THE ANATOMICAL RECORD 258:176185 (2000)

Activity-Induced Fiber Regeneration

in Rat Soleus Muscle
LINDA J. WANEK1* AND MIKEL H. SNOW2
1Graduate Program in Physical Therapy, University of California, San Francisco,
California, and San Francisco State University, San Francisco, California 94132
2Department of Anatomy, University of Wisconsin School of Medicine,

Madison, Wisconsin 53706

ABSTRACT
In an attempt to understand why muscle recovery is limited following
atrophy due to limb immobilization, satellite cell activity and muscle fiber
regeneration were analyzed in rat soleus muscles. Adult rat hindlimbs were
immobilized in plaster casts for a period of two to ten weeks. Soleus muscles
were examined by electron microscopy for evidence of fiber degeneration or
regeneration, and to quantify satellite cell nuclei. Immunocytochemical
localization of embryonic myosin was used to identify regenerating myofi-
bers. Soleus muscle wet weight to body weight ratios for the casted muscles
significantly decreased over the 10-week immobilization period. The casted
muscles displayed ultrastructural evidence of minor fiber damage, including
myofibrillar atrophy, Z-disc disruption, and abnormal triadic junctions. No
ultrastructural evidence of regeneration was seen in the casted animals.
The number of satellite cells in the casted muscles significantly decreased
from 6.4% to 3.3% by eight to 10 weeks of immobilization. Approximately
1.0% of extrafusal fibers in the control soleus muscles appeared to be
regenerating since they expressed embryonic myosin and were of a small
diameter, while in casted muscles, only 0.1% of the fibers were embryonic
myosin-positive. Following release from immobilization, a reappearance of
embryonic myosin-positive fibers was noted within four days of renewed
activity. In contrast to control muscles, embryonic myosin-positive fibers in
the recovery muscles included both small and large diameter fibers. Subtle
changes in functional activity influence muscle damage and subsequent
myofiber regeneration. Reduced activity reduces muscle fiber regeneration,
while increased activity, as seen by increased hindlimb weight bearing and
return to normal activity following immobilization, increase regenerating
fibers and also the expression of embryonic myosin in adult fibers. Anat Rec
258:176185, 2000. r 2000 Wiley-Liss, Inc.

Key words: skeletal muscle; regeneration; embryonic myosin; rat; immobi-

lization

Skeletal muscle is a highly differentiated tissue that has
the capacity to adapt to extreme fluctuations in its func-
tional state. Despite this high degree of plasticity, certain
adaptive changes are not completely reversible. Clinical
studies have confirmed that skeletal muscle atrophy and
strength reduction, secondary to procedures such as cast-
immobilization or surgery, cannot always be returned to
normal levels despite extensive rehabilitation (LoPresti et
al., 1988; Matthews and St. Pierre, 1996). Although a
variety of mechanisms may be involved in limiting the
functional recovery of skeletal muscle following these
procedures, little attention has been directed at examining
the possible role of the satellite cell population during
recovery from atrophy. Satellite cells have been shown to
form new, regenerating muscle fibers following muscle
injury (Snow, 1977; McGeachie and Allbrook, 1978), to
serve as a source of new nuclei during muscle fiber growth
and hypertrophy (Moss and LeBlond, 1970; Snow, 1990;
*Correspondence to: Linda Wanek, Graduate Program in Physi-
cal Therapy, San Francisco State University, 1600 Holloway, San
Francisco, CA 94132.
Received 14 August 1998; Accepted 8 October 1999

r 2000 WILEY-LISS, INC.

