1Department of Pharmacology and 2High Technology Research Center, Tsurumi University School of Dental Medicine, Yokohama, Japan; 3Brodie Laboratory for Craniofacial Genetics, Department of Orthodontics, University of Illinois College of Dentistry, Chicago, Illinois; and 4Department of Biology, The University of Tokyo, Tokyo, Japan
Submitted 29 November 2004 ; accepted in final form 3 February 2005
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ABSTRACT |
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dystrophy; pax7; m-cadherin; dystrophin-related proteins
Muscle satellite cells are mononucleated, quiescent stem cells that reside between the sarcolemma and the basal lamina of adult myofibers (4, 13). In response to stimuli such as mechanical loading, unloading, denervation, and injury, the satellite cells are activated to proliferate, differentiate into myoblasts, and fuse to preexisting myofibers. This activation is thought to induce adaptive changes of skeletal muscle such as hypertrophy, the alteration of fiber type, and regeneration (1, 4, 13, 23). These observations suggest that the activity and pool size of satellite cells are correlated with the degree of damage in mdx skeletal muscles.
Utrophin is a paralog of dystrophin and can functionally replace dystrophin (2, 7, 30). In normal adult myofibers, the expression of utrophin is confined to the neuromuscular and myotendinous junctions, whereas in mdx myofibers, utrophin is expressed throughout the sarcolemma instead of dystrophin, and the total amount of utrophin is markedly elevated compared with that in normal myofibers (2, 33). Moreover, transgenic mdx mice displaying fairly high amounts of utrophin show a complete correction of the dystrophic status (30). On the other hand, mdx mice in which the utrophin gene is inactivated present a catastrophic aggravation of myopathy, leading to early death (6). The situation in mdx muscles, however, lies between these latter two extremes. It seems important to know whether the spontaneous level of utrophin expression is negatively correlated with the degree of damage in mdx skeletal muscles.
In the present study, to determine whether muscle satellite cells and the expression of utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region relative to the total muscle area, an indicator of myofiber degeneration, in the masseter, gastrocnemius, soleus, and diaphragm muscles of the mdx mouse. Furthermore, we analyzed the mRNA expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (maker of muscle satellite cells), and utrophin and investigated the immunolocalization of m-cadherin and utrophin in the muscles of both normal and mdx mice.
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MATERIALS AND METHODS |
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Histological analysis of degenerative area.
Six mdx and six control B10 mice, aged 6 wk, were killed by cervical dislocation under ether anesthesia. The body weights of the mdx and B10 mice were 17.2 ± 1.9 g and 18.9 ± 1.5 g (mean ± SD), respectively. Whole portions of the left masseter, gastrocnemius, soleus, and diaphragm muscles were removed and fixed in Bouin's fixative for 1 h at 4°C. After being washed in phosphate-buffered saline (PBS), the sections were immersed in a graduated series of sucrose solutions (2040% wt/vol) in PBS at 4°C, embedded in Tissue-Tek OCT compound (Miles Laboratories, Elkhart, IN), and frozen. Middle portions of the muscles were cut at 10-µm thickness using a cryostat and air dried for 1 h at room temperature. The sections were stained with hematoxylin and eosin and observed under a light microscope. The total and degenerative areas of the muscle sections were measured using an image analyzer (Luzex 3U; Nikon, Tokyo, Japan). The degenerative area was normalized to the total area and expressed as a percentage of the total area of the muscles.
Immunohistochemistry for m-cadherin and utrophin.
To detect the satellite cells in the mdx and B10 muscles, the cryosections of the muscles were immunostained for a goat antibody against m-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA) as described previously (31). The number of m-cadherin-positive cells in the periphery of myofibers and in the degenerative area was counted as satellite cells in a total of 10 rectangular areas of 140 x 130 µm2 on several sections obtained from each mouse (21). The 10 rectangular areas, which did not overlap and were uniformly distributed on the sectional area of muscles, were selected throughout the several sections and composed 520% of the total sectional area of the B10 and mdx muscles. The number of total cell nuclei in muscle tissues was also counted, and the percentage of m-cadherin-positive cells to the total number of cell nuclei in the muscle tissues was calculated. The percentage values in the 10 rectangular areas were averaged to obtain the mean value for each mouse. This mean value was further averaged to obtain the mean values for six mdx and six B10 mice. To analyze the change in the expression of utrophin, immunostaining for utrophin was performed using a goat antibody against utrophin (Santa Cruz Biotechnology). For control staining, the primary antibody was replaced with PBS or normal goat IgG.
