ARTICLE |
Correspondence to: Zipora YablonkaReuveni, Dept. of Biological Structure, Box 357420, School of Medicine, U. of Washington, Seattle, WA 98195. E-mail: reuveni@u.washington.edu.
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Summary |
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Although the role of satellite cells in muscle growth and repair is well recognized, understanding of the molecular events that accompany their activation and proliferation is limited. In this study, we used the single myofiber culture model for comparing the proliferative dynamics of satellite cells from growing (3-week-old), young adult (8- to 10-week-old), and old (9- to 11-month-old) rats. In these fiber cultures, the satellite cells are maintained in their in situ position underneath the fiber basement membrane. We first demonstrate that the cytoplasm of fiber-associated satellite cells can be monitored with an antibody against the extracellular signal regulated kinases 1 and 2 (ERK1 and ERK2), which belong to the mitogen-activated protein kinase (MAPK) superfamily. With this immunocytological marker, we show that the satellite cells from all three age groups first proliferate and express PCNA and MyoD, and subsequently, about 24 hr later, exit the PCNA+/MyoD+ state and become positive for myogenin. For all three age groups, fibroblast growth factor 2 (FGF2) enhances by about twofold the number of satellite cells that are capable of proliferation, as determined by monitoring the number of cells that transit from the MAPK+ phenotype to the PCNA+/MAPK+ or MyoD+/MAPK+ phenotype. Furthermore, contrary to the commonly accepted convention, we show that in the fiber cultures FGF2 does not suppress the subsequent transition of the proliferating cells into the myogenin+ compartment. Although myogenesis of satellite cells from growing, young adult, and old rats follows a similar program, two distinctive features were identified for satellite cells in fiber cultures from the old rats. First, a large number of MAPK+ cells do not appear to enter the MyoDmyogenin expression program. Second, the maximal number of proliferating satellite cells is attained a day later than in cultures from the young adults. This apparent "lag" in proliferation was not affected by hepatocyte growth factor (HGF), which has been implicated in accelerating the first round of satellite cell proliferation. HGF and FGF2 were equally efficient in promoting proliferation of satellite cells in fibers from old rats. Collectively, the investigation suggests that FGF plays a critical role in the recruitment of satellite cells into proliferation. (J Histochem Cytochem 47:2342, 1999)
Key Words: satellite cells, PCNA, MyoD, myogenin, mitogen-activated protein, kinase, MAPK, ERK1, ERK2, fibroblast growth factor, hepatocyte growth factor
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Introduction |
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Satellite cells, the myogenic precursors in postnatal and adult skeletal muscle, are situated between the basement membrane and the plasma membrane of myofibers in growing and mature muscle (
After various kinds of muscle trauma, satellite cells enter a program that involves the expression of the myogenic transcription factors MyoD and myogenin (
Because satellite cells are believed to be the only source of myogenic precursors in postnatal and adult muscle, myogenic cultures of cells isolated from juvenile or adult muscles are commonly presumed to be cultures of satellite cells. A number of growth factor families are involved in controlling proliferation and differentiation in such satellite cell cultures (
We reported previously on the use of indirect immunofluorescence to trace myogenesis of satellite cells in the isolated fiber model in analyzing fibers from "young adult" rats (8 to 12 weeks old). We demonstrated that the satellite cells follow a highly coordinated, multistep program of regulatory and structural protein expression (, whose levels correlate with DNA synthesis during the cell cycle, becoming maximal during the S-phase (
The primary goal of the present study was to compare the proliferative dynamics of satellite cells from "growing" (3 weeks old), "young adult" (810 weeks old), and "old" (911 months old) rats. Whereas 3-week-old rats grow rapidly and their satellite cells are believed to be proliferative, older rats grow slowly and their satellite cells are believed to be quiescent. Experiments with primary cultures raised the possibility that quiescent satellite cells require, for their first round of cell proliferation, other growth factors then those needed during ongoing proliferation of satellite cells from growing animals (
To pursue the analysis of satellite cells in myofibers from the rats of different ages, we first needed to identify a cytoplasmic marker for satellite cells that allows, in combination with the nuclear expression of PCNA, MyoD, and myogenin, unequivocal identification of the cells. This was crucial for the present study; in various models of muscle injury and in different situations of muscle stress, the myofiber nuclei are also believed to express MyoD and/or myogenin, making the tracing of satellite cells on the basis of nuclear expression of the MRFs potentially problematic (
We found that the cytoplasm of satellite cells undergoing myogenesis on isolated fibers can be monitored by immunostaining with an antibody against the extracelluar signal-regulated kinases 1 and 2 (ERK1 and ERK2). These ERKs are members of the mitogen-activated protein kinase (MAPK) superfamily, which is involved in the transmission of extracellular signals to their intracellular targets. The ERK1/ERK2 signal transduction pathway is typically initiated by binding of growth factors to their receptors, generating an activation cascade of specific protein kinases and leading to the activation of ERK1/ERK2. Activated ERK1/ERK2 can phosphorylate regulatory targets in the cytosol or can translocate to the nucleus and phosphorylate transcription factors. Although activated ERK1 and ERK2 are dually phosphorylated on specific tyrosine and threonine residues, at any given time the cell may also contain unphosphorylated ERK1/ERK2, along with ERKs that are singly phophorylated either at the tyrosine or the threonine residue (for reviews see
The immunolabeling with the anti-MAPK antibody (which recognizes both the phosphorylated and nonphosphorylated forms of ERK1/2), combined with the immunolabeling of the nuclear proteins PCNA, MyoD, and myogenin, has provided a direct means for tracing satellite cells as they undergo proliferation and differentiation in fiber cultures, regardless of the age of the muscle from which the fibers are isolated. Using these cytological markers, we show in this study that FGF2 regulates proliferation of satellite cells from both young and old rats. The number of satellite cells entering proliferation and expressing MyoD, as well as the overall number of satellite cells subsequently transiting into the myogenin state, is enhanced in the presence of FGF2 regardless of the age of the muscle. This effect of FGF2 in the single fiber model differs from the commonly accepted convention that FGF suppresses MyoD expression and myogenic differentiation (
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Materials and Methods |
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Animals
Male rats (SpragueDawley) were used throughout the study and were purchased from B & K Universal (Kent, WA). Unless otherwise noted, the following three age groups were used: young or growing rats3 weeks old (5055 g, fast growers); young adult rats810 weeks old (225300 g, moderate growers); and old rats911 months old (retired breeders; 600750 g at 9 months, slow growers).