 ACTIVITY-INDUCED FIBER REGENERATION
McCormick and Schultz, 1994), and to increase in num-
bers during denervation-induced muscle fiber atrophy
(Schultz, 1978, Snow, 1983). Given these documented roles
during muscle regeneration, growth, and hypertrophy, it
seemed appropriate to analyze the effects of one model of
muscle disuse, cast-immobilization, on the satellite cell
population and to determine if the recovery of atrophied
muscle is correlated with satellite cell activity.
Previous attempts to examine satellite cells and muscle
regeneration during cast-immobilization have led to con-
flicting results. Appell (1986) reported the presence of
myoblasts and myotubes with immobilization of rat hind-
limb muscles, whereas Sjostrom and colleagues (1979)
observed regenerative activity only during the earliest
periods of immobilization. Cooper (1972), on the other
hand, observed no evidence of muscle regeneration during
any periods of rat hindlimb immobilization, but following
release from immobilization and return to activity, evi-
dence of regeneration was seen. Tomanek and Lund (1974),
using electron microscopic techniques, observed satellite
cells after three weeks of immobilization although they
neither quantified the population nor reported the appear-
ance of regenerating myotubes.
The above studies, relying exclusively on morphological
techniques, were unable to assess accurately the frequency
of satellite cells while also analyzing the extent of fiber
regeneration during a chronic period of hindlimb immobili-
zation. If full recovery of atrophic muscles following peri-
ods of cast-immobilization is limited by changes in the
satellite cell population, a more precise analysis of satellite
cells and myotube formation during the period of immobili-
zation is essential. The purpose of the present study was to
use electron microscopy to determine the frequency of
satellite cells in rat soleus muscles following various
intervals of cast-immobilization, and to determine the
extent of muscle repair by using immunocytochemistry to
localize a developmental myosin in regenerating myo-
tubes.
MATERIALS AND METHODS
Hindlimb immobilization was induced in Sprague-
Dawley rats (Harlan / Holtzman) by applying a plaster
cast. Two age groups of rats were used in this study.
Two-month-old male and female rats (11 animals) were
used to survey by electron microscopy the satellite cell
population, which has a high incidence in young animals.
Six-month-old male rats (10 animals) were used to monitor
changes in fiber cross-sectional areas and to survey for
muscle fiber regeneration employing immunocytochemical
markers. Each rat in both age groups had one hindlimb
immobilized in a plaster cast for two, four, six, eight, or 10
weeks (3 4 rats per interval). Six additional six-month-
old male rats were included to examine the recovery of
atrophied muscles following four weeks of cast-immobiliza-
tion. In this recovery group, two rats were allowed four
days and two were allowed ten days of cage activity
following cast removal, while the remaining two rats were
sacrificed immediately after cast removal. The non-casted
hindlimb plus 10 normal age- and sex-matched animals
served as controls. The University of Southern California
School of Medicine Institutional Animal Care and Use
Committee approved all experimental procedures. Ani-
mals were housed in individual, solid bottom cages in an
animal room with 12-hour light and dark cycles. Animals
were allowed food and water ad libitum.
177
Following anesthesia induced by an intraperitoneal
injection of sodium pentobarbital (2.5mg/100gm rat body
weight), one hindlimb was shaved and covered with a
protective layer of elastic tape. Several layers of moistened
plaster-of-Paris strips were applied from mid-thigh to the
toes, keeping the knee and ankle joints immobilized at a
90-degree angle. A plaster footplate was applied to the
plantar surface of the foot, leaving the dorsum of the toes
uncovered to check for edema or circulatory problems.
Plaster casts were covered with fiberglass tape to deter
animals from chewing. Casts were inspected daily and
changed as needed, or at least every seven to 10 days to
accommodate decreasing girth of the leg. At the conclusion
of each experimental period, bilateral soleus muscles were
excised under pentobarbital anesthesia, trimmed of connec-
tive tissue, wet-weighed, and then divided into proximal
and distal halves. One half was frozen unfixed in isopen-
tane cooled by liquid nitrogen for cryostat sectioning and
light microscopy or immunocytochemistry, while the other
half was processed for electron microscopy. Following
muscle removal, animals were euthanized with an over-
