Neutral sphingomyelinase 2 is required for cytokine-induced skeletal muscle calpain activation

Calpain contributes to infection-induced diaphragm dysfunction but the upstream mechanism(s) responsible for calpain activation are poorly understood. It is known, however, that cytokines activate neutral sphingomyelinase (nSMase) and nSMase has downstream effects with the potential to increase calpain activity. We tested the hypothesis that infection-induced skeletal muscle calpain activation is a consequence of nSMase activation. We administered cytomix (20 ng/ml TNF-α, 50 U/ml IL-1β, 100 U/ml IFN-γ, 10 μg/ml LPS) to C2C12 muscle cells to simulate the effects of infection in vitro and studied mice undergoing cecal ligation puncture (CLP) as an in vivo model of infection. In cell studies, we assessed sphingomyelinase activity, subcellular calcium levels, and calpain activity and determined the effects of inhibiting sphingomyelinase using chemical (GW4869) and genetic (siRNA to nSMase2 and nSMase3) techniques. We assessed diaphragm force and calpain activity and utilized GW4869 to inhibit sphingomyelinase in mice. Cytomix increased cytosolic and mitochondrial calcium levels in C2C12 cells ( P < 0.001); addition of GW4869 blocked these increases ( P < 0.001). Cytomix also activated calpain, increasing calpain activity ( P < 0.02), and the calpain-mediated cleavage of procaspase 12 ( P < 0.001). Procaspase 12 cleavage was attenuated by either GW4869 ( P < 0.001), BAPTA-AM ( P < 0.001), or siRNA to nSMase2 ( P < 0.001) but was unaffected by siRNA to nSMase3. GW4869 prevented CLP-induced diaphragm calpain activation and diaphragm weakness in mice. These data suggest that nSMase2 activation is required for the development of infection-induced diaphragm calpain activation and muscle weakness. As a consequence, therapies that inhibit nSMase2 in patients may prevent infection-induced skeletal muscle dysfunction.

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Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00312.2015 ◽

2016 ◽

Vol 310 (10) ◽

pp. L975-L984 ◽

Cited By ~ 16

Author(s):

Gerald S. Supinski ◽

Alexander P. Alimov ◽

Lin Wang ◽

Xiao-Hong Song ◽

Leigh A. Callahan

Keyword(s):

Skeletal Muscle ◽

Cecal Ligation ◽

C2c12 Cells ◽

Superoxide Generation ◽

Diaphragm Dysfunction ◽

Calpain Activation ◽

Calcium Dependent ◽

Mitochondrial Superoxide ◽

Cecal Ligation Puncture ◽

Diaphragm Weakness

Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A2 (cPLA2) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA2 activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-α 20 ng/ml, IL-1β 50 U/ml, IFN-γ 100 U/ml, LPS 10 μg/ml); 3) cytomix + AACOCF3, a cPLA2 inhibitor (10 μM); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA2 activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 μg/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 μM), a cPLA2 inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA2 activity ( P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation ( P < 0.001). Cytomix also activated calpain ( P < 0.001) and inactivated calpastatin ( P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction ( P < 0.001). cPLA2 modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA2 may prevent diaphragm weakness in infected, critically ill patients.

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Anti-Diabetic Effects and Anti-Inflammatory Effects of Laminaria japonica and Hizikia fusiforme in Skeletal Muscle: In Vitro and In Vivo Model

Nutrients ◽

10.3390/nu10040491 ◽

2018 ◽

Vol 10 (4) ◽

pp. 491 ◽

Cited By ~ 11

Author(s):

Sae-ym Kang ◽

Eunyoung Kim ◽

Inhae Kang ◽

Myoungsook Lee ◽

Yunkyoung Lee

Keyword(s):

Skeletal Muscle ◽

Laminaria Japonica ◽

In Vivo Model ◽

Hizikia Fusiforme ◽

Anti Inflammatory

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The in vitro effect of corticosterone on insulin binding and glucose metabolism in mouse skeletal muscles

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y85-185 ◽

1985 ◽

Vol 63 (9) ◽

pp. 1133-1138 ◽

Cited By ~ 10

Author(s):

M. H. Tan ◽

A. Bonen

Keyword(s):

