Satellite Cells on Isolated Myofibers from Normal and Denervated Adult Rat Muscle

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) (6-7 weeks old, 150–180 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 Bischoff (1986) and modified by Yablonka-Reuveni and Rivera (1994), whose detailed procedure we followed with the following slight modifications. Flexor digitorum brevis (FDB) muscles of both hind feet were used for each preparation of innervated muscle fibers. Only the FDB of the denervated hind foot was used for the denervation experiments. The outer connective tissue was removed and the muscles were immersed in a 5-ml solution of 0.2% collagenase (type P, lot 83834624; Boehringer, Mannheim, Germany) resuspended in Eagle's minimal essential medium (MEM) with 10% horse serum (PAN Systems; Aidenbach, Germany). Digestion was for 3 hr at 37C with occasional agitation. The collagenase-treated muscle was transferred into 5 ml of MEM containing 10% horse serum, teased, and carefully triturated (about 300 times) with a wide-mouth pipet. Fibers were then allowed to settle at 1 X g for 5–10 min at room temperature through 10 ml MEM containing 10% horse serum in 15-ml tubes. This procedure was repeated three times. The final fiber sediment was aliquotted and plated on coated cover-slides (placed in 35-mm plates). The slides were precoated with 0.004 mg poly-L-lysine (Sigma; Deisenhofen, Germany) in 0.5 ml H₂O for 8 min and air-dried. The slides were coated with 60 μl of Vitrogen solution which was made isotonic and buffered to pH 7.4 by the addition of 1 part of 7 X MEM to 6 parts of stock Vitrogen 100 (Collagen; Ismaning, Germany). Plates were incubated for 30 min at 37C, 5% CO₂ to allow formation of gel and adherence of fibers to the matrix. Cultures then received basal medium [MEM containing 20% controlled process serum replacement Type 2 or Type 1 (CPSR2 or CPSR1; Sigma), 1% horse serum, and 1% antibiotic antimycotic solution (Sigma)]. When compared under identical conditions, CPSR1 and CPSR2 yielded the same results. Medium was changed every day.

Fixation

Cultures were fixed after 0–4 days according to the modified method of Kurki et al. (1988). Normal innervated muscle fibers were also fixed after 16 hr. Briefly, cultures were incubated in paraformaldehyde 1% (v/v) in PBS, pH 7.4, washed in ice-cold PBS, and incubated in absolute methanol at −20C for 10 min. After this the cultures were washed in PBS containing 0.1% Triton X-100 (v/v) (Sigma) and were stored in PBS with 0.1% (w/v) sodium azide.

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 (Yablonka-Reuveni et al. 1999) ruled out the possibility that PCNA and/or myogenin were expressed by myonuclei. In that study, double-labeling experiments with a polyclonal antibody against two kinases of the mitogen-activated protein (MAP) kinase family (an intracytoplasmic enzyme) and either PCNA or myogenin revealed that all PCNA⁺ or myogenin⁺ nuclei were found within the cytoplasm of single cells but never within muscle fibers. Furthermore, comparative autoradiographic studies after [³H]-thymidine incorporation showed kinetics of cell proliferation on isolated fibers similar to the results obtained when the numbers of PCNA⁺ nuclei were quantified via immunofluorescence (Yablonka-Reuveni and Rivera 1997). The FITC-conjugated goat anti-mouse and TRITC-conjugated goat anti-rabbit antibodies were purchased from Dianova (Hamburg, Germany). DAPI (Boehringer) for nuclear counterstaining was added for 12 min after the secondary antibody at a concentration of 1 μg/ml.

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 2–3 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 Mann-Whitney U-test (rank sum test) was employed, with p ≤ 0.01.

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Figure 1

Fibers from normal innervated muscle 2 days after plating, double-labeled with anti-myogenin (green) and M-cadherin (red). (A) Two SCs are attached to the muscle fiber. The upper one shows an SC expressing both myogenin and M-cadherin; the lower one expresses only M-cadherin. (B) The single SC expresses myogenin but not M-cadherin. Bar = 50 μm.

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. Yablonka-Reuveni and Rivera 1994). The graph in Figure 2A also shows that not all cells that have expressed PCNA (i.e., have undergone the mitotic cycle) differentiate fully to express M-cadherin.