dose intraperitoneal injection of sodium pentobarbital.
Transverse frozen sections (4 6) were cut in a
cryostat and processed in one of three ways. For routine
light microscopic analysis, sections were fixed in methanol
followed by immersion in Richardsons stain (Richardson
et al., 1960). For identification of regenerating myotubes,
sections were incubated in humidified chambers with a
1:50 dilution of a monoclonal antibody (2B6) directed
against embryonic myosin (Gambke and Rubinstein, 1984).
For determining cross-sectional fiber areas, sections were
incubated with an antibody against laminin (1:100 dilu-
tion, Cal Biochem), which outlines individual muscle fi-
bers. Each primary antibody was incubated for one hour at
room temperature or overnight at 4C. Localization of
primary antibodies was accomplished by incubating sec-
tions with a fluorescein- or rhodamine-conjugated goat
anti-mouse secondary antibody (1:40 dilution, Sigma) at
room temperature in the dark. Negative controls included
sections treated only with BSA, goat anti-mouse fluores-
cein- or rhodamine-conjugated secondary antibody, or nor-
mal mouse serum. All sections were viewed with a light
microscope equipped for epifluorescence.
The frequency of regenerating myotubes (embryonic
myosin-positive extrafusal fibers) was determined by count-
ing the total number of embryonic myosin-positive fibers
seen in an entire cross-section. The percentage of embry-
onic myosin-positive fibers was expressed as a ratio of the
total number of muscle fibers (approximately 2,000 fibers
per muscle section), the latter being estimated by multiply-
ing the number of 20X fields per section by the mean
number of total muscle fibers counted in three to four
randomly selected 20X fields (Wanek and Snow, 1995).
Mean fiber cross-sectional areas for each group of ani-
mals were determined from four random fields per muscle
section stained for anti-laminin. Fiber outlines of a mini-
mum of 200 fibers per muscle were traced using a Summa-
graphics Digitizer. Wet-weights of the muscles were re-
corded as ratios of total body weights.
For ultrastructural examination, muscle samples were
pre-fixed in 4% glutaraldehyde in 0.1M cacodylate buffer
(pH 7.4), post-fixed in 1% osmium tetroxide, dehydrated in
an ethanol series, and embedded in epoxy resin. Thin
sections (90 100 nanometers) were placed on carbon
coated grids, stained with uranyl acetate and lead citrate
178 WANEK AND SNOW
(Reynolds, 1963), and examined using a 100C JEOL
electron microscope. Each cross-section was surveyed for
fiber pathology and /or muscle regeneration. The percent-
age of satellite cells was determined per section by count-
ing the number of satellite cell nuclei and myonuclei at a
magnification of 4,000 6,000, and dividing the number
of satellite cell nuclei by the total muscle nuclei (satellite
cell nuclei myonuclei) observed. Satellite cell nuclei
were distinguished from myonuclei by the identification of
a satellite cell membrane distinct from a sarcolemma. The
values for three to five sections per muscle were pooled to
calculate a mean percentage of satellite cells per muscle. A
minimum of 1,000 total muscle nuclei was counted per
muscle.
The quantitative data were analyzed using descriptive
statistics, the Wilcoxon Rank Sum test and the Kruskal
Wallis test with post hoc testing for differences between
groups. Alpha and F values were set at 0.05.
RESULTS
Based on muscle wet weight / body weight ratios, statisti-
cally significant muscle atrophy was seen at all intervals
in the casted soleus muscles (data not shown). No signifi-
cant differences were noted between normal soleus and
non-casted soleus muscles. Fiber atrophy within the cast-
immobilized soleus muscles was confirmed by measuring
cross-sectional fiber areas (Fig. 1). Frequency distribution
graphs of fiber cross-sectional areas demonstrated a pro-
gressive increase in the number of small fibers in the
soleus muscles over a six-week period of casting.
Light microscopic examination of normal, non-casted,
and casted soleus muscles revealed only an occasional fiber
that appeared degenerating based on the presence of
vacuolated cytoplasm and an invasion of large mononucle-
ated cells. At the ultrastructural level, myofibrillar atro-
phy and disorganization were observed in both casted and
non-casted muscles, although casted muscles displayed a
greater incidence of degenerative change. The myofibrillar
disorganization observed in casted muscles was usually
limited to a small segment of a fiber, with the remainder of
the fiber appearing relatively normal (Fig. 2). Alterations
in the Z-disc morphology were frequently observed in the