Skeletal Muscle ◽

Skeletal Muscles ◽

Extensor Digitorum Longus ◽

Insulin Binding ◽

In Vivo Model ◽

In Vitro System ◽

Vitro Effect ◽

Contralateral Muscle

We studied the in vitro effect of corticosterone on insulin binding, uptake of 2-deoxy-D-glucose, glycolysis, and glycogenesis in the soleus and extensor digitorum longus (EDL) of Swiss–Webster mice. In each experiment, one muscle (soleus/EDL) was incubated with corticosterone (0.1, 1, 50, and 100 μg/mL) and the respective contralateral muscle was incubated without corticosterone, but at the same insulin and pH levels. Corticosterone did not affect insulin binding in both muscles. However, corticosterone decreased the uptake of 2-deoxy-D-glucose and the rate of glycolysis and glycogenesis in both muscles when the dose was pharmacologic (50 and 100 μg/mL), but not when it was physiologic (0.1 and 1 μg/mL). For glycolysis and glycogenesis, the suppression was greater in the EDL when compared with the soleus. This suppression was seen in both basal and insulin-stimulated conditions. In this in vitro system, where the experimental muscle is not exposed to prior hyperinsulinemia as in the in vivo model, corticosterone, at pharmacologic doses, affects postreceptor events without altering the insulin binding in the skeletal muscle.

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The Effect of Relaxin Treatment on Skeletal Muscle Injuries

The American Journal of Sports Medicine ◽

10.1177/0363546505278701 ◽

2005 ◽

Vol 33 (12) ◽

pp. 1816-1824 ◽

Cited By ~ 73

Author(s):

Shinichi Negishi ◽

Yong Li ◽

Arvydas Usas ◽

Freddie H. Fu ◽

Johnny Huard

Keyword(s):

Skeletal Muscle ◽

Growth Factor ◽

Functional Recovery ◽

Muscle Regeneration ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

C2c12 Cells ◽

Muscle Injuries

Background Injured skeletal muscle can repair itself via spontaneous regeneration; however, the overproduction of extracellular matrix and excessive collagen deposition lead to fibrosis. Neutralization of the effect of transforming growth factor-β1, a key fibrotic cytokine, on myogenic cell differentiation after muscle injury can prevent fibrosis, enhance muscle regeneration, and thereby improve the functional recovery of injured muscle. Hypothesis The hormone relaxin, a member of the family of insulin-like growth factors, can act as an antifibrosis agent and improve the healing of injured muscle. Study Design Controlled laboratory study. Methods In vitro: Myoblasts (C2C12 cells) and myofibroblasts (transforming growth factor-β1-transfected myoblasts) were incubated with relaxin, and cell growth and differentiation were examined. Myogenic and fibrotic protein expression was determined by Western blot analysis. In vivo: Relaxin was injected intramuscularly at different time points after laceration injury. Skeletal muscle healing was evaluated via histologic, immunohistochemical, and physiologic tests. Results Relaxin treatment resulted in a dose-dependent decrease in myofibroblast proliferation, down-regulated expression of the fibrotic protein α-smooth muscle actin, and promoted the proliferation and differentiation of myoblasts in vitro. Relaxin therapy enhanced muscle regeneration, reduced fibrosis, and improved injured muscle strength in vivo. Conclusion Administration of relaxin can significantly improve skeletal muscle healing. Clinical Relevance These findings may facilitate the development of techniques to eliminate fibrosis, enhance muscle regeneration, and improve functional recovery after muscle injuries.

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6-Bromoindirubin-3′-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice

Scientific Reports ◽

10.1038/s41598-019-54574-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Elvira Ragozzino ◽

Mariarita Brancaccio ◽

Antonella Di Costanzo ◽

Francesco Scalabrì ◽

Gennaro Andolfi ◽

...

Keyword(s):

Skeletal Muscle ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Skeletal Muscle Differentiation ◽

Myoblast Proliferation ◽

Enhanced Expression ◽

Pharmacologically Active

AbstractDystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Thus, new drugs for restoring skeletal muscle deterioration are critically needed. To identify new and alternative compounds with a functional role in skeletal muscle myogenesis, we screened a library of pharmacologically active compounds and selected the small molecule 6-bromoindirubin-3′-oxime (BIO) as an inhibitor of myoblast proliferation. Using C2C12 cells, we examined BIO’s effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from cell proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO is able to promote myogenic differentiation in damaged myotubes in-vitro by enriching the population of newly formed skeletal muscle myotubes. Moreover, in-vivo experiments in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers number. Additionally, we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was observed in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was observed in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was observed upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a positive modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.

