Mouse Skeletal Muscle Cell Line Research Articles

Effect of 20-hydroxyecdysone and its metabolites in the absence or presence of IGF-1 on regulation of skeletal muscle cell growth

Purpose: To investigate the effect of 20-hydroxyecdysone (20E) and its metabolites and their synergistic effect with IGF-1 on regulation of skeletal muscle cell growth.
 Methods: Mouse skeletal muscle cell line (C2C12) was solely treated with 20E and its metabolites (14- deoxy- 20-hydroxyecdysone, poststerone, and 14-deoxypoststerone) at doses of 0.1, 1, and 10 µM or co-treated with IGF-1 (10 ng/ml). Cell viability and proliferative capacity were evaluated using MTT and BrdU incorporation assays, respectively. Myogenic differentiation proteins [embryonic myosin heavy chain (EbMHC) and MHC], androgen receptor (AR), and IGF-1 receptor (IGF-1R) protein expression were investigated using immunocytochemistry.
 Results: Treatments of 20E and its metabolites had no toxicity on skeletal muscle cells or induced AR/IGF-1R expression. In addition, solely treatment of 20E and its metabolites or co-treatment with IGF-1 had no significant effect on cell proliferation and myogenic differentiation capacity. In contrast, IGF-1 treatment alone significantly increased EbMHC expression (p<0.0001), MHC expression (p<0.05), and myotube number (p<0.05).
 Conclusion: These results indicate that 20E and its metabolites have no direct or synergistic effect with IGF-1 on skeletal muscle cell growth. Nevertheless, the pharmacological effects of 20E on skeletal muscle mass and strength in vivo that raises its therapeutic potential may associate with its indirect action.

TNF-α Induced Myotube Atrophy in C2C12 Cell Line Uncovers Putative Inflammatory-Related lncRNAs Mediating Muscle Wasting.

Background: Muscle atrophy is a complex catabolic condition developing under different inflammatory-related systemic diseases resulting in wasting of muscle tissue. While the knowledge of the molecular background of muscle atrophy has developed in recent years, how the atrophic conditions affect the long non-coding RNA (lncRNAs) machinery and the exact participation of the latter in the mediation of muscle loss are still unknown. The purpose of the study was to assess how inflammatory condition developing under the tumor necrosis factor alpha (TNF-α) treatment affects the lncRNAs’ expression in a mouse skeletal muscle cell line. Materials and method: A C2C12 mouse myoblast cell line was treated with TNF-α to develop atrophy, and inflammatory-related lncRNAs mediating muscle loss were identified. Bioinformatics was used to validate and analyze the discovered lncRNAs. The differences in their expression under different TNF-α concentrations and treatment times were investigated. Results: Five lncRNAs were identified in a discovery set as atrophy related and then validated. Three lncRNAs, Gm4117, Ccdc41os1, and 5830418P13Rik, were selected as being significant for inflammatory-related myotube atrophy. Dynamics changes in the expression of lncRNAs depended on both TNF-α concentration and treatment time. Bioinformatics analysis revealed the mRNA and miRNA target for selected lncRNAs and their putative involvement in the molecular processes related to muscle atrophy. Conclusions: The inflammatory condition developing in the myotube under the TNF-α treatment affects the alteration of lncRNAs’ expression pattern. Experimental and bioinformatics testing suggested the prospective role of lncRNAs in the mediation of muscle loss under an inflammatory state.

Interleukin-15 receptor subunit alpha regulates interleukin-15 localization and protein expression in skeletal muscle cells.

What is the central question of this study? How are the dynamics of interleukin (IL)-15 and its receptors altered during the differentiation of myoblasts into myotubes, and how is IL-15 regulated? What is the main finding and its importance? The mRNA levels of IL-15 and interleukin-2 receptor subunits beta and gamma increase during skeletal muscle differentiation, whereas interleukin-15 receptor subunit alpha (IL-15RA) exhibits different kinetics. IL-15RA regulates the localization and expression of IL-15 at the protein level. Interleukin-15 (IL-15) is a myokine in the interleukin-2 (IL-2) family that is generated in the skeletal muscle during exercise. The functional effect of IL-15 involves muscle regeneration and metabolic regulation in skeletal muscle. Reports have indicated that interleukin-15 receptor subunit alpha (IL-15RA) acts by regulating IL-15 localization in immune cells. However, the dynamics of IL-15 and its receptors, which regulate the IL-15 pathway in skeletal muscle differentiation, have not yet been clarified. In this study, we investigated the mechanism of IL-15 regulation using a mouse skeletal muscle cell line, C2C12 cells. We found that the mRNA expression of IL-15, interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132) increased, but that IL-15RA exhibited different kinetics as differentiation progressed. We also found that IL-15, mainly present in the cytosol, pre-assembled with IL-15RA in the cytosol and fused to the plasma membrane. Moreover, IL-15RA increased IL-15 protein levels. Our findings suggest that genes involved in the IL-15 signalling complex are enhanced with the differentiation of myotubes and that IL-15RA regulates the protein kinetics of IL-15 signalling in skeletal muscle.

