scholarly journals Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro

2022 ◽  
Vol 9 ◽  
Author(s):  
Philip Barrett ◽  
Tom J. Quick ◽  
Vivek Mudera ◽  
Darren J. Player
Keyword(s):  
Skeletal Muscle ◽  
Muscle Spindle ◽  
De Novo ◽  
C2c12 Myotubes ◽  
Muscle Fibres ◽  
Neuregulin 1 ◽  
Gene And Protein Expression ◽  
Intrafusal Fibres

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.

scholarly journals Generating intrafusal skeletal muscle fibres in vitro: Current state of the art and future challenges

2020 ◽  
Vol 11 ◽  
pp. 204173142098520
Author(s):  
Philip Barrett ◽  
Tom J Quick ◽  
Vivek Mudera ◽  
Darren J Player
Keyword(s):  
Skeletal Muscle ◽  
Muscle Spindle ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Ex Vivo ◽  
Muscle Fibres ◽  
Current State ◽  
Intrafusal Fibres ◽  
Proprioceptive Function

Intrafusal fibres are a specialised cell population in skeletal muscle, found within the muscle spindle. These fibres have a mechano-sensory capacity, forming part of the monosynaptic stretch-reflex arc, a key component responsible for proprioceptive function. Impairment of proprioception and associated dysfunction of the muscle spindle is linked with many neuromuscular diseases. Research to-date has largely been undertaken in vivo or using ex vivo preparations. These studies have provided a foundation for our understanding of muscle spindle physiology, however, the cellular and molecular mechanisms which underpin physiological changes are yet to be fully elucidated. Therefrom, the use of in vitro models has been proposed, whereby intrafusal fibres can be generated de novo. Although there has been progress, it is predominantly a developing and evolving area of research. This narrative review presents the current state of art in this area and proposes the direction of future work, with the aim of providing novel pre-clinical and clinical applications.

scholarly journals Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock

2010 ◽  
Vol 163 (5) ◽  
pp. 765-773 ◽  
Author(s):  
J Lado-Abeal ◽  
A Romero ◽  
I Castro-Piedras ◽  
A Rodriguez-Perez ◽  
J Alvarez-Escudero
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Septic Shock ◽  
Thyroid Hormone ◽  
Hormone Receptors ◽  
Serum Samples ◽  
Pathway Activation ◽  
Gene And Protein Expression

AimNon-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo.MethodsSerum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators.ResultsPatients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRβ (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48 h.ConclusionsPatients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

scholarly journals Trimetazidine Attenuates Dexamethasone-Induced Muscle Atrophy via Inhibiting NLRP3/GSDMD Pathway-Mediated Pyroptosis

2020 ◽  
Author(s):  
Li Wang ◽  
Ming-Qing He ◽  
Xi-Yu Shen ◽  
Kang-Zhen Zhang ◽  
Can Zhao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Ring Finger ◽  
Muscle Performance ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
High Dose ◽  
Therapeutic Compound

Skeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can also improve skeletal muscle performance both in human and mice. We here showed that dexamethasone induced atrophy, evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression , and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLRP3, Caspase-1 and GSDMD. Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine administration ameliorated dexamethasone-induced muscle atrophy both in vivo and in vitro. Moreover, trimetazidine improved exercise tolerance, as evidenced by increased running distance and running time, as well as increased skeletal muscle mass in dexamethasone-treated mice. Mechanically, trimetazidine could reverse dexamethasone-induced activation of pyroptosis both in C2C12 myotubes and in mice. Taken together, our present study demonstrated that NLRP3/GSDMD pathway-mediated pyroptosis was involved in dexamethasone-induced skeletal muscle atrophy. Trimetazidine could partially alleviate dexamethasone-induced skeletal muscle atrophy, and increase the diameter of C2C12 myotubes via inhibiting pyroptosis. Thus, trimetazidine might be a potential therapeutic compound for the prevention of muscle atrophy in glucocorticoid-treated patients.

Formation of skeletal muscle in vivo from the mouse C2 cell line

1992 ◽  
Vol 102 (4) ◽  
pp. 779-787 ◽  
Author(s):  
J.E. Morgan ◽  
S.E. Moore ◽  
F.S. Walsh ◽  
T.A. Partridge
Keyword(s):  
Skeletal Muscle ◽  
Cell Line ◽  
Muscle Cell ◽  
Muscle Fibres ◽  
Neural Cell Adhesion ◽  
Anterior Tibial ◽  
Undifferentiated Cells ◽  
Vicryl Suture