Isolation and Culture of Rat Muscle Fibers
Single muscle fibers with associated satellite cells were prepared from the flexor digitorum brevis (FDB) muscle of rat hind foot according to procedures previously described for 812-week-old rats (
Medium (± additives when indicated) was replenished daily to allow adequate supply of the reagents. FGF2 (human recombinant, produced in yeast; kindly provided by Dr. S. Hauschka, University of Washington) was added to the medium of the fiber cultures at 2 ng/ml. Higher FGF2 concentrations in the range of 510 ng/ml had identical mitogenic effect as that obtained with 2 ng/ml FGF2. HGF (human recombinant; R&D Systems, Minneapolis, MN) was added at 10 ng/ml; this concentration was based on the analysis of the effect of 5 to 40 ng/ml HGF. Cytosine arabinoside (Sigma) was added to the medium at 10 µM unless otherwise noted.
Immunolabeling and Counterstaining of Nuclei
Single and double immunolabeling of fiber cultures were performed using indirect immunofluorescence as previously described (
In studying the co-expression of various antigens (detected with monoclonal antibodies) along with MAPK (detected with a polyclonal antibody), the two primary antibodies were added to the cultures together and subsequently the two appropriate secondary antibodies were added together under the same conditions as for single antibody staining. For analysis of the co-expression of the various antigens (detected with monoclonal antibodies) along with MyoD (detected with a polyclonal antibody), the cultures were reacted first with the monoclonal antibody followed by a reaction with the secondary antibody, as discussed above. Cultures were then reacted overnight with both the specific monoclonal antibody and the polyclonal antibody against MyoD, followed by a reaction with both the fluorescein-conjugated goat anti-mouse IgG and the rhodamine-conjugated goat anti-rabbit IgG. The two cycles of reaction with the monoclonal antibody amplified the fluorescein signal, reducing eye fatigue when doubly stained nuclei were monitored in fiber cultures.
In many experiments, nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; 1 µg/ml in PBS) to allow detection of all nuclei in the myofiber cultures. After removal of the secondary antibodies, the cultures were stained with DAPI for 15 min at RT. Cultures were then rinsed three times with TBS-TW20 and mounted in VECTASHIELD as above. DAPI-stained nuclei were observed with Hoechst filters.
Primary Antibodies
The following primary antibodies diluted in TBS-NGS were used to study fiber cultures.
Anti-PCNA (MAb 19F4).
A mouse MAb against proliferating cell nuclear antigen (PCNA) was from Boehringer Mannheim (Indianapolis, IN). This antibody has been used by us in earlier studies to monitor proliferating rat satellite cells (
Anti-MyoD (MAb 5.8A).
A mouse MAb against murine MyoD (IgG fraction) was developed and kindly provided by Drs. P. Houghton and P. Dias (St. Jude Children's Research Hospital, Memphis) (
Anti-MyoD (Polyclonal Ab).
A rabbit polyclonal Ab against rodent MyoD was prepared and provided by Dr. S. Alemá (Inst. of Cell Biology, CNR, Rome, Italy). We described in previous studies additional characterizations of this antibody (
Anti-myogenin (MAb F5D).
A mouse MAb against rodent myogenin was used in hybridoma supernatant form. The F5D hybridoma was developed and kindly provided by Dr. W. Wright (University of Texas). The utilization of this anti-myogenin MAb to stain rat satellite cells on isolated fibers and mouse-derived myogenic cultures has been described in our previous publications (
Anti-developmental Myosin (MAb F1584C10).
A mouse MAb against developmental but not adult isoforms of sarcomeric myosin heavy chain (DEVmyosin) was developed and kindly provided by Drs. J.J. Leger and F. Pons (Faculty of Pharmacy, INSERM, Montpellier, France). This antibody recognizes developmental isoforms of myosin heavy chain in various species, including rodents. The antibody was originally prepared against fetal bovine myosin and was shown to recognize myosin heavy chain in embryonic and fetal but not adult human muscle (
Anti-MAPK (Polyclonal Ab).
