scholarly journals Expression Analysis of Irisin During Different Differentiation Stages of Skeletal Muscle Cells

2021 ◽  
Author(s):  
Yi Yan ◽  
Ding Yang ◽  
Pei Wen ◽  
Yilei Li ◽  
Yufang Ge ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Development ◽  
Developmental Stages ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Growth Stages ◽  
Glucose And Lipid Metabolism ◽  
Muscle Tissues ◽  
Sexually Mature

Abstract Background: As a newly discovered muscle factor secreted by skeletal muscle cells, irisin is a polypeptide fragment formed after hydrolysis by fibronectin type Ⅲ domain-containing protein 5 (FNDC5). Previous studies have shown that irisin has biological functions that promote beigeing of WAT, regulate glucose and lipid metabolism. However, the functions of irisin in muscle development and muscle fat metabolism remain unknown.Results: In order to study the expression of irisin in different growth stages of skeletal muscle, this study used SPF mice as experimental subjects to select skeletal muscle cells and muscle tissues of different developmental stages of mice. The expression of irisin precursor FNDC5 in different stages of cells and tissues was detected by western blotting and real-time fluorescent quantitative PCR, and the expression of FNDC5 in cells was detected by immunofluorescence. The results showed that FNDC5 was expressed in all stages of tissues and cells, but the expression was different at different stages. FNDC5 protein has the highest expression in muscle of sexually mature mice, followed by elderly mice and adolescent mice, and low expression in pups. Secondly, FNDC5 protein is mainly expressed in the cytoplasm and highest in muscle fibers. The myotubes were the second, and the lowest in C2C12 cells. Conclusions: This experiment can provide a theoretical basis for the subsequent study of irisin in skeletal muscle, and lay the foundation for targeted therapy of related diseases.

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.

scholarly journals 17β-Estradiol abrogates apoptosis in murine skeletal muscle cells through estrogen receptors: role of the phosphatidylinositol 3-kinase/Akt pathway

2007 ◽  
Vol 196 (2) ◽  
pp. 385-397 ◽  
Author(s):  
Andrea Vasconsuelo ◽  
Lorena Milanesi ◽  
Ricardo Boland
Keyword(s):  
Skeletal Muscle ◽  
Protective Action ◽  
Muscle Cells ◽  
Cellular Systems ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Akt Pathway ◽  
Akt Activation ◽  
17Β Estradiol ◽  
Murine Skeletal Muscle

Estrogens can regulate apoptosis in various cellular systems. The present study shows that 17β-estradiol (E2), at physiological concentrations, abrogates DNA damage, poly (ADP-ribose) polymerase cleavage, and mitochondrial cytochrome c release induced by H2O2 or etoposide in mouse skeletal muscle C2C12 cells. This protective action, which involved PI3K/Akt activation and Bcl-2 associated death agonist (BAD) phosphorylation, was inhibited by antibodies against the estrogen receptor (ER) α or β isoforms, or transfecting siRNA specific for each isoform. The inhibition of the antiapoptotic action of E2 at the mitochondrial level was more pronounced when ER-β was immunoneutralized or suppressed by mRNA silencing, whereas transfection of C2C12 cells with either ER-α siRNA or ER-β siRNA blocked the activation of Akt by E2, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway evaluated. These results indicate that E2 exerts antiapoptotic effects in skeletal muscle cells which are mediated by ER-β and ER-α and involve the PI3K/Akt pathway.

NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells

2008 ◽  
Vol 294 (3) ◽  
pp. C715-C725 ◽  
Author(s):  
Juan Antonio Valdés ◽  
Eduardo Gaggero ◽  
Jorge Hidalgo ◽  
Nancy Leal ◽  
Enrique Jaimovich ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factors ◽  
Electrical Stimulation ◽  
Stimulus Frequency ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Mrna Levels ◽  
Activated T Cells ◽  
Nfat Activation

Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c- fos, c- jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-κB activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-κB activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-κB and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels.

scholarly journals Alignment of Skeletal Muscle Cells Cultured in Collagen Gel by Mechanical and Electrical Stimulation

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Takara Tanaka ◽  
Noriko Hattori-Aramaki ◽  
Ayano Sunohara ◽  
Keisuke Okabe ◽  
Yoshiaki Sakamoto ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tissue Engineering ◽  
Electrical Stimulation ◽  
Collagen Gel ◽  
Muscle Cells ◽  
Mechanical Force ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Collagen Gels ◽  
Cells Cultured

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method.

scholarly journals Actions of 1,25(OH)2-vitamin D3 on the cellular cycle depend on VDR and p38 MAPK in skeletal muscle cells

2014 ◽  
Vol 53 (3) ◽  
pp. 331-343 ◽  
Author(s):  
Ana P Irazoqui ◽  
Ricardo L Boland ◽  
Claudia G Buitrago
Keyword(s):  
Skeletal Muscle ◽  
Vitamin D ◽  
P38 Mapk ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Cyclin D3 ◽  
C2c12 Myoblast ◽  
G1 Arrest ◽  
Dependent Manner

