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Am J Physiol Cell Physiol 279: C682-C690, 2000;
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Vol. 279, Issue 3, C682-C690, September 2000

Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat

Charles T. Putman, Sabine Düsterhöft, and Dirk Pette

Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Satellite cell proliferation was assessed in low-frequency-stimulated hypothyroid rat fast-twitch muscle by 5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent staining of labeled muscle nuclei, and by staining for proliferating cell nuclear antigen (PCNA). BrdU labeling and PCNA staining were highly correlated and increased approximately fourfold at 5 days of stimulation, decayed thereafter, but remained elevated over control in 10- and 20-day stimulated muscles. Myogenin mRNA was ~4-fold elevated at 5 days and 1.5-fold at 10 days. Staining for myogenin protein yielded results similar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequency stimulation. By 20 days, myogenin staining was observed in transforming fast fibers that coexpressed embryonic and adult myosin heavy chain isoforms. In the slower fiber populations (i.e., IIA and I), myogenin-positive transforming fibers that coexpressed embryonic myosin heavy chain, appeared already at 5 days. Thus the satellite cell progeny on slower fibers seemed to proliferate less and to fuse earlier to their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions.

chronic low-frequency stimulation; muscle fiber transformation; myogenin; myosin heavy chain isoforms


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHRONIC LOW-FREQUENCY STIMULATION (CLFS) induces fast-to-slow fiber type transitions in mammalian muscle (for reviews, see Refs. 17 and 18). The question has been raised as to a possible role of satellite cell progeny in this transformation process (19, 22). Satellite cells are quiescent muscle precursor cells located between the basal lamina and the sarcolemma of adult skeletal muscle fibers. They account for 2-7% of the nuclei associated with a particular fiber, while the proportion varies primarily with fiber type and age (24). Under steady-state conditions, satellite cells are mitotically inactive. In response to various exogenous stressors, however, they may be activated to enter the cell cycle, proliferate, and fuse with existing fibers or with each other to form new fibers (30). At the point where satellite cells leave their position between the basal lamina and the sarcolemma, such as occurs during migration or cell fusion, they are more appropriately referred to as muscle precursor cells (mpc). Quiescent myoblasts do not express detectable amounts of the myogenic regulatory factors at the protein level (3, 8, 16, 32), but on activation start to express MyoD in combination with proliferating cell nuclear antigen (PCNA), a specific proliferation marker (5). Subsequently, MyoD and PCNA are downregulated, and myogenin expression becomes manifest in cells committed to terminal differentiation.

A conceivable role of satellite cells in fast-to-slow fiber type conversions relates to an enhancement of the myonuclear content, because slow fibers display higher myonuclear contents than fast fibers (9). Another possible role of satellite cells in fiber type conversions is that reprogramming of fiber types occurs under the influence of specific satellite cell lineages. In an accompanying morphological and immunohistochemical study, we recently showed that CLFS of fast-twitch muscles in hypothyroid rats leads to a time-dependent increase in satellite cell content and myonuclear density (19). With that approach, we were able to show that satellite cell content rose 2.6-, 3.0-, and 3.7-fold over that of corresponding controls in 5-, 10-, and 20-day stimulated extensor digitorum longus (EDL) muscles. Compared with the total muscle nuclei, the relative satellite cell content increased from 3.8% in euthyroid control muscle to 7.9, 11.5, and 13.8% in the 5-, 10-, and 20-day stimulated hypothyroid muscles (19). These findings suggested satellite cell activation and proliferation, and widespread fusion of satellite cell progeny (i.e., mpc) to transforming fibers in response to CLFS.

