Guide Cells Support Muscle Regeneration and Affect Neuro-Muscular Junction Organization

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them ‘guide’ cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.

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Pim1 kinase positively regulates myoblast behaviors and skeletal muscle regeneration

Cell Death and Disease ◽

10.1038/s41419-019-1993-3 ◽

2019 ◽

Vol 10 (10) ◽

Cited By ~ 1

Author(s):

Yuantong Liu ◽

Yue Shang ◽

Zihan Yan ◽

Hao Li ◽

Zhen Wang ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Muscle Injury ◽

Myogenic Differentiation ◽

Skeletal Muscle Regeneration ◽

Myoblast Differentiation ◽

Adult Skeletal Muscle ◽

Skeletal Muscle Injury ◽

Pim1 Kinase

Abstract Adult skeletal muscle regeneration after injury depends on normal myoblast function. However, the intrinsic mechanisms for the control of myoblast behaviors are not well defined. Herein, we identified Pim1 kinase as a novel positive regulator of myoblast behaviors in vitro and muscle regeneration in vivo. Specifically, knockdown of Pim1 significantly restrains the proliferation and accelerates the apoptosis of myoblasts in vitro, indicating that Pim1 is critical for myoblast survival and amplification. Meanwhile, we found that Pim1 kinase is increased and translocated from cytoplasm into nucleus during myogenic differentiation. By using Pim1 kinase inhibitor, we proved that inhibition of Pim1 activity prevents myoblast differentiation and fusion, suggesting the necessity of Pim1 kinase activity for proper myogenesis. Mechanistic studies demonstrated that Pim1 kinase interacts with myogenic regulator MyoD and controls its transcriptional activity, inducing the expression of muscle-specific genes, which consequently promotes myogenic differentiation. Additionally, in skeletal muscle injury mouse model, deletion of Pim1 hinders the regeneration of muscle fibers and the recovery of muscle strength. Taken together, our study provides a potential target for the manipulation of myoblast behaviors in vitro and the myoblast-based therapeutics of skeletal muscle injury.

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MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽

10.3390/biomedicines9060630 ◽

2021 ◽

Vol 9 (6) ◽

pp. 630

Author(s):

Huili Lyu ◽

Cody M. Elkins ◽

Jessica L. Pierce ◽

C. Henrique Serezani ◽

Daniel S. Perrien

Keyword(s):

Heterotopic Ossification ◽

Muscle Injury ◽

Cell Types ◽

Fibrodysplasia Ossificans Progressiva ◽

Inflammatory Signaling ◽

Conditional Deletion ◽

Pathway Crosstalk ◽

Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

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Surface-directed engineering of tissue anisotropy in microphysiological models of musculoskeletal tissue

Science Advances ◽

10.1126/sciadv.abe9446 ◽

2021 ◽

Vol 7 (11) ◽

pp. eabe9446 ◽

Cited By ~ 1

Author(s):

Mark J. Mondrinos ◽

Farid Alisafaei ◽

Alex Y. Yi ◽

Hossein Ahmadzadeh ◽

Insu Lee ◽

...

Keyword(s):

Muscle Injury ◽

Cancer Cachexia ◽

Myogenic Differentiation ◽

Mechanical Anisotropy ◽

Musculoskeletal Tissue ◽

Boundary Constraints ◽

Cell Contractility ◽

Engineered Tissue ◽

Tissue Anisotropy

Here, we present an approach to model and adapt the mechanical regulation of morphogenesis that uses contractile cells as sculptors of engineered tissue anisotropy in vitro. Our method uses heterobifunctional cross-linkers to create mechanical boundary constraints that guide surface-directed sculpting of cell-laden extracellular matrix hydrogel constructs. Using this approach, we engineered linearly aligned tissues with structural and mechanical anisotropy. A multiscale in silico model of the sculpting process was developed to reveal that cell contractility increases as a function of principal stress polarization in anisotropic tissues. We also show that the anisotropic biophysical microenvironment of linearly aligned tissues potentiates soluble factor-mediated tenogenic and myogenic differentiation of mesenchymal stem cells. The application of our method is demonstrated by (i) skeletal muscle arrays to screen therapeutic modulators of acute oxidative injury and (ii) a 3D microphysiological model of lung cancer cachexia to study inflammatory and oxidative muscle injury induced by tumor-derived signals.

