scholarly journals Myoblast Adhesion, Proliferation and Differentiation on Human Elastin-Like Polypeptide (HELP) Hydrogels

2017 ◽  
Vol 15 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Paola D'Andrea ◽  
Deborah Civita ◽  
Michela Cok ◽  
Luisa Ulloa Severino ◽  
Francesca Vita ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Matrix Protein ◽  
Myogenic Differentiation ◽  
Adhesion Formation ◽  
Genetically Engineered ◽  
C2c12 Cells ◽  
Type Iv ◽  
Proliferation And Differentiation

Background The biochemical, mechanical and topographic properties of extracellular matrix are crucially involved in determining skeletal muscle cell morphogenesis, proliferation and differentiation. Human elastin-like polypeptides (HELPs) are recombinant biomimetic proteins designed to mimic some properties of the native matrix protein; when employed as myoblast adhesion substrates, they stimulate in vitro myogenesis. Given the influence that the biophysical properties of extracellular matrix have on skeletal muscle cells, the aim of this work was to investigate the effects of HELP hydrogels on myoblasts’ viability and functions. Methods We recently synthesized a novel polypeptide, HELPc, by fusing the elastin-like backbone to a 41aa sequence present in the α2 chain of type IV collagen, containing two arginyl-glycyl-aspartic acid (RGD) motifs. To obtain hydrogels, the enzymatic cross-linking of the HELPc was accomplished by transglutaminase. Here, we employed both non-cross-linked HELPc glass coatings and cross-linked HELPc hydrogels at different monomer densities, as adhesion substrates for C2C12 cells, used as a myoblast model. Results By comparing cell adhesion, proliferation and differentiation, we revealed several striking differences. Depending on support rigidity, adhesion to HELPc substrates dictated cell morphology, spreading, focal adhesion formation and cytoskeletal organization. Hydrogels greatly stimulated cell proliferation, particularly in low-serum medium, and partially inhibited myogenic differentiation. Conclusions On the whole, the results underline the potential of these genetically engineered polypeptides as a tool for dissecting crucial steps in myogenesis.

scholarly journals Spexin Promotes the Proliferation and Differentiation of C2C12 Cells In Vitro—The Effect of Exercise on SPX and SPX Receptor Expression in Skeletal Muscle In Vivo

Genes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 81
Author(s):  
Natalia Leciejewska ◽  
Ewa Pruszyńska-Oszmałek ◽  
Karolina Mielnik ◽  
Maciej Głowacki ◽  
Tomasz P. Lehmann ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Receptor Expression ◽  
C2c12 Cells ◽  
Receptor Subtype ◽  
Galanin Receptor ◽  
Differentiation Markers ◽  
Proliferation And Differentiation ◽  
The Impact

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.

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

2019 ◽  
Vol 9 (1) ◽  
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.

scholarly journals KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Qi Zhu ◽  
Feng Liang ◽  
Shufang Cai ◽  
Xiaorong Luo ◽  
Tianqi Duo ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Proliferation ◽  
Satellite Cells ◽  
Muscle Development ◽  
Kinase Inhibitor ◽  
Myogenic Differentiation ◽  
Myoblast Differentiation ◽  
Proliferation And Differentiation ◽  
H3k9me3 Level

AbstractHistone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

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

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.

scholarly journals Inositide-Dependent Phospholipase C Signaling Mimics Insulin in Skeletal Muscle Differentiation by Affecting Specific Regions of the Cyclin D3 Promoter

Endocrinology ◽  
2007 ◽  
Vol 148 (3) ◽  
pp. 1108-1117 ◽  
Author(s):  
Irene Faenza ◽  
Giulia Ramazzotti ◽  
Alberto Bavelloni ◽  
Roberta Fiume ◽  
Gian Carlo Gaboardi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Phospholipase C ◽  
Myogenic Differentiation ◽  
Muscle Differentiation ◽  
C2c12 Cells ◽  
Cyclin D3 ◽  
Expression Vectors ◽  
Skeletal Muscle Differentiation ◽  
Small Interfering Rnas

Our main goal in this study was to investigate the role of phospholipase C (PLC) β1 and PLCγ1 in skeletal muscle differentiation and the existence of potential downstream targets of their signaling activity. To examine whether PLC signaling can modulate the expression of cyclin D3, a target of PLCβ1 in erythroleukemia cells, we transfected C2C12 cells with expression vectors containing PLCβ1 or PLCγ1 cDNA and with small interfering RNAs from regions of the PLCβ1 or PLCγ1 gene and followed myogenic differentiation in this well-established cell system. Intriguingly, overexpressed PLCβ1 and PLCγ1 were able to mimic insulin induction of both cyclin D3 and muscle differentiation. By knocking down PLCβ1 or PLCγ1 expression, C2C12 cells almost completely lost the increase in cyclin D3, and the differentiation program was down-regulated. To explore the induction of the cyclin D3 gene promoter during this process, we used a series of 5′-deletions of the 1.68-kb promoter linked to a reporter gene and noted a 5-fold augmentation of promoter activity upon insulin stimulation. These constructs were also cotransfected with PLCβ1 or PLCγ1 cDNAs and small interfering RNAs, respectively. Our data indicate that PLCβ1 or PLCγ1 signaling is capable of acting like insulin in regard to both the myogenic differentiation program and cyclin D3 up-regulation. Taken together, this is the first study that hints at cyclin D3 as a target of PLCβ1 and PLCγ1 during myogenic differentiation in vitro and implies that up-regulation of these enzymes is sufficient to mimic the actions of insulin in this process.

