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 |
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 |
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 |
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 |
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.

View larger version (146K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
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.

View larger version (18K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
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.

View larger version (19K):
[in this window]
[in a new window]
|
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).

View larger version (70K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
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.
View this table:
[in this window]
[in a new window]
|
Table 1.
Alterations in MHC-based fiber types of the rat EDL exposed to
hypothyroidism and chronic low frequency electrical stimulation
|
|

View larger version (140K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
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 |
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 |
1.
Aigner, S,
and
Pette D.
In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA.
Histochemistry
95:
11-18,
1990[ISI][Medline].
2.
Allen, RE,
and
Boxhorn LK.
Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-
, insulin-like growth factor I, and fibroblast growth factor.
J Cell Physiol
138:
311-315,
1989[ISI][Medline].
3.
Beilharz, MW,
Lareu RR,
Garrett KL,
Grounds MD,
and
Fletcher S.
Quantitation of muscle precursor cell activity in skeletal muscle by northern analysis of MyoD and myogenin expression: application to dystrophic (mdx) mouse muscle.
Mol Cell Neurosci
3:
326-331,
1992[ISI].
4.
Bischoff, R.
Proliferation of muscle satellite cells on intact myofibers in culture.
Dev Biol
115:
129-139,
1986[ISI][Medline].
5.
Bravo, R,
Frank R,
Blundell PA,
and
MacDonald-Bravo H.
Cyclin/PCNA is the auxiliary protein of DNA polymerase-
.
Nature
326:
515-517,
1987[ISI][Medline].
6.
Düsterhöft, S,
and
Pette D.
Satellite cells from slow rat muscle express slow myosin under appropriate culture conditions.
Differentiation
53:
25-33,
1993[ISI][Medline].
7.
Eppley, ZA,
Kim J,
and
Russell B.
A myogenic regulatory gene, qmf1, is expressed by adult myonuclei after injury.
Am J Physiol Cell Physiol
265:
C397-C405,
1993[Abstract/Free Full Text].
8.
Füchtbauer, E-M,
and
Westphal H.
MyoD and myogenin are coexpressed in regenerating skeletal muscle of the mouse.
Dev Dynamics
193:
34-39,
1992[ISI][Medline].
9.
Gibson, MC,
and
Schultz E.
The distribution of satellite cells and their relationship to specific fiber types in soleus and extensor digitorum longus muscles.
Anat Rec
202:
329-337,
1982[ISI][Medline].
10.
Goblet, C,
and
Whalen RG.
Modifications of gene expression in myotonic murine skeletal muscle are associated with abnormal expression of myogenic regulatory factors.
Dev Biol
170:
262-273,
1995[ISI][Medline].
11.
Goldberg, AL.
Protein synthesis in tonic and phasic skeletal muscles.
Nature
216:
1219-1220,
1967[ISI][Medline].
12.
Harris, AJ,
Fitzsimons RB,
and
McEwan JC.
Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles.
Development
107:
751-769,
1989[Abstract].
13.
Hinterberger, TJ,
and
Barald KF.
Fusion between myoblasts and adult muscle fibers promotes remodeling of fibers into myotubes in vitro.
Development
109:
139-148,
1990[Abstract].
14.
Jacobs-El, J,
Zhou M-Y,
and
Russell B.
MRF4, myf-5, and myogenin responses of mature rat muscle.
Am J Physiol Cell Physiol
268:
C1045-C1052,
1995[Abstract/Free Full Text].
15.