The antibody against mitogen activated protein kinase was made in a rabbit immunized with a peptide that represents residues 307327 of the ERK gene product. The peptide sequence is conserved in both ERK1 and ERK2 and the antibody recognizes both ERKs (phosphorylated and nonphosphorylated forms) at the same sensitivity. The antibody was first produced by Drs. R. Seger and E. Krebs and is characterized in
Counting Positive Cells on Isolated Fibers
Fibers were monitored for the number of fiber-associated nuclei and/or cells positive for the different antibodies. In initial studies, the immunocytochemical observations were made with a Zeiss epifluorescence microscope using a x25 or x40 objective (depending on the intensity of the immunoreagent). More recent experiments were analyzed with a Nikon Optiphot 2 fluorescence microscope using a x20 or x40 objective. Two to three parallel 35-mm plates were used for each time point of an individual experiment. Positive cells were scored as the number of positives on each individual fiber, analyzing 30 fibers per plate. The total number of positive cells for 30 fibers was then averaged for the duplicate or triplicate plates. This value is eventually expressed per 10 fibers as shown in the figures. For some experiments (see Table 1), the data are also shown as the total number of positive cells per the 60 fibers analyzed. Although the data discussed under Results represent individual experiments, each experiment was repeated several times.
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Immunoblot Analysis of Fiber Cultures with Anti-MAPK
Fiber cultures (two 35-mm plates per time point) were rinsed three times with MEM and received 100 µl of 2 x SDS sample buffer (4% SDS, 20% glycerol, 10% ß-mercaptoethanol, 0.016% bromophenol blue, 50 mM Tris, pH 6.8). Cultures were then collected with a cell scraper, which led to the extraction of both the fibers and the Vitrogen into the SDS sample buffer. The culture extracts were then frozen at -20C until analysis by SDS-polyacrylamide gels. Samples were heated at 95100C for 5 min just before the analysis. SDS gels, prepared with 12% polyacrylamide and 0.12% bisacrylamide, were run at 185 V until the dye front reached the bottom edge of the gel. Phosphorylation of MAPKs on the regulatory tyrosine and threonine residues changes the protein mobility in SDS-PAGE (
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Results |
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Reactivity of Satellite Cells on Isolated Fibers from Young Adult Rats with the Anti-MAPK Antibody
Figure 1 shows fiber cultures from the FDB muscles of 8-week-old rats stained via double immunofluorescence with the polyclonal antibody against MAPK along with the monoclonal antibodies against PCNA (Figure 1A and Figure 1A'), MyoD (Figure 1B and Figure 1B'), or myogenin Figure 1C and Figure 1C'). Cultures were also reacted with DAPI to trace the multiple nuclei within the myofiber and the nuclei of the fiber-associated satellite cells (Figure 1A''1C''). In agreement with our earlier studies, the antibodies against PCNA, MyoD, or myogenin recognized a small number of nuclei which, by the DAPI counterstain, were indistinguishable from the rest of the myofiber nuclei. In contrast, MAPK immunostaining distinguished the cytoplasm of individual cells associated with the fibers and demonstrated that the nuclei stained with the antibodies against PCNA, MyoD, or myogenin are always co-localized to these MAPK-positive cells. This localization proves that the fiber-associated nuclei stained with PCNA, MyoD, or myogenin are within satellite cells (and are not myofiber nuclei). The MAPK immunostaining shown in Figure 1 represent results with fibers that were cultured for 2 or 3 days; these are the days on which the maximal number of PCNA+/MyoD+ or myogenin+ cells peaks (see Figure 2). Satellite cells in Time 0 cultures can also be recognized with the antibody against MAPK. However, at this early stage the satellite cell cytoplasm occupies just the periphery of the nucleus and shows a far lower staining intensity than at the later stages (not shown). For all time points, we cannot rule out the possibility that the cytoplasm of the myofiber itself is also positive for MAPK. Nevertheless, the staining of the satellite cells with the anti-MAPK is far more intense than that of the fiber cytoplasm, providing a means for tracing the cells.
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Figure 2 demonstrates further quantification of the fiber-associated MAPK+ cells. Data in all three panels are based on experiments with fiber cultures prepared from 8-week-old rats and each panel represent an independent experiment. Similar results were obtained with cultures of fibers isolated from 612-week-old rats.
