Effects of hyperoxia and hypoxia on the proliferation of C2C12 myoblasts

2021 ◽  
Author(s):  
Misa Horiike ◽  
Yoshiko Ogawa ◽  
Shigeo Kawada
Keyword(s):  
Cell Proliferation ◽  
Cell Differentiation ◽  
Muscle Regeneration ◽  
Culture Medium ◽  
C2c12 Cells ◽  
Skeletal Muscle Regeneration ◽  
Hypoxic Conditions ◽  
Injured Area ◽  
Myoblast Proliferation ◽  
Contrast Exposure

Hyperoxic conditions are known to accelerate skeletal muscle regeneration after injuries. In the early phase of regeneration, macrophages invade the injured area and subsequently secrete various growth factors, which regulate myoblast proliferation and differentiation. Although hyperoxic conditions accelerate muscle regeneration, it is unknown whether this effect is indirectly mediated by macrophages. Here, using C2C12 cells, we show that not only hyperoxia but also hypoxia enhance myoblast proliferation directly, without accelerating differentiation into myotubes. Under hyperoxic conditions (95% O2 + 5% CO2), the cell membrane was damaged because of lipid oxidization, and a disrupted cytoskeletal structure, resulting in suppressed cell proliferation. However, a culture medium containing vitamin C (VC), an antioxidant, prevented this lipid oxidization and cytoskeletal disruption, resulting in enhanced proliferation in response to hyperoxia exposure of ≤4 h/day. In contrast, exposure to hypoxic conditions (95% N2 + 5% CO2) for ≤8 h/day enhanced cell proliferation. Hyperoxia did not promote cell differentiation into myotubes, regardless of whether the culture medium contained VC. Similarly, hypoxia did not accelerate cell differentiation. These results suggest that regardless of hyperoxia or hypoxia, changes in oxygen tension can enhance cell proliferation directly, but do not influence differentiation efficiency in C2C12 cells. Moreover, excess oxidative stress abrogated the enhancement of myoblast proliferation induced by hyperoxia. The present research will contribute to basic data for applying the effects of hyperoxia or hypoxia to muscle regeneration therapy.

scholarly journals PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells

2015 ◽  
Vol 309 (3) ◽  
pp. C159-C168 ◽  
Author(s):  
Tsung-Chuan Ho ◽  
Yi-Pin Chiang ◽  
Chih-Kuang Chuang ◽  
Show-Li Chen ◽  
Jui-Wen Hsieh ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Proliferation ◽  
Cyclin D1 ◽  
Satellite Cell ◽  
Muscle Regeneration ◽  
Muscle Fibers ◽  
Skeletal Muscle Regeneration ◽  
Satellite Cell Proliferation

In response injury, intrinsic repair mechanisms are activated in skeletal muscle to replace the damaged muscle fibers with new muscle fibers. The regeneration process starts with the proliferation of satellite cells to give rise to myoblasts, which subsequently differentiate terminally into myofibers. Here, we investigated the promotion effect of pigment epithelial-derived factor (PEDF) on muscle regeneration. We report that PEDF and a synthetic PEDF-derived short peptide (PSP; residues Ser93-Leu112) induce satellite cell proliferation in vitro and promote muscle regeneration in vivo. Extensively, soleus muscle necrosis was induced in rats by bupivacaine, and an injectable alginate gel was used to release the PSP in the injured muscle. PSP delivery was found to stimulate satellite cell proliferation in damaged muscle and enhance the growth of regenerating myofibers, with complete regeneration of normal muscle mass by 2 wk. In cell culture, PEDF/PSP stimulated C2C12 myoblast proliferation, together with a rise in cyclin D1 expression. PEDF induced the phosphorylation of ERK1/2, Akt, and STAT3 in C2C12 myoblasts. Blocking the activity of ERK, Akt, or STAT3 with pharmacological inhibitors attenuated the effects of PEDF/PSP on the induction of C2C12 cell proliferation and cyclin D1 expression. Moreover, 5-bromo-2′-deoxyuridine pulse-labeling demonstrated that PEDF/PSP stimulated primary rat satellite cell proliferation in myofibers in vitro. In summary, we report for the first time that PSP is capable of promoting the regeneration of skeletal muscle. The signaling mechanism involves the ERK, AKT, and STAT3 pathways. These results show the potential utility of this PEDF peptide for muscle regeneration.

scholarly journals Effects of intramuscular administration of 1α,25(OH)2D3 during skeletal muscle regeneration on regenerative capacity, muscular fibrosis, and angiogenesis

2016 ◽  
Vol 120 (12) ◽  
pp. 1381-1393 ◽  
Author(s):  
Ratchakrit Srikuea ◽  
Muthita Hirunsai
Keyword(s):  
Skeletal Muscle ◽  
Vitamin D ◽  
Protein Synthesis ◽  
Cell Differentiation ◽  
Satellite Cell ◽  
Muscle Regeneration ◽  
Fiber Type ◽  
Fiber Formation ◽  
Skeletal Muscle Regeneration ◽  
Type Composition