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The LIM domain protein nTRIP6 modulates the dynamics of myogenic differentiation

Scientific Reports ◽

10.1038/s41598-021-92331-8 ◽

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.

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MiR-27b Promotes Muscle Development by Inhibiting MDFI Expression

Cellular Physiology and Biochemistry ◽

10.1159/000489595 ◽

2018 ◽

Vol 46 (6) ◽

pp. 2271-2283 ◽

Cited By ~ 12

Author(s):

Lianjie Hou ◽

Jian Xu ◽

Yiren Jiao ◽

Huaqin Li ◽

Zhicheng Pan ◽

...

Keyword(s):

Skeletal Muscle ◽

Western Blot ◽

Satellite Cells ◽

Muscle Regeneration ◽

Muscle Injury ◽

Target Gene ◽

Muscle Development ◽

Muscle Satellite Cells ◽

Qrt Pcr

Background/Aims: Skeletal muscle plays an essential role in the body movement. However, injuries to the skeletal muscle are common. Lifelong maintenance of skeletal muscle function largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation, differentiation, and myoblast fusion play an important role in muscle regeneration after injury. Therefore, understanding of the mechanisms associated with muscle development during muscle regeneration is essential for devising the alternative treatments for muscle injury in the future. Methods: Edu staining, qRT-PCR and western blot were used to evaluate the miR-27b effects on pig muscle satellite cells (PSCs) proliferation and differentiation in vitro. Then, we used bioinformatics analysis and dual-luciferase reporter assay to predict and confirm the miR-27b target gene. Finally, we elucidate the target gene function on muscle development in vitro and in vivo through Edu staining, qRT-PCR, western blot, H&E staining and morphological observation. Result: miR-27b inhibits PSCs proliferation and promotes PSCs differentiation. And the miR-27b target gene, MDFI, promotes PSCs proliferation and inhibits PSCs differentiation in vitro. Furthermore, interfering MDFI expression promotes mice muscle regeneration after injury. Conclusion: our results conclude that miR-27b promotes PSCs myogenesis by targeting MDFI. These results expand our understanding of muscle development mechanism in which miRNAs and genes work collaboratively in regulating skeletal muscle development. Furthermore, this finding has implications for obtaining the alternative treatments for patients with the muscle injury.

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Chlorella vulgaris Improves the Regenerative Capacity of Young and Senescent Myoblasts and Promotes Muscle Regeneration

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/3520789 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 5

Author(s):

Nurhazirah Zainul Azlan ◽

Yasmin Anum Mohd Yusof ◽

Ekram Alias ◽

Suzana Makpol

Keyword(s):

Chlorella Vulgaris ◽

Muscle Regeneration ◽

Myogenic Differentiation ◽

Myoblast Differentiation ◽

Regenerative Capacity ◽

Maturation Index ◽

Myoblast Cells ◽

And Function ◽

Pharmaceutical Uses

Sarcopenia is characterized by the loss of muscle mass, strength, and function with ageing. With increasing life expectancy, greater attention has been given to counteracting the effects of sarcopenia on the growing elderly population. Chlorella vulgaris, a microscopic, unicellular, green alga with the potential for various pharmaceutical uses, has been widely studied in this context. This study is aimed at determining the effects of C. vulgaris on promoting muscle regeneration by evaluating myoblast regenerative capacity in vitro. Human skeletal myoblast cells were cultured and underwent serial passaging into young and senescent phases and were then treated with C. vulgaris, followed by the induction of differentiation. The ability of C. vulgaris to promote myoblast differentiation was analysed through cellular morphology, real-time monitoring, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation, myogenin expression, and cell cycle profiling. The results obtained showed that senescent myoblasts exhibited an enlarged and flattened morphology, with increased SA-β-gal expression, reduced myogenic differentiation, decreased expression of myogenin, and an increased percentage of cells in the G0/G1 phase. Treatment with C. vulgaris resulted in decreased SA-β-gal expression and promotion of myogenic differentiation, as observed via an increased fusion index, maturation index, myotube size, and surface area and an increased percentage of cells that stained positive for myogenin. In conclusion, C. vulgaris improves the regenerative capacity of young and senescent myoblasts and promotes myoblast differentiation, indicating its potential to promote muscle regeneration.