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Jianpi Qinghua Formula Reduced Intramyocellular Lipids (IMCLs) by Inhibiting Excessive Activation of mTORC1

10.21203/rs.3.rs-136215/v1 ◽

2021 ◽

Author(s):

Xv Han ◽

Qingguang Chen ◽

Yahua Liu ◽

Junfei Xv ◽

Hao Lu

Keyword(s):

Skeletal Muscle ◽

Insulin Sensitivity ◽

Palmitic Acid ◽

High Fat Diet ◽

C2c12 Cells ◽

Akt Pathway ◽

High Fat ◽

Akt Phosphorylation

Abstract Background:IMCLs are an important factor in skeletal muscle insulin resistance. This study aimed to explore the effect of Jianpi Qinghua formula (JPQHF) on IMCLs and its mechanism, as well as the relationship between IMCLs and other skeletal muscle insulin sensitivity factors, thereby elucidating the mechanism by which JPQHF improves insulin sensitivity.Methods: In an in vivo experiment, JPQHF and pioglitazone (PIO) were individually used to treat C57 mice with high-fat diet-induced obesity. In an in vitro experiment, JPQHF and rapamycin in serum were individually used to treat C2C12 cells induced with palmitic acid. The IMCLs of tissue and cells were subjected to oil red O staining. The RNA and protein expression of PPARγ, myogenin, mTORC1 and members of the PI3K/AKT pathway in skeletal muscle tissue and C2C12 cells was examined. Differences between the different intervention groups were determined.Results: IMCLs were significantly increased in mice with obesity induced by a high-fat diet and the C2C12 cell line treated with palmitic acid compared to the corresponding controls. mTORC1 phosphorylation and PPARγ levels were also increased, and AKT phosphorylation and myogenin levels were decreased. Intervention with JPQHF reversed the above changes. In addition, the PPARγ level in C2C12 cells was reduced after intervention with rapamycin, an inhibitor of mTORC1. However, AKT phosphorylation and myogenin levels did not recover after rapamycin intervention.Conclusion: IMCLs were significantly increased in obese C57 mice and palmitic acid-treated C2C12 cells. JPQHF reduced IMCLs both in vivo and in vitro. Mechanistically, this effect likely occurred through JPQHF-mediated inhibition of the overactivation of mTORC1 and a subsequent reduction in the expression of PPARγ. However, the function of JPQHF in elevating myogenin levels and the PI3K/AKT pathway may not be entirely dependent on mTORC1.

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Prion Infection of Muscle Cells In Vitro

Journal of Virology ◽

10.1128/jvi.02628-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4615-4624 ◽

Cited By ~ 21

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.

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Ascorbate inhibits iNOS expression and preserves vasoconstrictor responsiveness in skeletal muscle of septic mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00564.2002 ◽

2003 ◽

Vol 285 (1) ◽

pp. R50-R56 ◽

Cited By ~ 50

Author(s):

Feng Wu ◽

John X. Wilson ◽

Karel Tyml

Keyword(s):

Skeletal Muscle ◽

Arterial Pressure ◽

Vascular Reactivity ◽

Cecal Ligation ◽

Interferon Γ ◽

Inos Expression ◽

Inos Mrna ◽

Valuable Adjunct

Inducible nitric oxide synthase (iNOS) expression in blood vessels contributes to the vascular hyporeactivity characteristic of sepsis. Our previous work demonstrated in vitro that ascorbate inhibits iNOS expression in lipopolysaccharide- and interferon-γ-stimulated skeletal muscle endothelial cells (ECs) through an antioxidant mechanism. The present study evaluated in vivo the hypothesis that administration of ascorbate decreases oxidative stress, prevents endothelial iNOS expression, and improves vascular reactivity in septic skeletal muscle. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Plasma nitrite and nitrate (NOx) levels were elevated by 6 h after CLP. Prior ascorbate bolus injection (200 mg/kg body wt iv) blocked the elevation of plasma NOx and abolished the expression of iNOS protein and activity in the septic skeletal muscle. We also demonstrated that iNOS mRNA determined by RT-PCR was induced in the microvascular ECs of the muscle at 3 h after CLP. This induction was attenuated by prior ascorbate administration. Ascorbate inhibition of iNOS expression was associated with decreased oxidant levels in the septic muscle. Moreover, ascorbate administration restored partially the baseline arterial pressure and preserved completely the microvascular constriction and arterial pressure responses to norepinephrine in CLP mice. These results suggest that early administration of ascorbate may be a valuable adjunct treatment of sepsis.