casted muscles, with Z-disc disruptions and dense clumps
or rods as the most common occurrences (Fig. 3). Myofibril-
lar disorganization and Z-disc disruptions were most notice-
able in casted muscles sampled from the 8 to 10-week
immobilization period. Pyknotic satellite cell nuclei or
myonuclei were not observed in any of the muscles sampled.
One interesting observation, noted only in cast-immobi-
lized soleus fibers, was the presence of unusual triadic
profiles. Such abnormal triads surrounded areas of Z-disc
disruption and appeared to be linked in series to form
polyads (Fig. 4). These multiple triadic profiles are
referred to as polyads to distinguish them from the
pentads (two transverse tubules and three terminal cister-
nae) previously observed by others (Revel, 1962; Mair and
Tome, 1972; Tomanek and Lund, 1973; Cullen and Pluskal,
1977; Ovalle, 1987). Polyads were seen in both longitudi-
nal and transverse sections, suggesting they had no orien-
tation to the longitudinal axis of the myofibers.
Infrequently, regenerating myotubes were noted in the
non-casted soleus muscles (Fig. 5). These myotubes con-
tained a Golgi apparatus, enlarged endoplasmic reticu-
lum, immature myofibrils, and polyribosomes. Regardless
of the duration of immobilization, no evidence of regenerat-
ing myotubes was encountered in any of the cast-
immobilized muscles. The smallest diameter fibers ob-
served in the cast-immobilized muscles routinely exhibited
mature organization and morphology, suggesting fibers
were atrophic rather than regenerating fibers.
The frequency of satellite cells in casted soleus muscles
remained constant or decreased slightly during the shorter
periods of immobilization (Fig. 6). However, by 8 to 10
weeks of immobilization, the mean percentage of satellite
cells in casted soleus muscles had decreased significantly
(P .05) from control values of 6.4% (range 4.1% to 11.7%)
to 3.3% (range 1.0 to 6.0%). To differentiate whether this
decrease in satellite cell frequency was due to a decrease in
the absolute number of satellite cells, or to an increase in
the number of myonuclei, the ratio of myonuclei per
myofiber was determined. No statistically significant differ-
ence was seen between casted and non-casted muscles
with respect to the number of myonuclei per myofiber
(data not shown). Thus, the observed decrease in the
percentage of satellite cells appears to be a decrease in the
absolute numbers of satellite cells.
Approximately one out of every 100 (1.0%) extrafusal
muscle fibers appeared positively stained for embryonic
myosin in both the normal and non-casted soleus muscles
(Fig. 7). Nearly all such fibers were of a small diameter
(500 2). Although intrafusal fibers also expressed embry-
onic myosin, such fibers were excluded from the data. By
two and four weeks of cast-immobilization, the number of
extrafusal fibers staining positively for embryonic myosin
was reduced to 0.21% and 0.15%, respectively. By six
weeks of immobilization, the frequency of extrafusal fibers
staining positively for embryonic myosin was significantly
reduced to 0.1% of the total number of fibers (P .05). The
majority of these embryonic myosin-positive fibers ob-
served in the casted muscles tended to be larger and closer
to the size of normal muscle fibers ( 1000 2), in contrast
to the small diameter, embryonic myosin-positive fibers
seen in normal and non-casted soleus muscles.
Although six weeks of cast-immobilization essentially
eliminated the small number of embryonic myosin-
positive fibers seen in normal soleus muscles, a return to
just four days of cage activity resulted in the reappearance
of fibers that stained positively for embryonic myosin (Fig.
8A). Based on size and staining intensity, two populations
of embryonic myosin-positive fibers were observed in the
muscles of the recovery group. One population resembled
regenerating myotubes since they were intensely fluores-
cent for embryonic myosin, had a small cross-sectional
area, and often displayed a central nucleus. Other fibers
were of a much larger diameter with a less intense
embryonic myosin staining that typically appeared re-
stricted to the periphery of the fiber (Fig. 8B). After ten
days of recovery, the frequency of staining for embryonic
myosin in both small and large diameter extrafusal fibers
appeared to increase (Fig. 8B).
Fig. 1. Frequency distribution of changes in fiber size in the soleus
muscle immobilized from two to six weeks. Controls include measure-
ments from normal and noncasted muscles. Between 1,063 and 2,319
fibers were measured for each group at each interval. Note increased
incidence of small fibers over time.
ACTIVITY-INDUCED FIBER REGENERATION 179

