ARTICLE |
Correspondence to: Antje Bornemann, Inst. of Brain Research, University of Tübingen, Calwerstr. 3, D-72076 Tübingen, Germany.
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Summary |
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Satellite cells (SCs) in normal adult muscle are quiescent. They can enter the mitotic program when stimulated with growth factors such as basic FGF. Short-term denervation stimulates SC to enter the mitotic cycle in vivo, whereas long-term denervation depletes the SC pool. The molecular basis for the neural influence on SCs has not been established. We studied the phenotype and the proliferative capacity of SCs from muscle that had been denervated before being cultured in vitro. The expression of PCNA, myogenin, and muscle (M)-cadherin in SCs of normal and denervated muscle fibers was examined at the single-cell level by immunolabeling in a culture system of isolated rat muscle fibers with attached SCs. Immediately after plating (Day 0), neither PCNA nor myogenin was present on normal muscle fibers, but we detected an average of 0.5 M-cadherin+ SCs per muscle fiber. The number of these M-cadherin+ cells (which are negative for PCNA and myogenin) increased over the time course examined. A larger fraction of cells negative for M-cadherin underwent mitosis and expressed PCNA, followed by myogenin. The kinetics of SCs from muscle fibers denervated for 4 days before culturing were similar to those of normal controls. Denervation from 1 to 32 weeks before plating, however, suppressed PCNA and myogenin expression almost completely. The fraction of M-cadherin+ (PCNA-/myogenin-) SCs was decreased after 1 week of denervation, increased above normal after denervation for 4 or 8 weeks, and decreased again after denervation for 16 or 32 weeks. We suggest that the M-cadherin+ cells are nondividing SCs because they co-express neither PCNA or myogenin, whereas the cells positive for PCNA or myogenin (and negative for M-cadherin) have entered the mitotic cycle. SCs from denervated muscle were different from normal controls when denervated for 1 week or longer. The effect of denervation on the phenotypic modulation of SCs includes resistance to recruitment into the mitotic cycle under the conditions studied here and a robust extension of the nonproliferative compartment. These characteristics of SCs deprived of neural influence may account for the failure of denervated muscle to fully regenerate. (J Histochem Cytochem 47:13751383, 1999)
Key Words: PCNA, myogenin, M-cadherin, immunolabeling, myogenesis, in vitro
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Introduction |
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Satellite cells (SCs) are mononucleated myogenic cells that reside in the G0-phase of the cell cycle in normal adult muscle. In response to various stimuli, SCs can enter the mitotic cycle, proliferate, and fuse, thereby contributing to growth, repair, and hypertrophy of postnatal skeletal muscle. When undergoing mitosis and transiting to the differentiative compartment, SCs transiently express muscle-specific molecules such as transcription factors of the MyoD family, the developmentally regulated isoforms of myosin heavy chain, and the intermediate filament protein desmin both in vitro and in vivo in experimentally induced muscle regeneration. They are closely attached to the plasma membrane of the adjacent muscle fiber and express the adhesion molecules NCAM and M-cadherin in the quiescent state (
We examined SCs by cultivating isolated intact muscle fibers with attached SCs (
Short-term denervation induces the SCs to enter the mitotic cycle, whereas long-term denervation depletes the SC pool (McGeachie and Albrook 1978;
In this study we characterized the proliferative and differentiative potential of SCs from denervated muscle. We also characterized the expression of M-cadherin protein because SCs in denervated muscle in vivo have been shown to express M-cadherin (
Collectively, the study demonstrates that in this culture system a second SC phenotype exists in addition to the proliferatingdifferentiating compartment, which is M-cadherin+/PCNA-/myogenin- and which is therefore nondividing, or very slowly dividing. This compartment may contain quiescent SCs, differentiated cells, or both. Denervation fully suppresses the proliferativedifferentiative SC phenotype and leads to a robust increase in the number of M-cadherin+ (nondividing) cells. This switch in the SCs' phenotype may account for the inability of denervated muscle to generate fully matured muscle fibers when stimulated to regenerate.
