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Cells Aug 2023Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization,...
Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.
Topics: Aged; Animals; Humans; Mice; Forkhead Box Protein O3; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts; Quality of Life; Sarcopenia; Myogenin; MyoD Protein
PubMed: 37681900
DOI: 10.3390/cells12172167 -
American Journal of Physiology. Cell... Oct 2013There is a global epidemic of obesity, and obesity is known to inhibit AMP-activated protein kinase (AMPK) activity and impairs myogenesis. Myogenin mediates the fusion...
There is a global epidemic of obesity, and obesity is known to inhibit AMP-activated protein kinase (AMPK) activity and impairs myogenesis. Myogenin mediates the fusion of myoblasts into myotubes, a critical step in myogenesis. We observed that inhibition of AMPKα1 downregulates myogenin expression and myogenesis, but the underlying mechanisms are unclear. We postulated that AMPK regulates myogenin expression through phosphorlytion of histone deacetylase 5 (HDAC5). In C2C12 cells, HDAC5 knockdown increased while HDAC5 stablization by MC1568 reduced myogenin expression. Consistently, using luciferase assay, we observed that myogenin promoter activity was negatively regulated by HDAC5. Using RNA interference and primary myoblasts prepared from wild-type and AMPKα1 knockout mice, we further demonstrate that AMPKα1 regulates HDAC5 phosphorylation at Ser 259 and 498. Mutation of these two Ser to Ala in HDAC5 abolished the regulatory role of AMPKα1 on myogenin expression, clearly showing the necessity of these phosphorylation sites in mediating myogenin expression. In aggregate, these data show that AMPK inhibition downregulates myogenin transcription and myogenesis through phosphorylation of HDAC5, mediated mainly by AMPKα1. These data demonstrate that AMPK is a key molecular target for promoting myogenesis and muscular regeneration. Because drugs activating AMPK activity, such as metformin, are widely available, our finding has critical clinical implications to ensure proper muscle development and regeneration in obese subjects and under other pathophysiological conditions where AMPK activity is attenuated.
Topics: AMP-Activated Protein Kinases; Animals; Cell Differentiation; Cells, Cultured; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts; Myogenin; Phosphorylation; Promoter Regions, Genetic; Pyrroles; RNA Interference; RNA, Small Interfering
PubMed: 23926128
DOI: 10.1152/ajpcell.00124.2013 -
The Journal of Biological Chemistry Dec 2016Protein 4.1R (4.1R) isoforms are expressed in both cardiac and skeletal muscle. 4.1R is a component of the contractile apparatus. It is also associated with dystrophin...
Protein 4.1R (4.1R) isoforms are expressed in both cardiac and skeletal muscle. 4.1R is a component of the contractile apparatus. It is also associated with dystrophin at the sarcolemma in skeletal myofibers. However, the expression and function of 4.1R during myogenesis have not been characterized. We now report that 4.1R expression increases during C2C12 myoblast differentiation into myotubes. Depletion of 4.1R impairs skeletal muscle differentiation and is accompanied by a decrease in the levels of myosin heavy and light chains and caveolin-3. Furthermore, the expression of myogenin at the protein, but not mRNA, level is drastically decreased in 4.1R knockdown myocytes. Similar results were obtained using MyoD-induced differentiation of 4.1R mouse embryonic fibroblast cells. von Hippel-Lindau (VHL) protein is known to destabilize myogenin via the ubiquitin-proteasome pathway. We show that 4.1R associates with VHL and, when overexpressed, reverses myogenin ubiquitination and stability. This suggests that 4.1R may influence myogenesis by preventing VHL-mediated myogenin degradation. Together, our results define a novel biological function for 4.1R in muscle differentiation and provide a molecular mechanism by which 4.1R promotes myogenic differentiation.
Topics: Animals; Cell Differentiation; Cell Line; Cytoskeletal Proteins; Membrane Proteins; Mice; Mice, Knockout; MyoD Protein; Myoblasts, Skeletal; Myogenin; Protein Stability; Proteolysis; Von Hippel-Lindau Tumor Suppressor Protein
PubMed: 27780863
DOI: 10.1074/jbc.M116.761296 -
Postepy Higieny I Medycyny... May 2014The commitment of myogenic cells in skeletal muscle differentiation requires earlier irreversible interruption of the cell cycle. At the molecular level, several key... (Review)
Review
The commitment of myogenic cells in skeletal muscle differentiation requires earlier irreversible interruption of the cell cycle. At the molecular level, several key regulators of the cell cycle have been identified: cyclin-dependent kinases and their cyclins stimulate the cell cycle progress and its arrest is determined by the activity of cdk inhibitors (Cip/Kip and INK protein families) and pocket protein family: Rb, p107 and p130. The biological activity of cyclin/cdk complexes allows the successive phases of the cell cycle to occur. Myoblast specialization, differentiation and fusion require the activity of myogenic regulatory factors, which include MyoD, myogenin, Myf5 and MRF4. MyoD and Myf5 play a role in muscle cell specialization, myogenin controls the differentiation process, whereas MRF4 is involved in myotube maturation. The deregulation of the cell cycle leads to uncontrolled proliferation, which antagonizes the functions of myogenic factors and it explains the lack of differentiation-specific gene expression in dividing cells. Conversely, the myogenic factor MyoD seems to cooperate with cell cycle inhibitors leading to inhibition of cell cycle progress and commitment to the differentiation process. The hypophosphorylated form of Rb and cdk inhibitors play an important role in permanent arrest of the cell cycle in differentiated myotubes. Furthermore, cyclin/cdk complexes not only regulate cell division by phosphorylation of several substrates, but may also control other cellular processes such as signal transduction, differentiation and apoptosis. Beyond regulating the cell cycle, Cip/Kip proteins play an important role in cell death, transcription regulation, cell fate determination, cell migration and cytoskeletal dynamics. The article summarizes current knowledge concerning the interactions of intracellular signaling pathways controlling crucial stages of fetal and regenerative myogenesis.
Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cyclins; DNA-Binding Proteins; Humans; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; MyoD Protein; Myoblasts; Myogenic Regulatory Factors; Myogenin; Phosphorylation; Signal Transduction
PubMed: 24864103
DOI: 10.5604/17322693.1101617 -
FASEB Journal : Official Publication of... Jan 2021Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages...
Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages of myogenesis are orchestrated and regulated by myogenic regulatory factors and various downstream cellular signaling. Here we identify phosphatase orphan 1 (Phospho1) as a new player in myogenesis. During activation, proliferation, and differentiation of quiescent satellite cells, the expression of Phospho1 gradually increases. Overexpression of Phospho1 inhibits myoblast proliferation but promotes their differentiation and fusion. Conversely, knockdown of Phospho1 accelerates myoblast proliferation but impairs myotube formation. Moreover, knockdown of Phospho1 decreases the OXPHO protein levels and mitochondria density, whereas overexpression of Phospho1 upregulates OXPHO protein levels and promotes mitochondrial oxygen consumption. Finally, we show that Phospho1 expression is controlled by myogenin, which binds to the promoter of Phospho1 to regulate its transcription. These results indicate a key role of Phospho1 in regulating myogenic differentiation and mitochondrial function.
Topics: Animals; Cell Differentiation; Cell Proliferation; Gene Expression Regulation, Enzymologic; Mice; Mitochondria, Muscle; Muscle Development; Myoblasts, Skeletal; Myogenin; Phosphoric Monoester Hydrolases
PubMed: 33140469
DOI: 10.1096/fj.202001672R -
Cell Oct 2010Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an...
Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.
Topics: Animals; Atrophy; Histone Deacetylases; Mice; Mice, Knockout; Muscle Proteins; Muscle, Skeletal; Myogenin; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 20887891
DOI: 10.1016/j.cell.2010.09.004 -
International Journal of Molecular... Dec 2022Hyperphosphatemia can occur as a result of reduced phosphate (P) excretion in cases of kidney dysfunction, which can induce muscle wasting and suppress myogenic...
Hyperphosphatemia can occur as a result of reduced phosphate (P) excretion in cases of kidney dysfunction, which can induce muscle wasting and suppress myogenic differentiation. Higher P suppresses myogenic differentiation and promotes muscle atrophy through canonical (oxidative stress-mediated) and noncanonical (p62-mediated) activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. However, the crosstalk between myogenin and Nrf2/p62 and potential drug(s) for the regulation of myogenin expression needed to be addressed. In this study, we further identified that myogenin may negatively regulate Nrf2 and p62 protein levels in the mouse C2C12 muscle cell line. In the drug screening analysis, we identified N-acetylcysteine, metformin, phenformin, berberine, 4-chloro-3-ethylphenol, cilostazol, and cilomilast as ameliorating the induction of Nrf2 and p62 expression and reduction in myogenin expression that occur due to high P. We further elucidated that doxorubicin and hydrogen peroxide reduced the amount of myogenin protein mediated through the Kelch-like ECH-associated protein 1/Nrf2 pathway, differently from the mechanism of high Pi. The dual functional roles of L-ascorbic acid (L-AA) were found to be dependent on the working concentration, where concentrations below 1 mM L-AA reversed the effect of high P on myogenin and those above 1 mM L-AA had a similar effect of high P on myogenin when used alone. L-AA exacerbated the effect of hydrogen peroxide on myogenin protein and had no further effect of doxorubicin on myogenin protein. In summary, our results further our understanding of the crosstalk between myogenin and Nrf2, with the identification and verification of several potential drugs that can be applied in rescuing the decline of myogenin due to high P in muscle cells.
Topics: Animals; Mice; Ascorbic Acid; Doxorubicin; Hydrogen Peroxide; Kelch-Like ECH-Associated Protein 1; Muscle Fibers, Skeletal; Muscular Atrophy; Myogenin; NF-E2-Related Factor 2; Oxidative Stress; Sequestosome-1 Protein; Signal Transduction
PubMed: 36499650
DOI: 10.3390/ijms232315324 -
Cells Dec 2022During aging, muscle cell apoptosis increases and myogenesis gradually declines. The impaired myogenic and survival potential of the aged skeletal muscle can be...