Jaschinski, F,
Schuler M,
Peuker H,
and
Pette D.
Transitions in myosin heavy chain mRNA and protein isoforms of rat muscle during forced contractile activity.
Am J Physiol Cell Physiol
274:
C365-C371,
1998[Abstract/Free Full Text].
16.
Kuschel, R,
Yablonka-Reuveni Z,
and
Bornemann A.
Satellite cells on isolated myofibers from normal and denervated adult rat muscle.
J Histochem Cytochem
47:
1375-1383,
1999[Abstract/Free Full Text].
17.
Pette, D,
and
Staron RS.
Mammalian skeletal muscle fiber type transitions.
Int Rev Cytol
170:
143-223,
1997[Medline].
18.
Pette, D,
and
Vrbová G.
Adaptation of mammalian skeletal muscle fibers to chronic electrical stimulation.
Rev Physiol Biochem Pharmacol
120:
116-202,
1992.
19.
Putman, CT,
Düsterhöft S,
and
Pette D.
Changes in satellite cell content and myosin isoforms in low-frequency stimulated fast muscle of hypothyroid rat.
J Appl Physiol
86:
40-51,
1999[Abstract/Free Full Text].
20.
Schiaffino, S,
Gorza L,
Sartore S,
Saggin L,
Ausoni S,
Vianello M,
Gundersen K,
and
Lömo T.
Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.
J Muscle Res Cell Motil
10:
197-205,
1989[ISI][Medline].
21.
Schimmelpfeng, J,
Jockusch H,
and
Heimann P.
Increased density of satellite cells in the absence of fibre degeneration in muscle of myotonic mice.
Cell Tissue Res
249:
351-357,
1987[ISI][Medline].
22.
Schultz, E,
and
Darr KC.
The role of satellite cells in adaptive or induced fiber transformations.
In: The Dynamic State of Muscle Fibers, edited by Pette D.. New York: de Gruyter, 1990, p. 667-679.
23.
Schultz, E,
Darr KC,
and
Macius A.
Acute effects of hindlimb unweighting on satellite cells of growing skeletal muscle.
J Appl Physiol
76:
266-270,
1994[Abstract/Free Full Text].
24.
Schultz, E,
and
McCormick KM.
Skeletal muscle satellite cells.
Rev Physiol Biochem Pharmacol
123:
213-257,
1994[ISI][Medline].
25.
Simoneau, J-A,
and
Pette D.
Species-specific effects of chronic nerve stimulation upon tibialis anterior muscle in mouse, rat, guinea pig, and rabbit.
Pflügers Arch
412:
86-92,
1988[ISI][Medline].
26.
Tamaki, T,
Akatsuka A,
Tokunaga M,
Ishige K,
Uchiyama S,
and
Shiraishi T.
Morphological and biochemical evidence of muscle hyperplasia following weight-lifting exercise in rats.
Am J Physiol Cell Physiol
273:
C246-C256,
1997[Abstract/Free Full Text].
27.
Tamaki, T,
Akutsuka A,
Tokunga MY,
Uchihama S,
and
Shiraisi T.
Characteristics of compensatory hypertrophied muscle in the rat. I. Electron microscopic and immunohistochemical studies.
Anat Rec
246:
325-334,
1996[ISI][Medline].
28.
Wehrle, U,
Düsterhöft S,
and
Pette D.
Effects of chronic electrical stimulation on myosin heavy chain expression in satellite cell cultures derived from rat muscles of different fiber-type composition.
Differentiation
58:
37-46,
1994[ISI][Medline].
29.
Weis, J.
Jun, Fos, Myod1, and myogenin proteins are increased in skeletal muscle fiber nuclei after denervation.
Acta Neuropathol (Berl)
87:
63-70,
1994[ISI][Medline].
30.
Yablonka-Reuveni, Z,
and
Rivera AJ.
Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated adult rat fibers.
Dev Biol
164:
588-603,
1994[ISI][Medline].
31.
Yablonka-Reuveni, Z,
Rudnicki MA,
Rivera AJ,
Primig M,
Anderson JE,
and
Natanson P.
The transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD.
Dev Biol
210:
440-455,
1999[ISI][Medline].
32.
Yablonka-Reuveni, Z,
Seger R,
and
Rivera AJ.
Fibroblast growth factor promotes recruitment of skeletal muscle satellite cells in young and old rats.
J Histochem Cytochem
47:
23-42,
1999[Abstract/Free Full Text].
Am J Physiol Cell Physiol 279(3):C682-C690
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society