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Materials and Methods |
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Animal Experiments
All animal experiments were performed in adherence to the standards of the German law for the care and use of laboratory animals. Male Wistar rats (Charles River; Kisslegg, Germany) (67 weeks old, 150180 g body weight) were used. For comparison, two animals aged 35 weeks were also examined. All experiments were performed under thiopental anesthesia (50 mg/kg body weight). The right sciatic nerve was cut at midthigh and a 10-mm segment was resected. For denervation periods longer than 4 weeks, the sciatic nerve was transected. The proximal end was sutured to the skin to prevent reinnervation. Four animals were sacrificed at each of the following time points after denervation: 4 days, 1 week, 4, 8, 16, and 32 weeks.
Isolation and Culture of Muscle Fibers
Single muscle fibers with associated SCs were prepared from the FDB according to the procedure elaborated by
Fixation
Cultures were fixed after 04 days according to the modified method of
Antibodies
A polyclonal rabbit antibody to a bacterial fusion protein encoding the EC1, EC2, and part of the EC3 domains of M-cadherin was prepared and immunoaffinity purified as described by Rose and co-workers (1994). Control experiments included incubation with an antibody solution that had been preabsorbed with the antigen and omission of the primary antibody. The immunoaffinity IgG fraction was diluted at 1:50. Monoclonal anti-myogenin (clone F5D) was purchased from Pharmingen (Hamburg, Germany) and used at a concentration of 1:100. Anti-PCNA (clone 19 F4) was acquired from Boehringer and used at 1:50. An experiment performed in a previous work (
Antibody Staining and Counting of Positive Cells on Isolated Fibers
The cultures were blocked with 0.1% bovine serum albumin (fraction V; Boehringer Ingelheim, Heidelberg, Germany) (BSA) for at least 10 min and washed with PBS and 0.1% Triton X-100. Double labeling of fiber cultures was performed using indirect immunofluorescence. Primary and secondary antibodies were dissolved in PBS containing 0.1% BSA and 0.5% Tween-20 (Boehringer Ingelheim). Each culture was labeled overnight with anti-M-cadherin (1:50) and either anti-PCNA (1:50) or anti-myogenin (1:100). Cultures were then rinsed three times with PBS and incubated for 23 hr with both FITC-conjugated goat anti-mouse immunoglobulins (1:100) to visualize the monoclonal and TRITC-conjugated goat anti-rabbit (1:100) to visualize anti-M-cadherin. All incubations were performed at room temperature.
Cultures were rinsed again with PBS and mounted in Vectashield mounting medium (Boehringer Ingelheim). Observations were made with the Olympus BX60 microscope equipped for epifluorescence. The laser microscope LSM 410 was used for photography (Zeiss; Oberkochen, Germany).
For each time point of an individual experiment, a minimum of 20 fibers on three coverslides were used to count fiber-associated cells or nuclei positive for the different antibodies. Exceptionally, only two coverslides were analyzed. Counting of fiber cultures was done using a x40 objective. Positive cells were scored as the number of positives on each individual fiber. Experiments were always conducted in parallel with control cultures maintained in basal medium. For the statistical analysis, the MannWhitney U-test (rank sum test) was employed, with p0.01.
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Results |
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The numbers of positive nuclei (for anti-PCNA and anti-myogenin staining) or cells (for anti-M-cadherin staining) are given as the average per a single fiber. In the text and in the figures the term "cells" is used indifferently in conjunction with all antibodies. The specificity of the antibodies used has been established (see Materials and Methods). Figure 1 shows a fiber from normal innervated muscle double-stained for M-cadherin and myogenin.
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Fiber Cultures from Normal Innervated Muscle
PCNA was not present at the time of plating but appeared shortly thereafter, reached its peak at 16 hr, and decreased gradually (Figure 2A). Myogenin expression started, peaked, and declined approximately 1 day later than PCNA (Figure 2A). This suggests that the SCs depicted here first went through the mitotic cycle and then differentiated to express myogenin (cf.
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M-cadherin was already present at the time of plating (Day 0) in a small number of SCs (Figure 2A). These SCs cannot possibly have passed through a mitotic cycle after plating. Their number increased over the first 2 days examined in culture (Figure 2A).
