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METHODS IN CELL PHYSIOLOGY
1Faculty of Veterinary Medicine and 2Department of Biology, Academic Biomedical Centre, Utrecht University, Utrecht, The Netherlands; and 3Institute of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Submitted 7 April 2005 ; accepted in final form 12 September 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused C2C12 myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant C2C12 myotubes. Therefore, C2C12 myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C2C12 cells produce atrophy-inducible and atrophy-resistant myotubes. glucocorticoids; nuclear receptors; atrogin
, are likely to be major initiators of protein breakdown. Interestingly, thyroid hormones can cause muscle wasting due to both excess and deficiency by yet-unknown mechanisms (24).
Glucocorticoid-induced skeletal muscle atrophy encompasses enhanced proteolysis (2, 11, 39), altered gene expression (16), growth inhibition by myostatin (19), and inhibition of muscle differentiation (30). New insights into molecular programs that govern muscle atrophy suggest that activation of the Foxo family of transcription factors, which induce gene expression of the key ubiquitin ligase atrogin-1, triggers protein breakdown by the proteasome system (10, 16). Microarray analysis of glucocorticoid-induced myopathy in vivo was described recently (13). Proteolysis is also enhanced in hyperthyroidism (1, 4, 7), explaining the body weight loss of thyrotoxic patients (20); but in contrast to glucocorticoids, thyroid hormones act as positive regulators of muscle development (9). Microarray analysis of 3,3',5-triiodo-L-thyronine (T3)-treated human skeletal muscle confirmed the wide range of T3 targets (5). The effects of T3 are mediated by nuclear receptors (T3R), of which three have been identified in skeletal muscle, T3R1 and -2 and T3R1, the latter being the major receptor (27).
The mouse C2C12 and rat L6 myogenic cell lines, both developed by Yaffe (37, 38), have been used extensively in muscle wasting research. The commonly used growth medium is supplemented with 10% FCS, whereas differentiation medium contains 2% adult horse serum. Successful modifications using the serum substitute Ultroser G (3) and detailed analysis of differentiation markers in both cell lines (22) have been performed in the laboratories of J. H. Veerkamp. Examination of muscle wasting in vitro is based mostly on determination of enhanced proteolysis or 3-methylhistidine release, both reflecting myofibrillar protein degradation. The final outcome of muscle wasting in vivo is characterized by myotube atrophy as determined using histological methods. However, this end point evaluation has not been performed successfully with myogenic cells, probably because of the disordered growth of myotubes in vitro.
The present study was performed to determine and quantify hormone-induced size changes of large myotubes derived from C2C12 and L6 cells. Therefore, a novel method was developed to measure myotube size. This approach included cultivation of large myotubes and their assessment using digital quantification of periodic acid Schiff (PAS)-stained myotubes. Herein we report the successful application of this method and its correlation with changes at the molecular level.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture. Myoblasts from the muscle-derived mouse C2C12 and rat L6 cell lines were obtained from the European Collection of Cell Cultures (Salisbury, UK) and maintained as proliferating myoblasts in DMEM-high-glucose medium supplemented with 10% FCS, 4 mM L-glutamine, 1 mM pyruvate, and 100 U/ml penicillin-100 µg/ml streptomycin at 37°C in the presence of 5% CO2. The seeding density used for the experiments was 1 x 104 cells/cm2 for C2C12 and 2 x 104 cells/cm2 for L6 on 1% gelatin-coated dishes. To induce fusion of myoblasts, 90–100% confluent cultures were switched to DMEM supplemented either with 2% adult horse serum or with 0.4% Ultroser G. Myoblast growth and myotube formation were examined using phase-contrast microscopy. The medium was changed every 2 days, and differentiation was allowed to continue for up to 2 wk before the experimentation period, depending on cell line and differentiation medium. Both cell lines were used for up to a maximum of 10 passages to preserve their characteristics. The experiments lasted from 24 to 72 h, and test chemicals were added to serum- and antibiotic-free medium and renewed every 24 h. Dexamethasone and thyroid hormone were dissolved in DMSO, with a final concentration of 10 and 100 nM, respectively, keeping DMSO end concentration below 0.01%. Cycloheximide was used at a nontoxic concentration of 1 µg/ml and curcumin at 1 µM.