The present study was undertaken to investigate satellite cell activation under the same conditions used in the preceding study (19), namely, CLFS-induced fast-to-slow fiber type transition in EDL and tibialis anterior (TA) muscle of hypothyroid rat. Satellite cell proliferation was assessed in vivo by two different methods: 1) 5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent immunolocalization of labeled nuclei and 2) immunohistochemical staining for PCNA, which is an acidic nonhistone auxiliary protein of DNA polymerase and is expressed solely during DNA synthesis (5). Immunohistochemical staining of mpc nuclei for myogenin was also completed to evaluate changes in the number of mpc that committed to terminal differentiation during CLFS. Changes in myogenin expression were also assessed at the mRNA level by RNase protection assay. Finally, to investigate whether or not the fusion of satellite cell progeny was related to a specific muscle fiber type, myogenin and myosin heavy chain (MHC) isoform expression patterns were studied in individual fibers of muscles exposed to CLFS for different time periods.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and hypothyroidism. Twenty-four adult (4 mo old) male Wistar rats (Thomae, Biberach, Germany) were utilized in this study. All animal experiments were approved by the local government (Regierungsprösidium Freiburg). The rats were treated in accordance with established principles of care and use. They were housed in the Animal Research Center of the University of Konstanz in a thermally controlled room maintained at 22°C with 12-h dark cycles alternating with 12-h light cycles. Before stimulation was initiated, hypothyroidism was induced in 21 rats by 7 wk of feeding an iodine-poor diet (Altromin C1042; Altromin, Lage, Germany) and by the addition of propylthiouracil to the drinking water. The remaining euthyroid controls received a regular diet (Altromin 1314). The euthyroid rats weighed 506 ± 35 (SD) g. The hypothyroid rats weighed 309 ± 7 g at the beginning of CLFS and lost 47 ± 25 g (P < 0.0001) during 20 days of CLFS. The EDL muscles of the euthyroid rats weighed 227 ± 33 (SD) mg, whereas EDL muscles of the hypothyroid rats weighed significantly less (P < 0.0001), being only 128 ± 14 mg. The TA muscles of the euthyroid rats weighed 770 ± 59 mg, whereas hypothyroid TA muscles were significantly (P < 0.0001) smaller, weighing only 482 ± 57 mg. Weights were not different between stimulated and control muscles, within groups.

Chronic low-frequency electrical stimulation, BrdU pulse labeling, and muscle sampling. Electrodes were implanted laterally to the peroneal nerve of the left hindlimb (25). After 1 wk of recovery, stimulation was started (continuously for 10 h daily at 10 Hz, impulse width 0.3 ms). The following groups were studied: 0-day euthyroid controls (n = 3); 0-day hypothyroid controls (n = 4), 5-day (n = 5), 10-day (n = 6), and 20-day (n = 6) stimulated hypothyroid animals. The left leg of the 0-day animals was sham-operated. After stimulation, rats were injected intraperitoneally with BrdU (Boehringer Mannheim, 45 mg/kg body wt) and 1 h later were euthanized. The EDL and TA muscles of the stimulated (left) and contralateral control (right) legs were excised, weighed, and frozen in melting (-159°C) isopentane. Muscles were stored in liquid nitrogen until analyzed.

Antibodies. To examine changes in fiber type distribution, the following monoclonal antibodies directed against adult MHC isoforms were used: MHCI [NOQ7.5.4D (12) and 7HCS15 (28)], MHCI + MHCIIa [7HCS11 (6)], MHCIIa [SC-71 (20)], MHCIIb (IgM) [BF-F3 (20)], and all fast MHC isoforms with the exception of MHCIIx/d [BF-35 (20)]. Developmental (embryonic) MHCemb was detected by NCL-D (Novocastra, Newcastle, UK). Mouse monoclonal anti-BrdU (containing nuclease enzymes; clone BMC 9318) and anti-PCNA (clone 19F4) were obtained from Boehringer Mannheim. Rabbit polyclonal anti-myogenin antibody (IgG; M-225) was obtained from Santa Cruz Biochemicals (Santa Cruz, CA). Biotinylated horse anti-mouse IgG (rat absorbed, affinity purified), biotinylated goat anti-rabbit IgG, and biotinylated goat anti-mouse IgM were obtained from Vector Laboratories (Burlingame, CA). Nonspecific control mouse IgG antibodies were obtained from Santa Cruz Biochemical.