Antioxidative Activity of Soy, Wheat and Pea Protein Isolates Characterized by Multi-Enzyme Hydrolysis.

Hydrolysis of protein by proteases produces small molecular weights (MWs) peptides as nanomaterials that are easily absorbed. This study investigated the physicochemical properties and antioxidant activity of three plant protein isolates (PIs) including soy, wheat and pea after multi-enzyme hydrolysis. The MWs, particle size and microstructure of PI hydrolysate (PIH) were determined by SDS-PAGE and MALDI-TOF-MS mass spectrometry, dynamic light scattering and transmission electron microscopy, respectively. Cell viability was determined in vitro using a mouse skeletal muscle cell line (C2C12) and crystal violet staining. The MWs and particle sizes of the three plant PIs were reduced after hydrolysis by three proteases (bromelain, Neutrase and Flavourzyme). The MWs of soy, wheat and pea PIH were 103.5–383.0 Da, 103.5–1146.5 Da and 103.1–1937.7 Da, respectively, and particle size distributions of 1.9–2.0 nm, 3.2–5.6 nm and 1.3–3.2 nm, respectively. All three plant PIHs appeared as aggregated nanoparticles. Soy PIH (100 μg/mL) provided better protection against H2O2-induced oxidative damage to C2C12 than wheat or pea PIH. In summary, soy PIH had the best antioxidant activity, and particle size than wheat PIH and pea PIH. Therefore, soy PIH might be a dietary supplement for healthy diet and medical applications.

Molecular Toxicological Mechanisms of Synthetic Cathinones on C2C12 Myoblasts.

Synthetic cathinones are popular psychoactive substances that may cause skeletal muscle damage. In addition to indirect sympathomimetic myotoxicity, these substances could be directly myotoxic. Since studies in myocytes are currently lacking, the aim of the present study was to investigate potential toxicological effects by synthetic cathinones on C2C12 myoblasts (mouse skeletal muscle cell line). We exposed C2C12 myoblasts to 3-methylmethcathinone, 4-methylmethcathinone (mephedrone), 3,4-methylenedioxymethcathinone (methylone), 3,4-methylenedioxypyrovalerone (MDPV), alpha-pyrrolidinovalerophenone (α-PVP), and naphthylpyrovalerone (naphyrone) for 1 or 24 h before cell membrane integrity, ATP content, mitochondrial oxygen consumption, and mitochondrial superoxide production was measured. 3,4-Methylenedioxymethamphetamine (MDMA) was included as a reference compound. All investigated synthetic cathinones, as well as MDMA, impaired cell membrane integrity, depleted ATP levels, and increased mitochondrial superoxide concentrations in a concentration-dependent manner in the range of 50–2000 μM. The two pyrovalerone derivatives α-PVP and naphyrone, and MDMA, additionally impaired basal and maximal cellular respiration, suggesting mitochondrial dysfunction. Alpha-PVP inhibited complex I, naphyrone complex II, and MDMA complex I and III, whereas complex IV was not affected. We conclude that, in addition to sympathetic nervous system effects and strenuous muscle exercise, direct effects of some cathinones on skeletal muscle mitochondria may contribute to myotoxicity in susceptible synthetic cathinone drugs users.

Ameliorative effect of Ruellia tuberosa L. on hyperglycemia in type 2 diabetes mellitus and glucose uptake in mouse C2C12 myoblasts.