PAS staining. Cells were rinsed with cold PBS and fixed for 5 min in 3.7% formaldehyde buffered in PBS. After undergoing further PBS washing, cells were incubated in 1% periodic acid for 15 min and washed three times for 5 min in distilled water. Samples were then incubated for 30 min in Schiff's reagent, followed by three incubations with 0.5% potassium bisulfite-0.05 M HCl for 5 min each. If necessary, nuclei were counterstained with Meyer's hematoxylin. For PAS image analysis (see below), nuclear staining was omitted. Cells were mounted in Gurr medium (BDH).
Image analysis. Quantitative PAS analysis was standardized using 12-well plates and four 4-cm2 wells per condition. Digital red-green-blue (RGB) photographs were taken with a Nikon DXM 1200 camera attached to a Zeiss Axioskop. From each well, five images were made with a x5 objective and an image frame of 1,024 x 768 pixels.
To quantify the absolute area of myotubes as recognized by their PAS-positive staining and its relative area to the image field, a PC-based image analysis system was used. A dedicated program was developed using the KS400 software package (version 3.0; Zeiss Vision, Oberkochen, Germany).
Because the image names are standardized, the images were loaded from disks automatically. Before each session, the system was geometrically calibrated with an image containing an object micrometer. The myotubes were selected on the basis of their color in the RGB image by interactive thresholding to measure their area. Differences in illumination or brightness (caused by, e.g., different areas of the well that refract light differently) must be equalized to make the thresholding work properly. To that end, the RGB color image is converted to hue-luminance-saturation (HLS). The luminance image is shading corrected with its low-pass image (low-pass matrix 49 x 49 pixels, 17 times). This operation results in an image displaying the light distribution of the original image without structural elements. This image is used to carry out shading correction that results in equalization of the brightness over the whole image, without changing the color or saturation. A median filter, size 7 x 7, carried out on the saturation image prevented small, separate PAS-positive objects from being considered as myotubes. After reconversion of the image to an RGB image, the myotubes were selected on the basis of their color. Although the thresholding was carried out interactively, a trained user/microscopist easily obtained reproducible results. Objects smaller than 250 pixels were rejected. All objects selected were displayed over the image with a yellow/blue contour to enable interactive corrections if necessary.
Western blot.
Cells and tissue were lysed in sample buffer under denaturating conditions and separated by SDS-electrophoresis using 10% and 7.5% polyacrylamide minigels (Protean System III; Bio-Rad). After transfer to a nitrocellulose membrane, equal protein loading was confirmed and documented using Ponceau S staining. Membranes were blocked with 2.5% dry milk powder (Roth, Karlsruhe, Germany) and 1% BSA (Sigma) in Tris-buffered saline containing 0.1% Tween 20 (TBS-Tween) for 2 h. Subsequently, primary monoclonal antibodies for the developmental isoform (MHC-dev) of myosin heavy chain (MHC) (Ref. 29; clone 47A, 1:50 dilution), and for the Z-disk protein -actinin (clone EA-53, 1:1,000 dilution) were added and incubated overnight in blocking solution at 4°C. Monoclonal antibodies against thyroid hormone receptor 1 (clone J52, 1:500 dilution; Santa Cruz Biotechnology) and polyclonal antibodies against actin (A2066, 1:20,000 dilution; Sigma) were incubated for 2 h in blocking solution at room temperature. After several washing steps in TBS-Tween, peroxidase-coupled secondary antibody (Jackson ImmunoResearch Laboratories) was used for 45 min. Detection was performed with the chemiluminescence substrate SuperSignal West Dura (Pierce).
Immunohistochemistry.
Cells were fixed in 3.7% formaldehyde buffered in PBS for 5 min, permeabilized in PBS containing 0.1% Tween 20 (PBS-t, pH 7.4), and blocked for 2 h in PBS-t that contained 2% BSA and 10% horse serum. Monoclonal antibodies for desmin (clone DE-U-10; Sigma) and T3R1 (Ref. 17; clone J52; Santa Cruz Biotechnology) were diluted 1:100 in block solution and incubated overnight at 4°C. After undergoing three washing steps in PBS-t, cells were incubated for 45 min with biotinylated secondary antibodies, washed, subsequently incubated for 60 min with a biotin-avidin-horseradish peroxidase complex (Vectastain ABC elite reagent; Vector), and finally incubated for 5 min with staining solution (3,3'-diaminobenzidine tetrahydrochloride peroxidase substrate kit SK-4100; Vector).