Although the initial work was done with 67-week-old rats, which are still growing and may contain some proliferating SCs in vivo, results with 35-week-old rats, in which the SCs are believed to be quiescent, were similar (Figure 2B). For the proliferativedifferentiative compartment of SCs from old animals, the number of PCNA+ SC did not differ from those of young adult controls except for Days 2 and 3 in culture (p<0.001); the number of myogenin+ SCs differed only at Day 3 in culture (p<0.001). The number of nondividing (M-cadherin+) SCs did not differ significantly from the young adult controls. This indicates a general pattern of two pools of SCs regardless of the age of the animal.
SCs co-expressing M-cadherin and PCNA were practically non-existent (Figure 3A). Around Day 1, a fraction of cells co-expressing M-cadherin and myogenin appeared (Figure 3A). These cells may have been derived from two sources. First, they have passed the proliferative compartment and are now further differentiating. Second, they have started as M-cadherin+/myogenin- cells and are now expressing myogenin as well.
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Fiber Cultures from Denervated Muscle
In cultures from fibers denervated for 4 days, SCs expressing PCNA or myogenin were still present. They differed only little from normal controls (Figure 2A, Figure 2C, Figure 3A, and Figure 3B) [differences were present for Days 2 and 3 for both PCNA and myogenin (p<0.001)]. In cultures from muscle denervated for 1 week or longer, PCNA and myogenin were hardly expressed at all by SCs, regardless of the time point examined (Figure 2D2H, Figure 3C, and Figure 3D). Therefore, a mitogenic cycle had probably not happened after fiber plating. This was not due to an age effect, because control animals whose muscle fibers were cultured at age 35 weeks still provided SCs that proliferated in culture (Figure 2B). Similarly,
The number of M-cadherin+-only cells differed depending on the denervation period that had elapsed before culturing. In cultures from muscle denervated for 4 days, it was not significantly different from normal controls (Figure 2A, Figure 2C, Figure 3A, and Figure 3B). In fibers cultured 1 week after denervation, a small fraction of M-cadherin+-only cells was present, which remained stable over the 4 days at a low level and never reached the level it had reached in the control cultures from normal innervated muscle (Figure 2D)(p<0.001 Days 14). In SCs derived from muscle denervated for 4 weeks, however, M-cadherin+ cells were more abundant than in the other conditions (Figure 2E)(p<0.001 Days 04 in comparison with normal muscle and with muscle denervated for 1 week). At 8 weeks after denervation, the number of M-cadherin+ cells was also elevated at Day 0 in comparison with normal fibers (Figure 2F) (p<0.001), similar to the 4-week value at Day 0, but remained stable over the 4 days in culture, such that the difference to 4 weeks denervation was significant for the values at Days 2 and 3 (p<0.002). Sixteen and 32 weeks after denervation, the SC numbers did not exceed an average of 0.5 cells/fiber over the 4 days in culture, without a significant difference from one another (Figure 2G and Figure 2H).
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Discussion |
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This present study was undertaken to compare the dynamics of SCs in normal and denervated muscle because long-term denervation is known to interfere with regeneration in the adult muscle. One of the muscle-specific molecules that are expressed in quiescent SCs is M-cadherin, a member of the multigene family of the cadherins (
Characterization of SC Compartments from Innervated Muscle
Our study of normal muscle revealed a compartment of cells in which PCNA expression started shortly after plating and peaked at 16 hr (Figure 2A). Myogenin expression started and peaked approximately 1 day later (Figure 2A). We suggest that these cells have undergone the proliferative compartment first and the differentiative compartment second (
Another fraction of cells associated with the myofibers already expressed M-cadherin when fixed immediately after culturing (Figure 2A). These cells almost never co-expressed PCNA or myogenin (Figure 3A). Their numbers increased between Days 0 and 2. This expression pattern corresponds to the pattern of quiescent SCs in vivo, which express M-cadherin (
The nondividing compartment of SCs has not been identified with the antibodies applied in the study by
A fraction of the myogenin+ cells was M-cadherin- (Figure 2A). M-cadherin expression is part of the differentiative program (
A population of SCs co-expressing M-cadherin and myogenin appeared around Day 1 (Figure 3A). They may be quiescent SCs that have additionally assumed myogenin expression. It is interesting to note that Rantanen and co-workers (1995), in an experimental study on regenerating rat muscle in vivo, found cells that were myogenin+ soon after injury, without having passed the mitotic cycle. Our M-cadherin+/myogenin+ cells may correspond to these cells.
