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Myosatellite cells growth and regeneration in murine dystrophic muscleA quantitative study.

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THE ANATOMICAL RECORD 208:159-174 (1984)
Myosatellite Cells, Growth, and Regeneration in Murine
Dystrophic Muscle: A Quantitative Study
MARCIA ONTELL, K.C. FENG, KATHLEEN KLUEBER, ROBERT F.
DUNN, AND FLOYD TAYLOR
Department of Anatomy and Cell Lliology, University of Pittsburgh School of
Medicine, Pittsburgh, PA 15261 (M.O., K. C.F., K,K), Department of
Otolaryngology, Eye and Ear Hospital, University of Pittsburgh School of
Medicine, Pittsburgh, PA 15213 (R.RD), and Department of Community
Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA
15261 (I? T)
ABSTRACT
Patterns of growth and regeneration in 2-, 4-, 8-,and 17-weekold murine dystrophic (129 ReJ dy/dy) extensor digitorum longus muscles have
been determined. Necrosis and myofiber loss, hypertrophy, and regeneration
result in a reduced population of myofibers whose diameter distribution is
more extensive than that found in the extensor digitorum longus muscles of
age-matched normal mice. At the onset of dystrophic symptoms (2 weeks
postnatal), the ratio of myosatellite cell nuclei to the total sublaminal nuclear
population (myonuclei + myosatellite cells) is similar to that found in 2-weekold control muscles. The frequency of finding myosatellite cells decreases with
age in both control and dystrophic muscles. Myosatellite cells account for 11%,
6%, 5%,and 3%of the total sublaminal nuclear population in control muscle
and 12%, 8%, 6%, and 5% of the total sublaminal nuclear population in
dystrophic muscle a t 2, 4, 8, and 17 weeks, respectively. No preferential
association of myosatellite cells with myofibers of a particular diameter is
found in control muscle or in the two youngest dystrophic groups. At 8 and 17
weeks, myosatellite cells are less frequently encountered on small-diameter,
regenerating myofibers of dystrophic muscle, and they are preferentially associated with large diameter, hypertrophied myofibers. The labeling index of
myosatellite cells decreases with age in both normal and dystrophic muscle.
At all ages the myosatellite cell labeling index is higher in dystrophic muscle
(23%,7%, 5%, and 2% a t 2, 4,8, and 17 weeks, respectively) than in normal
muscle (5%, < 1%at 2 and 4 weeks, respectively), with no labeled myosatellite
cells being found in 8- and 17-week-old normal muscles. It is suggested that
the magnitude of the regenerative response of dystrophic murine muscle decreases with age and that this factor may be responsible for the inability of the
regenerative response of dystrophic muscle to keep pace with the rapid muscle
deterioration.
Murine muscular d strophies (dy, Michel- Bradley et al., 1977; Bray et al., 1979; Kuno,
son et al., 1955; dy2 , Meier and Southard, 19791, and there remains considerable con1970) are characterized by the early involve- troversy as to whether the primary lesion
ment of hindlimb musculature with other involves the muscle or the nervous system
muscles becoming affected as the disease pro- (cf. Harris and Ribchester, 1979).
gresses. While the disorders were originally
In the dy mutant, clinical systems (i.e.,
believed to be primary myopathies, morpho- hindlimb dragging) occur 2 weeks postnatal,
logic and physiologic abnormalities in the
peripheral nervous system have been reported (Bradley and Jaros, 1973; Salafsky and
Received April 6, 1983; accepted August 30, 1983
Stirling, 1973; Bradley and Jenkison, 1975;
Address reprint requests to Dr. Marcia Ontell.
Y
0 1984 ALAN R. LISS, INC.
.
160
M. ONTELL ET AL.
concurrent with moderate histologic changes
within hindlimb muscles (Michelson et al.,
1955). The disease is progressive, and ultimately these muscles may be replaced, in
part, by connective tissue. During the course
of the disease, however, there is evidence of
regenerative attempts of the muscle fibers
(West and Murphy, 1960; Banker, 1967,1969;
Summers and Parsons, 1978). The myosatellite cell, first described by Mauro (1961) is
believed to be the source of the regenerating
myotubes in other regenerating systems
(Snow, 1978; Lipton and Schultz, 1979).There
have been reports of elevated populations of
myosatellite cells in murine muscular dystrophy (Summers and Parsons, 1981) and in
human muscle diseases (Laguens, 1963;
Shafiq et al., 1967; Aloisi, 1970; Conen and
Bell, 1970; Mastaglia et al., 1970; Van Haelst,
1970; Nonaka et al., 1973; Wakayama, 1976;
Chou and Nonaka, 1977; Wakayama et al.,
1979), some of which are similar to but not
identical with the mouse model. There are,
however, few quantitative studies. It has
been suggested that this elevated population
of myosatellite cells is preferentially located
in regenerative foci (Shafiq et al., 1967; Summers and Parsons, 1981). Despite the reported elevation of the myosatellite cell
population and the existence of regenerating
myotubes, the dystrophic muscle continues
to deteriorate.