Figure 2A summarizes an experiment in which parallel plates were reacted via double immunofluorescence with the antibody against MAPK in combination with the anti-PCNA, the anti-myogenin, or the anti-DEVmyosin. The immunostaining with the different antibodies (excluding anti-DEVmyosin) is as in Figure 1. Micrographs depicting immunostaining of satellite cells with the antibody against DEVmyosin are included in
The decline in the number of MAPK+ cells at late time points probably reflects a reduction in the level of MAPK expressed by the cells as they differentiate. This is supported by the appearance of the DEVmyosin+ cells, which are negative for MAPK. The possibility that the decline in MAPK+ cells is due to fusion with the myofiber is not favored in view of the studies of
A second independent experiment, shown in Figure 2B, summarizes a comparison of the accumulation of MyoD+/MAPK+ cells vs PCNA+/MAPK+ cells when cultures are maintained in the absence or presence of FGF2. Regardless of the presence of FGF2, all PCNA+ nuclei and all MyoD+ nuclei were within cell cytoplasm positive for MAPK. Although the overall kinetics of PCNA+/MAPK+ and MyoD+/MAPK+ shown in Figure 2B are similar, there are slight variations in the actual values of PCNA+/MAPK+ or MyoD+/PCNA+ cells, as noted by the error bars at the different time points. However, similar values were revealed for PCNA+ or MyoD+ cells when fiber cultures were doubly stained with the anti-PCNA and anti-MyoD antibodies. Almost all PCNA+ cells were also positive for MyoD and vice versa (
Figure 2C summarizes the results from a third experiment in which we analyzed, via double antibody labeling, the effect of FGF2 on the total number of MAPK+ cells along with the effect on the PCNA+/MAPK+ cells. The results show that, at the time of culture establishment, the number of PCNA+/MAPK+ cells is markedly lower than the number of total MAPK+ cells. The increase in PCNA+/MAPK+ cells promoted by FGF2 correlates with an increase in the total number of MAPK+ cells. Regardless of the presence of FGF2, the increases in the number of PCNA+/MAPK+ cells or in total MAPK+ cells occur mainly after the first day in culture and peak by culture Day 2. As in Figure 2A, the total number of MAPK+ cells in Day 2 cultures is greater than the number of PCNA+/MAPK+ cells. This additional number of MAPK+ cells is primarily due to the emergence of myogenin+ cells (which are also MAPK+) by Day 2 (data not shown). Overall, the total number of MAPK+ cells is increased by 2.8-fold in cultures receiving FGF2 and only by 1.8-fold in control cultures. A similar relationship in the increase of the number of MAPK+ cells in the presence or absence of FGF2 is further demonstrated in Figure 3. The results suggest that the addition of FGF2 to the cultures allows more (or perhaps all) satellite cells present on the fibers to undergo cell division. Alternatively, FGF2 may accelerate the cell cycle time of the satellite cells which, without the addition of FGF2, proliferate at a far slower rate.
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In the experiments shown in Figure 2C and Figure 2A, there is a decline in the number of MAPK+ cells at later time points. This decline is more rapid in the experiment shown in Figure 2C than in the experiment shown in Figure 2A. The decline in the number of MAPK+ cells for Day 3 cultures (Figure 2C) and the plateau of MAPK+ cells for Days 2 and 3 (Figure 2A) have both been reproduced in many experiments. These slight variations in the timing of the decline in MAPK+ cells reflect how synchronous the cells are as they transit into the myogenin+ compartment.
Effect of the Mitotic Drug Cytosine Arabinoside on MAPK+ Cells
Culturing rat fibers with cytosine arabinoside led to the elimination of the fiber-associated satellite cells, as estimated by regular microscopic evaluation (
Immunoblot Analysis of the MAPK Isoforms Expressed by Satellite Cells
Immunoblot analyses of routine cell cultures with the anti-MAPK antibody used in the present study showed that the antibody recognizes ERK1 and ERK2 (44- and 42-kD molecular weight, respectively) in their inactive and active forms (
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In addition to the specific ERK polypeptides, fiber extracts show one more intense band when analyzed by immunoblotting (Figure 4AC). The migration position of this extra band, just above the 47-kD molecular weight marker, is marked with an arrowhead at the left of each panel. This is a nonspecific band contributed by the horse serum included in the fiber isolation medium and in the basal medium used for fiber cultures. This 47-kD protein cannot be washed away from the Vitrogen even after excessive rinses and is also present in extracts of sham fiber cultures that are devoid of fibers (data not shown). As shown in Figure 4D, extracts of fibers isolated and cultured without horse serum did not exhibit this band.
The immunoblot analysis in Figure 4A and Figure 4B has shown a marked decline in the intensity of the ERK2p band between the initial 30 min in culture (i.e., 30 min in basal medium after the initial 20 min of fiber adherence to the Vitrogen) and the Day 1 time point. Figure 4C shows a more detailed analysis of the early time points after culture establishment. At time point 0 the prevalent forms of ERK1 and ERK2 are the shifted ones (ERK1p and ERK2p). ERK1p is drastically reduced by 30 min in culture, whereas ERK2p is drastically reduced in intensity between the 60-min and the 24-hr time points. These transitions are not influenced by the presence of FGF2. We further investigated the possibility that the horse serum included in the fiber preparation protocol (after the collagenase digestion) might provide the signals leading to the active ERK1 and ERK2 detected at the early time points. The horse serum, present routinely at 10% in the fiber isolation medium, was replaced with serum replacement (CPSR2, 10%) and the horse serum present in the culture medium was omitted. Figure 4D summarizes the immunoblot analysis of this horse serum-free protocol and shows that, in the absence of horse serum, ERK2p was present at high levels throughout the early time points tested, regardless of the presence or absence of FGF2. ERK2p declined to a lower level by 24 hr in culture. The pattern shown for the 5-, 10-, and 20-min time points was maintained for the 60- and 120-min time points as well (data not shown). Time points between 120 min and Day 1 were not analyzed. ERK1p was not detected in any of the extracts of horse serum-free fiber cultures. At present we do not have means for distinguishing whether the "collapses" in ERK1p and ERK2p seen by the first day in culture are specific to the satellite cells. Obviously, these transitions may reflect changes occurring in the myofiber itself on culturing.