Immunohistochemistry for myosin and myogenin. For myosin staining, 9-µm-thick frozen sections of EDL muscles were air dried, washed once in PBS with 0.1% Tween 20 (PBS-Tween), twice in PBS, and incubated for 30 min in 3% H2O2 in methanol. Sections were subsequently washed and incubated for 1 h in a blocking solution (BS-1; 1% BSA, 10% horse serum, and 0.1% Tween 20 in PBS, pH 7.4). For BF-F3 (IgM), goat serum was substituted for horse serum in the blocking solution (BS-2). Primary anti-mouse IgG monoclonal antibodies were diluted in BS-1 as follows: NCL-D culture supernatant 1:20; NOQ7.5.4D culture supernatant 1:400; 7HCS15 culture supernatant 1:40; 7HCS11 culture supernatant 1:400; SC-71 3.8 µg/ml; BF-35 1.7 µg/ml. BF-F3 was diluted 1:400 in BS-2. Control sections were completed in parallel in which 1) the primary antibody was substituted with nonspecific control mouse IgG and 2) the primary antibody was omitted. Sections were washed as before and reacted for 30 min with biotinylated horse anti-mouse IgG or biotinylated goat anti-mouse IgM (BF-F3). Sections were then washed, incubated with biotin-avidin-horseradish peroxidase (HRP) complex for 30 min, washed again, and reacted for 4 min with the substrate solution containing diaminobenzidine, H2O2, and NiCl2 in 50 mM Tris · HCl, pH 7.5 (Vector Laboratories Substrate Kit). The reaction was stopped by washing several times with distilled water. After dehydration in ethanol, sections were cleared and mounted with Entellan (Merck, Darmstadt, Germany).

For myogenin staining, sections were air dried, fixed for 10 min in cold acetone (-20°C), and excess acetone was allowed to evaporate. Sections were then washed once in PBS-Tween, twice in PBS, incubated for 30 min in 3% H2O2 in methanol, and washed again and incubated for 1 h in BS-2. Excess blocking solution was removed, and sections were incubated overnight at 4°C with the primary anti-myogenin antibody, diluted to a concentration of 0.3 µg/ml in BS-2. Sections were washed as before and reacted for 30 min with biotinylated goat anti-rabbit IgG. After washing, sections were incubated for 30 min with biotin-avidin-HRP complex (Vectastain Elite; Vector Laboratories), washed, and reacted for 6 min with the substrate solution (see above). After stopping the reaction with distilled water, sections were counter-stained with Nuclear Fast Red (Merck), dehydrated, cleared, and mounted in Entellan.

Serial sections, stained for the various MHC isoforms and myogenin, were examined to determine muscle fiber type, fiber cross-sectional area, and fiber type patterns of myogenin expression, using a computer program. The stimulated (mean ± SD: 264 ± 25 fibers/muscle) and control legs (268 ± 3 fibers/muscle) of three euthyroid rats were examined, while the stimulated and control legs of four rats (265 ± 21 fibers/muscle) were studied in each of the hypothyroid conditions. A second series of sections was also stained for myogenin, and the number of positive nuclei, which were clearly associated with a muscle fiber at high magnification (see below), were evaluated per unit cross-sectional area. The EDL muscles of stimulated and control legs were examined in three euthyroid rats and in four rats from each of the remaining conditions. For each individual muscle studied, 802 ± 206 fibers (mean ± SD) were examined, corresponding to a cross-sectional area of 1.95 ± 0.99 mm2 (mean ± SD) per muscle.

Immunohistochemistry for BrdU and PCNA. Frozen sections (9 µm thick) of EDL muscles were air dried. Sections to be incubated with the anti-BrdU antibody were fixed in a solution of 70% ethanol in 50 mM glycine buffer (pH 2.0) for 40 min at -20°C. Sections to be incubated with the anti-PCNA antibody were fixed in 1% paraformaldehyde at room temperature, according to the manufacturer's instructions. All sections were washed once in PBS-Tween, twice in PBS, and incubated for 30 min in 3% H2O2 in methanol. Working stocks of anti-BrdU and anti-PCNA antibodies were prepared by diluting to 10 and 25 µg/ml, respectively, in BS-1. Sections were subsequently washed and blocked for 1 h in BS-1 and washed again. The primary antibody was overlaid, and sections were incubated overnight at 4°C. Sections were then washed, reacted for 30 min with biotinylated horse anti-mouse IgG, washed again, and incubated with biotin-avidin-HRP complex (Vectastain Elite) for 30 min. Immunoreactivity was localized by incubating sections for 6 min with the substrate solution (see Immunohistochemistry for myosin and myogenin). Sections were counterstained with Nuclear Fast Red, dehydrated, cleared, and mounted with Entellan. Control sections, in which the primary antibody was omitted, were completed in parallel. Sections were analyzed by counting the number of positive nuclei per unit area that were clearly associated with a muscle fiber at high magnification (see below). EDL muscles of the stimulated and control legs were examined in three euthryoid rats and in four rats from each of the remaining conditions. For each individual muscle studied, 348 ± 69 fibers and 532 ± 89 fibers were examined for the BrdU and PCNA evaluations, respectively. This corresponded to mean cross-sectional areas of 0.69 ± 0.10 and 1.14 ± 0.13 mm2 for each individual muscle studied. All microscopic analyses were done using a magnification of ×787 or ×1,250.

