Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

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Nano-graphene oxide/polyurethane nanofibers: mechanically flexible and myogenic stimulating matrix for skeletal tissue engineering

Journal of Tissue Engineering ◽

10.1177/2041731419900424 ◽

2020 ◽

Vol 11 ◽

pp. 204173141990042 ◽

Cited By ~ 7

Author(s):

Seung Bin Jo ◽

Uyanga Erdenebileg ◽

Khandmaa Dashnyam ◽

Guang-Zhen Jin ◽

Jae-Ryung Cha ◽

...

Keyword(s):

Skeletal Muscle ◽

Graphene Oxide ◽

Myogenic Differentiation ◽

C2c12 Cells ◽

Dynamic Force ◽

Mrna Levels ◽

Differentiation Markers ◽

Nanofibrous Scaffolds ◽

Initial Adhesion ◽

Nano Graphene

For skeletal muscle engineering, scaffolds that can stimulate myogenic differentiation of cells while possessing suitable mechanical properties (e.g. flexibility) are required. In particular, the elastic property of scaffolds is of importance which helps to resist and support the dynamic conditions of muscle tissue environment. Here, we developed highly flexible nanocomposite nanofibrous scaffolds made of polycarbonate diol and isosorbide-based polyurethane and hydrophilic nano-graphene oxide added at concentrations up to 8%. The nano-graphene oxide incorporation increased the hydrophilicity, elasticity, and stress relaxation capacity of the polyurethane-derived nanofibrous scaffolds. When cultured with C2C12 cells, the polyurethane–nano-graphene oxide nanofibers enhanced the initial adhesion and spreading of cells and further the proliferation. Furthermore, the polyurethane–nano-graphene oxide scaffolds significantly up-regulated the myogenic mRNA levels and myosin heavy chain expression. Of note, the cells on the flexible polyurethane–nano-graphene oxide nanofibrous scaffolds could be mechanically stretched to experience dynamic tensional force. Under the dynamic force condition, the cells expressed significantly higher myogenic differentiation markers at both gene and protein levels and exhibited more aligned myotubular formation. The currently developed polyurethane–nano-graphene oxide nanofibrous scaffolds, due to their nanofibrous morphology and high mechanical flexibility, along with the stimulating capacity for myogenic differentiation, are considered to be a potential matrix for future skeletal muscle engineering.

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Human Resistin Inhibits Myogenic Differentiation and Induces Insulin Resistance in Myocytes

BioMed Research International ◽

10.1155/2013/804632 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Chun Hua Sheng ◽

Zhen Wu Du ◽

Yang Song ◽

Xiao Dong Wu ◽

Yu Cheng Zhang ◽

...

Keyword(s):

Insulin Resistance ◽

Cell Proliferation ◽

Insulin Sensitivity ◽

Glucose Uptake ◽

Myogenic Differentiation ◽

C2c12 Cells ◽

C2c12 Myotubes ◽

Mrna Levels ◽

Uptake Ratio ◽

Human Resistin

This study is aimed to investigate the effect of human resistin on myocyte differentiation and insulin resistance. The human resistin eukaryotic expression vector was stable transfected into C2C12 myocyte cells and was transiently transfected into COS7 cells. The effects of human resistin on cell proliferation, cell cycle, and myogenic differentiation of C2C12 cells were examined. Glucose uptake assays was performed on C2C12 myotubes by using [3H] 2-deoxy-D-glucose. The mRNA levels of insulin receptor (IR) and glucose transporter 4 (GLUT4) were evaluated by semiquantitative RT-PCR. Results showed by the C2C12 cells transfected with human resistin gene compared with that without transfecting gene are as follows: (1) cell proliferation was significantly promoted, (2) after inducing differentiation, the myotube’s diameters and expression of desmin and myoglobin decreased, and (3) glucose uptake ratio was lowered and expression of IR and GLUT4 decreased. However, there was no significant difference in the glucose uptake ratio between C2C12 myotubes treated with a human resistin conditioned medium of COS7 cells and treated with control medium. These results suggest that maybe human resistin has not a direct role on insulin sensitivity of myocytes. However, maybe it impaired the insulin sensitivity of myocytes through suppressing myogenesis and stimulating proliferation of myoblasts.

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A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells

Cells ◽

10.3390/cells8111340 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1340 ◽

Cited By ~ 7

Author(s):

Kurgan ◽

Whitley ◽

Maddalena ◽

Moradi ◽

Stoikos ◽

...

Keyword(s):

Bipolar Disorder ◽

Therapeutic Dose ◽

Specific Activity ◽

Glycogen Synthase ◽

Myogenic Differentiation ◽

Myoblast Fusion ◽

C2c12 Cells ◽

Glycogen Synthase Kinase 3 ◽

C2c12 Myotubes ◽

Control Myotubes

: Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/β-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3β) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5–1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3β (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2–2.5 fold, p < 0.05), a result associated with an increase in total β-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (−86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.

