Multiscale-Engineered Muscle Constructs: PEG Hydrogel Micro-Patterning on an Electrospun PCL Mat Functionalized with Gold Nanoparticles

The development of new, viable, and functional engineered tissue is a complex and challenging task. Skeletal muscle constructs have specific requirements as cells are sensitive to the stiffness, geometry of the materials, and biological micro-environment. The aim of this study was thus to design and characterize a multi-scale scaffold and to evaluate it regarding the differentiation process of C2C12 skeletal myoblasts. The significance of the work lies in the microfabrication of lines of polyethylene glycol, on poly(-caprolactone) nanofiber sheets obtained using the electrospinning process, coated or not with gold nanoparticles to act as a potential substrate for electrical stimulation. The differentiation of C2C12 cells was studied over a period of seven days and quantified through both expression of specific genes, and analysis of the myotubes’ alignment and length using confocal microscopy. We demonstrated that our multiscale bio-construct presented tunable mechanical properties and supported the different stages skeletal muscle,as well as improving the parallel orientation of the myotubes with a variation of less than 15°. These scaffolds showed the ability of sustained myogenic differentiation by enhancing the organization of reconstructed skeletal muscle. Moreover, they may be suitable for applications in mechanical and electrical stimulation to mimic the muscle’s physiological functions.

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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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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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Alignment of Skeletal Muscle Cells Cultured in Collagen Gel by Mechanical and Electrical Stimulation

International Journal of Tissue Engineering ◽

10.1155/2014/621529 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 5

Author(s):

Takara Tanaka ◽

Noriko Hattori-Aramaki ◽

Ayano Sunohara ◽

Keisuke Okabe ◽

Yoshiaki Sakamoto ◽

...

Keyword(s):

Skeletal Muscle ◽

Tissue Engineering ◽

Electrical Stimulation ◽

Collagen Gel ◽

Muscle Cells ◽

Mechanical Force ◽

C2c12 Cells ◽

Skeletal Muscle Cells ◽

Collagen Gels ◽

Cells Cultured

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method.

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6-Bromoindirubin-3′-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice

Scientific Reports ◽

10.1038/s41598-019-54574-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Elvira Ragozzino ◽

Mariarita Brancaccio ◽

Antonella Di Costanzo ◽

Francesco Scalabrì ◽

Gennaro Andolfi ◽

...

Keyword(s):

Skeletal Muscle ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Skeletal Muscle Differentiation ◽

Myoblast Proliferation ◽

Enhanced Expression ◽

Pharmacologically Active

AbstractDystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Thus, new drugs for restoring skeletal muscle deterioration are critically needed. To identify new and alternative compounds with a functional role in skeletal muscle myogenesis, we screened a library of pharmacologically active compounds and selected the small molecule 6-bromoindirubin-3′-oxime (BIO) as an inhibitor of myoblast proliferation. Using C2C12 cells, we examined BIO’s effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from cell proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO is able to promote myogenic differentiation in damaged myotubes in-vitro by enriching the population of newly formed skeletal muscle myotubes. Moreover, in-vivo experiments in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers number. Additionally, we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was observed in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was observed in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was observed upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a positive modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.

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Sympathetic activity is correlated with satellite cell aging and myogenesis via β2-adrenoceptor

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02571-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shiguo Yuan ◽

Sheng Zheng ◽

Kai Zheng ◽

Yanping Gao ◽

Meixiong Chen ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Sympathetic Activity ◽