The C2 muscle cell line is myogenic in vitro and has been extensively used in studies of muscle cell differentiation. Here, we have investigated the myogenicity in vivo of C2 cells implanted into suitable sites in the mouse. Large amounts of new muscle were formed when C2 cells were implanted into sites in nude mice which were undergoing regeneration following whole muscle grafting and in scaffolding of freeze-killed muscle or vicryl suture in the anterior tibial compartment. When implanted into regenerating muscle, C2 cells fused with the host muscle to form mosaic fibres; when implanted into inert sites, they formed muscle of largely donor origin. C2-derived muscle fibres appeared to become innervated, but the progression of N-CAM (neural cell adhesion molecule) isoform changes in such regenerates indicated that they did not become fully mature. Proliferating, undifferentiated cells of C2 origin form tumours in older grafts; however, this was more pronounced in the absence of competition from host muscle cells. In the short term, C2 cells can form large amounts of muscle in vivo for biochemical analysis. In addition, C2 cells are easily manipulable in vitro; genes of interest may be transfected into them prior to implantation of the cells into skeletal muscle and the effects of these genes in vivo may thus be examined.

scholarly journals A Fish-Derived Protein Hydrolysate Induces Postprandial Aminoacidaemia and Skeletal Muscle Anabolism in an In Vitro Cell Model Using Ex Vivo Human Serum

Nutrients ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 647
Author(s):  
Matthew J. Lees ◽  
David Nolan ◽  
Miryam Amigo-Benavent ◽  
Conor J. Raleigh ◽  
Neda Khatib ◽  
...  
Keyword(s):  
Older Adults ◽  
Skeletal Muscle ◽  
Human Serum ◽  
Body Mass ◽  
Ex Vivo ◽  
Protein Hydrolysate ◽  
C2c12 Myotubes ◽  
The Impact

Fish-derived proteins, particularly fish protein hydrolysates (FPH), offer potential as high-quality sources of dietary protein, whilst enhancing economic and environmental sustainability. This study investigated the impact of a blue whiting-derived protein hydrolysate (BWPH) on aminoacidaemia in vivo and skeletal muscle anabolism in vitro compared with whey protein isolate (WPI) and an isonitrogenous, non-essential amino acid (NEAA) control (0.33 g·kg−1·body mass−1) in an ex vivo, in vitro experimental design. Blood was obtained from seven healthy older adults (two males, five females; age: 72 ± 5 years, body mass index: 24.9 ± 1.6 kg·m2) in three separate trials in a randomised, counterbalanced, double-blind design. C2C12 myotubes were treated with ex vivo human serum-conditioned media (20%) for 4 h. Anabolic signalling (phosphorylation of mTOR, p70S6K, and 4E-BP1) and puromycin incorporation were determined by immunoblotting. Although BWPH and WPI both induced postprandial essential aminoacidaemia in older adults above the NEAA control, peak and area under the curve (AUC) leucine and essential amino acids were more pronounced following WPI ingestion. Insulin was elevated above baseline in WPI and BWPH only, a finding reinforced by higher peak and AUC values compared with NEAA. Muscle protein synthesis, as measured by puromycin incorporation, was greater after incubation with WPI-fed serum compared with fasted serum (P = 0.042), and delta change was greater in WPI (P = 0.028) and BWPH (P = 0.030) compared with NEAA. Myotube hypertrophy was greater in WPI and BWPH compared with NEAA (both P = 0.045), but was similar between bioactive conditions (P = 0.853). Taken together, these preliminary findings demonstrate the anabolic potential of BWPH in vivo and ex vivo, thus providing justification for larger studies in older adults using gold-standard measures of acute and chronic MPS in vivo.

scholarly journals DHA/EPA Improves GLUT4 Translocation, Glycogen Synthesis and Aerobic Glycolysis in Skeletal Muscle of db/db Mice

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 591-591
Author(s):  
Haoyu Li ◽  
Wenqiao Wang ◽  
Pan Zhuang ◽  
Jingjing Jiao ◽  
Yu Zhang
Keyword(s):  
Skeletal Muscle ◽  
Aerobic Glycolysis ◽  
Control Diet ◽  
Glycogen Synthesis ◽  
Glucose Consumption ◽  
Underlying Mechanism ◽  
C2c12 Myotubes ◽  
Glut4 Translocation