RNA extraction, reverse transcription, and competitive polymerase chain reaction amplification. Six mdx and six B10 mice, aged 6 wk, were killed by cervical dislocation while they were under ether anesthesia. The body weights of the mdx and B10 mice were 19.8 ± 0.8 g and 24.3 ± 0.5 g (means ± SD), respectively. Whole portions of the left masseter, gastrocnemius, soleus, and diaphragm muscles were removed, immediately frozen, and stored at 80°C until use.
Total RNA extraction, reverse transcription, and competitive polymerase chain reaction (PCR) amplification were performed as previously described (34, 36). Briefly, total RNA extraction was performed according to the manufacturer's specifications (rapid total RNA isolation kit; 5 Prime-3 Prime, Boulder, CO). The RNA was treated with 2 U of ribonuclease-free deoxyribonuclease I (Life Technologies, Gaithersburg, MD) and was then reverse transcribed with 200 U of reverse transcriptase (SuperScript II; Life Technologies).
In the conventional PCR technique, a small difference in the starting amount of target DNA can result in a large change in the yield of the final product, owing to the exponential nature of the PCR reaction. A plateau effect after many cycles can lead to an inaccurate estimation of final product yield. Furthermore, because the PCR amplification depends on the reaction efficiency, small changes in efficiency can lead to major differences in the final product yield. To overcome these problems, the competitor (an internal standard), which has the same primer sequences as those of the target DNA at the 5' and 3' ends, was amplified simultaneously with the target (11, 25, 34, 36). Competitors for the competitive PCR amplification were constructed according to the manufacturer's instructions (Competitive DNA Construction Kit; Takara, Shiga, Japan). The sequences of primers for pax7, m-cadherin, and utrophin were as follows: pax7, FW, 5'-CCACAGCTTCTCCAGCTACTCTG-3' and BW, 5'-CACTCGGGTTGCTAAGGATGCTC-3' (29); m-cadherin, FW, 5'-ATGTGCCACAGCCACATCG-3' and BW, 5'-YCCATACATGCTCGCCAGC-3' (14); utrophin, FW, 5'-AAACTCCTATCACGCTCATCA-3' and BW, 5'-CTCATCCTCCACGCTTCCT-3' (9). Those for S16 were identical to those used in a previous report (18). The amplification products were isolated by performing electrophoresis with an agarose gel containing ethidium bromide. The fluorescence intensities of the bands of the target genes (Fig. 1A, top bands) and their respective competitors (Fig. 1A, bottom bands) were measured using an image analyzer (Molecular Imager FX; Bio-Rad, Hercules, CA). We then calculated the ratios of the fluorescence intensities of the target gene bands to those of their respective competitors. The logarithmic value of the fluorescence intensity ratio was used to calculate the amount of endogenous target mRNA on the basis of the line formula derived from a standard curve for each target gene. The standard curve was generated as described previously (18, 35). Figure 1B shows the standard curve for m-cadherin calculated using the image analysis data of electrophoretic bands shown in Fig. 1A. The slope of the regression line was 0.772, and the correlation coefficient was 0.994, which was significantly different from zero (P < 0.001). The quantity of each target gene was normalized to the quantity of S16 (ribosomal protein). The resulting ratio value in each sample was expressed as a percentage of the mean value for the B10 masseter muscle. Because each percentage value relative to the mean value for the B10 masseter muscle (% of B10 masseter value) was an arbitrary unit, it was used in the scatterplots shown in Figs. 35, although the scatterplots contain no data regarding the B10 muscles.