In their study of mRNA with Marcaine-killed isolated mouse muscle fibers,
There are two compartments of SCs in cultures from normal innervated muscle fibers, one proliferativedifferentiative (PCNA+/myogenin-/M-cadherin- or PCNA-/myogenin+/M-cadherin-) and the other one non-dividing (M-cadherin+/PCNA-/myogenin-).
Compartments of SCs from Denervated Muscle
SCs from muscle fibers denervated for 4 days were still able to proliferate and differentiate (i.e., expressed PCNA and myogenin; Figure 2C). However, expression of both PCNA and myogenin was virtually absent in SCs from muscle denervated for 132 weeks (Figure 2D2H). Therefore, none of the SCs entered the mitotic cycle in fiber cultures from muscles that were denervated in vivo for 132 weeks. This was in striking contrast to the findings with cultures from innervated muscle, in which the SCs passing the mitotic cycle outnumbered the M-cadherin+ only cells (Figure 2A).
The number of M-cadherin+ SCs varied, depending on the time that had elapsed after denervation. Muscle fibers denervated for 4 days before culturing had numbers of M-cadherin+ cells that did not differ from those of normal controls (Figure 2C). Muscle fibers denervated 1 week before culturing had a reduced number of M-cadherin+ only cells compared to control cultures from the innervated muscle (Figure 2D). There are two possibilities to explain this reduction. First, the SCs may be lost from the fiber altogether. This is probably not the case in short-term (1 week) denervated muscle. Previous investigators could not detect a reduction in the SC ratio in mouse or rat muscle denervated for up to 30 or 70 days, respectively (
The number of M-cadherin+ cells is increased in fibers cultured after having been denervated for 4 or 8 weeks. In both cases, these may be cells that enter the quiescent or differentiated compartment after having been placed in culture. In view of the finding that proliferative SCs are hardly present, if at all (Figure 2E and Figure 2F), and that the number of SCs in muscle denervated for this time period is not significantly reduced in vivo (
The loss of M-cadherin+ cells from fibers that had been denervated 16 or 32 weeks before culturing may reflect the general decrease in SCs observed after long-time denervation (
M-cadherin+ SCs in muscles denervated for 1 week or more hardly assume myogenin expression, as opposed to innervated muscle fibers (Figure 3A, Figure 3C, and Figure 3D). It has been shown before in vivo that both mRNA and protein myogenin is elevated after denervation, but only transiently so (
The shift in the phenotypic composition of SCs demonstrated here, in conjunction with their resistance to proliferation when placed in culture, may be part of the molecular basis for the inability of muscle tissue to fully differentiate when deprived of the neural influence.
Taken together, our findings demonstrate that SCs from muscle denervated for 132 weeks shift their phenotype towards a molecular pattern that resembles that of nondividing SCs (PCNA-/myogenin-/M-cadherin+). They cannot be recruited into the mitotic cycle under the conditions employed here. The factors that make this phenotype of SCs more vulnerable to degeneration, such that they do not survive long-term denervation, remain to be established. We are now investigating the effect of factors derived from the lesioned peripheral nerve during myogenesis of SCs to try to develop culture conditions in which the influence of the denervation on the robust increase in M-cadherin+ SCs can be reproduced. This will allow future analysis of the mechanisms underlying the phenotypic switch in SCs to M-cadherin+ cells.
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Acknowledgments |
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Suported by the Deutsche Forschungsgemeinschaft (Bo 992/4-1; AB), the intramural fortune program of the University of Tübingen(#229; AB), Boehringer Ingelheim Fonds (travel grant; RK), Cooperative State Research Service US Department of Agriculture (#93-37206-9301 and 95-37206-2356; ZYR), and by the National Institutes of Health (#AG13798;ZYR).
Received for publication March 22, 1999; accepted June 29, 1999.
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