RNase protection assay for myogenin mRNA. TA muscles were used for investigating changes in myogenin mRNA. Control and stimulated muscles from five animals stimulated for 5 days and six animals stimulated for 10 days were investigated. Muscles were pulverized under liquid nitrogen, and total RNA was extracted as previously described (15). RNase protection assays were performed with the RPA II RNase protection assay kit from Ambion (Austin, TX) according to the manufacturer's instructions (standard procedure). The cRNA probe specific to myogenin was a 228-bp PCR product (position 511-838 of the myogenin gene; GenBank accession no. M24393) cloned into the SP6/T7 vector pGEM-7ZF(-) (Promega, Heidelberg, Germany) in an orientation that SP6 RNA polymerase transcribed the cRNA. In the case of MyoD, a 489-bp PCR product covering parts of exons 1 and 3 of the MyoD gene (GenBank accession no. M84176) was cloned in the same vector as above in an orientation that T7 RNA polymerase transcribed the cRNA. cRNA probes were digoxigenin labeled as previously described (1). For the RNase protection assay, 5 or 10 µg of total RNA were used. As negative controls, the same amount of yeast RNA was included in the assay. The protected fragments were separated on 5% PAA gels according to the manufacturer's instructions. Chemiluminescence detection was performed after electroblotting to a nylon membrane (Hybond N+, Amersham). The digoxigenin-labeled protected fragments were visualized by an antibody-linked assay, followed by an alkaline phosphatase catalyzed chemiluminescence reaction with CSPD (Tropix, MA). Signals were photographically documented (Hyperfilm ECL; Amersham) and evaluated by integrating densitometry.

Statistical analyses. Data are presented as means ± SD. Differences between group means were assessed by an independent samples Student's t-test. Differences between control and stimulated conditions were assessed by a paired dependent samples Student's t-test. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CLFS increases satellite cell proliferation. To assess satellite cell proliferation, we used an established method (23, 26, 27) in which BrdU is injected intraperitoneally 1 h before the animals are killed. Serial cross sections of control and stimulated EDL muscles were stained with an anti-BrdU antibody, and all BrdU-positive intrafiber nuclei were counted. Being aware that cells other than satellite cells may also replicate in response to CLFS, precautions were taken by analyzing at high magnification and counting only those stained nuclei that were unambiguously fused to existing muscle fibers. An example of a BrdU-stained nucleus is shown in Fig. 1. Euthyroid, hypothyroid control, and sham-operated muscles exhibited similar low numbers of BrdU-positive nuclei (Fig. 2). In 5- and 10-day stimulated muscles, the number of positive nuclei was elevated approximately fourfold. After 20 days of CLFS the number of BrdU-labeled nuclei was somewhat lower but remained 2.5-fold elevated over control.


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  		Fig. 1.   Representative photographs of 5-bromo-2'-deoxyuridine (BrdU; A), proliferating cell nuclear antigen (PCNA; B), and myogenin (C) stains, and a negative control (D) from a 5-day stimulated extensor digitorum longus (EDL) muscle. Note that sections were counterstained with Nuclear Fast Red. Unreactive nuclei appear red, while immunoreactive cells appear dark brown-black. Bar = 20 µm.



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  		Fig. 2.   Number of BrdU-positive nuclei per unit cross-sectional area in control and stimulated EDL muscles. Statistical symbols: a, different from euthyroid (euthyr) controls; b, different from hypothyroid control; c, different from contralateral control; d, different from 5-day stimulated; e, different from 10-day stimulated (P < 0.05).

The same muscles investigated for BrdU incorporation were analyzed for PCNA expression with the use of an anti-PCNA antibody (Fig. 1). PCNA reactivity identifies cells in the S phase of the cell cycle at the time point when the animal is killed. Therefore, it was not surprising that the number of PCNA-positive nuclei was lower than the number of the BrdU-positive nuclei. The number of PCNA-positive nuclei amounted to ~25% of the latter. Nevertheless, the results were similar to the BrdU data and showed pronounced increases in DNA synthesis in the 5- and 10-day stimulated muscles (Fig. 3). Contrary to the BrdU data, however, PCNA reactivity returned to control values in 20-day stimulated muscles. The strong correlation (r2 = 0.83) between BrdU labeling and PCNA staining is depicted in Fig. 4.


