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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.
Skeletal muscle is a highly differentiated tissue that has the possible role of the satellite cell population during
the capacity to adapt to extreme fluctuations in its func- recovery from atrophy. Satellite cells have been shown to
tional state. Despite this high degree of plasticity, certain form new, regenerating muscle fibers following muscle
adaptive changes are not completely reversible. Clinical injury (Snow, 1977; McGeachie and Allbrook, 1978), to
studies have confirmed that skeletal muscle atrophy and serve as a source of new nuclei during muscle fiber growth
strength reduction, secondary to procedures such as cast- and hypertrophy (Moss and LeBlond, 1970; Snow, 1990;
immobilization or surgery, cannot always be returned to
normal levels despite extensive rehabilitation (LoPresti et
al., 1988; Matthews and St. Pierre, 1996). Although a *Correspondence to: Linda Wanek, Graduate Program in Physi-
variety of mechanisms may be involved in limiting the cal Therapy, San Francisco State University, 1600 Holloway, San
functional recovery of skeletal muscle following these Francisco, CA 94132.
procedures, little attention has been directed at examining Received 14 August 1998; Accepted 8 October 1999
(Reynolds, 1963), and examined using a 100C JEOL tained a Golgi apparatus, enlarged endoplasmic reticu-
electron microscope. Each cross-section was surveyed for lum, immature myofibrils, and polyribosomes. Regardless
fiber pathology and /or muscle regeneration. The percent- of the duration of immobilization, no evidence of regenerat-
age of satellite cells was determined per section by count- ing myotubes was encountered in any of the cast-
ing the number of satellite cell nuclei and myonuclei at a immobilized muscles. The smallest diameter fibers ob-
magnification of 4,000 6,000, and dividing the number served in the cast-immobilized muscles routinely exhibited
of satellite cell nuclei by the total muscle nuclei (satellite mature organization and morphology, suggesting fibers
cell nuclei myonuclei) observed. Satellite cell nuclei were atrophic rather than regenerating fibers.
were distinguished from myonuclei by the identification of The frequency of satellite cells in casted soleus muscles
a satellite cell membrane distinct from a sarcolemma. The remained constant or decreased slightly during the shorter
values for three to five sections per muscle were pooled to periods of immobilization (Fig. 6). However, by 8 to 10
calculate a mean percentage of satellite cells per muscle. A weeks of immobilization, the mean percentage of satellite
minimum of 1,000 total muscle nuclei was counted per cells in casted soleus muscles had decreased significantly
muscle. (P .05) from control values of 6.4% (range 4.1% to 11.7%)
The quantitative data were analyzed using descriptive to 3.3% (range 1.0 to 6.0%). To differentiate whether this
statistics, the Wilcoxon Rank Sum test and the Kruskal decrease in satellite cell frequency was due to a decrease in
the absolute number of satellite cells, or to an increase in
Wallis test with post hoc testing for differences between
the number of myonuclei, the ratio of myonuclei per
groups. Alpha and F values were set at 0.05.
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
RESULTS (data not shown). Thus, the observed decrease in the
Based on muscle wet weight / body weight ratios, statisti- percentage of satellite cells appears to be a decrease in the
cally significant muscle atrophy was seen at all intervals absolute numbers of satellite cells.
in the casted soleus muscles (data not shown). No signifi- Approximately one out of every 100 (1.0%) extrafusal
cant differences were noted between normal soleus and muscle fibers appeared positively stained for embryonic
non-casted soleus muscles. Fiber atrophy within the cast- myosin in both the normal and non-casted soleus muscles
immobilized soleus muscles was confirmed by measuring (Fig. 7). Nearly all such fibers were of a small diameter
cross-sectional fiber areas (Fig. 1). Frequency distribution (500 2). Although intrafusal fibers also expressed embry-
graphs of fiber cross-sectional areas demonstrated a pro- onic myosin, such fibers were excluded from the data. By
two and four weeks of cast-immobilization, the number of
gressive increase in the number of small fibers in the
extrafusal fibers staining positively for embryonic myosin
soleus muscles over a six-week period of casting.
was reduced to 0.21% and 0.15%, respectively. By six
Light microscopic examination of normal, non-casted,
weeks of immobilization, the frequency of extrafusal fibers
and casted soleus muscles revealed only an occasional fiber
staining positively for embryonic myosin was significantly
that appeared degenerating based on the presence of
reduced to 0.1% of the total number of fibers (P .05). The
vacuolated cytoplasm and an invasion of large mononucle- majority of these embryonic myosin-positive fibers ob-
ated cells. At the ultrastructural level, myofibrillar atro- served in the casted muscles tended to be larger and closer
phy and disorganization were observed in both casted and to the size of normal muscle fibers ( 1000 2), in contrast
non-casted muscles, although casted muscles displayed a to the small diameter, embryonic myosin-positive fibers
greater incidence of degenerative change. The myofibrillar seen in normal and non-casted soleus muscles.
disorganization observed in casted muscles was usually Although six weeks of cast-immobilization essentially
limited to a small segment of a fiber, with the remainder of eliminated the small number of embryonic myosin-
the fiber appearing relatively normal (Fig. 2). Alterations positive fibers seen in normal soleus muscles, a return to
in the Z-disc morphology were frequently observed in the just four days of cage activity resulted in the reappearance
casted muscles, with Z-disc disruptions and dense clumps of fibers that stained positively for embryonic myosin (Fig.
