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Metabolomic analysis of primary human skeletal muscle cells during myogenic progression.

ABSTRACT: Skeletal muscle constitutes more than 30% of total body mass using substrates such as glycogen, glucose, free fatty acids, and creatinine phosphate to generate energy. Consequently, multinucleated myofibers and resident mononucleated stem cells (satellite cells) generate several metabolites, which enter into circulation affecting the function of other organs, especially during exercise and atrophy. The present study was aimed at building a comprehensive profile of metabolites in primary human skeletal muscle cells during myogenic progression in an untargeted metabolomics approach using a high resolution Orbitrap Fusion Tribrid Mass Spectrometer. Identification of metabolites with multivariate statistical analyses showed a global shift in metabolomic profiles between myoblasts undergoing proliferation and differentiation along with distinctly separable profiles between early and late differentiating cultures. Pathway analyses of 71 unique metabolites revealed that Pantothenate metabolism and Coenzyme A biosynthesis and Arginine Proline metabolism play dominant roles in proliferating myoblasts, while metabolites involved in vitamin B6, Glyoxylate and Dicarboxylate, Nitrogen, Glutathione, and Tryptophan metabolism were upregulated during differentiation. We found that early and late differentiating cultures displayed differences in Phenylalanine, Tyrosine, Glycine, Serine and Threonine metabolism. Our results identify metabolites during maturation of muscle from progenitor myoblasts that have implications in muscle regeneration and pathophysiology.

SUBMITTER: Kumar A

PROVIDER: S-EPMC7366914 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Metabolomic analysis of primary human skeletal muscle cells during myogenic progression.

Kumar Ashok A Kumar Yashwant Y Sevak Jayesh Kumar JK Kumar Sonu S Kumar Niraj N Gopinath Suchitra Devi SD

Scientific reports 20200716 1

Skeletal muscle constitutes more than 30% of total body mass using substrates such as glycogen, glucose, free fatty acids, and creatinine phosphate to generate energy. Consequently, multinucleated myofibers and resident mononucleated stem cells (satellite cells) generate several metabolites, which enter into circulation affecting the function of other organs, especially during exercise and atrophy. The present study was aimed at building a comprehensive profile of metabolites in primary human sk ...[more]

PMID: 32678274

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Project description:BackgroundSkeletal muscle in livestock develops into meat, an important source of protein and other nutrients for human consumption. The muscle is largely composed of a fixed number of multinucleated myofibers determined during late gestation and remains constant postnatally. A population of postnatal muscle stem cells, called satellite cells, gives rise to myoblast cells that can fuse with the existing myofibers, thus increasing their size. This requires a delicate balance of transcription and growth factors and specific microRNA (miRNA) expressed by satellite cells and their supporting cells from the muscle stem cell niche. The role of transcription and growth factors in bovine myogenesis is well-characterized; however, very little is known about the miRNA activity during this process. We have hypothesized that the expression of miRNA can vary between primary cultures of skeletal muscle cells isolated from the semitendinosus muscles of different cattle breeds and subjected to myogenic differentiation.ResultsAfter a 6-day myogenic differentiation of cells isolated from the muscles of the examined cattle breeds, we found statistically significant differences in the number of myotubes between Hereford (HER)/Limousine (LIM) beef breeds and the Holstein-Friesian (HF) dairy breed (p ≤ 0.001). The microarray analysis revealed differences in the expression of 23 miRNA among the aforementioned primary cultures. On the basis of a functional analysis, we assigned 9 miRNA as molecules responsible for differentiation progression (miR-1, -128a, -133a, -133b, -139, -206, -222, -486, and -503). The target gene prediction and functional analysis revealed 59 miRNA-related genes belonging to the muscle organ development process.ConclusionThe number of myotubes and the miRNA expression in the primary cultures of skeletal muscle cells derived from the semitendinosus muscles of the HER/LIM beef cattle breeds and the HF dairy breed vary when cells are subjected to myogenic differentiation. The net effect of the identified miRNA and their target gene action should be considered the result of the breed-dependent activity of satellite cells and muscle stem cell niche cells and their mutual interactions, which putatively can be engaged in the formation of a larger number of myotubes in beef cattle-related cells (HER/LIM) during in vitro myogenesis.

Project description:IntroductionHuman amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and their regenerative potential for muscle injury remain to be elucidated.MethodsIn order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and transduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression of molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western blotting. For in vivo muscle regeneration, MYOD transduced hAFS cells were injected into left tibialis anterior (TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxylin and eosin, immunocytochemistry and formation of neuro-muscular junction.ResultsMYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells. Consistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD and MYH, was induced by MYOD. Analysis of pre-myogenic factors showed that expression of PAX3, MEOX1 and EYA2 was significantly increased by MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells, MYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in myogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them into injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA muscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more numbers of neuro-muscular junctions compared to controls, indicating functional restoration of muscle injury by MYOD-hAFS cells.ConclusionsCollectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal myogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.

Project description:BackgroundhTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research, being used as therapeutically pertinent models of various neuromuscular disorders and in numerous fundamental studies of muscle cell function. However, the cell cycle is linked to other cellular processes such as integrin regulation, the PI3K/Akt pathway, and microtubule stability, raising the question as to whether genetic modification related to the cell cycle results in secondary effects that could undermine the validity of these cell models.ResultsHere we subjected five healthy and disease muscle cell isolates to transcriptomic analysis, comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states, and testing their myogenic character by comparison with non-myogenic (CD56-negative) cells. Principal component analysis of global gene expression showed tight clustering of immortalized myoblasts to their parent primary populations, with clean separation from the non-myogenic reference. Comparison was made to publicly available transcriptomic data from studies of muscle human pathology, cell, and animal models, including to derive a consensus set of genes previously shown to have altered regulation during myoblast differentiation. Hierarchical clustering of samples based on gene expression of this consensus set showed that immortalized lines retained the myogenic expression patterns of their parent primary populations. Of 2784 canonical pathways and gene ontology terms tested by gene set enrichment analysis, none were significantly enriched in immortalized compared to primary cell populations. We observed, at the whole transcriptome level, a strong signature of cell cycle shutdown associated with senescence in one primary myoblast population, whereas its immortalized clone was protected.ConclusionsImmortalization had no observed effect on the myogenic cascade or on any other cellular processes, and it was protective against the systems level effects of senescence that are observed at higher division counts of primary cells.

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Metabolomic analysis of primary human skeletal muscle cells during myogenic progression.

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Metabolomic analysis of primary human skeletal muscle cells during myogenic progression.

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