Kinetics of Satellite Cell Myogenesis in Cultures of Fibers from Young Rats
Double immunostaining of fiber cultures from 3-week-old rats demonstrated that all nuclei positive for PCNA, MyoD, or myogenin are contained within MAPK+ cytoplasm of single cells (not shown; see Figure 1 for a similar staining). This indicates that, as in cultures from young adult rats, all positive nuclei in fibers from 3-week-old rats are within satellite cells and do not represent myofiber nuclei.
Figure 5 shows the results of two independent studies in which satellite cells in fibers from 3-week-old rats were monitored by their immunoreactivity with the antibodies against PCNA, MyoD, myogenin, and MAPK. Data in Figure 5 are based on analyses of fibers that were cultured in the presence of FGF2. Fibers cultured in the absence of FGF2 demonstrated only a small number of PCNA+ nuclei (Figure 6) and a similar small number of MyoD+, and subsequently myogenin+, nuclei (data not shown). In the experiment shown in Figure 5A, cultures were reacted with individual antibodies againt MAPK, PCNA, and MyoD. In the experiment shown in Figure 5B, cultures were reacted with the antibody against MAPK alone or with the polyclonal antibody against MyoD in combination with the anti-myogenin. The studies in Figure 5 demonstrate that satellite cells from 3-week-old rats follow a similar program as that seen with satellite cells from the young adult rats, first becoming positive for MyoD and PCNA and subsequently entering the myogenin+ state. Although the overall program of transition through proliferation and differentiation for satellite cells from 3-week-old rats is similar to that for satellite cells from the 810-week-old rats, the increase in the number of proliferating cells and the transition into the myogenin+ state is more rapid for satellite cells from the younger animals. Despite the presence of FGF2, the maximal number of proliferating satellite cells is lower for fibers from the 3-week-old rats compared to fibers from the young adult animals. Analysis of MAPK+ cells at the time of culture establishment is not shown because of the difficulty in differentiating between the cells and the myofiber nuclei in working with Time 0 fibers from the young rats. After about 12 hr in culture, satellite cell analysis by the antibody against MAPK becomes feasible.
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Both panels in Figure 5 show a surplus of MAPK+ cells by Day 1, when the total number of MAPK+ cells is compared to the number of PCNA+ or MyoD+ cells. This surplus (as in the Day 1 fiber cultures from young adult rats) may indicate that the satellite cells become detectable by the anti-MAPK antibody before their detectability with the other antibodies. However, it is possible that some of the surplus cells represent additional satellite-like cells that do not enter the MyoD expression program. We further analyzed this issue in the study summarized in Table 1. Parallel plates were monitored by double antibody staining for the presence of MyoD±/PCNA± nuclei and MyoD±/myogenin± nuclei, or by single antibody staining for MAPK+ cells. At each time point, the total number of positive nuclei consisted of all MyoD+ cells (determined by averaging the number of MyoD+ cells from the aforementioned pairs), combined with the number of PCNA+ cells negative for MyoD, and the number of myogenin+ cells negative for MyoD. Indeed, the data in Table 1 show a surplus of MAPK+ cells in both Day 1 and Day 2 cultures (which amounts to 27 and 42 cells, respectively). This surplus may represent a subpopulation of cells that are incapable of entering the PCNA/MyoD/myogenin program under the culture conditions used (and may even be nonmyogenic). The discrepancy shown in Table 1 between the number of MAPK+ cells and the number of total positive nuclei in Days 3 and 4 is probably primarily due to the fact that most of the satellite cells are no longer traceable at late time points via the nuclear antigens studied.
As mentioned above, fiber cultures from 3-week-old rats exhibited only a small number of PCNA+ nuclei (and a similarly small number of MyoD+ or myogenin+ nuclei) without the addition of FGF2 to the basal medium. Figure 6 summarizes typical results of such analyses on the effect of FGF2 in fiber cultures from 3-week-old rats, comparing the number of PCNA+ nuclei and MAPK+ cells. Figure 6 shows that the addition of FGF2 led to an increase in the number of PCNA+ nuclei and MAPK+ cells by Day 1 in culture (no effect was observed by culture Day 0.5; data not shown). On Day 2, the peak day for PCNA+ or MAPK+ nuclei, there is a discrepancy between the number of MAPK+ cells and PCNA+ nuclei. In the FGF2-treated cultures, a large portion of this discrepancy can be explained by the presence of myogenin+ cells (which are also MAPK+), but additional surplus MAPK+ cells are likely to be present too, as shown in Table 1. However, in the Day 2 control cultures, the number of myogenin+ cells is small and cannot account for most of the surplus MAPK+ cells. We therefore suggest that there is a larger number of surplus cells in control cultures and that the addition of FGF2 allows more of the surplus MAPK+ cells to enter the proliferative pathway (which is subsequently followed by myogenic differentiation).