Muscle Injury ◽

Myogenic Differentiation ◽

Sympathetic Nerves ◽

C2c12 Cells ◽

Muscle Repair ◽

Aged Mice ◽

Young Mice

Abstract Background and objective Sympathetic activity plays an important role in the proliferation and differentiation of stem cells, and it changes over time, thereby exerting differential effects on various stem cell types. Aging causes sympathetic hyperactivity in aged tissues and blunts sympathetic nerves regulation, and sympathetic abnormalities play a role in aging-related diseases. Currently, the effect of sympathetic activity on skeletal muscle stem cells, namely satellite cells (SCs), is unclear. This study aimed to investigate the effects of skeletal muscle sympathetic activity on SC aging and skeletal muscle repair. Materials and methods To evaluate skeletal muscle and fibrotic areas, numbers of SCs and myonuclei per muscle fiber, β2-adrenoceptor (β2-ADR) expression, muscle repair, and sympathetic innervation in skeletal muscle, aged mice, young mice that underwent chemical sympathectomy (CS) were utilized. Mice with a tibialis anterior muscle injury were treated by barium chloride (BaCl2) and clenbuterol (CLB) in vivo. SCs or C2C12 cells were differentiated into myotubes and treated with or without CLB. Immunofluorescence, western blot, sirius red, and hematoxylin–eosin were used to evaluate SCs, myogenic repair and differentiation. Results The number of SCs, sympathetic activity, and reparability of muscle injury in aged mice were significantly decreased, compared with those in young mice. The above characteristics of young mice that underwent CS were similar to those of aged mice. While CLB promoted the repair of muscle injury in aged and CS mice and ameliorated the reduction in the SC number and sympathetic activity, the effects of CLB on the SCs and sympathetic nerves in young mice were not significant. CLB inhibited the myogenic differentiation of C2C12 cells in vitro. We further found that NF-κB and ERK1/2 signaling pathways were activated during myogenic differentiation, and this process could be inhibited by CLB. Conclusion Normal sympathetic activity promoted the stemness of SCs to thereby maintain a steady state. It also could maintain total and self-renewing number of SCs and maintain a quiescent state, which was correlated with skeletal SCs via β2-ADR. Normal sympathetic activity was also beneficial for the myogenic repair of injured skeletal muscle.

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TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake

Cell Death and Disease ◽

10.1038/s41419-021-04383-3 ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Barbara Toffoli ◽

Federica Tonon ◽

Veronica Tisato ◽

Giorgio Zauli ◽

Paola Secchiero ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucose Uptake ◽

Therapeutic Potential ◽

Myogenic Differentiation ◽

Body Weight Gain ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Acid Oxidation ◽

Skeletal Muscle Differentiation ◽

Akt Phosphorylation

AbstractTNF-related apoptosis-inducing ligand (TRAIL) is a protein that induces apoptosis in cancer cells but not in normal ones, where its effects remain to be fully understood. Previous studies have shown that in high-fat diet (HFD)-fed mice, TRAIL treatment reduced body weight gain, insulin resistance, and inflammation. TRAIL was also able to increase skeletal muscle free fatty acid oxidation. The aim of the present work was to evaluate TRAIL actions on skeletal muscle. Our in vitro data on C2C12 cells showed that TRAIL treatment significantly increased myogenin and MyHC and other hallmarks of myogenic differentiation, which were reduced by Dr5 (TRAIL receptor) silencing. In addition, TRAIL treatment significantly increased AKT phosphorylation, which was reduced by Dr5 silencing, as well as glucose uptake (alone and in combination with insulin). Our in vivo data showed that TRAIL increased myofiber size in HFD-fed mice as well as in db/db mice. This was associated with increased myogenin and PCG1α expression. In conclusion, TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake. These data shed light onto a pathway that might hold therapeutic potential not only for the metabolic disturbances but also for the muscle mass loss that are associated with diabetes.

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Coordinated Vascular Endothelial Growth Factor Expression and Signaling During Skeletal Myogenic Differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0856 ◽

2008 ◽

Vol 19 (3) ◽

pp. 994-1006 ◽

Cited By ~ 76

Author(s):

Brad A. Bryan ◽

Tony E. Walshe ◽

Dianne C. Mitchell ◽

Josh S. Havumaki ◽

Magali Saint-Geniez ◽

...