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TWIST1 and TWIST2 regulate glycogen storage and inflammatory genes in skeletal muscle

Journal of Endocrinology ◽

10.1530/joe-14-0474 ◽

2015 ◽

Vol 224 (3) ◽

pp. 303-313 ◽

Cited By ~ 7

Author(s):

Jonathan M Mudry ◽

Julie Massart ◽

Ferenc L M Szekeres ◽

Anna Krook

Keyword(s):

Skeletal Muscle ◽

Metabolic Diseases ◽

Glycogen Synthesis ◽

C2c12 Cells ◽

Acid Oxidation ◽

Diabetic Patients ◽

Mrna Levels ◽

Intact Mouse ◽

Mouse Muscle ◽

The Impact

TWIST proteins are important for development of embryonic skeletal muscle and play a role in the metabolism of tumor and white adipose tissue. The impact of TWIST on metabolism in skeletal muscle is incompletely studied. Our aim was to assess the impact of TWIST1 and TWIST2 overexpression on glucose and lipid metabolism. In intact mouse muscle, overexpression of Twist reduced total glycogen content without altering glucose uptake. Expression of TWIST1 or TWIST2 reducedPdk4mRNA, while increasing mRNA levels ofIl6,Tnfα, andIl1β. Phosphorylation of AKT was increased and protein abundance of acetyl CoA carboxylase (ACC) was decreased in skeletal muscle overexpressing TWIST1 or TWIST2. Glycogen synthesis and fatty acid oxidation remained stable in C2C12 cells overexpressing TWIST1 or TWIST2. Finally, skeletal muscle mRNA levels remain unaltered inob/obmice, type 2 diabetic patients, or in healthy subjects before and after 3 months of exercise training. Collectively, our results indicate that TWIST1 and TWIST2 are expressed in skeletal muscle. Overexpression of these proteins impacts proteins in metabolic pathways and mRNA level of cytokines. However, skeletal muscle levels of TWIST transcripts are unaltered in metabolic diseases.

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Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle cells: role of the STAT3 pathway

AJP Cell Physiology ◽

10.1152/ajpcell.00025.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. C128-C136 ◽

Cited By ~ 55

Author(s):

Miriam Hoene ◽

Heike Runge ◽

Hans Ulrich Häring ◽

Erwin D. Schleicher ◽

Cora Weigert

Keyword(s):

Skeletal Muscle ◽

Mrna Expression ◽

Myogenic Differentiation ◽

Muscle Cells ◽

C2c12 Cells ◽

Skeletal Muscle Cells ◽

Wild Type ◽

C2c12 Myoblasts ◽

Sequence Of Events ◽

Primary Mouse

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6−/− skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6−/− myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6−/− cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6−/− cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

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Tenogenic Contribution to Skeletal Muscle Regeneration: The Secretome of Scleraxis Overexpressing Mesenchymal Stem Cells Enhances Myogenic Differentiation In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21061965 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1965

Author(s):

Maximilian Strenzke ◽

Paolo Alberton ◽

Attila Aszodi ◽

Denitsa Docheva ◽

Elisabeth Haas ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Skeletal Muscles ◽

Myogenic Differentiation ◽

Muscular Contraction ◽

Myoblast Fusion ◽

Skeletal Muscle Regeneration ◽

Potential Candidate ◽

Musculoskeletal Tissue

Integrity of the musculoskeletal system is essential for the transfer of muscular contraction force to the associated bones. Tendons and skeletal muscles intertwine, but on a cellular level, the myotendinous junctions (MTJs) display a sharp transition zone with a highly specific molecular adaption. The function of MTJs could go beyond a mere structural role and might include homeostasis of this musculoskeletal tissue compound, thus also being involved in skeletal muscle regeneration. Repair processes recapitulate several developmental mechanisms, and as myotendinous interaction does occur already during development, MTJs could likewise contribute to muscle regeneration. Recent studies identified tendon-related, scleraxis-expressing cells that reside in close proximity to the MTJs and the muscle belly. As the muscle-specific function of these scleraxis positive cells is unknown, we compared the influence of two immortalized mesenchymal stem cell (MSC) lines—differing only by the overexpression of scleraxis—on myoblasts morphology, metabolism, migration, fusion, and alignment. Our results revealed a significant increase in myoblast fusion and metabolic activity when exposed to the secretome derived from scleraxis-overexpressing MSCs. However, we found no significant changes in myoblast migration and myofiber alignment. Further analysis of differentially expressed genes between native MSCs and scleraxis-overexpressing MSCs by RNA sequencing unraveled potential candidate genes, i.e., extracellular matrix (ECM) proteins, transmembrane receptors, or proteases that might enhance myoblast fusion. Our results suggest that musculotendinous interaction is essential for the development and healing of skeletal muscles.