Ruellia tuberosa L. (RTL) exhibits a wide range of phytochemical activities, for example, on treatment of diabetes mellitus (DM), in Orient. There is, however, few study regarding the effect of RTL on glycemic‐related homeostasis in type 2 DM (T2DM). We investigated the effect of RTL aqueous and ethanolic extracts on hypoglycemia in high‐fat diet (HFD)‐fed plus streptozotocin (STZ)‐induced T2DM rats, and examined the effect of RTL on glucose uptake in tumor necrosis factor‐α‐induced insulin‐resistant mouse C2C12 myoblasts, a mouse skeletal muscle cell line. The administration of 100 or 400 mg kg BW−1 day−1 of RTL aqueous or ethanolic extracts once a day for 4 weeks significantly ameliorated hyperglycemia, hyperinsulinemia, and the insulin resistance (IR) index in diabetic rats. RTL either aqueous or ethanolic extract at a concentration of 25–800 μg/ml significantly improved glucose uptake in insulin‐resistant mouse C2C12 myoblasts, indicating inhibiting the IR in skeletal muscles. These evidences suggest that RTL ameliorates hyperglycemia in HFD/STZ‐induced T2DM rats may be attributed to the alleviation of IR in skeletal muscles.

Expression of Thyroid Stimulating Hormone Receptor mRNA in Mouse C2C12 Skeletal Muscle Cells

BackgroundWe analyzed whether thyroid stimulating hormone receptor (TSH-R) is expressed in a skeletal muscle cell line and if TSH has influence on the differentiation of muscle cells or on the determination of muscle fiber types.MethodsTSH-R gene expression was detected with nested real-time polymerase chain reaction (RT-PCR) in C2C12, a mouse skeletal muscle cell line. The effect of TSH on myotube differentiation was assessed by microscopic examination of myotube formation and through the measurement of expression of muscle differentiation markers, i.e., myogenin and myoD, and muscle type-specific genes, i.e., MyHC1, MyHC2a, and MyHC2b, with quantitative RT-PCR before and after incubation of C2C12 myotube with TSH.ResultsTSH-R was expressed in the mouse skeletal muscle cell line. However, treatment with TSH had little effect on the differentiation of muscle cells, although the expression of the muscle differention marker myogenin was significantly increased after TSH treatment. Treatment of TSH did not affect the expression of muscle type-specific genes.ConclusionTSH-R is expressed in a mouse skeletal muscle cell line, but the role of TSH receptor signaling in skeletal muscle needs further investigation.

The extract G115 of Panax ginseng C.A. Meyer enhance energy production in mammals

A number of studies demonstrated that Ginseng increases endurance and vitality.The mechanism of action in these fields has not been clarified. We aimed to evaluate the ability of a standardized extract of Ginseng (G115) to improve energy production and metabolic activity in C2C12 mouse skeletal muscle cell line and male C3H/He mice. In vitro, digested G115 was used at concentration of 10–50–100–200µg/ml for 24, 48 and 72 hours. Animals were treated with G115at 25mg/kg daily by oral administration. Gene expression of PGC-1a (gene involved in mitochondria biogenesis), AMPK and SIRT1 was evaluated by Real Time PCR. ATP production and O2 consumption were evaluated. The same parameters were evaluated in the gastrocnemius muscle collected from mice after two weeks of treatment. A concentration/effect relationship was observed in in vitro experiments. At higher concentration the effects declined to basal values. Gene expression of PGC-1a increased by 40% after 72 hours of incubation with 50 mcg/ml of G115. Similar behaviour was observed of AMPK and SIRT1 gene expression. ATP production was 21.6±2.3 nmol/mg proteins and 33.6±5.8 nmol/mg proteins in the control and treated cells respectively and O2 consumption was 11.3±1.5 mmol/min/mg proteins and 27.3±6.1 mmol/min/mg proteins in the control and treated cells respectively, after 72 hours incubation. The in vivo experiments confirmed the results observed in vitro. This study shows that the extract of Panax ginseng C.A. Meyer G115 is able to increase mitochondrial function and this may explain the its ability to increase endurance and vitality.