Analysis of mRNA expression. Total RNA was isolated from cells and tissue with an RNA extraction kit (TRIzol; Invitrogen) according to the manufacturer's protocol. RNA concentration was assessed spectrophotometrically, and its integrity was checked by electrophoresis in 1% agarose. Reverse transcription of total RNA (1 µg) was performed with the reverse transcriptase SuperScript II (Invitrogen), random hexamer, and oligo(dT) primers.
Glucocorticoid receptor (GR), T3R1, atrogin-1, and GAPDH primers (Table 1) were designed using the free software Primer3 (25). T3R1 and -2 primer sequences have been reported for mouse and rat skeletal muscle (27). BLASTN searches were conducted for all primer nucleotide sequences to ensure gene specificity.
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Statistical analysis. Results for individual experiments were replicated in three to six independent experiments and presented as means ± SD. Student's t-test was used to determine whether differences existed between results from different cells and experimental conditions. The acceptable level of significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Fusion and myotube growth were compared with the use of fusion medium based on 2% adult horse serum or 0.4% Ultroser G. Horse serum-induced myotube formation produced large myotubes in the second week of culture at a fusion rate of 
20–30% (data not shown). The slow fusion rate and the low percentage of fusion of C2C12 cells could be enhanced by using the serum substitute Ultroser G. A rapid fusion onset could be observed after 3 days, leading to large myotubes by 4–5 days (Fig. 1A). Spontaneous contraction occurred as in horse serum-fused myotubes, but proliferating myoblasts could not be observed. C2C12 myotubes obtained in this way retained their morphology under serum-free conditions for an additional 3 days. Improvement of myotube formation of the rat L6 cell line could not be achieved. Ultroser G-based medium did not lead to myotube formation in L6 cells. Maximal fusion rate of these cells was obtained after 10 days with a 2% adult horse serum-based medium, and myotube cultures could be kept for another week in serum-free medium (data not shown).
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-actinin could not be detected after 3 days of fusion in horse serum-based medium (Fig. 1B), whereas MHC-dev was detectable at this time point in myotubes derived with Ultroser G-based medium (Fig. 1C), confirming the morphological observations. After optimization of fusion conditions, we used the PAS stain to visualize myotubes. Figure 2 demonstrates a typical PAS stain of C2C12-derived myotubes obtained using Ultroser G-based fusion medium. As can be seen, PAS-positive staining not only was restricted to myotubes and excluded from nonfused myoblasts but also helped to distinguish easily between myotubes of different sizes. To quantify PAS-positive areas, we set up a digital analysis system, allowing serial quantification of large experiments. Furthermore, we included a size limitation to exclude reactions of growing small myotubes, thus allowing us to focus on large myotube changes. The digital imaging program is illustrated in Fig. 3 in its major single steps until PAS-positive C2C12 and L6 myotube area is expressed.
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25% in C2C12 myotubes and to a lesser extent in L6 myotubes (Fig. 4A). The synthetic glucocorticoid dexamethasone (10 nM) reduced myotube area by 30% in L6 myotubes after a 3-day treatment (Fig. 4B). Surprisingly, C2C12 myotubes did not react to dexamethasone at either 10 or 100 nM. T3 (10 nM) reduced myotube area in both cell lines, but the reduction was not statistically significant (Fig. 4B).
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1 and T3R2 did not differ significantly at the mRNA level between C2C12 and L6 myotubes (Fig. 6, A and B, respectively). Figure 6C shows the results of the major skeletal muscle T3R, namely, T3R1. L6 myotubes expressed an mRNA level about fivefold higher than that of C2C12 cells. PCR efficiency and product size (data not shown) confirmed our results with respect to the comparison of T3R mRNA between the cell lines.