BACKGROUND
During aging, muscle cell apoptosis increases and myogenesis gradually declines. The impaired myogenic and survival potential of the aged skeletal muscle can be ameliorated by its mechanical loading. However, the molecular responses of aged muscle cells to mechanical loading remain unclear. This study examined the effect of mechanical loading of aged, proliferating, and differentiated myoblasts on the gene expression and signaling responses associated with their myogenic lineage progression and survival.
METHODS
Control and aged C2C12 cells were cultured on elastic membranes and underwent passive stretching for 12 h at a low frequency (0.25 Hz) and different elongations, varying the strain on days 0 and 10 of myoblast differentiation. Activation of ERK1/2 and Akt, and the expression of focal adhesion kinase (FAK) and key myogenic regulatory factors (MRFs), MyoD and Myogenin, were determined by immunoblotting of the cell lysates derived from stretched and non-stretched myoblasts. Changes in the expression levels of the MRFs, muscle growth, atrophy, and pro-apoptotic factors in response to mechanical loading of the aged and control cells were quantified by real-time qRT-PCR.
RESULTS
Mechanical stretching applied on myoblasts resulted in the upregulation of FAK both in proliferating (day 0) and differentiated (day 10) cells, as well as in increased phosphorylation of ERK1/2 in both control and aged cells. Moreover, Akt activation and the expression of early differentiation factor MyoD increased significantly after stretching only in the control myoblasts, while the late differentiation factor Myogenin was upregulated in both the control and aged myoblasts. At the transcriptional level, mechanical loading of the proliferating myoblasts led to an increased expression of IGF-1 isoforms and MRFs, and to downregulation of muscle atrophy factors mainly in control cells, as well as in the upregulation of pro-apoptotic factors both in control and aged cells. In differentiated cells, mechanical loading resulted in an increased expression of the IGF-1Ea isoform and Myogenin, and in the downregulation of atrophy and pro-apoptotic factors in both the control and aged cells.
CONCLUSIONS
This study revealed a diminished beneficial effect of mechanical loading on the myogenic and survival ability of the senescent muscle cells compared with the controls, with a low strain (2%) loading being most effective in upregulating myogenic/anabolic factors and downregulating atrophy and pro-apoptotic genes mainly in the aged myotubes.
Topics: Myogenin; Proto-Oncogene Proteins c-akt; Myogenic Regulatory Factors; Myoblasts; Muscle Development
PubMed: 36552743
DOI: 10.3390/cells11243979 -
Oxidative Medicine and Cellular... 2016Hypoxic training is believed to increase endurance capacity in association with hypoxia inducible factor-1 (HIF-1), a modulator of vascular endothelial growth factor-A...
Hypoxic training is believed to increase endurance capacity in association with hypoxia inducible factor-1 (HIF-1), a modulator of vascular endothelial growth factor-A (VEGF-A), and to influence activation of satellite cells (SCs). However, the effect of hypoxic training on SC activation and its relation to angiogenesis has not been thoroughly investigated. Eight Thoroughbred horses were subjected to normoxic (F = 21%) or hypoxic (F = 15%) training for 3 days/week (100% [Formula: see text]) for 4 weeks. Incremental exercise tests (IET) were conducted on a treadmill under normoxia and the maximal oxygen consumption ([Formula: see text]) and running distance were measured before and after each training session. Muscle biopsy samples were obtained from the gluteus medius muscle at 6 scheduled times before, during, and one week after IET for immunohistochemical analysis and real-time RT-PCR analysis. Running distance and [Formula: see text], measured during IET, increased significantly after hypoxic training compared with normoxic training. Capillary density and mRNA expression related to SC activation (e.g., myogenin and hepatocyte growth factor) and angiogenesis (VEGF-A) increased only after hypoxic training. These results suggest that increases in mRNA expression after training enhance and prolong SC activation and angiogenesis and that nitric oxide plays an important role in these hypoxia-induced training effects.
Topics: Animals; Body Weight; Cross-Over Studies; Female; Hepatocyte Growth Factor; Horses; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Male; Muscle, Skeletal; Myogenin; Oxygen Consumption; Physical Endurance; RNA, Messenger; Satellite Cells, Skeletal Muscle; Vascular Endothelial Growth Factor A
PubMed: 27721912
DOI: 10.1155/2016/1535367 -
The Journal of Biological Chemistry Oct 2008Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD,...
Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.
Topics: Animals; COS Cells; Calcineurin; Cell Line; Chlorocebus aethiops; Gene Expression Regulation, Developmental; Humans; Mice; Mice, Transgenic; Models, Biological; MyoD Protein; Myogenin; NFATC Transcription Factors; Promoter Regions, Genetic; Rats
PubMed: 18676376
DOI: 10.1074/jbc.M801297200