Keyword(s):

Skeletal Muscle ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Myogenic Differentiation ◽

Es Cells ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Vascular Endothelial ◽

Skeletal Muscle Differentiation ◽

Endothelial Growth Factor

Angiogenesis is largely controlled by hypoxia-driven transcriptional up-regulation and secretion of vascular endothelial growth factor (VEGF) and its binding to the endothelial cell tyrosine receptor kinases, VEGFR1 and VEGFR2. Recent expression analysis suggests that VEGF is expressed in a cell-specific manner in normoxic adult tissue; however, the transcriptional regulation and role of VEGF in these tissues remains fundamentally unknown. In this report we demonstrate that VEGF is coordinately up-regulated during terminal skeletal muscle differentiation. We reveal that this regulation is mediated in part by MyoD homo- and hetero-dimeric transcriptional mechanisms. Serial deletions of the VEGF promoter elucidated a region containing three tandem CANNTG consensus MyoD sites serving as essential sites of direct interaction for MyoD-mediated up-regulation of VEGF transcription. VEGF-null embryonic stem (ES) cells exhibited reduced myogenic differentiation compared with wild-type ES cells, suggesting that VEGF may serve a role in skeletal muscle differentiation. We demonstrate that VEGFR1 and VEGFR2 are expressed at low levels in myogenic precursor cells and are robustly activated upon VEGF stimulation and that their expression is coordinately regulated during skeletal muscle differentiation. VEGF stimulation of differentiating C2C12 cells promoted myotube hypertrophy and increased myogenic differentiation, whereas addition of sFlt1, a VEGF inhibitor, resulted in myotube hypotrophy and inhibited myogenic differentiation. We further provide evidence indicating VEGF-mediated myogenic marker expression, mitogenic activity, migration, and prosurvival functions may contribute to increased myogenesis. These data suggest a novel mechanism whereby VEGF is coordinately regulated as part of the myogenic differentiation program and serves an autocrine function regulating skeletal myogenesis.

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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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Multi-Staged Regulation of Lipid Signaling Mediators during Myogenesis by COX-1/2 Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms20184326 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4326

Author(s):

Chenglin Mo ◽

Zhiying Wang ◽

Lynda Bonewald ◽

Marco Brotto

Keyword(s):

Skeletal Muscle ◽

Myogenic Differentiation ◽

Gene Array ◽

Fine Tuning ◽

C2c12 Cells ◽

Lipid Mediator ◽

Expression Of Genes ◽

Primary Myoblasts ◽

Cox 1

Cyclooxygenases (COXs), including COX-1 and -2, are enzymes essential for lipid mediator (LMs) syntheses from arachidonic acid (AA), such as prostaglandins (PGs). Furthermore, COXs could interplay with other enzymes such as lipoxygenases (LOXs) and cytochrome P450s (CYPs) to regulate the signaling of LMs. In this study, to comprehensively analyze the function of COX-1 and -2 in regulating the signaling of bioactive LMs in skeletal muscle, mouse primary myoblasts and C2C12 cells were transfected with specific COX-1 and -2 siRNAs, followed by targeted lipidomic analysis and customized quantitative PCR gene array analysis. Knocking down COXs, particularly COX-1, significantly reduced the release of PGs from muscle cells, especially PGE2 and PGF2α, as well as oleoylethanolamide (OEA) and arachidonoylethanolamine (AEA). Moreover, COXs could interplay with LOXs to regulate the signaling of hydroxyeicosatetraenoic acids (HETEs). The changes in LMs are associated with the expression of genes, such as Itrp1 (calcium signaling) and Myh7 (myogenic differentiation), in skeletal muscle. In conclusion, both COX-1 and -2 contribute to LMs production during myogenesis in vitro, and COXs could interact with LOXs during this process. These interactions and the fine-tuning of the levels of these LMs are most likely important for skeletal muscle myogenesis, and potentially, muscle repair and regeneration.

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