The recent discovery of the vitamin D receptor (VDR) in regenerating muscle raises the question regarding the action of vitamin D3 on skeletal muscle regeneration. To investigate the action of vitamin D3 on this process, the tibialis anterior muscle of male C57BL/6 mice (10 wk of age) was injected with 1.2% BaCl2 to induce extensive muscle injury. The bioactive form of vitamin D3 [1α,25(OH)2D3] was administered daily via intramuscular injections during the regenerative phase (days 4-7 postinjury). Physiological and supraphysiological doses of 1α,25(OH)2D3 relative to 1 μg/kg muscle wet weight and mouse body weight were investigated. Muscle samples were collected on day 8 postinjury to examine proteins related to vitamin D3 metabolism (VDR, CYP24A1, and CYP27B1), satellite cell differentiation and regenerative muscle fiber formation [myogenin and embryonic myosin heavy chain (EbMHC)], protein synthesis signaling (Akt, p70 S6K1, 4E-BP1, and myostatin), fiber-type composition (fast and slow MHCs), fibrous formation (vimentin), and angiogenesis (CD31). Administration of 1α,25(OH)2D3 at physiological and supraphysiological doses enhanced VDR expression in regenerative muscle. Moreover, CYP24A1 and vimentin expression was increased, accompanying decreased myogenin and EbMHC expression at the supraphysiological dose. However, there was no change in CYP27B1, Akt, p70 S6K1, 4E-BP1, myostatin, fast and slow MHCs, or CD31 expression at any dose investigated. Taken together, administration of 1α,25(OH)2D3 at a supraphysiological dose decreased satellite cell differentiation, delayed regenerative muscle fiber formation, and increased muscular fibrosis. However, protein synthesis signaling, fiber-type composition, and angiogenesis were not affected by either 1α,25(OH)2D3 administration at a physiological or supraphysiological dose.

scholarly journals P0707UREMIC TOXINS IMPAIR SKELETAL MUSCLE REGENERATION PROCESS INDUCING CELL CYCLE ARREST AND APOPTOSIS IN CULTURED MYOBLASTS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Elena Alcalde-Estévez ◽  
Ana Asenjo-Bueno ◽  
Patricia Sosa ◽  
Patricia Plaza ◽  
Diego Rodríguez-Puyol ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Muscle Regeneration ◽  
Uremic Toxins ◽  
C2c12 Cells ◽  
Cyclin B ◽  
Skeletal Muscle Regeneration ◽  
Regeneration Process ◽  
Cycle Arrest ◽  
Advanced Stages ◽  
And Function

Abstract Background and Aims The loss of muscle mass and function has been related to chronic kidney disease (CKD). About 37% of dialysis patients show symptoms of sarcopenia and this has been related to an increased risk of mortality. Changes in sarcopenic muscle include the loss of its regenerative capacity due to a reduction in the number and function of satellite cells, the muscle stem cells. The concentration of serum uremic toxins (UT) increases in parallel to a decline in the glomerular filtration rate in patients with CKD and this uremia may be involved in the development of sarcopenia. Previous studies showed as serum concentration of UT found in the early stages of CKD inhibits myogenic differentiation of cultured myoblasts. Nevertheless, the effect of those concentrations found in the advanced stages of CKD has not been described. The study aimed to analyse whether UT affect the muscular regeneration process by modifying the proliferation capacity of myoblasts (activated satellite cells). Method Cultured mouse myoblasts C2C12 cells were used for all experiments. Cells were grown with 0% or 10% FBS culture media in the presence or absence of indoxyl sulphate and para-cresol at doses of 100µg/ml each one, which are similar to ones found in the advanced stages of CKD. Proliferation was evaluated by scratch wound healing and cell cycle by flow cytometry with propidium iodide and the fluorescent probe CFSE, an intracellular protein binding dye that is divided equally between daughter cells, allowing the discrimination of successive rounds of cell division. Chromosome condensation was assessed by immunofluorescence staining by confocal microscopy. Apoptosis was analysed by annexin V staining. Results C2C12 cells treated with UT shown a significant decrease in the proliferation rate. A significant delay in wound closure was observed in cells treated with UT compared to control cells. Myoblasts treated with UT suffered a significant decrease in the proliferation rate since the probe remained higher than in the vehicle-treated cells. Proliferating cells treated with UT suffered a dramatic cell cycle arrest between the phases S and G2/M. Chromosome condensation was also analysed, finding that in the presence of colcemid, vehicle-treated cells condensed their chromosomes, as expected, whereas UT-treated cells did not, suggesting that UT stop the cell cycle at any point before the entry of cells in the mitosis phase. Besides, there was strong phosphorylation of cdc2 in the presence of UT indicating that cdc2 and the complex cdc2-cyclin B were inactive. This result explains why cells did not enter in the mitosis phase under UT exposition. Finally, UT induced the death of proliferating C2C12 cells by apoptosis. Conclusion In the advanced stages of CKD, uremic toxins concentration increases, thereby inducing a dramatic arrest in the cell cycle of myoblasts, inactivating the cdc2-cyclin B complex, interrupting their proliferation and leading them towards cell apoptosis. These results point to a role of uremic toxins impairing the skeletal muscle regeneration process, which could be involved in CKD-related sarcopenia and frailty.