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  		Fig. 3.   Number of PCNA-positive nuclei per unit cross-sectional area in control and EDL muscles of hypothyroid rat stimulated for various time periods. Statistical symbols as in Fig. 2.



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  		Fig. 4.   Relationship between the number of PCNA- and BrdU-positive nuclei in control and stimulated (5, 10, 20 days) EDL muscles. Dashed lines are the prediction limits of the equation.

The staining of nuclei for myogenin was used to detect satellite cell progeny that had become committed to or were in the later stages of terminal differentiation (Fig. 1). Only strong positive nuclei that were clearly fused to an existing fiber were counted. Similar to the BrdU and PCNA data, the number of positive nuclei was elevated in the 5-day stimulated muscles (Fig. 5). Compared with the 0-day control and sham-operated animals, the increase was approximately fivefold. However, due to a slightly elevated level in the unstimulated contralateral muscles, the increase was less pronounced (i.e., ~2-3-fold). In 10- and 20-day stimulated muscles, the number of positive mpc nuclei tended to decrease, but remained elevated over the corresponding contralateral controls.


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  		Fig. 5.   Number of myogenin-positive nuclei per unit cross-sectional area in control and stimulated EDL muscles of hypothyroid rats. Statistical symbols as in Fig. 2.

The upregulation of myogenin, at the protein level, in the stimulated muscles was mirrored by increased myogenin mRNA, as determined by RNase protection assay (Figs. 6 and 7). Myogenin mRNA was transiently elevated by 4-fold and 1.5-fold in 5- and 10-day stimulated muscles, respectively. Conversely, CLFS had no effect on the transcript levels of MyoD mRNA in both 5- and 10-day stimulated muscles (Fig. 7).


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  		Fig. 6.   Blot of a representative RNase protection assay for myogenin mRNA in control and stimulated (5, 10, and 20 days) tibialis anterior (TA) muscles of hypothyroid rats. For the RNase protection assay, 5 µg of total RNA was used for the control muscles, and 5 or 10 µg of total RNA were used for the stimulated muscles. For negative control, yeast RNA was included in the assay. Numbers denote µg of total RNA applied. co, Contralateral control; st, stimulated; M, marker V (Boehringer Mannheim); Y, yeast RNA.



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  		Fig. 7.   Relative MyoD and myogenin contents in stimulated and control TA muscles after 5 and 10 days of CLFS. * Stimulated leg significantly elevated over control leg (P < 0.05).

Satellite cell proliferation in relation to specific fiber types. To establish the fate of satellite cell progeny during CLFS-induced fiber type transitions, myogenin-positive muscle nuclei were counted in fibers identified by their immunohistochemically identified MHC isoform complement (Tables 1 and 2 and Fig. 8). According to these analyses, increases in myogenin-positive nuclei in 5-day stimulated muscle were most pronounced in pure type IIB and type IID(X) fibers (Table 2 and Fig. 9). At this time point, the percentages of myogenin-positive pure type IIA and type I fibers remained unaltered but were elevated in transforming slow hybrid fibers that coexpressed MHCemb (Table 2). This pattern shifted in 10-day stimulated muscles: the percentage of type IIB fibers that contained myogenin-positive nuclei decreased somewhat but remained elevated over controls. A similar distribution of myogenin-positive nuclei was found in 20-day stimulated muscles.

                              
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  		Table 1.   Alterations in MHC-based fiber types of the rat EDL exposed to hypothyroidism and chronic low frequency electrical stimulation


                              
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  		Table 2.   The proportion of fibers that stained positive for myogenin



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  		Fig. 8.   Localization of myogenin-positive nuclei in immunohistochemically classified fiber types of a 5-day-stimulated EDL muscle of a hypothyroid rat. A: BF-F3 antibody staining type IIB. B: NOQ7.5.4D antibody staining type I. C: BF-35 staining all fibers types, except IID(X). D: NCL-D antibody staining embryonic myosin. E: SC-71 antibody staining type IIA. F: IgG control. G and H: antibody M-225 specific to myogenin. Note the strong staining for myogenin in type IIB fibers numbered 1-3. Bars in G and H are 40 µm.



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  		Fig. 9.   Percentage distribution myogenin-positive intrafiber nuclei in immunohistochemically classified fiber types. A: control; B: stimulated. Statistical symbols are the same as in Fig. 2. MHC, myosin heavy chain.