1α,25‐dihydroxyvitamin D3 enhances fast‐myosin heavy chain expression in differentiated C2C12 myoblasts

We investigated the effect of VD3 (1α,25-dihydroxyvitamin D3) on the proliferating, differentiating and differentiated phases of C2C12 myoblasts, a mouse skeletal muscle cell line. VD3 treatment in 10% FBS (fetal bovine serum) inhibited the proliferation and viability of the cells in a dose-dependent manner. It also dose-dependently increased the percentage of cells in the G0/G1 phase as shown by flow cytometry. In the differentiating phase, VD3 treatment inhibited the formation of myotubes and the expression of total myosin heavy chain at both the mRNA and protein levels. In the differentiated phase, treatment had no significant effect on the amount of total myosin heavy chain, as Western blot analysis with MF20 antibody [DSHB (Developmental Studies Hybridoma Bank)] showed. However, significantly greater expression of fast myosin heavy chain in 1 nM VD3 was found by Western blot analysis with MY-32 (Sigma). Thus VD3 inhibited the proliferation of myoblasts during proliferating and differentiating phases, whereas it increased the expression of the fast myosin heavy chain isoform in the differentiated phase. The data indicate that an adequate concentration of VD3 might have an anabolic effect on differentiated skeletal muscle.

Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis.

NMR Based Metabonomics Study of DAG Treatment in a C2C12 Mouse Skeletal Muscle Cell Line Myotube Model of Burn-Injury

After severe burn injury, proinflammatory cytokine levels are elevated in serum and skeletal muscle, which in turn increases protein breakdown and decreases protein synthesis. In this study, C2C12 mouse skeletal muscle cell line myotubes were exposed to proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) as an in vitro cell-line model of catabolic response to burn injury and then treated with des-acyl ghrelin (DAG), a 28 amino acid polypeptide hormone thought to inhibit protein breakdown and increase protein synthesis, to assess its therapeutic potential. Nuclear magnetic resonance-based metabonomics was used to monitor metabolic activity of C2C12 myotubes under four treatment conditions: (1) control, (2) TNF-α/IFN-γ (TI), (3) DAG (DA), and (4) TNF-α/IFN-γ followed by DAG (TIDA) to assess the effect of DAG treatment on cellular metabolic response during basal or catabolic conditions. Twelve metabolites showed significant changes in concentrations following treatments in the hydrophilic cell extracts. Lactate (P < 10−4) and citrulline (P < 10−9) increased with TNF-α/IFN-γ treatment, indicating increased protein degradation, and returned to control levels in the TIDA group. Adenosine nucleotide levels had decreased trends in TI myotubes that returned to baseline levels after DAG treatment (P < 10−4). Guanidinoacetate and pantothenate, metabolites involved in protein synthesis and cell proliferation, had increased concentration trends following DAG treatment in both the DA and TIDA groups. Our metabonomics analysis provides further evidence that DAG counteracts the catabolic response caused by elevated muscle TNF-α/IFN-γ cytokine levels following severe burns and can play a potential therapeutic role in treatment of burn injury.

Identification of Differentially Regulated Secretome Components During Skeletal Myogenesis

Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.

Troponin T3 Regulates Calcium Channel Beta1a Subunit Nuclear Translocation in Skeletal Muscle

Voltage-dependent calcium channel (VDCC) beta subunits (CaVβ) are critical for CaVα subunit membrane expression and gating properties. While CaVβ interacts with a variety of other molecular partners, nonchannel functions of skeletal muscle CaVβ1a have not been reported. In a yeast two hybrid (Y2H) screening of the mouse tibialis anterior (TA) muscle cDNA library, troponin T3 (TNNT3), specific to fast skeletal muscle, was identified using full-length CaVβ1a as the bait. Further studies revealed that TNNT3, but not the slow skeletal muscle TNNT1, co-immunoprecipitated with CaVβ1a in the mouse skeletal muscle cell line C2C12 and mouse skeletal muscle. Consistently, both CaVβ1a and TNNT3 were highly expressed in mouse fast, but not slow, skeletal muscle. Surprisingly, when DsRed-tagged TNNT3 and YFP tagged CaVβ1a (CaVβ1a -YFP) were transiently co-expressed in C2C12 cells or the mouse flexor digitorum brevis (FDB) muscle, both showed a punctuate distribution pattern in the cytoplasm and nucleus. In comparison, CaVβ1a-YFP alone localized uniformly in the cytoplasm. Further truncation analysis revealed that TNNT3 C-terminus (aa 161-244) localized exclusively in FDB nuclei and enriched CaVβ1a -YFP accordingly. In contrast, the TNNT3 middle region (aa 55-160) localized only in the cytoplasm, while the N-terminus (aa 1-54) was found in both cytoplasm and nucleus. Note that the N-terminus could also recruit CaVβ1a-YFP in the nucleus. Y2H assay verified that the TNNT3 C-terminus has the strongest interaction with CaVβ1a. Immunoblotting with an antibody targeting the TNNT3 N-terminus detected an increased TNNT3 fragment in the aging mouse skeletal muscle. We conclude that TNNT3 interacts with CaVβ1a and regulates its nuclear translocation mainly through its TNNT3 C- or N-terminus. These findings strongly support a transcription-regulation function for CaVβ1a and TNNT3.

