Recapitulating pathophysiology of skeletal muscle diseases in vitro using primary mouse myoblasts on a nanofibrous platform

A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed “mechano-growth factor” (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.

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Recent Trends in Biofabrication Technologies for Studying Skeletal Muscle Tissue-Related Diseases

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.782333 ◽

2021 ◽

Vol 9 ◽

Author(s):

Seungyeun Cho ◽

Jinah Jang

Keyword(s):

Skeletal Muscle ◽

Muscle Tissue ◽

Motor Neurons ◽

Close Contact ◽

Human Muscle ◽

Skeletal Muscle Tissue ◽

Mechanism Study ◽

Muscle Diseases ◽

Muscle Models

In native skeletal muscle, densely packed myofibers exist in close contact with surrounding motor neurons and blood vessels, which are embedded in the fibrous connective tissue. In comparison to conventional two-dimensional (2D) cultures, the three-dimensional (3D) engineered skeletal muscle models allow structural and mechanical resemblance with native skeletal muscle tissue by providing geometric confinement and physiological matrix stiffness to the cells. In addition, various external stimuli applied to these models enhance muscle maturation along with cell–cell and cell–extracellular matrix interaction. Therefore, 3D in vitro muscle models can adequately recapitulate the pathophysiologic events occurring in tissue–tissue interfaces inside the native skeletal muscle such as neuromuscular junction. Moreover, 3D muscle models can induce pathological phenotype of human muscle dystrophies such as Duchenne muscular dystrophy by incorporating patient-derived induced pluripotent stem cells and human primary cells. In this review, we discuss the current biofabrication technologies for modeling various skeletal muscle tissue-related diseases (i.e., muscle diseases) including muscular dystrophies and inflammatory muscle diseases. In particular, these approaches would enable the discovery of novel phenotypic markers and the mechanism study of human muscle diseases with genetic mutations.

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A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions

Cell Death Discovery ◽

10.1038/s41420-020-00328-5 ◽

2020 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Brandon J. Gheller ◽

Jamie E. Blum ◽

Ern Hwei Hannah Fong ◽

Olga V. Malysheva ◽

Benjamin D. Cosgrove ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cell ◽

Muscle Regeneration ◽

Transcriptional Profile ◽

Skeletal Muscle Regeneration ◽

State Transitions ◽

C2c12 Myoblasts ◽

Primary Mouse

Abstract Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

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Human induced pluripotent stem cell models for the study and treatment of Duchenne and Becker muscular dystrophies

Therapeutic Advances in Neurological Disorders ◽

10.1177/1756286419833478 ◽

2019 ◽

Vol 12 ◽

pp. 175628641983347 ◽

Cited By ~ 11

Author(s):

Daniela Piga ◽

Sabrina Salani ◽

Francesca Magri ◽

Roberta Brusa ◽

Eleonora Mauri ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Muscle Development ◽

Primary Cultures ◽

Early Death ◽

Muscle Disease ◽

Muscular Dystrophies ◽

Muscle Diseases ◽

Induced Pluripotent

Duchenne and Becker muscular dystrophies are the most common muscle diseases and are both currently incurable. They are caused by mutations in the dystrophin gene, which lead to the absence or reduction/truncation of the encoded protein, with progressive muscle degeneration that clinically manifests in muscle weakness, cardiac and respiratory involvement and early death. The limits of animal models to exactly reproduce human muscle disease and to predict clinically relevant treatment effects has prompted the development of more accurate in vitro skeletal muscle models. However, the challenge of effectively obtaining mature skeletal muscle cells or satellite stem cells as primary cultures has hampered the development of in vitro models. Here, we discuss the recently developed technologies that enable the differentiation of skeletal muscle from human induced pluripotent stem cells (iPSCs) of Duchenne and Becker patients. These systems recapitulate key disease features including inflammation and scarce regenerative myogenic capacity that are partially rescued by genetic and pharmacological therapies and can provide a useful platform to study and realize future therapeutic treatments. Implementation of this model also takes advantage of the developing genome editing field, which is a promising approach not only for correcting dystrophin, but also for modulating the underlying mechanisms of skeletal muscle development, regeneration and disease. These data prove the possibility of creating an accurate Duchenne and Becker in vitro model starting from iPSCs, to be used for pathogenetic studies and for drug screening to identify strategies capable of stopping or reversing muscular dystrophinopathies and other muscle diseases.

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A system to monitor statin-induced myopathy in individual engineered skeletal muscle myobundles

Lab on a Chip ◽

10.1039/c8lc00654g ◽

2018 ◽

Vol 18 (18) ◽

pp. 2787-2796 ◽

Cited By ~ 8

Author(s):

Xu Zhang ◽

Sungmin Hong ◽

Ringo Yen ◽

Megan Kondash ◽

Cristina E. Fernandez ◽

...

Keyword(s):

Skeletal Muscle ◽

Tissue Engineering ◽

Human Skeletal Muscle ◽

Elastic Membrane ◽

Muscle Diseases ◽

In Series ◽

Engineering Models

Microphysiological tissue engineering models of human skeletal muscle (myobundles) in series with a calibrated elastic membrane provide a platform to investigate the mechanism of muscle diseases and to study the response to drugs and toxins in vitro.

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Ultrastructural autoradiography of L6 skeletal-muscle cells exposed to a tritiated macrolide antibiotic (LY237216) in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123465 ◽

1992 ◽

Vol 50 (1) ◽

pp. 612-613

Author(s):

S.L. White ◽

C.B. Jensen ◽

D.D. Giera ◽

D.A. Laska ◽

M.N. Novilla ◽

...

Keyword(s):

Skeletal Muscle ◽

Quantitative Analysis ◽

Macrolide Antibiotic ◽

Circle Method ◽

Apical Surface ◽

Polycarbonate Membrane ◽

L6 Cells ◽

Low Viscosity ◽

Erythromycin A

In vitro exposure to LY237216 (9-Deoxo-11-deoxy-9,11-{imino[2-(2-methoxyethoxy)ethylidene]-oxy}-(9S)-erythromycin), a macrolide antibiotic, was found to induce cytoplasmic vacuolation in L6 skeletal muscle myoblast cultures (White, S.L., unpubl). The present study was done to determine, by autoradiographic quantitative analysis, the subcellular distribution of 3H-LY237216 in L6 cells.L6 cells (ATCC, CRL 1458) were cultured to confluency on polycarbonate membrane filters (Millipore Corp., Bedford, MA) in M-199 medium (GIBCO® Labs) with 10% fetal bovine serum. The cells were exposed from the apical surface for 1-hour to unlabelled-compound (0 μCi/ml) or 50 (μCi/ml of 3H-LY237216 at a compound concentration of 0.25 mg/ml. Following a rapid rinse in compound-free growth medium, the cells were slam-frozen against a liquid nitrogen cooled, polished copper block in a CF-100 cryofixation unit (LifeCell Corp., The Woodlands, TX). Specimens were dried in the MDD-C Molecular Distillation Drier (LifeCell Corp.), vapor osmicated and embedded in Spurrs low viscosity resin. Ultrathin sections collected on formvar coated stainless steel grids were counter-stained, then individually mounted on corks. A monolayer of Ilford L4 nuclear emulsion (Polysciences, Inc., Warrington, PA) was placed on the sections, utilizing a modified “loop method”. The emulsions were exposed for 7-weeks in a light-tight box at 4°C. Autoradiographs were developed in Microdol-X developer and examined on a Philips EM410LS transmission electron microscope. Quantitative analysis of compound localization employed the point and circle approach of Williams; incorporating the probability circle method of Salpeter and McHenry.

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