In view of our previous observation (19) that a high percentage of fibers in 20-day stimulated muscle coexpressed MHCemb in combination with adult MHC isoforms, we were interested to elucidate possible relationships between myogenin upregulation and MHCemb expression. This was relevant because satellite cell progeny may partially recapitulate the myogenic program after fusion to their associated fibers (13, 19, 30). MHCemb-containing fibers were rarely detectable in 5-day stimulated muscle (Table 1). They were, however, more numerous in 10-day stimulated muscle and further increased after 20 days of CLFS (Table 1). Myogenin-positive fibers with the combinations MHCemb+MHCI or MHCemb+MHCI+MHCIIa were already detected after 5 days of CLFS (Table 2). Myogenin-positive hybrid fibers displaying the combinations MHCemb+ MHCIIb, as well as MHCemb+ MHCIIb + MHCIIa + MHCI were only found in 20-day stimulated muscles. Interestingly, this occurred long after the maximum increase in the number of myogenin-positive nuclei in the type IIB fiber population at day 5 (Table 2). Approximately 25% of the fibers coexpressing MHCemb stained for myogenin. Due to the transient expression of myogenin, which decays earlier than the MHCemb (30), we may have failed to detect a greater number of myogenin-positive hybrid fibers at the time points studied.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study unambiguously show that CLFS of rat fast-twitch muscle leads to an activation and proliferation of satellite cells and to myogenic differentiation of satellite cell progeny. These results are consistent with our previous findings (19) where, under the same experimental conditions, we observed absolute and relative increases in satellite cell content that paralleled the increase in myonuclear content (19). The proliferation of satellite cell progeny is reflected in the elevated number of BrdU-labeled nuclei and, in addition, by the enhanced and strongly correlated expression of PCNA. BrdU labeling and PCNA expression clearly distinguish proliferating satellite cell progeny from myonuclei (16, 30, 32). Moreover, the almost synchronous elevations in BrdU- and myogenin-positive nuclei also suggest that the satellite cell progeny progress directly from the proliferative to the differentiative compartments as they undergo a program of myogenesis (16, 30, 31).

The pronounced rise in myogenin mRNA in 5-day stimulated muscle is interpreted as the onset of a significant period of commitment to terminal differentiation after proliferation. The return of myogenin mRNA to near basal levels in 10-day stimulated muscles suggests that most mpc had fused to their associated fibers. At this time, however, myogenin remained elevated at the protein level. This apparent discrepancy could relate to the fact that immunohistochemical studies were performed on EDL muscles, whereas mRNA analyses were conducted on TA muscle, which is known to respond to a slightly lesser extent to CLFS than EDL muscle (18). Alternatively, this temporal relationship might also indicate that myogenin expression is under transcriptional as well as posttranscriptional control.

The finding that only myogenin but not MyoD mRNA is upregulated is similar to observations of myotonic mouse muscle, which exhibits similar changes in the expression of myogenic regulatory factors (10). The myotonic condition is comparable to the chronic low-frequency electrical stimulation model used in the present study, in that satellite cell numbers are elevated in the absence of fiber degeneration (21). Additionally, early muscle regulatory factors (i.e., MRF4 and myf-5), but not myogenin, are reportedly transiently elevated in response to 2 h of CLFS, with much of the increase attributable to an increased number of activated satellite cells (14).

Our previous studies on muscles under the same experimental conditions as in the present study failed to detect signs of fiber damage at the light microscopic level, as well as invading mononuclear cells. Fiber regeneration could be excluded because myotubes or fibers with central nuclei were absent in the stimulated rat muscle (19). An additional sign indicating the absence of fiber injury was increased desmin immunoreactivity in the stimulated muscle. Taken together, the rat model used in the present study has the advantage that adaptive changes, resulting from enhanced contractile activity, occur independently of fiber injury.