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Conditional Deletion of Dicer in Adult Mice Impairs Skeletal Muscle Regeneration

International Journal of Molecular Sciences ◽

10.3390/ijms20225686 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5686 ◽

Cited By ~ 2

Author(s):

Satoshi Oikawa ◽

Minjung Lee ◽

Takayuki Akimoto

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Myogenic Differentiation ◽

Tibialis Anterior Muscle ◽

Critical Factor ◽

Skeletal Muscle Regeneration ◽

Small Noncoding Rnas ◽

Adult Mice

Skeletal muscle has a remarkable regenerative capacity, which is orchestrated by multiple processes, including the proliferation, fusion, and differentiation of the resident stem cells in muscle. MicroRNAs (miRNAs) are small noncoding RNAs that mediate the translational repression or degradation of mRNA to regulate diverse biological functions. Previous studies have suggested that several miRNAs play important roles in myoblast proliferation and differentiation in vitro. However, their potential roles in skeletal muscle regeneration in vivo have not been fully established. In this study, we generated a mouse in which the Dicer gene, which encodes an enzyme essential in miRNA processing, was knocked out in a tamoxifen-inducible way (iDicer KO mouse) and determined its regenerative potential after cardiotoxin-induced acute muscle injury. Dicer mRNA expression was significantly reduced in the tibialis anterior muscle of the iDicer KO mice, whereas the expression of muscle-enriched miRNAs was only slightly reduced in the Dicer-deficient muscles. After cardiotoxin injection, the iDicer KO mice showed impaired muscle regeneration. We also demonstrated that the number of PAX7+ cells, cell proliferation, and the myogenic differentiation capacity of the primary myoblasts did not differ between the wild-type and the iDicer KO mice. Taken together, these data demonstrate that Dicer is a critical factor for muscle regeneration in vivo.

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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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Methylation and expression of the Myo D1 determination gene

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1990.0011 ◽

1990 ◽

Vol 326 (1235) ◽

pp. 277-284 ◽

Cited By ~ 22

Keyword(s):

De Novo ◽

Cell Types ◽

Model System ◽

Parental Line ◽

Molecular Control ◽

Embryo Cells ◽

End Stage ◽

Derivatives Of

Mouse embryo cells induced to differentiate with the demethylating agent 5- azacytidine represent an excellent model system to investigate the molecular control of development. Clonal derivatives of 10T1/2 cells that have become determined to the myogenic or adipogenic lineages can be isolated from the multipotential parental line after drug treatment. These determined derivatives can be cultured indefinitely and will differentiate into end-stage phenotypes on appropriate stimulation. A gene called Myo D1, recently isolated from such a myoblast line, will confer myogenesis when expressed in 10T1/2 or other cell types (Davis et al. 1987). The cDNA for Myo D1 contains a large number of CpG sequences and the gene is relatively methylated in 10T1/2 cells and an adipocyte derivative, but is demethylated in myogenic derivatives. Myo D1 may therefore be subject to methylation control in vitro . On the other hand, preliminary observations suggest that Myo D1 is not methylated at CCGG sites in vivo so that a de novo methylation event may have occurred in vitro . These observations may have significance in the establishment of immortal cell lines and tumours.

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Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

Molecular Biology of the Cell ◽

10.1091/mbc.5.7.819 ◽

1994 ◽

Vol 5 (7) ◽

pp. 819-828 ◽

Cited By ~ 46

Author(s):

Y Wang ◽

G M Fuller

Keyword(s):

Protein Synthesis ◽

Cell Surface ◽

De Novo ◽

Cell Types ◽

Surface Expression ◽

Cell Surface Expression ◽

Protein Synthesis Inhibitors ◽

Different Cell Types ◽

Kinetics Of

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

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