scholarly journals Extracellular Matrix-Derived Hydrogels as Biomaterial for Different Skeletal Muscle Tissue Replacements

Materials ◽  
2020 ◽  
Vol 13 (11) ◽  
pp. 2483 ◽  
Author(s):  
Daniele Boso ◽  
Edoardo Maghin ◽  
Eugenia Carraro ◽  
Mattia Giagante ◽  
Piero Pavan ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Muscle Tissue ◽  
Myogenic Differentiation ◽  
Skeletal Muscle Tissue ◽  
Tissue Microenvironment ◽  
Topographical Cues

Recently, skeletal muscle represents a complex and challenging tissue to be generated in vitro for tissue engineering purposes. Several attempts have been pursued to develop hydrogels with different formulations resembling in vitro the characteristics of skeletal muscle tissue in vivo. This review article describes how different types of cell-laden hydrogels recapitulate the multiple interactions occurring between extracellular matrix (ECM) and muscle cells. A special attention is focused on the biochemical cues that affect myocytes morphology, adhesion, proliferation, and phenotype maintenance, underlining the importance of topographical cues exerted on the hydrogels to guide cellular orientation and facilitate myogenic differentiation and maturation. Moreover, we highlight the crucial role of 3D printing and bioreactors as useful platforms to finely control spatial deposition of cells into ECM based hydrogels and provide the skeletal muscle native-like tissue microenvironment, respectively.

Formation of highly organized skeletal muscle fibers in vitro. Comparison with muscle development in vivo

1992 ◽  
Vol 102 (3) ◽  
pp. 643-652 ◽  
Author(s):  
S. Swasdison ◽  
R. Mayne
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Muscle Fiber ◽  
Muscle Development ◽  
Muscle Fibers ◽  
Myotendinous Junction ◽  
Type Iv

Two methods were developed in which long-term cultures of quail skeletal muscle were established so that all of the muscle fibers develop in a highly oriented manner. The muscle fibers became spontaneously and vigorously contractile and established strong connections with the extracellular matrix at their ends that closely duplicate the structure of the myotendinous junction. A continuous basal lamina was formed around each muscle fiber that contained type IV collagen, laminin and heparan sulfate proteoglycan. With one of the methods, an extensive extracellular matrix developed around each muscle fiber that was highly organized with the formation of a distinctive epimysium, perimysium and endomysium. Analysis of the cultures by both methods for different isoforms of myosin showed expression of an adult form of myosin by some of the muscle cells. The results therefore demonstrate that muscle development in the present culture systems proceeds extensively for several weeks. It will now be possible to investigate directly the structure of the connections between muscle fibers and the extracellular matrix.

scholarly journals Loss of splicing factor IK impairs normal skeletal muscle development

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hye In Ka ◽  
Hyemin Seo ◽  
Youngsook Choi ◽  
Joohee Kim ◽  
Mina Cho ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Development ◽  
Myogenic Differentiation ◽  
Mrna Splicing ◽  
C2c12 Cells ◽  
Splicing Factor ◽  
Skeletal Muscle Development

Abstract Background IK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo. Results The ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells. Conclusion This study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

scholarly journals The LIM domain protein nTRIP6 modulates the dynamics of myogenic differentiation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tannaz Norizadeh Abbariki ◽  
Zita Gonda ◽  
Denise Kemler ◽  
Pavel Urbanek ◽  
Tabea Wagner ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cells ◽  
Muscle Regeneration ◽  
Myogenic Differentiation ◽  
Skeletal Muscle Regeneration ◽  
Temporal Control ◽  
Lim Domain ◽  
Lim Domain Protein ◽  
Domain Protein

AbstractThe process of myogenesis which operates during skeletal muscle regeneration involves the activation of muscle stem cells, the so-called satellite cells. These then give rise to proliferating progenitors, the myoblasts which subsequently exit the cell cycle and differentiate into committed precursors, the myocytes. Ultimately, the fusion of myocytes leads to myofiber formation. Here we reveal a role for the transcriptional co-regulator nTRIP6, the nuclear isoform of the LIM-domain protein TRIP6, in the temporal control of myogenesis. In an in vitro model of myogenesis, the expression of nTRIP6 is transiently up-regulated at the transition between proliferation and differentiation, whereas that of the cytosolic isoform TRIP6 is not altered. Selectively blocking nTRIP6 function results in accelerated early differentiation followed by deregulated late differentiation and fusion. Thus, the transient increase in nTRIP6 expression appears to prevent premature differentiation. Accordingly, knocking out the Trip6 gene in satellite cells leads to deregulated skeletal muscle regeneration dynamics in the mouse. Thus, dynamic changes in nTRIP6 expression contributes to the temporal control of myogenesis.

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

2013 ◽  
Vol 304 (2) ◽  
pp. C128-C136 ◽  
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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