Previously, we have reported that 1,25(OH)2-vitamin D3(1,25D) activates p38 MAPK (p38) in a vitamin D receptor (VDR)-dependent manner in proliferative C2C12 myoblast cells. It was also demonstrated that 1,25D promotes muscle cell proliferation and differentiation. However, we did not study these hormone actions in depth. In this study we have investigated whether the VDR and p38 participate in the signaling mechanism triggered by 1,25D. In C2C12 cells, the VDR was knocked down by a shRNA, and p38 was specifically inhibited using SB-203580. Results from cell cycle studies indicated that hormone stimulation prompts a peak of S-phase followed by an arrest in the G0/G1-phase, events which were dependent on VDR and p38. Moreover, 1,25D increases the expression of cyclin D3 and the cyclin-dependent kinase inhibitors, p21Waf1/Cip1and p27Kip1, while cyclin D1 protein levels did not change during G0/G1 arrest. In all these events, p38 and VDR were required. At the same time, a 1,25D-dependent acute increase in myogenin expression was observed, indicating that the G0/G1 arrest of cells is a pro-differentiative event. Immunocytochemical assays revealed co-localization of VDR and cyclin D3, promoted by 1,25D in a p38-dependent manner. When cyclin D3 expression was silenced, VDR and myogenin levels were downregulated, indicating that cyclin D3 was required for 1,25D-induced VDR expression and the concomitant entrance into the differentiation process. In conclusion, the VDR and p38 are involved in control of the cellular cycle by 1,25D in skeletal muscle cells, providing key information on the mechanisms underlying hormone regulation of myogenesis.

scholarly journals Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors.

1991 ◽  
Vol 11 (1) ◽  
pp. 267-280 ◽  
Author(s):  
H Lin ◽  
K E Yutzey ◽  
S F Konieczny
Keyword(s):  
Skeletal Muscle ◽  
Muscle Development ◽  
Troponin I ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Regulatory Factors ◽  
Helix Loop Helix ◽  
Muscle Regulatory Factors

The quail fast skeletal troponin I (TnI) gene is a member of the contractile protein gene set and is expressed exclusively in differentiated skeletal muscle cells. TnI gene transcription is controlled by an internal regulatory element (IRE), located within the first intron, that functions as a muscle-specific enhancer. Recent studies have shown that the TnI IRE may interact directly with the muscle regulatory factors MyoD, myogenin, and Myf-5 to produce a muscle-specific expression pattern, since these factors trans-activate cotransfected TnI gene constructs in C3H10T1/2 fibroblasts. In this study, we have examined the protein-IRE interactions that are responsible for transcriptionally activating the TnI gene during skeletal muscle development. We demonstrate that the helix-loop-helix muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4, when complexed with the immunoglobulin enhancer-binding protein E12, interact with identical nucleotides within a muscle regulatory factor-binding site (MRF site) located in the TnI IRE. The nuclear proteins that bind to the MRF site are restricted to skeletal muscle cells, since protein extracts from HeLa, L, and C3H10T1/2 fibroblasts do not contain similar binding activities. Importantly, the TnI MRF site alone is not sufficient to elicit the full enhancer activity associated with the IRE. Instead, two additional regions (site I and site II) are required. The proteins that interact with site I and site II are expressed in both muscle and nonmuscle cell types and by themselves are ineffective in activating TnI gene expression. However, when the MRF site is positioned upstream or downstream of site I and site II, full enhancer activity is restored. We conclude that helix-loop-helix muscle regulatory factors must interact with ubiquitously expressed proteins to generate the active TnI transcription complex that is present in differentiated muscle fibers.

scholarly journals MiR-22-3p Inhibits Proliferation and Promotes Differentiation of Skeletal Muscle Cells by Targeting IGFBP3 in Hu Sheep

Animals ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 114
Author(s):  
Shan Wang ◽  
Xiukai Cao ◽  
Ling Ge ◽  
Yifei Gu ◽  
Xiaoyang Lv ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Skeletal Muscle ◽  
Growth And Development ◽  
Muscle Cells ◽  
Bioinformatic Analysis ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Hu Sheep

The growth and development of skeletal muscle require a series of regulatory factors. MiRNA is a non-coding RNA with a length of about 22 nt, which can inhibit the expression of mRNA and plays an important role in the growth and development of muscle cells. The role of miR-22-3p in C2C12 cells and porcine skeletal muscle has been reported, but it has not been verified in Hu sheep skeletal muscle. Through qPCR, CCK-8, EdU and cell cycle studies, we found that overexpression of miR-22-3p inhibited proliferation of skeletal muscle cells (p < 0.01). The results of qPCR and immunofluorescence showed that overexpression of miR-22-3p promoted differentiation of skeletal muscle cells (p < 0.01), while the results of inhibiting the expression of miR-22-3p were the opposite. These results suggested that miR-22-3p functions in growth and development of sheep skeletal muscle cells. Bioinformatic analysis with mirDIP, miRTargets, and RNAhybrid software suggested IGFBP3 was the target of miR-22-3p, which was confirmed by dual-luciferase reporter system assay. IGFBP3 is highly expressed in sheep skeletal muscle cells. Overexpression of IGFBP3 was found to promote proliferation of skeletal muscle cells indicated by qPCR, CCK-8, EdU, and cell cycle studies (p < 0.01). The results of qPCR and immunofluorescence experiments proved that overexpression of IGFBP3 inhibited differentiation of skeletal muscle cells (p < 0.01), while the results of interfering IGFBP3 with siRNA were the opposite. These results indicate that miR-22-3p is involved in proliferation and differentiation of skeletal muscle cells by targeting IGFBP3.

Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors

1991 ◽  
Vol 11 (1) ◽  
pp. 267-280 ◽  
Author(s):  
H Lin ◽  
K E Yutzey ◽  
S F Konieczny
Keyword(s):  
Skeletal Muscle ◽  
Muscle Development ◽  
Troponin I ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Regulatory Factors ◽  
Helix Loop Helix ◽  
Muscle Regulatory Factors

The quail fast skeletal troponin I (TnI) gene is a member of the contractile protein gene set and is expressed exclusively in differentiated skeletal muscle cells. TnI gene transcription is controlled by an internal regulatory element (IRE), located within the first intron, that functions as a muscle-specific enhancer. Recent studies have shown that the TnI IRE may interact directly with the muscle regulatory factors MyoD, myogenin, and Myf-5 to produce a muscle-specific expression pattern, since these factors trans-activate cotransfected TnI gene constructs in C3H10T1/2 fibroblasts. In this study, we have examined the protein-IRE interactions that are responsible for transcriptionally activating the TnI gene during skeletal muscle development. We demonstrate that the helix-loop-helix muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4, when complexed with the immunoglobulin enhancer-binding protein E12, interact with identical nucleotides within a muscle regulatory factor-binding site (MRF site) located in the TnI IRE. The nuclear proteins that bind to the MRF site are restricted to skeletal muscle cells, since protein extracts from HeLa, L, and C3H10T1/2 fibroblasts do not contain similar binding activities. Importantly, the TnI MRF site alone is not sufficient to elicit the full enhancer activity associated with the IRE. Instead, two additional regions (site I and site II) are required. The proteins that interact with site I and site II are expressed in both muscle and nonmuscle cell types and by themselves are ineffective in activating TnI gene expression. However, when the MRF site is positioned upstream or downstream of site I and site II, full enhancer activity is restored. We conclude that helix-loop-helix muscle regulatory factors must interact with ubiquitously expressed proteins to generate the active TnI transcription complex that is present in differentiated muscle fibers.

Developmental regulation of the mouse IGF-I exon 1 promoter region by calcineurin activation of NFAT in skeletal muscle

2007 ◽  
Vol 292 (5) ◽  
pp. C1887-C1894 ◽  
Author(s):  
Christina M. Alfieri ◽  
Heather J. Evans-Anderson ◽  
Katherine E. Yutzey
Keyword(s):  
Skeletal Muscle ◽  
Signaling Pathways ◽  
Muscle Development ◽  
Developmental Regulation ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Conserved Regions ◽  
Igf I ◽  
Exon 1 ◽  
I Gene

Skeletal muscle development and growth are regulated through multiple signaling pathways that include insulin-like growth factor I (IGF-I) and calcineurin activation of nuclear factor of activated T cell (NFAT) transcription factors. The developmental regulation and molecular mechanisms that control IGF-I gene expression in murine embryos and in differentiating C2C12 skeletal myocytes were examined. IGF-I is expressed in developing skeletal muscle, and its embryonic expression is significantly reduced in embryos lacking both NFATc3 and NFATc4. During development, the IGF-I exon 1 promoter is active in multiple organ systems, including skeletal muscle, whereas the alternative exon 2 promoter is expressed predominantly in the liver. The IGF-I exon 1 promoter flanking sequence includes two highly conserved regions that contain NFAT consensus binding sequences. One of these conserved regions contains a calcineurin/NFAT-responsive regulatory region that is preferentially activated by NFATc3 in C2C12 skeletal muscle cells and NIH3T3 fibroblasts. This NFAT-responsive region contains three clustered NFAT consensus binding sequences, and mutagenesis experiments demonstrated the requirement for two of these in calcineurin or NFATc3 responsiveness. Chromatin immunoprecipitation analyses demonstrated that endogenous IGF-I genomic sequences containing these conserved NFAT binding sequences interact preferentially with NFATc3 in C2C12 cells. Together, these experiments demonstrated that a NFAT-rich regulatory element in the IGF-I exon 1 promoter flanking region is responsive to calcineurin signaling and NFAT activation in skeletal muscle cells. The identification of a calcineurin/NFAT-responsive element in the IGF-I gene represents a potential mechanism of intersection of these signaling pathways in the control of muscle development and homeostasis.

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