or rods as the most common occurrences (Fig. 3). Myofibril- 8A). Based on size and staining intensity, two populations
lar disorganization and Z-disc disruptions were most notice- of embryonic myosin-positive fibers were observed in the
able in casted muscles sampled from the 8 to 10-week muscles of the recovery group. One population resembled
immobilization period. Pyknotic satellite cell nuclei or regenerating myotubes since they were intensely fluores-
myonuclei were not observed in any of the muscles sampled. cent for embryonic myosin, had a small cross-sectional
One interesting observation, noted only in cast-immobi- area, and often displayed a central nucleus. Other fibers
lized soleus fibers, was the presence of unusual triadic were of a much larger diameter with a less intense
profiles. Such abnormal triads surrounded areas of Z-disc embryonic myosin staining that typically appeared re-
disruption and appeared to be linked in series to form stricted to the periphery of the fiber (Fig. 8B). After ten
polyads (Fig. 4). These multiple triadic profiles are days of recovery, the frequency of staining for embryonic
referred to as polyads to distinguish them from the myosin in both small and large diameter extrafusal fibers
pentads (two transverse tubules and three terminal cister- appeared to increase (Fig. 8B).
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-
Fig. 1. Frequency distribution of changes in fiber size in the soleus
nal and transverse sections, suggesting they had no orien- muscle immobilized from two to six weeks. Controls include measure-
tation to the longitudinal axis of the myofibers. ments from normal and noncasted muscles. Between 1,063 and 2,319
Infrequently, regenerating myotubes were noted in the fibers were measured for each group at each interval. Note increased
non-casted soleus muscles (Fig. 5). These myotubes con- incidence of small fibers over time.
ACTIVITY-INDUCED FIBER REGENERATION 179
Figure 1.
180 WANEK AND SNOW
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 183
Observations reported here support the concept that for four weeks (Fig. 8A). Similar fibers expressing develop-
cage restricted levels of weight-bearing activity might be mental myosin isoforms have also been described in rat
sufficient to induce the regeneration of new fibers in the soleus muscles that were reloaded after a period of hind-
soleus muscle. Previous work established that postural limb suspension (St. Pierre and Tidball, 1994). The appear-
muscles of normal, adult rats contain a small number of ance of such regenerating fibers suggests that atrophic
regenerating myotubes, whereas non-postural muscles do skeletal muscle fibers in the soleus may be particularly
not (Wanek and Snow, 1995). Presumably, the regenerat- vulnerable to the reintroduction of limb activity immedi-
ing fibers form in response to fibers that became damaged ately following periods of chronic immobilization. This
secondary to their relatively frequent recruitment within finding could be clinically significant when developing
postural muscles. Examination of normal, adult soleus exercise protocols for rehabilitating muscles following long
muscles in the present study confirmed the presence of a periods of immobilization.
small number of small diameter, embryonic myosin- In summary, this study provides evidence supporting
positive fibers. Interestingly, the non-casted control soleus the concept that the degree of skeletal muscle fiber degen-
muscles had twice as many embryonic myosin-positive eration and new fiber formation might be related to subtle
fibers as did the normal soleus muscles from control changes in activity levels. In normal, adult, rat soleus
animals that ambulated on both hindlimbs (Fig. 7). This muscles, approximately 0.5% to 1% of the fibers appeared
difference might be due to the fact that casted animals to be regenerating. The number of regenerating fibers all
tended to hold the casted limb up, thus increasing weight- but disappeared when hindlimb activity was reduced by
bearing on the contralateral, non-casted limb. The pres- cast-immobilization for up to ten weeks, whereas it in-
ence of an increase in the number of embryonic myosin- creased in muscles of the contralateral, non-casted limbs,
positive fibers in non-casted muscles is consistent with the presumably due to the increased weight-bearing role of
concept that an increased workload leads to increased fiber this limb. Finally, a return to cage activity for only four
damage, followed by an increase in the number of regener- days, following four weeks of cast-immobilization, resulted
ating new fibers. in a reappearance of new, regenerating muscle fibers. This
Activity-induced muscle regeneration was also seen in study also suggests that a return to weight-bearing activ-
the soleus muscles from animals that had been returned to ity following a period of casting can induce the re-
normal cage activity following four weeks of hindlimb expression of embryonic myosin at the periphery of some
immobilization. A significant number of small, embryonic muscle fibers that otherwise appear mature. The mecha-
myosin-positive fibers were seen within four days of cage nism for this embryonic myosin re-expression and its
activity following removal of casts that had been in place possible relationship to satellite cells remains unknown.
184 WANEK AND SNOW
Fig. 8. A. Recovery soleus muscle demonstrating small, embryonic diameter fibers (arrows) after 10 days of cage activity following removal of
myosin-positive fibers after four days of cage activity following removal of a cast that had been on for four weeks. Note concentration of embryonic
a cast that had been on for four weeks. Note central nuclei (dark profiles) myosin at the periphery of the larger fibers. The presence of small,
in the presumably regenerating myotubes. 700. B. Recovery soleus intensely fluorescent, embryonic myosin-positive fibers are also seen in
muscle demonstrating the expression of embryonic myosin in large this section. 360.
ACTIVITY-INDUCED FIBER REGENERATION 185
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