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1 expression in the two myogenic cell lines, we performed protein analysis using a monoclonal antibody directed against the A/B domain of T3R1. Figure 7A shows the immunohistochemistry of T3R1 in L6 myotubes, verifying positive staining of the nuclei. For protein quantification, we performed Western blot analysis of T3R1 in C2C12 and L6 myotubes (Fig. 7B). Liver nuclei and total homogenates from porcine and mouse liver served as positive controls. The T3R1 antibody reacted with a 55-kDa band in myotube and liver samples. A second, slightly smaller band (52 kDa) was detected in the liver samples, representing a truncated form of the receptor (17). Densitometric analysis and standardization to actin levels of the same blot after T3R1 detection (Fig. 7C) revealed threefold higher expression of the receptor in L6-derived myotubes than in C2C12 myotubes (Fig. 7D).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Optimization of fusion conditions for C2C12 cells could be achieved by replacing horse serum with the serum substitute Ultroser G as described by Benders et al. (3). Serum-free medium formulations have also been reported for C2C12 cells, but, as in our study, improvement of L6 cultures could not be achieved (15). Interestingly, it has been suggested that endogenous IGF expression determines differentiation capacity rather than serum depletion (39). Despite the long fusion time course of L6 cells, their high fusion rate and their stability in simple serum-free media made them suitable for long-term studies of hormones and cytokines.
Myotube atrophy representing the in vitro equivalent to myofiber atrophy can be analyzed only using histochemical or immunohistochemical methods, the latter not being suitable for large-scale analysis. Therefore, we used the classic PAS stain, which reacts with glycogen, glycolipids, and glycoproteins, producing a magenta stain that is proportional, rapid, and economical. Our results demonstrate the advantages of this stain, because it excluded nonfused myoblasts and allowed quantification using routine digital image analysis because of strong contrast, which can be seen even macroscopically. The NF-
B inhibitor curcumin, which has been shown to enhance muscle mass and to accelerate myogenesis (31), induced myotube size enhancement as measured in our system. The catabolic hormone dexamethasone reduced myotube size by 30% in L6 myotubes during 3 days of exposure, which is in good agreement with in vivo studies (16). Surprisingly, C2C12 myotube size did not change under the same conditions. Divergent reactions of these two cell lines have also been reported for dexamethasone-induced gene expression of the glucose transporter GLUT4 (35).
The presence of a functional nuclear GR has been shown in L6 myotubes (14). Our quantitative receptor analysis data revealed a higher expression of GR in L6 than in C2C12 large myotubes, which could be part of the explanation of the dissimilar responsiveness to glucocorticoids of the myotubes. It is a matter of discussion whether a twofold difference of receptor expression could explain our observations, but it has been shown for thyroid hormones in endothelial cells that a specific receptor expression level is necessary to elicit a biological response (8).
Treatment with T3 did not lead to the same extent of myotube size reduction as did treatment with dexamethasone. Both cell lines did not react with a significant myotube atrophy after a 3-day treatment, and T3 receptor analysis excluded receptor deficiency as a cause. The higher expression of the major skeletal muscle receptor T3R1 at the mRNA and protein levels in L6 myotubes supports a former report of functional T3R in L6 myotubes (12). In both cell lines, metabolic effects of T3 have been reported, e.g., upregulation of the sarco(endo)plasmic reticulum Ca2+-ATPases in C2C12 myotubes and induction of uncoupling protein 3 in L6 myotubes (21, 40). Furthermore, in both myogenic cell lines, cross talk between T3-induced gene expression and contractile activity determining muscle phenotype has been reported (32, 33). This shows not only the importance of T3 in skeletal muscle physiology but also the responsiveness of myogenic cell lines to this hormone. Thyroid hormones cannot be categorized as direct-acting catabolic factors like glucocorticoids. However, indirect or permissive effects of T3 may contribute to a catabolic state induced by other factors.
It remains unclear whether C2C12 myotubes contain levels of endogenous GR that are too low or whether the time point of treatment is decisive for the atrophic response, because this cell line has been used successfully in various studies investigating gene expression of glucocorticoid-induced catabolism (6, 26, 28, 34). Experimental conditions are often the cause of contradictory results. The differentiation state of the myogenic cells and the composition of the culture medium are important factors that may affect the outcome of the experiment.
The physiological effects of a combination of multiple catabolic agents, as mostly occur in vivo, are certain to deviate from the results obtained by studying the effects of single compounds. Protein breakdown was reported in C2C12 myotubes within 6 h when thyroid hormones were added to dexamethasone; this is a result of permissive and synergistic effects of both hormones (26). The objective of the present study was to determine the catabolic potential of the single hormones. Therefore, a long-term exposure (3 days) and serum-free culture conditions were chosen.
