differ before and after the race, whereas dickkopf-1 levels were significantly decreased. At follow-up a decrement of sclerostin and dickkopf-1 levels was seen. Serum cathepsin K levels did not change.ConclusionThe increase of serum levels of myostatin appears to reflect muscle catabolic processes induced by overstrenuous exercise. After the short-term uncoupling of bone turnover participation in an ultradistance race seems to initiate a long-term positive effect on bone indicated by the low-level inhibition of the Wnt/β-catenin signaling pathway.

MTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation.

Transcriptional Dominance of Pax7 in Adult Myogenesis Is Due to High-Affinity Recognition of Homeodomain Motifs

Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrate distinct attributes of Pax7 function and provide mechanistic insight into the nonredundancy of Pax3 and Pax7 in muscle development.

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.

Extracellular signal‐regulated kinase 1/2 mitogen‐activated protein kinase pathway is involved in inhibition of myogenic differentiation of myoblasts by hypoxia

Oxygen conditions influence a variety of biological processes of cells, including alterations in cellular growth, survival and differentiation. However, there have been few studies on the effects of hypoxia on proliferation and differentiation of myoblasts. In this study, we observed the effects of hypoxia (3% O(2)) on myogenic differentiation of C2C12 myoblasts and sought a possible mechanism involved in the regulation of myogenic differentiation of myoblasts by hypoxia. The expression of myosin heavy chain was detected by immunocytochemistry staining and Western blot analysis. The expression of muscle regulatory transcription factors was examined by RT-PCR analysis and Western blot analysis. The activity of extracellular signal-regulated kinases (ERK1/2) was investigated by forced expression of mitogen-activated protein kinase kinase 1 (MEK1(E)) and using Western blot analysis. We found that hypoxia inhibited myogenic differentiation of myoblasts. The expression of muscle regulatory transcription factors was downregulated by hypoxia in C2C12 myoblasts. During the inhibition of myogenic differentiation by hypoxia, ERK1/2 activation was suppressed, and increasing ERK1/2 activity by forced expression of MEK1(E) could partly reverse the inhibition of myogenic differentiation by hypoxia. These results suggest that the ERK1/2-mitogen-activated protein kinase pathway might be a possible mechanism involved in the inhibition of myogenic differentiation by hypoxia.

S100B protein in myoblasts modulates myogenic differentiation via NF‐κB‐dependent inhibition of MyoD expression

S100B, a Ca(2+)-binding protein of the EF-hand type, is expressed in myoblasts, the precursors of skeletal myofibers, and muscle satellite cells (this work). S100B has been shown to participate in the regulation of several intracellular processes including cell cycle progression and differentiation. We investigated regulatory activities of S100B within myoblasts by stable overexpression of S100B and by inhibition of S100B expression. Overexpression of S100B in myoblast cell lines and primary myoblasts resulted in inhibition of myogenic differentiation, evidenced by lack of expression of myogenin and myosin heavy chain (MyHC) and absence of myotube formation. S100B-overexpressing myoblasts showed reduced MyoD expression levels and unchanged Myf5 expression levels, compared with control myoblasts, and transient transfection of S100B-overexpressing myoblasts with MyoD, but not Myf5, restored differentiation and fusion in part. The transcriptional activity of NF-kappaB, a negative regulator of MyoD expression, was enhanced in S100B-overexpressing myoblasts, and blocking NF-kappaB activity resulted in reversal of S100B's inhibitory effects. Yin Yang1, a transcriptional repressor that is induced by NF-kappaB (p65) and mediates NF-kappaB inhibitory effects on several myofibrillary genes, also was upregulated in S100B-overexpressing myoblasts. Conversely, silencing S100B expression in myoblast cell lines by RNA interference resulted in reduced NF-kappaB activity and enhanced MyoD, myogenin and MyHC expression and myotube formation. Thus, intracellular S100B might modulate myoblast differentiation by interfering with MyoD expression in an NF-kappaB-dependent manner.

EM.P.1.08 Low temperature-induced inhibition of myogenic differentiation is cancelled by IGF-I and vitamin C

EM.P.1.08 Low temperature-induced inhibition of myogenic differentiation is cancelled by IGF-I and vitamin C

Necdin is expressed in cachectic skeletal muscle to protect fibers from tumor-induced wasting

Skeletal muscles of subjects with advanced cancer undergo progressive wasting, referred to as cachexia. Cachexia is an important area for medical research because strategies proposed until now have yielded little benefit. We have recently identified necdin as a key player in fetal and postnatal physiological myogenesis and in muscle regeneration. Here we show that necdin is selectively expressed in muscles of cachetic mice and prove that its expression is causally linked to a protective response of the tissue against tumor-induced wasting, inhibition of myogenic differentiation and fiber regeneration. Necdin carries out this role mainly via interference with TNFalpha signaling at various levels, including regulation of expression of TNFR1 and p53, and regulation of the activity of caspase 3 and caspase 9. These data suggest that inhibition of muscle wasting using necdin is a feasible approach to treat cachexia in neoplastic patients.

The omega-3 fatty acid, eicosapentaenoic acid (EPA), prevents the damaging effects of tumour necrosis factor (TNF)-alpha during murine skeletal muscle cell differentiation

BackgroundEicosapentaenoic acid (EPA) is a ώ-3 polyunsaturated fatty acid with anti-inflammatory and anti-cachetic properties that may have potential benefits with regards to skeletal muscle atrophy conditions where inflammation is present. It is also reported that pathologic levels of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α are associated with muscle wasting, exerted through inhibition of myogenic differentiation and enhanced apoptosis. These findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by the actions of TNF-α.ResultsThe deleterious effects of TNF-α on C2C12 myogenesis were completely inhibited by co-treatment with EPA. Thus, EPA prevented the TNF-mediated loss of MyHC expression and significantly increased myogenic fusion (p < 0.05) and myotube diameter (p < 0.05) indices back to control levels. EPA protective activity was associated with blocking cell death pathways as EPA completely attenuated TNF-mediated increases in caspase-8 activity (p < 0.05) and cellular necrosis (p < 0.05) back to their respective control levels. EPA alone significantly reduced spontaneous apoptosis and necrosis of differentiating myotubes (p < 0.001 and p < 0.05, respectively). A 2 hour pre-treatment with EPA, prior to treatment with TNF alone, gave similar results.ConclusionIn conclusion, EPA has a protective action against the damaging effects of TNF-α on C2C12 myogenesis. These findings support further investigations of EPA as a potential therapeutic agent during skeletal muscle regeneration following injury.