scholarly journals Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration

2018 ◽  
Vol 32 (3) ◽  
pp. 1452-1467 ◽  
Author(s):  
Yukino Hatazawa ◽  
Yusuke Ono ◽  
Yuma Hirose ◽  
Sayaka Kanai ◽  
Nobuharu L. Fujii ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Differentiation ◽  
Satellite Cell ◽  
Muscle Regeneration ◽  
Skeletal Muscle Regeneration

Adenosine and hypoxia stimulate proliferation and migration of endothelial cells

1988 ◽  
Vol 255 (3) ◽  
pp. H554-H562 ◽  
Author(s):  
C. J. Meininger ◽  
M. E. Schelling ◽  
H. J. Granger
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Culture Medium ◽  
Culture Conditions ◽  
Hypoxic Conditions ◽  
Cell Counts ◽  
And Migration ◽  
Stimulate Cell Proliferation ◽  
Proliferation And Migration

The proliferation of bovine aortic or coronary venular endothelial cells (EC) in vitro was stimulated by the addition of adenosine (0.5 or 5.0 microM) to the culture medium. Cell counts of adenosine-treated aortic EC were 23–76% and coronary venular EC 19–52% greater than nontreated controls. Because adenosine is known to be released by hypoxic tissues, cell proliferation was quantitated when aortic EC were grown at 2% O2. Cell counts were 41–102% greater under hypoxic conditions than when cells were grown at standard tissue culture conditions (approximately 20% O2). When culture medium conditioned by coronary EC grown at 2% O2 was added to EC growing at standard conditions, cell counts were 24–69% greater than controls with medium conditioned by coronary EC grown at 20% O2. This suggests that hypoxia causes endothelial cells to release a factor(s) into the medium that can stimulate cell proliferation. The addition of the adenosine receptor blocker 8-phenyltheophylline (10(-5) M) prevented the stimulation of proliferation caused by hypoxia-conditioned medium, 2% O2 or 5.0 microM adenosine, suggesting that adenosine mediates its effect via an external membrane receptor. Adenosine also stimulated EC chemotaxis. Taken together, these results suggest that adenosine, released as a result of tissue hypoxia, may act as an angiogenic stimulus for the growth of new blood vessels.

scholarly journals MASTR directs MyoD-dependent satellite cell differentiation during skeletal muscle regeneration

2012 ◽  
Vol 26 (2) ◽  
pp. 190-202 ◽  
Author(s):  
M. H. Mokalled ◽  
A. N. Johnson ◽  
E. E. Creemers ◽  
E. N. Olson
Keyword(s):  
Skeletal Muscle ◽  
Cell Differentiation ◽  
Satellite Cell ◽  
Muscle Regeneration ◽  
Skeletal Muscle Regeneration

scholarly journals The circular RNA circCPE regulates myoblast development by sponging miR-138

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wenxiu Ru ◽  
Ao Qi ◽  
Xuemei Shen ◽  
Binglin Yue ◽  
Xiaoyan Zhang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Proliferation ◽  
Muscle Regeneration ◽  
Noncoding Rna ◽  
Muscle Development ◽  
Circular Rna ◽  
Inhibitory Effect ◽  
Skeletal Muscle Regeneration ◽  
Bovine Muscle

Abstract Background Skeletal muscle development, a long-term and complex process, is controlled by a set of the myogenic genes. Circular RNAs (circRNAs), a class of noncoding RNA, have been shown to regulate various biological processes. Recent studies indicate circRNAs may be involved in myogenesis, but the role and regulatory mechanism of circRNAs in myogenesis is largely unknown. In the present study, circCPE was firstly found to promote the bovine myoblast proliferation and inhibit cell apoptosis and differentiation by influencing the expression of FOXC1 in a miR138-mediated manner. And in vivo experiments revealed that overexpression of circCPE attenuates skeletal muscle regeneration. Results We identified a novel circular RNA circCPE by analyzing circRNAs sequencing data of bovine muscle tissue. Sequencing verification, RNase R treatment and Actinomycin D treatment confirmed the circular nature of circCPE in bovine muscle. Functional assays showed that overexpression of circCPE could inhibit bovine myoblast apoptosis and differentiation, as well as facilitate cell proliferation. Moreover, in vivo experiments revealed that overexpression of circCPE attenuates skeletal muscle regeneration. In consideration of circRNA action as miRNAs sponge, we found that circCPE harbors miR-138 binding sites and absorbed miR-138. Mechanistically, the rescue experiments showed that the overexpression of circCPE can counteract the inhibitory effect of miR-138 on the cell proliferation and the accelerated effects on the differentiation and apoptosis. Subsequently, we found that circCPE sequester the inhibitory effect of miR-138 on FOXC1 so as to involve in myogenesis. Conclusions Collectively, we constructed a novel circCPE/miR-138/FOXC1 regulatory network in bovine myogenesis, which further provide stronger evidence that circRNA involved in muscle development acting as miRNA sponge.

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