Figure 1.
180 WANEK AND SNOW
DISCUSSION
The ultrastructural and immunocytochemical results
presented in this study indicate that chronically reduced
activity of the rat soleus muscle neither induces the
regeneration of new fibers, nor leads to an increase in
satellite cells when hindlimbs are subjected to cast-
immobilization for up to ten weeks. Thus, the absence of
muscle regeneration during long-term limb immobiliza-
tion reported here supports the work of Cooper (1972) and
is contrary to the conclusions made in the studies by Appell
(1986) and Sjostrom and associates (1979).
It is conceivable that variations in the presence or
absence of regenerative activity noted in muscles undergo-
ing atrophy due to limb immobilization might be corre-
lated with the angle of joint fixation. Increased fiber
degeneration has been shown to occur in muscles immobi-
lized in either a shortened or lengthened position relative
to the resting or neutral position (Sjostrom et al., 1979).
Since muscle damage induces a regenerative response, it
follows that muscle regeneration would be expected in
muscles immobilized in either a shortened or lengthened
position. Loughna and co-workers (1990) examined embry-
onic myosin mRNA expression in muscles immobilized in
either a neutral or a stretched position and found embry-
onic myosin only in stretched muscles. Although these
molecular biology techniques do not allow specific localiza-
tion of the embryonic myosin expression, it is logical to
speculate that embryonic myosin would be present within
regenerating myotubes and /or in new sarcomeres being
added in response to the stretched muscle length. The lack
of regenerative activity in immobilized soleus muscles in
the present study may be due to the ankle joint being fixed
in a neutral position that avoided stretching or shortening
the soleus muscles.
It might also be speculated that the presence or absence
of muscle regeneration observed in muscles from cast-
immobilized limbs is related to the duration of immobiliza-
tion. Perhaps regeneration was not observed in the pre-
sent study because muscles were not examined earlier
than two weeks after the casts were applied. Other studies
have reported muscle regeneration within five to seven
days of hindlimb immobilization (Sjostrom et al., 1979;
Appell, 1986; Loughna et al., 1990). However, if myotubes
had formed within the two-week period immediately follow-
ing cast application in the present study, they would have
been observed when the muscles were sampled at two
weeks since embryonic myosin expression in myotubes has
been shown to remain detectable for at least 14 days
(Butler-Browne and Whalen, 1984; LaFramboise et al.,
1990; Wanek and Snow, 1995). St. Pierre and Tidball
(1994), using the hindlimb suspension model of immobili-
zation, also failed to detect developmental myosin expres-
sion in soleus muscles from hindlimbs that had been
suspended for as few as ten days.
Finally, differences in a muscles contractile activity
(slow-twitch versus fast-twitch) might account for differ-
ences in the extent of muscle regeneration observed by
others. While the present study examined both soleus
(slow-twitch) and plantaris (fast-twitch) muscles, no evi-
dence of embryonic myosin expression was ever observed
in the immobilized or contralateral, non-immobilized plan-
taris muscles (data not included). This suggests that with
normal usage, slow-twitch postural muscles have a higher
incidence of fiber degeneration-regeneration than do fast-