Ecdysteroids elicit a rapid Ca 2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells

Phytoecdysteroids, structurally similar to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. In skeletal muscle cells, phytoecdysteroids increase protein synthesis. In this study we show that in a mouse skeletal muscle cell line, C 2C 12, 20-hydroxyecdysone (20HE), a common phytoecdysteroid in both insects and plants, elicited a rapid elevation in intracellular calcium, followed by sustained Akt activation and increased protein synthesis. The effect was inhibited by a G-protein coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, and a phosphoinositide kinase-3 (PI3K) inhibitor.

Selenoprotein W depletion in vitro might indicate that its main function is not as an antioxidative enzyme

Examination of the antioxidative homeostasis in skeletal muscle cells in the presence or absence of selenoprotein W (SelW) is necessary to understand the importance of SelW in the antioxidative system. Depletion of SelW by RNA interference was achieved by introducing a synthetic small interfering RNA into the mouse skeletal muscle cell line C2C12 (C3H). Transfectant screening was performed by real-time reverse transcription-PCR, Western blotting, flow cytometry, fluorescence staining, cell viability, and glutathione assays. SelW expression and mRNA levels were downregulated by 62.1 and 72.4%, respectively. In addition, acute cytotoxicity and an apoptosis rate of approximately 36% in SelW-depleted cells demonstrated that RNA interference was successful. As compared with non-SelW-depleted cells, the enzyme activities of glutathione peroxidase, superoxide dismutase, and catalase and total antioxidative capability and glutathione level increased by 47.6, 103.0, 31.0, 205.6, and 30.0%, respectively (P < 0.05). Thus, SelW is important for the antioxidative system of muscle cells. Depletion of SelW, however, could be compensated by other intracellular antioxidative enzymes because oxidative stress was not the causative factor for apoptosis in SelW-depleted cells. Thus, the main function of SelW in muscle cells is not in the antioxidative system.

The Alterations of Store-Operated Calcium Entry in TRPC1-Overexpressing C2C12 Myotubes

When the endoplasmic reticulum (ER) calcium store is depleted, a Ca2+ influx is activated from the extracellular milieu to refill the intracellular stores. This well-regulated Ca2+ uptake mechanism, called store-operated Ca2+ entry (SOCE), depends on the cooperation of several proteins as STIM1, Orai1 and TRPC1. The role of STIM1 as the calcium sensor of the ER and Orai1 as the Ca2+ influx channel is well-known from the recent publications, but the function of TRPC1 as a store-operated channel remains elusive.Here TRPC1 was overexpressed by liposome-mediated transfection in C2C12 mouse skeletal muscle cell line. Overexpression was confirmed at mRNA level by RT-PCR and at protein level by immunostaining and Western-blot. The SOCE mechanism was studied by measuring the changes in [Ca2+]i evoked by the re-addition of 1.8 mM [Ca2+]e following the SERCA-inhibition by thapsigargin. As a result of TRPC1 overexpression, the amplitude and the maximum of the derivative of SOCE was significantly increased. When YM-58483, the antagonist of TRPC1 was used, these differences were eliminated, moreover in TRPC1-overexpressing myotubes the SOCE was slightly but not significantly lower, suggesting the downregulation of the STIM1-Orai1 system. This decrease in the expression level of STIM1 was confirmed by Western-blot together with the downregulation of SERCA. As a consequence a reduction in maximal Ca2+ uptake, and a higher resting [Ca2+]i following the transients evoked by 120 mM KCl were detected. Morphological changes caused by the overexpression of TRPC1 were also observed. The differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures.Our results suggest that enhancing the expression level of TRPC1 increases SOCE and has a negative feedback effect on the STIM1-Orai1 system, suggesting a cooperation between these proteins.