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Interaction of Fibromodulin and Myostatin to Regulate Skeletal Muscle Aging: An Opposite Regulation in Muscle Aging, Diabetes, and Intracellular Lipid Accumulation

Cells ◽

10.3390/cells10082083 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2083

Author(s):

Eun-Ju Lee ◽

Syed-Sayeed Ahmad ◽

Jeong-Ho Lim ◽

Khurshid Ahmad ◽

Sibhghatulla Shaikh ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Lipid Accumulation ◽

C2c12 Cells ◽

Gene Expressions ◽

Mstn Gene ◽

Muscle Aging ◽

Skeletal Muscle Aging

The objective of this study was to investigate fibromodulin (FMOD) and myostatin (MSTN) gene expressions during skeletal muscle aging and to understand their involvements in this process. The expressions of genes related to muscle aging (Atrogin 1 and Glb1), diabetes (RAGE and CD163), and lipid accumulation (CD36 and PPARγ) and those of FMOD and MSTN were examined in CTX-injected, aged, MSTN−/−, and high-fat diet (HFD) mice and in C2C12 myoblasts treated with ceramide or grown under adipogenic conditions. Results from CTX-injected mice and gene knockdown experiments in C2C12 cells suggested the involvement of FMOD during muscle regeneration and myoblast proliferation and differentiation. Downregulation of the FMOD gene in MSTN−/− mice, and MSTN upregulation and FMOD downregulation in FMOD and MSTN knockdown C2C12 cells, respectively, during their differentiation, suggested FMOD negatively regulates MSTN gene expression, and MSTN positively regulates FMOD gene expression. The results of our in vivo and in vitro experiments indicate FMOD inhibits muscle aging by negatively regulating MSTN gene expression or by suppressing the action of MSTN protein, and that MSTN promotes muscle aging by positively regulating the expressions of Atrogin1, CD36, and PPARγ genes in muscle.

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Improved Muscle Healing after Contusion Injury by the Inhibitory Effect of Suramin on Myostatin, a Negative Regulator of Muscle Growth

The American Journal of Sports Medicine ◽

10.1177/0363546508322886 ◽

2008 ◽

Vol 36 (12) ◽

pp. 2354-2362 ◽

Cited By ~ 65

Author(s):

Masahiro Nozaki ◽

Yong Li ◽

Jinhong Zhu ◽

Fabrisia Ambrosio ◽

Kenji Uehara ◽

...

Keyword(s):

Skeletal Muscle ◽

Functional Recovery ◽

Muscle Regeneration ◽

Inhibitory Effect ◽

Negative Regulator ◽

C2c12 Cells ◽

Dependent Manner ◽

Contusion Injury

Background Muscle contusions are the most common muscle injuries in sports medicine. Although these injuries are capable of healing, incomplete functional recovery often occurs. Hypothesis Suramin enhances muscle healing by both stimulating muscle regeneration and preventing fibrosis in contused skeletal muscle. Study Design Controlled laboratory study. Methods In vitro: Myoblasts (C2C12 cells) and muscle-derived stem cells (MDSCs) were cultured with suramin, and the potential of suramin to induce their differentiation was evaluated. Furthermore, MDSCs were cocultured with suramin and myostatin (MSTN) to monitor the capability of suramin to neutralize the effect of MSTN. In vivo: Varying concentrations of suramin were injected in the tibialis anterior muscle of mice 2 weeks after muscle contusion injury. Muscle regeneration and scar tissue formation were evaluated by histologic analysis and functional recovery was measured by physiologic testing Results In vitro: Suramin stimulated the differentiation of myoblasts and MDSCs in a dose-dependent manner. Moreover, suramin neutralized the inhibitory effect of MSTN on MDSC differentiation. In vivo: Suramin treatment significantly promoted muscle regeneration, decreased fibrosis formation, reduced myostatin expression in injured muscle, and increased muscle strength after contusion injury. Conclusion Intramuscular injection of suramin after a contusion injury improved overall skeletal muscle healing. Suramin enhanced myoblast and MDSC differentiation and neutralized MSTN's negative effect on myogenic differentiation in vitro, which suggests a possible mechanism for the beneficial effects that this pharmacologic agent exhibits in vivo. Clinical Relevance These findings could contribute to the development of biological treatments to aid in muscle healing after experiencing a muscle injury.

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