In view of these properties of our model, we assume that proliferation of satellite cell progeny occurs on intact and transforming muscle fibers. Because myotube formation is undetectable, the satellite cell progeny appears to first proliferate and then fuse to their associated fibers. Similar results were reported for avian muscle in which, following a period of stretch, the MyoD homologue qmf1 was expressed by muscle nuclei (7). MyoD and myogenin proteins were also detected in nuclei of denervated rat diaphragm (29). It should be mentioned that fusion of proliferated satellite cell progeny to intact fibers was not observed in some studies using the single fiber tissue culture model (4). The difference between those findings and our observations in vivo may relate to the fact that muscle fibers in vitro are aneural and inactive, whereas fibers in the stimulated muscle are innervated and contracting. Contractile activity may facilitate fusion, perhaps due to the release of growth factors or other soluble compounds. Indeed, it has been shown that myoblast fusion is enhanced in vitro by exogenous insulin-like growth factor I in combination with fibroblast growth factor (FGF) (2) and by FGF and hepatocyte growth factor alone (32). It has also been reported that, in the presence of fetal serum containing essential growth factors, widespread fusion of mpc to intact fibers occurs in culture, leading to fiber remodeling (13, 30). Furthermore, remodeling by satellite cell progeny was inhibited when fusion was blocked (13, 30) and accelerated when FGF was introduced into cultures (30, 32).

A conspicuous observation relates to both the time course and fiber type distribution of activated mpc, as indicated by the detection of myogenin in their nuclei. Myogenin-positive satellite cell progeny first appeared in pure type IIB and IID(X) fibers at 5 days, whereas the proportion present in pure type IIA and type I fibers did not change. It may be assumed that satellite cells associated with fast fibers are of the fast lineage. Therefore, it appears unlikely that fusion of their progeny to fibers triggers reprogramming of phenotypic properties in the fast-to-slow direction. Rather, the early onset of increased myogenin expression in satellite cell progeny of fast fibers suggests that this process primarily serves to expand the myonuclear pool of the fast fibers. This assumption is in line with our observation that satellite cell proliferation and increases in myonuclear content precede the changes in MHC isoform expression. Thus it appears that higher myonuclear densities are a prerequisite for a transition to slower phenotypes. It has been suggested that smaller nuclear domains, i.e., a smaller volume of cytoplasm under control of a nucleus, are a requirement for higher biosynthetic activities in slow fibers (11). Moreover, higher myonuclear contents of slower fiber types have been discussed with regard to the maintenance of the slow phenotype. This interpretation is in line with the reduction in myonuclear content of slow-to-fast transforming rat soleus muscle during unloading by hindlimb suspension (23).

Reports on satellite cell activation in single fiber cultures show that the expression of developmental MHC isoforms follows the upregulation of myogenin (30). Similarly, in the present study the number of fibers coexpressing MHCemb and adult MHC isoforms in combination with myogenin increased with the duration of CLFS. In light of recent reports that myogenin is detected in satellite cell progeny but not in mature myonuclei (16, 32) and that killing satellite cells with cytosine arabinoside inhibits the expression of myogenin and embryonic myosin (32), our findings suggest that satellite cell progeny partially recapitulate the myogenic program and transiently express developmental MHC isoforms after fusion to their associated fibers. In the case of the IIB fibers, the relatively late appearance of MHCemb-expressing hybrid fibers is consistent with this interpretation. The observation that the appearance of MHCemb in the type IIA and type I fibers precedes that of the type IIB fibers suggests that satellite cell recruitment, and the subsequent fusion of their progeny, occurs in a fiber-type-specific manner. Perhaps the more numerous satellite cells on type I and type IIA fibers undergo fewer proliferation cycles than those on type IIB fibers before fusing to their associated fibers.

In summary, we show that CLFS-induced fast-to-slow fiber type transitions in rat muscle are accompanied by pronounced activation, proliferation, and fusion of mpc to intact transforming fibers. Muscle precursor cell proliferation on pure fast IIB and IID(X) fibers appears to precede that on type IIA and type I fibers. These findings suggest that the activation and fusion of mpc to transforming muscle fibers serves to increase the myonuclear content in fast fibers, which seems to be a prerequisite for fiber type transition.


    ACKNOWLEDGEMENTS

C. T. Putman thanks the Canadian Medical Research Council and Muscular Dystrophy Association for a fellowship.


    FOOTNOTES

This study was supported by the Deutsche Forschungsgemeinschaft, Du 260/2-1.

Address for reprint requests and other correspondence: C. T. Putman, Skeletal Muscle Research Group, Faculty of Physical Education, Univ. of Alberta, Edmonton, Alberta, Canada, T6G 2H9 (E-mail: tputman{at}per.ualberta.ca).

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. §1734 solely to indicate this fact.

Received 15 July 1999; accepted in final form 9 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 279(3):C682-C690
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