twitch muscles, possibly due to the more frequent and
chronic recruitment of postural muscles during stance and
gait. Since neither cast-immobilization, nor increased use
of the contralateral, weight-bearing limb showed evidence
of new muscle fiber formation in fast-twitch muscles in the
present study, these results are at variance with Appell
(1986) who reported fiber regeneration in the cast-
immobilized tibialis anterior muscle, another predomi-
nately fast-twitch muscle.
Although minor abnormalities were noted within a
small number of fibers in the casted muscles in the present
study, such changes did not appear to be severe enough to
lead to cell death since sarcoplasmic fragmentation and
pyknotic nuclei were not observed. Apparently these
changes were also not severe enough to lead to muscle
regeneration. This finding is consistent with the report of
Teravainen (1970) who showed that a minor compression
injury to a muscle is not always sufficient to induce a
regenerative response involving the formation of new
myotubes.
The occasional embryonic myosin-positive fiber seen in
muscles subjected to cast-immobilization for six weeks or
longer tended to have a large cross-sectional fiber area (
1000 2) and moderate to weak staining for embryonic
myosin. Although such fibers did not appear to be newly
formed regenerating fibers, it was not possible to deter-
mine if these fibers represented mature fibers re-express-
ing embryonic myosin, or mature fibers with residual
embryonic myosin from an earlier developmental stage.
Soleus muscles from the recovery animals also displayed
large diameter fibers that expressed embryonic myosin. In
these fibers, however, embryonic myosin was detected only
at the periphery of the fiber (Figure 8B). St. Pierre and
Tidball (1994), using animals that were allowed recovery
following hindlimb suspension, reported similar fibers
with peripheral staining for embryonic myosin. Perhaps a
return to muscle activity, following long periods of disuse,
leads to the peripheral replacement of the lost contractile
proteins, including the possible expression of embryonic
myosin. This concept is consistent with the addition of
newly formed myofibrillar proteins being predominantly
at the periphery of fibers undergoing growth (Morkin,
1970; Patterson and Goldspink, 1976).
It also seems conceivable that the observed peripheral
staining could be related to the fusion of satellite cells with
adjacent atrophic fibers in an attempt to reverse the
atrophy. If satellite cells were to fuse with atrophic fibers,
it seems logical to expect the occurrence of myofibrillogen-
esis, including embryonic myosin expression, at the fibers
periphery and within the domain of the satellite cell
nucleus. Fusion of satellite cells with muscle fibers, in the
absence of satellite cell proliferation, would lead to a net
decrease in the number of satellite cells, which is consis-
tent with the data presented here, particularly for the
muscles immobilized for 8 to 10 weeks (Fig. 7). A similar
decrease in satellite cells has been reported in the soleus
muscles of rats subjected to hindlimb suspension (Darr
and Schultz, 1989). However, if this decrease were due to
satellite cells fusing with adjacent muscle fibers, one
would expect to find an increase in the ratio of myonuclei
per muscle fiber, but such was not case in the present
study. Further studies are needed to clarify the possibility
of satellite cells fusing with adult fibers that express
embryonic myosin.
 ACTIVITY-INDUCED FIBER REGENERATION 181