We also analyzed the influence of cytosine arabinoside on the kinetics of PCNA+ nuclei and MAPK+ cells in fibers from the 3-week-old rats. Regardless of the absence or presence of FGF2, almost all of the PCNA+ nuclei were eliminated by Day 1 in culture. Some residual MAPK+ cells were still present by Day 1 in culture (about 2025% of the cells) but were almost all eliminated by Day 2 in culture. Cultures maintained for 3 or 4 days in the basal medium (±FGF2) in the presence of cytosine arabinoside were practically devoid of PCNA+ nuclei or MAPK+ cells (data not shown). These findings suggest that all MAPK+ cells (PCNA+/MAPK+ or surplus MAPK+ cells) are sensitive to the anti-mitotic drug.
Monitoring Satellite Cells on Isolated Fibers from Old Rats
With the ability to co-localize nuclear antigens within MAPK+ cytoplasm of fiber-associated cells, we set out to investigate the dynamics of satellite cells on isolated fibers from the old (911-month-old) rats. Immunostaining of the fibers with the antibodies against MAPK and the nuclear proteins revealed more fiber-associated MAPK+ cells compared to fibers from the younger rats. Furthermore, a high proportion of these cells did not stain with the antibodies against the nuclear proteins MyoD, PCNA, and myogenin. Figure 7 shows two examples of fibers from the old rats stained with the antibodies against MAPK and MyoD (Figure 7/A' and 7B/B'), demonstrating a larger number of cells together in single sites on the fibers compared to what we observed with the younger rats (Figure 1). The quantification of fiber-associated cells in cultures from the old rats is summarized in Figure 8 for isolated fibers maintained with or without FGF2. Figure 8A and Figure 8B show the results of one experiment in which parallel plates were reacted via double immunofluorescence with the antibodies against PCNA and MAPK, or MyoD and MAPK. Figure 8C shows the results of a similar study with the anti-myogenin/anti-MAPK antibody combination. The analyses demonstrate that all PCNA+, MyoD+, and myogenin+ nuclei associated with fibers from old rats are always contained within MAPK+ cytoplasm, indicating that our method of monitoring satellite cell myogenesis is valid for the old rats as well. The kinetics of the PCNA+ cells are similar to those of the MyoD+ cells in either the presence or absence of FGF2, indicating that both PCNA and MyoD immunostaining mark the satellite cells (as shown above for fiber cultures from younger rats). Without the addition of FGF2, only a basal level of cells undergoes myogenesis, whereas in the presence of FGF2 a larger number of cells are detected. In addition, fibers from the old rats demonstrate a higher number of surplus MAPK+ cells than those seen in cultures from the younger ages. It is possible that some of the surplus MAPK+ cells reside outside of the myofiber basement membrane (especially in view of the elaborated connective tissue network between the myofibers in the older rats). However, similar to the MyoD+ and PCNA+ cells, these surplus cells also respond to FGF2.
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We further analyzed the effect of cytosine arabinoside in FGF2-treated fiber cultures from the old rats. Cytosine arabinoside in the range of 1050 µM did not influence the total number of MAPK+ cells or the number of MyoD+ cells by the first day in culture. However, by the second culture day about 84% of the MyoD+ cells (and 40% of the total MAPK+ cells) were eliminated compared to FGF2-treated control cultures. By the third culture day, about 96% of the MyoD+ cells (and 81% of the total MAPK+ cells) were eliminated compared to the FGF2-treated controls. Therefore, some of the MAPK+/MyoD- cells are insensitive to cytosine arabinoside even by culture Day 3, whereas in the younger rats all MAPK+ cells were sensitive to the drug by this day. In addition, the effect of the drug on the elimination of MyoD+ satellite cells requires one additional day in culture from old rats compared to the younger rats.
Comparison of the Effects of FGF2 and HGF on Proliferation of Satellite Cells from Old Rats
The data in Figure 8 indicate that the satellite cells from the old animals spend a longer time in the proliferative compartment than satellite cells from younger animals. Furthermore, the delayed effect of cytosine arabinoside on the MyoD+ cells from the old rats suggests that the onset of satellite cell proliferation in fiber cultures from old rats lags behind the onset of proliferation in cultures from younger animals. Analyzing myogenic cultures from 9-month-old rats,
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Discussion |
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This study was undertaken to evaluate the proliferative potential of satellite cells from growing and old rats. Satellite cells are believed to be proliferative in growing rats, whereas in older animals satellite cells are believed to be quiescent. Therefore, it is possible that proliferation of satellite cells from younger and older animals are regulated differently by growth factors. We employed cultures of single myofibers, in which the original position of the satellite cells by the myofiber is preserved. This association between the satellite cells and the myofibers is potentially important because the myofibers might contribute some of the growth factors involved in controlling proliferation and differentiation of satellite cells. Furthermore, this association between the satellite cells and the myofibers provides a means for localizing the myogenic precursors even before they express myogen-specific traits.