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RESULTS |
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To investigate the relationship between utrophin expression and the degree of damage in the mdx skeletal muscles, we analyzed the expression levels of utrophin mRNA and the immunolocalization of utrophin (Fig. 5). In all muscles studied, the mean values of the expression levels for utrophin mRNA were higher in the mdx mice than in the B10 mice, and the 1.4- and 3.0-fold increases in the soleus and diaphragm muscles, respectively, were statistically significant (P < 0.05 and P < 0.001) (Fig. 5A). In the mdx mice, the expression level of utrophin mRNA in the diaphragm muscle was 1.4- to 2.7-fold (P < 0.05 and P < 0.01) the levels in the other three muscles, and that in the gastrocnemius muscle was
2-fold (P < 0.01) the levels in the masseter and soleus muscles. The correlation coefficient between the degenerative areas of the skeletal muscles and the expression levels for utrophin mRNA in the mdx mice was 0.231, which was not significantly different from zero (Fig. 5B). In the B10 mice, immunostaining for utrophin was sporadically found in the periphery of myofibers (black arrowheads in Fig. 5, C and E). In the mdx masseter muscle, the periphery (black arrowheads) and the whole sarcoplasm of regenerative myofibers with central nuclei were immunostained for utrophin (Fig. 5D). The periphery of normal masseter myofibers without central nuclei (white arrowheads) was slightly stained, but the sarcoplasm (arrows) was not stained (Fig. 5D). In the mdx diaphragm muscle, immunostaining for utrophin was observed in both the whole sarcoplasm and the periphery of normal and degenerative myofibers, and the immunostaining in the periphery (sarcolemma) of myofibers (black arrowheads) was more intense than that in the sarcoplasm (Fig. 5F). The immunostaining patterns for utrophin in the gastrocnemius and soleus muscles were similar to those in the masseter muscle (data not shown).
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DISCUSSION |
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In the mdx mice, we found no significant correlation between muscle damage and expression level of pax7 (Fig. 3C) and no correlation between muscle damage and the ratio of satellite cells to the total number of cell nuclei in the muscle tissues (Fig. 4F), suggesting that the levels of activation and proliferation of muscle satellite cells are not correlated with the degree of damage in mdx skeletal muscles. However, because the correlation between muscle damage and the ratio of satellite cells to the total number of cell nuclei in the muscle tissues was nearly significant (when the t value for the correlation is >2.07, the correlation is statistically significant; the t value in the present study was 2.01), muscle satellite cells seem to be one of several factors influencing the degree of damage in mdx skeletal muscles, but not a great influencing factor. Gillis (12) proposed the following three factors that can lead to severe myofiber damage in the mdx diaphragm muscle: a large proportion of fast oxidative fibers having a large diameter, lifelong sustained activity, and forced lengthening during each contraction. Further studies are necessary to elucidate the factors that determine the degree of damage in mdx skeletal muscles other than the diaphragm muscle.
In both the B10 and mdx mice, the ratios of m-cadherin-positive cells to the total number of cell nuclei in the muscle tissues were greatest in the masseter muscles. This result indicates that the masseter muscle contains the largest pool of satellite cells, suggesting that the regeneration potential of the masseter muscle is much larger than that of the other muscles. If the same situation existed in the mdx muscles before the first episode of degeneration, it would not be surprising for the mdx masseter muscle to show much less damage than the mdx diaphragm muscle (Fig. 2E). In the present study, however, a clear and statistically significant negative correlation between muscle damage and the percentage of satellite cells could not be obtained (Fig. 4F). This result is probably due to the existence of other factors more influential than satellite cells. Masseter muscle reportedly has several unique characteristics (3, 5, 17, 26, 28, 32), and to these we may now add the characteristic of having a large pool of satellite cells and a large potential for regeneration.
Previous studies have reported that utrophin can functionally replace dystrophin and that the transgene expression of utrophin can prevent muscular dystrophy in mdx mice (2, 7, 30). Thus we had expected a high negative correlation between damage to the skeletal muscles and the expression level of utrophin mRNA in the mdx mice. Contrary to our expectation, the correlation coefficient between muscle damage and expression level was low and not significantly different from zero (Fig. 5B). In particular, the mdx diaphragm muscle exhibited the highest expression level for utrophin mRNA, although it was the most severely damaged by dystrophy. To determine whether upregulated utrophin cannot functionally replace dystrophin because it is not incorporated into the sarcolemma instead of dystrophin, we investigated the immunolocalization of utrophin (Fig. 5, CF). Because intense immunostaining for utrophin was observed in the periphery of mdx myofibers (Fig. 5, D and F), we presumed that it was incorporated into the sarcolemma. In the present study, the expression level of utrophin mRNA in the mdx diaphragm muscle was 1.4- to 2.7-fold the levels in the other three mdx muscles studied (Fig. 5A). To obtain complete disappearance of muscle damage, transgene expression is needed to reach
11- and 25-fold the utrophin expression in mdx and normal mice, respectively (22). Thus it is most likely that the difference in the spontaneous upregulation of utrophin among different mdx muscles is too small to produce a difference in the degree of damage among different mdx muscles.
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GRANTS |
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ACKNOWLEDGMENTS |
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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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