Abstract Objectives The aim of this study was to investigate the effects of DHA and EPA on glucose metabolism including glucose uptake and disposal in skeletal muscle and C2C12 myotubes. Methods Four-week-old db/db diabetic mice were fed with control diet enriched with DHA/EPA (purity > 99%,  1% wt/wt) for 10 weeks. To further explore the underlying mechanism, C2C12 myotubes were induced insulin resistance by palmitate and treated with 25 and 50 μM DHA/EPA for 24 h after differentiation. Results The untargeted metabolome of skeletal muscle showed BCAAs and other metabolites associated with glycolysis and TCA cycle were altered by DHA/EPA treatment. Further detections revealed DHA/EPA treatment promoted the translocation of GLUT4 via increasing Rab8a and SNAP23 expression, and enhanced the activity of GS and PDH. In vitro, the glucose consumption was improved coupled with promoted Rab8a or SNAP23, and GS and PDH were also activated under DHA/EPA intervention increased glucose consumption via promoted Rab8a and SNAP23. The GS and PDH were also activated, which were in line with the results in vivo. Conclusions Long-term intake of DHA and EPA may have a protective effect on diabetes through promoted GLUT4 translocation, glycogen synthesis and aerobic glycolysis in skeletal muscle. Funding Sources This work was supported by the National Natural Science Foundation of China (grant number 81773419 and 81300309), Chinese Institute of Nutrition DSM Research Fund (grant number CNS-DSM-2017–035), China National Program for Support of Top-notch Young Professionals and China Postdoctoral Science Foundation (grant number 2020M681869).

scholarly journals Prion Infection of Muscle Cells In Vitro

2007 ◽  
Vol 81 (9) ◽  
pp. 4615-4624 ◽  
Author(s):  
Wendy M. Dlakic ◽  
Eric Grigg ◽  
Richard A. Bessen
Keyword(s):  
Skeletal Muscle ◽  
Cell Line ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
C2c12 Myotubes ◽  
C2c12 Myoblasts ◽  
Peripheral Cell ◽  
Prion Infection

ABSTRACT The prion agent has been detected in skeletal muscle of humans and animals with prion diseases. Here we report scrapie infection of murine C2C12 myoblasts and myotubes in vitro following coculture with a scrapie-infected murine neuroblastoma (N2A) cell line but not following incubation with a scrapie-infected nonneuronal cell line or a scrapie brain homogenate. Terminal differentiation of scrapie-infected C2C12 myoblasts into myotubes resulted in an increase in the expression of the disease-specific prion protein, PrPSc. The amount of scrapie infectivity or PrPSc in C2C12 myotubes was comparable to the levels found in scrapie-infected N2A cells, indicating that a high level of infection was established in muscle cells. Subclones of scrapie-infected C2C12 cells produced high levels of PrPSc in myotubes, and the C-terminal C2 polypeptide fragment of PrPSc was found based on deglycosylation and PrPSc-specific immunoprecipitation of cell lysates. This is the first report of a stable prion infection in muscle cells in vitro and of a long-term prion infection in a nondividing, differentiated peripheral cell type in culture. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.

Melatonin modifies tumor hypoxia and metabolism by inhibiting HIF-1α and energy metabolic pathway in the in vitro and in vivo models of breast cancer

2019 ◽  
Vol 2 (4) ◽  
pp. 83-98 ◽  
Author(s):  
André De Lima Mota ◽  
Bruna Vitorasso Jardim-Perassi ◽  
Tialfi Bergamin De Castro ◽  
Jucimara Colombo ◽  
Nathália Martins Sonehara ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Glucose Metabolism ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Carbonic Anhydrases ◽  
In Vivo Models ◽  
Ca Ix ◽  
Gene And Protein Expression

Breast cancer is the most common cancer among women and has a high mortality rate. Adverse conditions in the tumor microenvironment, such as hypoxia and acidosis, may exert selective pressure on the tumor, selecting subpopulations of tumor cells with advantages for survival in this environment. In this context, therapeutic agents that can modify these conditions, and consequently the intratumoral heterogeneity need to be explored. Melatonin, in addition to its physiological effects, exhibits important anti-tumor actions which may associate with modification of hypoxia and Warburg effect. In this study, we have evaluated the action of melatonin on tumor growth and tumor metabolism by different markers of hypoxia and glucose metabolism (HIF-1α, glucose transporters GLUT1 and GLUT3 and carbonic anhydrases CA-IX and CA-XII) in triple negative breast cancer model. In an in vitro study, gene and protein expressions of these markers were evaluated by quantitative real-time PCR and immunocytochemistry, respectively. The effects of melatonin were also tested in a MDA-MB-231 xenograft animal model. Results showed that melatonin treatment reduced the viability of MDA-MB-231 cells and tumor growth in Balb/c nude mice (p <0.05). The treatment significantly decreased HIF-1α gene and protein expression concomitantly with the expression of GLUT1, GLUT3, CA-IX and CA-XII (p <0.05). These results strongly suggest that melatonin down-regulates HIF-1α expression and regulates glucose metabolism in breast tumor cells, therefore, controlling hypoxia and tumor progression. 

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

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