Fig. 2. Electron micrograph of a longitudinal section of an 8- to Fig. 3. Electron micrograph of a transverse section of an 8- to
10-week cast-immobilized soleus muscle fiber showing Z-disc streaming 10-week cast-immobilized soleus muscle fiber showing Z-disc clumping
(solid arrow) and abnormal triadic junctions (open arrows) surrounded by (Z) and a central nucleus (N). 9,500.
normal appearing myofibrils. 15,300.
182 WANEK AND SNOW

Fig. 4. Electron micrograph of a transverse section of an 810 week Fig. 5. Electron micrograph of noncasted soleus muscle seen in
cast-immobilized soleus muscle fiber. Note presence of multiple triads transverse section and showing immature, presumably regenerating
(polyads) linked in series. Polyads were seen in casted muscles only. myotubes (MT). Note presence of immature myofibrils (arrows) and
22,900. central nucleus. 11,100.
 ACTIVITY-INDUCED FIBER REGENERATION
Fig. 6. Frequency of satellite cells observed in casted and noncasted
soleus muscles. Note decrease in satellite cell frequency over time of
hindlimb immobilization. Percentage of satellite cells equals number of
observed satellite cell nuclei divided by number of observed myonuclei
plus satellite cell nuclei. Vertical bars represent group means 1 SEM.
The asterisk indicates statistically different values at the .05 level of
confidence using the nonparametric Wilcoxon Rank Sum test. Values
within the bars represent the number of animals per group. Data from the
8- and 10-week animals were pooled.
Observations reported here support the concept that
cage restricted levels of weight-bearing activity might be
sufficient to induce the regeneration of new fibers in the
soleus muscle. Previous work established that postural
muscles of normal, adult rats contain a small number of
regenerating myotubes, whereas non-postural muscles do
not (Wanek and Snow, 1995). Presumably, the regenerat-
ing fibers form in response to fibers that became damaged
secondary to their relatively frequent recruitment within
postural muscles. Examination of normal, adult soleus
muscles in the present study confirmed the presence of a
small number of small diameter, embryonic myosin-
positive fibers. Interestingly, the non-casted control soleus
muscles had twice as many embryonic myosin-positive
fibers as did the normal soleus muscles from control
animals that ambulated on both hindlimbs (Fig. 7). This
difference might be due to the fact that casted animals
tended to hold the casted limb up, thus increasing weight-
bearing on the contralateral, non-casted limb. The pres-
ence of an increase in the number of embryonic myosin-
positive fibers in non-casted muscles is consistent with the
concept that an increased workload leads to increased fiber
damage, followed by an increase in the number of regener-
ating new fibers.
Activity-induced muscle regeneration was also seen in
the soleus muscles from animals that had been returned to
normal cage activity following four weeks of hindlimb
immobilization. A significant number of small, embryonic
myosin-positive fibers were seen within four days of cage
activity following removal of casts that had been in place
183
Fig. 7. The frequency of observed embryonic myosin-positive fibers
relative to the total number of fibers counted in each section of casted and
noncasted soleus muscles. Vertical bars are group means 1 SEM.
Asterisk indicates statistical significance (P 0.5) using Kruskal-Wallis
nonparametric test followed by post hoc testing for minimum significant
differences between groups. n four rats per group for the two-week
animals. n three rats per group for the four- and six-week animals.
for four weeks (Fig. 8A). Similar fibers expressing develop-
mental myosin isoforms have also been described in rat
soleus muscles that were reloaded after a period of hind-
limb suspension (St. Pierre and Tidball, 1994). The appear-
ance of such regenerating fibers suggests that atrophic
skeletal muscle fibers in the soleus may be particularly
vulnerable to the reintroduction of limb activity immedi-
ately following periods of chronic immobilization. This
finding could be clinically significant when developing
exercise protocols for rehabilitating muscles following long
periods of immobilization.
In summary, this study provides evidence supporting
the concept that the degree of skeletal muscle fiber degen-
eration and new fiber formation might be related to subtle
changes in activity levels. In normal, adult, rat soleus
muscles, approximately 0.5% to 1% of the fibers appeared
to be regenerating. The number of regenerating fibers all
but disappeared when hindlimb activity was reduced by
cast-immobilization for up to ten weeks, whereas it in-
creased in muscles of the contralateral, non-casted limbs,
presumably due to the increased weight-bearing role of
this limb. Finally, a return to cage activity for only four
days, following four weeks of cast-immobilization, resulted
in a reappearance of new, regenerating muscle fibers. This
study also suggests that a return to weight-bearing activ-
ity following a period of casting can induce the re-
expression of embryonic myosin at the periphery of some
muscle fibers that otherwise appear mature. The mecha-
nism for this embryonic myosin re-expression and its
possible relationship to satellite cells remains unknown.
184 WANEK AND SNOW

Fig. 8. A. Recovery soleus muscle demonstrating small, embryonic
myosin-positive fibers after four days of cage activity following removal of
a cast that had been on for four weeks. Note central nuclei (dark profiles)
in the presumably regenerating myotubes. 700. B. Recovery soleus
muscle demonstrating the expression of embryonic myosin in large
diameter fibers (arrows) after 10 days of cage activity following removal of
a cast that had been on for four weeks. Note concentration of embryonic
myosin at the periphery of the larger fibers. The presence of small,
intensely fluorescent, embryonic myosin-positive fibers are also seen in
this section. 360.
 ACTIVITY-INDUCED FIBER REGENERATION
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