Lipopolysaccharide‐induced monocyte chemotactic protein‐1 is enhanced by suppression of nitric oxide production, which depends on poor CD14 expression on the surface of skeletal muscle

It is known that lipopolysaccharide (LPS)-induced monocyte chemotactic protein (MCP)-1 secretion from tissues recruits monocytes from the circulation, but the mechanism of the LPS-induced MCP-1 production in skeletal muscle is largely unexplained. To clarify the effect of LPS on MCP-1 production in skeletal muscle cells, C2C12 cells from a mouse skeletal muscle cell line, and RAW 264.7 cells from a mouse macrophage cell line, were used to assess production of LPS-induced MCP-1, nitric oxide (NO) and interferon (IFN)-beta. In addition, we evaluated inducible NO synthases (iNOS) mRNA expression using RT-PCR, and cell surface expression of CD14 and toll-like receptor (TLR) 4 using flow cytometry. In C2C12 cells, LPS stimulation increased MCP-1 production (p < 0.01), but combined treatment with LPS and NO inducer, diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (NONOate), significantly inhibited its production (p < 0.01). LPS stimulation neither induced production of NO nor of IFN-beta, which is an NO inducer. Recombinant IFN-beta stimulation, on the other hand, enhanced LPS-induced NO production (p < 0.01). Interestingly, we found that surface expression of CD14, which regulates IFN-beta production, in C2C12 cells was much lower than that in RAW 264.7 cells, although TLR4 expression on C2C12 cells was similar to that on RAW 264.7 cells. These data suggest that the reduced NO production in response to LPS may depend on low expression of CD14 on the cell surface of skeletal muscle, and that it may enhance LPS-induced MCP-1 production. Together, these functions of skeletal muscle could decrease the risk of bacterial infection by recruitment of monocytes.

Small-Molecule Fluorophores To Detect Cell-State Switching in the Context of High-Throughput Screening

A small molecule capable of distinguishing the distinct states resulting from cellular differentiation would be of enormous value, for example, in efforts aimed at regenerative medicine. We screened a collection of fluorescent small molecules for the ability to distinguish the differentiated state of a mouse skeletal muscle cell line. High-throughput fluorescence-based screening of C2C12 myoblasts and myotubes resulted in the identification of six compounds with the desired selectivity, which was confirmed by high-content screening in the same cell states. The compound that resulted in the greatest fluorescence intensity difference between the cell states was used as the screening agent in a pilot screen of 84 kinase inhibitors, each present in four doses, for inhibition of myogenesis. Of the kinase inhibitors, 17 resulted in reduction of fluorescence at one or more concentrations; among the "hits" included known inhibitors of myogenesis, confirming that this compound is capable of detecting the differentiated myotube state. We suggest that the strategy of screening for screening agents reported here may be extended more broadly in the future.

Muscle specific Ring finger‐1 (MuRF‐1) siRNA in C2C12 cells leads to an increase in Troponin I protein

We have previously shown that the E3 ubiquitin ligase, MuRF-1, is elevated in animal models of muscle atrophy, providing evidence for the involvement of MuRF-1 in divergent models of skeletal muscle wasting. MuRF-1 knockout mice show a resistance to denervation-induced atrophy (Bodine et al. 2001), further implicating MuRF-1 activity in the regulation of muscle wasting. Our laboratory is investigating the molecular pathways associated with MuRF-1 action in skeletal muscle. Here we show the effects of MuRF-1 siRNA on Troponin I protein levels in C2C12 cells, a mouse skeletal muscle cell line. C2C12 cells express muscle specific proteins and can be differentiated from myoblasts to myotubes by starving the cells in media with 2% horse serum in place of fetal bovine serum. When C2C12 myoblasts were transfected with MuRF-1 siRNA duplexes (Dharmacon) and allowed to differentiate to myotubes, Troponin I protein levels increased relative to other muscle markers. MuRF-1 overexpression caused a reciprocal decrease in Troponin I levels. These data are consistent with recent reports that cardiac troponin I is a direct ubiquitination target of MuRF-1 (Kedar et al. 2004). Current work is focusing on determining whether direct ubiquitination of Troponin I is related to MuRF-1’s role in muscle catabolism.

N-WASP and WAVE2 Acting Downstream of Phosphatidylinositol 3-Kinase Are Required for Myogenic Cell Migration Induced by Hepatocyte Growth Factor

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.