In most common protocols, a 4-day differentiation period is used, during which C2C12 cells are incubated in 2% horse serum before experiments (6, 18, 34). Variance in protocols consists of shortening the differentiation time to 3 days (28, 36). Alternatively, cells are differentiated in the presence of dexamethasone to obtain a high dexamethasone-induced gene expression response of C2C12 myotubes (35). It remains unclear to what extent glucocorticoids affect myogenesis, myotube growth, and mature myotubes of C2C12 cells. It is likely that C2C12 myoblasts and the differentiation process are more sensitive than the mature myotubes to hormonal treatments that were used in the present study. We studied effects on already-formed myotubes, with a 4- and 10-day differentiation period for C2C12 and L6 cells, respectively.
Other important variances in protocols, besides differentiation time, are the differentiation conditions. In addition to 2% horse serum being the most common way to differentiate, 10% horse serum (34) and 2% calf serum (35) have been used. Ultroser G-based fusion medium was chosen because it resulted in a higher fusion rate and a more even fusion time course as described previously (3). Interestingly, the Ultroser G-fused C2C12 myotubes did not show any size reduction by dexamethasone treatment, in contrast to L6 myotubes. The dexamethasone-induced increase in atrogin-1 expression in L6, but not in C2C12 myotubes, was in line with these observations. Furthermore, Ultroser G-fused C2C12 myotubes expressed less atrogin-1 mRNA than did myotubes that were fused in horse serum.
It was expected that glucocorticoids would upregulate atrogin-1 expression in both types of C2C12 myotubes, despite the different atrogin-1 mRNA levels, because treatments were performed under serum-free conditions. Interestingly, no dexamethasone-induced changes could be detected in Ultroser G-fused myotubes. However, dexamethasone increased atrogin-1 mRNA in horse serum-fused C2C12 myotubes, which is in line with two recent reports (6, 26). The atrophy resistance of Ultroser G-fused myotubes and the observation that IGF-I overrides the effects of glucocorticoids (6) suggest a role of IGF-I in Ultroser G-fused myotubes. It is tempting to speculate that IGF-I-mediated low atrogin-1 levels lead to an atrophy-resistant condition in skeletal muscle. However, it cannot be ruled out that Ultroser G prevents the upregulation of atrogin-1 expression and the induction of atrophy by other, unknown mechanisms. Even at up to 3 days in serum-free medium, Ultroser G-fused myotubes maintained their atrophy resistance. This observation suggests that the intrinsic atrophy resistance is acquired by altered gene expression rather than direct interference with atrophy-inducing factors as described for IGF. It will be interesting to investigate which components in Ultroser G affect the signaling pathways that result in atrophy resistance. Studies of the differences between Ultroser G- and horse serum-fused C2C12 myotubes regarding the interplay among protein degradation (ubiquitin ligases such as atrogin-1), protein synthesis (mammalian target of rapamycin), and their regulation by growth factors such as IGF-I and myostatin may lead to ways to prevent cachexia.
In conclusion, the present study provides a useful in vitro model to study atrophy in muscle fibers in which myotube size can be compared with changes at the molecular level. The results confirm dexamethasone, in contrast to thyroid hormones, acting primarily as a catabolic factor. Furthermore, C2C12 myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C2C12 cells produce atrophy-inducible and atrophy-resistant myotubes.
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ACKNOWLEDGMENTS |
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Parts of this work were performed in the Institute of Biochemistry and Molecular Biology-III: Biochemical Endocrinology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, with the support of Prof. Dr. Hans-Joachim Seitz.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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  J. Yin, Z. Gao, D. Liu, Z. Liu, and J. Ye Berberine improves glucose metabolism through induction of glycolysis Am J Physiol Endocrinol Metab, January 1, 2008; 294(1): E148 - E156. [Abstract] [Full Text] [PDF]
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 K. Nakanishi, N. Dohmae, and N. Morishima Endoplasmic reticulum stress increases myofiber formation in vitro FASEB J, September 1, 2007; 21(11): 2994 - 3003. [Abstract] [Full Text] [PDF]
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3 independent experiments. **P < 0.001 vs. untreated myotubes.