We first demonstrated that satellite cells undergoing myogenesis in fiber cultures can be traced immunohistochemically by their cytoplasmic expression of ERK1 and ERK2, members of the MAPK superfamily involved in the transmission of extracellular signals to their intracellular targets. The antibody we used reacts with both the phosphorylated and nonphosphorylated forms of the two ERKs. Although phosphorylated, active ERKs are believed to be able to translocate to the nucleus, we were unable to detect ERK1/2-positive nuclei throughout the study. The immunohistochemical staining with the anti-MAPK antibody was further employed for analyzing the proliferative dynamics of satellite cells from young (3-week-old), young adult (810-week-old), and old (911-month-old) animals. We demonstrated that satellite cells from all three age groups undergo a similar program, first becoming positive for PCNA and MyoD and later for myogenin. In addition, irrespective of the age of the muscle studied, FGF2 promotes the number of proliferating, MyoD+ satellite cells and does not suppress the transition of the cells into the myogenin+ state. Collectively, the present studies have led us to propose that skeletal muscle from both young and old animals contains satellite cells whose recruitment into active myogenesis, characterized by rapid proliferation and differentiation, is regulated by FGF2.
Analysis of Myofibers from Young Adult Rats Suggests that FGF2 Promotes an Increase in the Number of Satellite Cells Entering Replication
Monitoring satellite cells on fibers from 810-week-old rats by their expression of MAPK has provided an overall measure of the number of fiber-associated cells. As the number of PCNA+ cells rises to a maximal level, the number of MAPK+ cells rises as well. Interestingly, the co-localization of PCNA and MAPK at the earlier days in culture (Time 0 and Day 1) demonstrated a far higher number of MAPK+ cells compared to PCNA+ cells. This indicates that satellite cells can be monitored by their cytoplasmic MAPK before cell replication. This number of initial MAPK+ cells increases minimally, if at all, between Days 0 and 1, regardless of the presence or absence of FGF2. Between culture Days 1 and 2 there is an increase in the total number of MAPK+ cells, representing an increase in the total number of satellite cells associated with the fibers. The majority of these MAPK+ cells are positive for PCNA but a smaller fraction of the cells have already transited to the myogenin+ state. Because this increase in MAPK+ cells between Days 1 and 2 is far more robust when FGF2 is added to the cultures, we considered the possibility that FGF2 promotes an increase in the number of satellite cells recruited from quiescence to proliferation. If this indeed is how FGF2 influences the satellite cells, then the proliferation of some satellite cells without the addition of FGF2 may indicate that a subpopulation of the satellite cells requires a lower mitogen level (FGF2 and/or other mitogens) to enter replication. This low-level mitogen(s) is perhaps contributed by the myofibers themselves, either naturally or because of the stress induced by the isolation/culturing procedure. However, at this stage we can not exclude an alternative recruitment mechanism in which the exogenously added FGF2 accelerates satellite cell proliferation, generating altogether more satellite cells. In this instance, satellite cells may proliferate more slowly without the addition of FGF2, generating a smaller number of satellite cells. In fact, the finding that the mitotic drug cystosine arabinoside rapidly eliminated all fiber-associated satellite cells provides support in favor of the second recruitment mechanism, i.e., the satellite cells can proliferate without the additional FGF2 but the addition of FGF2 facilitates more rapid proliferation of the cells.
The FGF-promoted increase in satellite cells was observed in the present fiber study by the second day in culture. It is noteworthy that, in another study of fibers from young adult rats, we showed that the effect of FGF2 on increasing the number of PCNA+ cells is already taking place by 36 hr in culture. At this 36-hr time point, the number of PCNA+ cells was about 75% of the maximal number seen at 48 hr in culture (
The Transition of MAPK+ Cells to the Proliferative State in Fiber Cultures from 3-week-old Rats Is Highly Dependent on Exogenous FGF2
The quantification of fiber-associated cells in isolated fibers from 3-week-old rats revealed that, once the cells have been recruited into proliferation, myogenesis progresses in a similar fashion to that described for myofibers from young adult rats. In addition, as in the young adult rats, the FGF2-promoted increase in the number of PCNA+ cells in fibers from 3-week-old rats leads to a consequent increase in the number of cells entering the myogenin+ state. There are, however, distinct differences between the two age groups. First, the peak number of proliferating/differentiating satellite cells is lower in control or FGF-treated cultures from 3-week-old rats compared to cultures from young adults. Second, without the addition of FGF2, only a small number of MAPK+ cells are positive for PCNA in cultures from the 3-week rats (see Figure 5A, Day 2). On adding FGF2, the number of total MAPK+ cells increases along with a substantial increase in the number of PCNA+/MAPK+ cells and a decrease in the proportion of the surplus MAPK+ cells (negative for PCNA). The finding that satellite cells from 3-week-old rats demonstrate higher dependency on exogenous FGF2 for proliferation than satellite cells from 810-week-old rats is somewhat enigmatic, given the common convention that satellite cells from younger rats are proliferative, adding nuclei to the enlarging myofibers. The satellite cells in the 3-week-old fiber culture might be highly dependent on exogenous FGF2 because the culture system may lack endogenous FGF2 (and/or other FGFs). In contrast, FGF2 (or other FGFs) might be stored or produced in fibers from young adult rats, allowing some proliferation of the satellite cells without the addition of exogenous FGF2. Earlier studies have shown that FGF2 is present in the extracellular matrix surrounding the muscle fibers (