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Zfp422 promotes skeletal muscle differentiation by regulating EphA7 to induce appropriate myoblast apoptosis

Cell Death and Differentiation ◽

10.1038/s41418-019-0448-9 ◽

2019 ◽

Vol 27 (5) ◽

pp. 1644-1659 ◽

Cited By ~ 1

Author(s):

Yaping Nie ◽

Shufang Cai ◽

Renqiang Yuan ◽

Suying Ding ◽

Xumeng Zhang ◽

...

Keyword(s):

Skeletal Muscle ◽

Zinc Finger ◽

Muscle Development ◽

Zinc Finger Protein ◽

Critical Role ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Skeletal Muscle Differentiation ◽

Finger Protein

Abstract Zinc finger protein 422 (Zfp422) is a widely expressed zinc finger protein that serves as a transcriptional factor to regulate downstream gene expression, but until now, little is known about its roles in myogenesis. We found here that Zfp422 plays a critical role in skeletal muscle development and regeneration. It highly expresses in mouse skeletal muscle during embryonic development. Specific knockout of Zfp422 in skeletal muscle impaired embryonic muscle formation. Satellite cell-specific Zfp422 deletion severely inhibited muscle regeneration. Myoblast differentiation and myotube formation were suppressed in Zfp422-deleted C2C12 cells, isolated primary myoblasts, and satellite cells. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) revealed that Zfp422 regulated ephrin type-A receptor 7 (EphA7) expression by binding an upstream 169-bp DNA sequence, which was proved to be an enhancer of EphA7. Knocking EphA7 down in C2C12 cells or deleting Zfp422 in myoblasts will inhibit cell apoptosis which is required for myoblast differentiation. These results indicate that Zfp422 is essential for skeletal muscle differentiation and fusion, through regulating EphA7 expression to maintain proper apoptosis.

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Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

Journal of Applied Physiology ◽

10.1152/jappl.1993.75.6.2471 ◽

1993 ◽

Vol 75 (6) ◽

pp. 2471-2477 ◽

Cited By ~ 38

Author(s):

F. Haddad ◽

R. E. Herrick ◽

G. R. Adams ◽

K. M. Baldwin

Keyword(s):

Skeletal Muscle ◽

Muscle Protein ◽

Vastus Lateralis ◽

Mrna Level ◽

Relative Content ◽

Type I ◽

Zero Gravity ◽

Mrna Levels ◽

Heavy Chains ◽

Vastus Intermedius

This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

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NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00195.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. C715-C725 ◽

Cited By ~ 26

Author(s):

Juan Antonio Valdés ◽

Eduardo Gaggero ◽

Jorge Hidalgo ◽

Nancy Leal ◽

Enrique Jaimovich ◽

...

Keyword(s):

Skeletal Muscle ◽

Transcription Factors ◽

Electrical Stimulation ◽

Stimulus Frequency ◽

Muscle Cells ◽

C2c12 Cells ◽

Skeletal Muscle Cells ◽

Mrna Levels ◽

Activated T Cells ◽

Nfat Activation

Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c- fos, c- jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-κB activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-κB activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-κB and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels.

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The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc–dependent mechanism

Molecular Biology of the Cell ◽

10.1091/mbc.e14-04-0895 ◽

2014 ◽

Vol 25 (23) ◽

pp. 3765-3778 ◽

Cited By ~ 17

Author(s):

Aymeric Ravel-Chapuis ◽

Tara E. Crawford ◽

Marie-Laure Blais-Crépeau ◽

Guy Bélanger ◽

Chase T. Richer ◽

...

Keyword(s):

Skeletal Muscle ◽

Similar Pattern ◽

Rna Binding ◽

Myogenic Differentiation ◽

Rna Binding Protein ◽

C2c12 Cells ◽

Muscle Degeneration ◽

Low Levels ◽

Dependent Mechanism

Recent work has shown that Staufen1 plays key roles in skeletal muscle, yet little is known about its pattern of expression during embryonic and postnatal development. Here we first show that Staufen1 levels are abundant in mouse embryonic muscles and that its expression decreases thereafter, reaching low levels in mature muscles. A similar pattern of expression is seen as cultured myoblasts differentiate into myotubes. Muscle degeneration/regeneration experiments revealed that Staufen1 increases after cardiotoxin injection before returning to the low levels seen in mature muscles. We next prevented the decrease in Staufen1 during differentiation by generating stable C2C12 muscle cell lines overexpressing Staufen1. Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay. However, levels of c-myc, a factor known to inhibit differentiation, were increased in C2C12 cells overexpressing Staufen1 through enhanced translation. By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts. Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.

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