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 6-7-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 proliferative-differentiative 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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Figure 2

Graphs of cells/fiber stained for PCNA and M-cadherin or for myogenin and M-cadherin. Double-staining was performed with a polyclonal anti-M-cadherin antibody in combination with either PCNA or myogenin (both monoclonal). The graph shows the results of the three molecules individually, regardless of whether or not co-expression was present. (A) Normal controls 6–7 weeks of age. (B) Normal controls 35 weeks of age. (C-G) SCs from muscle that had been denervated 4 days (C), 1 week (D), 4 (E), 8 (F), 16 (G), and 32 weeks (H) before denervation. The standard error is indicated.

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Figure 3

Graphs showing the number of doubly stained SCs (PCNA/M-cadherin and myogenin/M-cadherin). (A) Normal controls. (B-D) SCs from muscle that had been denervated for 4 days (B), 1 week (C), or 4 weeks (D). As in the graphs for the fibers of the muscle that had been denervated for 1 or 4 weeks, there werehardly any doubly stained SCs in muscle fibers denervated 8, 16, or 32 weeks before plating (not shown).

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 (Figures 2A, 2C, 3A, and 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 (Figures 2D-2H, 3C, and 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, Yablonka-Reuveni et al. (1999) demonstrated that SCs in myofibers from young (3-week-old), adult (8- to 12-week-old), and old (9- to 12-month-old) rats all could undergo proliferation and differentiation when their attached fibers were subjected to isolation and culture.

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 (Figures 2A, 2C, 3A, and 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 1-4). 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 0–4 in comparison with normal muscle and with muscle denervated for 1 week). At 8 weeks after de-nervation, 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 (Figures 2G and 2H).

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 (Yablonka-Reuveni and Rivera 1994).

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 (Bornemann and Schmalbruch 1994; Irintchev et al. 1994) but do not divide or express myogenin (Grounds et al. 1992). It is therefore feasible that these cells are quiescent SCs. However, the possibility that they are differentiated cells cannot be ruled out. Their increase between Days 0 and 2 could be due to the fact that some SCs completed their entry into the quiescent or differentiated state during culture Day 1.

The nondividing compartment of SCs has not been identified with the antibodies applied in the study by Yablonka-Reuveni and Rivera (1994), which used the same culture model as we did. However, Yablonka et al. (1999) did provide evidence for the existence of a cell population that was different from the proliferative-differentiative compartment identified with antibodies against PCNA and myogenin. They visualized cells by use of an antibody against the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) which belong to the mitogen-activated protein kinase (MAPK) superfamily. This antibody labeled the cells’ cytoplasm and showed that they were single cells attached to but distinct from the myofiber and, hence, SCs. They were present as early as Day 0 (Yablonka-Reuveni et al. 1999). At least part of the MAPK⁺ cells may correspond to nondividing M-cadherin⁺ SCs.

A fraction of the myogenin⁺ cells was M-cadherin⁻ (Figure 2A). M-cadherin expression is part of the differentiative program (Rose et al. 1994; Cifuentes-Diaz et al. 1995; Zeschnigk et al. 1995). Therefore, the finding that the majority of the myogenin⁺ cells do not become subsequently positive for M-cadherin in the fiber cultures was unexpected. Continuous expression of M-cadherin, as found on SCs in vivo, may require conditions of cell-cell contacts and the opportunity to adhere to one another or to newly formed myotubes, conditions that are provided for in muscle tissue and also in cell lines and mass cultures (Donalies et al. 1991; Pouliot et al. 1994; Zeschnigk et al. 1995) but not in our system, in which SCs are kept at a distance from one another. A primary rat SC culture may even lose its M-cadherin protein expression if the cells are made unable to fuse by deprivation of Ca⁺⁺ ions in the culture medium (Eng et al. 1997).