It is possible that many of the PCNA-/MAPK+ cells detected in the absence of FGF2 in fibers from 3-week-old rats are slowly dividing cells that do not accumulate sufficient PCNA for tracing via immunocytochemistry (but are sensitive to the mitotic drug cytosine arabinoside). The addition of FGF2 may shift many of these cells from the slow-dividing to the rapid-dividing phenotype. Fibers isolated from young adult rats may also contain such surplus PCNA-/MAPK+ cells, although at lower numbers than the fibers from the young rats (see, e.g., the Day 2 time point in Figure 2A, where the discrepency between the total number of MAPK+ cells compared to the PCNA+ cells cannot be solely explained by the small number of myogenin+ cells present). The possible existence of rapid-dividing and slow-dividing satellite cells in growing rats has been previously proposed by
Recruitment, Proliferation, and Differentiation of Satellite Cells in Fiber Cultures from Old Rats
Our studies demonstrated that FGF2 influences myogenesis of satellite cells from old animals in a similar manner to that seen for the younger animals. In the presence of FGF2, there is an increase in the number of PCNA+ or MyoD+ cells by the second day in culture, reaching maximal numbers by culture Day 3 and reduced to nearly baseline level by culture Day 5. An increase in myogenin+ cells commences 24 hr after the increase in the PCNA+ or MyoD+ cells, indicating that the myogenic precursors rapidly transit from proliferation to differentiation. In the absence of FGF2, the number of cells that transit through the program is significantly lower. We also conclude that cultured myofibers from old rats support a higher number of proliferating MyoD+ satellite cells than cultured myofibers from the rapidly growing young rats. This finding suggests that the number of myogenic precursors increases with the age of the animal. This increase in the number of satellite cells from older animals was unexpected because muscle regeneration was found to be impaired in older animals (
As in the younger rats, the addition of FGF2 to fibers from old rats allowed a larger number of satellite cells to undergo myogenesis compared to control cultures. However, the peak of proliferating cells is broadened to include culture Days 2, 3, and 4 (see Figure 8 and Figure 9). It is interesting to note that the in vivo studies of
We detected many surplus cells which were PCNA-/MAPK+ or MyoD-/MAPK+ in fibers from the old rats. The kinetics of the total number of MAPK+ cells closely parallel the kinetics of the PCNA+/MAPK+ cells or MyoD+/MAPK+ cells in control, FGF2-treated, and HGF-treated cultures. This correlation suggests that the surplus MAPK+ cells in the old animals could be related to the satellite cells undergoing active myogenesis. Perhaps satellite cells from the old animals give rise to such surplus cells more frequently than do satellite cells from the younger animals. Myogenic and nonmyogenic phenotypes are routinely present in myogenic clones derived from a single progenitor (see discussion in
How Might FGF2 Operate in Influencing Satellite Cell Proliferation in the Isolated Fiber Model?
The results of the present study indicate that FGF2 can influence the number of satellite cells undergoing myogenesis in fiber cultures from growing, young adult, and old rats. For all three age groups, the increase in the number of satellite cells is due to the effect of FGF2 on enhancing the number of proliferating satellite cells, whereas the transition of the cells into the differentiative state is not suppressed by the growth factor. The kinetics of proliferation in the absence and presence of FGF2 suggest that for all three age groups FGF2 is likely to act during the first round of cell replication after fiber isolation. Although the results indicate that FGF2 is involved in generating more satellite cells, the specific mode of action of FGF2 is not resolved by the present study. FGF2 may recruit more satellite cells by accelerating the cell cycle of cells that have already been enabled by other factors to enter the mitogenicmyogenic program, and/or by potentiating the entry of slowly dividing (possibly almost quiescent) precursor cells into a regular progression through the cell cycle and MyoD expression. Additional experiments with delayed exposures to exogenous FGF2 (done with fibers from young adult rats) demonstrated that the addition of FGF2 as late as 1518 hr after culture establishment results in a maximal number of fiber-associated PCNA+ satellite cells, which is similar to the number of cells seen when FGF2 is added at Time 0 (
In agreement with our proposal regarding the role of FGF2 in supporting recruitment of satellite cells,
Studies of routine myogenic cultures have reported that FGF2 (and other FGFs) can support continuous proliferation while delaying differentiation of myoblasts (
Experiments are under way in our laboratory to determine which FGF receptors are involved in the recruitment of satellite cells into the proliferative PCNA+/MyoD+ state seen in the fiber model and whether the same (or additional) FGF receptors are operating in culture systems in which FGF extends proliferation and suppresses differentiation.
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Acknowledgments |
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Supported in part by grants to ZYR from the Cooperative State Research ServiceUS Department of Agriculture (agreements nos. 93-37206-9301 and 95-37206-2356) and the National Institutes of Health (AR39677 and AG13798).
We thank Priscilla Natanson, Stephanie Kästner, and Maria Elias for their important contributions to the study. We also thank many colleagues for providing us with valuable reagents: Dr S. Hauschka (FGF2), Dr S. Alemá (anti-MyoD, polyclonal), Drs P. Houghton and P. Dias (anti-MyoD, monoclonal), Dr W. Wright (anti-myogenin), and Drs L.L. Leger and F. Pons (anti-DEVmyosin).
Received for publication June 12, 1998; accepted September 15, 1998.
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