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, Cornelison and Wold (1997) found, as we did, that only a fraction of SCs expressed M-cadherin immediately after plating. SCs co-expressing myogenin and M-cadherin started to appear after 48 hr. In their culture system, it was predominantly the M-cadherin⁺/myogenin⁺ subset that increased in number, whereas only few M-cadherin⁺ cells co-expressed neither myogenin or MyoD. Their fraction of M-cadherin⁺ cells increased with time to include 100% of the cells investigated, whereas ours never reached the number of PCNA⁺ or myogenin⁺ cells (Figure 2A). These investigators used a serum-rich medium, and therefore may have stimulated the proliferation of SCs more than we did. Furthermore, it is possible that only high levels of mRNA correspond to protein expression. However, the single-cell analysis of Cornelison and Wold (1997) did not assess possible changes in mRNA levels and rather focused on whether or not M-cadherin was expressed. The discrepancy in M-cadherin⁺ cell numbers between the two studies may also be explained by species differences or by the finding that M-cadherin mRNA and protein expression are not necessarily tightly regulated. The protein appears 1–2 days later than its mRNA in mouse embryos (Rose et al. 1994). However, Eng and co-workers (1997) found that, after Ca⁺⁺ deprivation in primary SC cultures, M-cadherin mRNA was still present when the protein had already disappeared.

There are two compartments of SCs in cultures from normal innervated muscle fibers, one proliferative-differentiative (PCNA⁺/myogenin⁻/M-cadherin⁻ or PCNA⁻/myogenin⁺/M-cadherin⁻) and the other one non-dividing (M-cadherin⁺/PCNA⁻/myogenin⁻). Schultz (1996), in a detailed autoradiographic study of growing rat muscle, detected two compartments of SCs that differed regarding their cell cycle duration. Using [³H]-thymidine and bromodeoxyuridine, he found that 80% of SCs divided with a 32-hr cell cycle duration and that the remaining 20% divided more slowly, if at all. Schultz suggested that the rapidly dividing cells were responsible chiefly for providing myonuclei to growing fibers (producer compartment) and that the slowly dividing cells probably entered a G₀-phase between mitotic divisions and generated the SC population (reserve compartment). The cells going through the PCNA-myogenin program of the present study might be the rapidly dividing SCs of Schultz (1996), and the M-cadherin⁺/PCNA⁻/myogenin⁻ cells might be the slowly dividing cells.

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 1–32 weeks (Figures 2D-2H). Therefore, none of the SCs entered the mitotic cycle in fiber cultures from muscles that were denervated in vivo for 1–32 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 (Snow 1983; Schmalbruch and Lewis 1994). If loss of SCs occurs at this early time point after denervation, it is compensated for by proliferation which has been detected autoradiographically (McGeachie and Allbrook 1978; Murray and Robbins 1982; McGeachie 1989). SCs from muscle fibers denervated for 1 week have most probably not undergone a mitotic cycle after plating in vitro (Figure 2D), but at least some of the SCs may have undergone mitosis in vivo before plating, because SCs from muscle denervated for 4 days are still able to proliferate (Figure 2C). Perhaps they have proliferated at the expense of the nondividing SC pool, which is now reduced (Figure 2D). Alternatively, SCs may be present but fail to synthesize M-cadherin.

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 (Figures 2E and 2F), and that the number of SCs in muscle denervated for this time period is not significantly reduced in vivo (Schmalbruch and Lewis 1994; Rodrigues and Schmalbruch 1995), we can speculate that a shift in the phenotypic composition of the SCs has taken place and that the SCs have transited from the proliferative-differentiative compartment to the quiescent, or perhaps differentiated, compartment. It is unclear whether the M-cadherin⁺ cells are unable to enter proliferation or are already differentiated before isolation of the muscle fibers. Preliminary results indicated that basic (b) FGF did not recruit SCs from myofibers denervated for 1–32 weeks into the mitotic cycle (unpublished observations). This finding is in contrast to the behavior of SCs from normal innervated muscle, which respond to bFGF by enhanced proliferation (Yablonka-Reuveni and Rivera 1994; Yablonka-Reuveni et al. 1999), and could support the notion that SCs from denervated muscle are indeed postmitotic. However, it still does not rule out the possibility that SCs from denervated muscle are quiescent but require a greater stimulus to enter the mitotic cycle. Billington and Carlson (1996) found that SCs in long-term denervated muscle are in effect able to enter the mitotic program 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 longtime denervation (Rodrigues and Schmalbruch 1995).

M-cadherin⁺ SCs in muscles denervated for 1 week or more hardly assume myogenin expression, as opposed to innervated muscle fibers (Figures 3A, 3C, and 3D). It has been shown before in vivo that both mRNA and protein myogenin is elevated after denervation, but only transiently so (Voytik et al. 1993; Weis 1994; Adams et al. 1995).

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 1–32 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.