The present study extends the morphometric analyses of patterns of growth of normal
and dystrophic murine muscles by Rowe and
Goldspink (1969a,b).Particular attention has
been focused on determining alterations in
the myonuclei and the myosatellite cell populations, the labeling index of the myosatellite cells, and the possible selective distribution of these cells to regenerating myofibers.
2.0% glutaraldehyde in 0.125 M cacodylate
buffer (pH 7.3). After removal, the muscle
was placed in fresh buffered 2.0% glutaraldehyde for 1 hour. The muscle was weighed
prior to postfixation in cacodylate-buffered
1.0% osmium tetroxide. The entire muscle
was dehydrated and embedded in Epon 812
(Luft, 1961). The Epon block containing the
muscle was placed on a sliding microtome
equipped with a steel knife. A 6-pm-thick
section was cut, mounted in Permount, and
observed in a phase contrast microscope to
ensure that the plane of sectioning was perpendicular to the long axis of the muscle
fibers.’ Then, the muscle was serially sectioned from origin to insertion into sets consisting of ten 15-pm-thick sections and one 6pm-thick section. The 15-pm-thick sections
were cleared in drops of Epon between two
layers of polystyrene film and cured in a n
oven a t 60°C (Davidowitz et al., 1976).The 6pm-thick sections were mounted on glass
slides and studied using a Leitz microscope
equipped with phase contrast optics. Selected
15-pm-thick sections, from known regions of
the muscle, were cut out of the polystyrene
sandwich and attached to preformed Epon
blocks. The reembedded sections were cut
into semithin (0.5 pm) or ultrathin (60-90
nm) sections with a n LKB-Huxley microtome. Ultrathin sections were collected on
50-mesh copper grids, stained with uranyl
acetate and lead citrate (Reynolds, 1963),and
observed using a Philips 300 electron microscope. Semithin sections were stained with
toluidine blue.
Maximal Girth of the Muscle2
A phase contrast microscope equipped with
an eyepiece reticule, consisting of 100 equal
squares calibrated with a stage micrometer
(one square = 0.014 mm2), was used to determine which of the 6-pm-thick sections perpendicular to the long axis of the muscle
MATERIALS AND METHODS
fiber contained the maximal cross-sectional
A minimum of four 2-, 4-, 8-, and 17-week- area of muscle. The maximal cross-sectional
old female 129 ReJ dyldy mice and four gen- area was calculated.
otypically normal female 129 ReJ mice, deNumber of Myofibers at the Muscles’
rived from the dystrophic colony and the
Maximal Girths
normal colony maintained at The Jackson
The 15-pm-thick sections closest to the
Laboratory, were injected (at 11 AM) with 2
pCi of 13H]thymidine per gram body weight muscles’ maximal girths were remounted,
(specific activity 20 Ci mmol) and killed 2
hours after injection. Additional mice in each
‘The extensor digitorum longus is a pennate muscle, and secage group were allowed to survive for 24 tions perpendicular to the myofibers are not perpendicular to
the
long axis of the muscle belly.
hours after t3H]thymidine injection. The ex‘The term “muscle’s widest (maximal) girth” refers to that
tensor digitorum longus muscle was exposed section,
perpendicular to the long axis of the muscle fibers, that
and fixed in situ for 1 hour by emersion in contains the largest section of the series.
MURINE DYSTROPHY
161
and alternate semithin and ultrathin sections were cut. By comparison of montages of
light micrographs with serial, ultrathin sections, it was determined that all of the myofibers found in the normal muscles were
readily identifiable in the light microscope.
The number of myofibers in the control muscles, therefore, was determined from montages of micrographs ( X 1,675) of the Od-pmthick sections passing through the muscles’
widest girths. Comparison of ultrathin and
semithin sections of dystrophic muscle revealed that some fibers were too small or too
necrotic to be easily discernible in the light
microscope. Therefore, the number of myofibers was determined using the electron
microscope.
Direct comparisons of montaged light micrographs of toluidine blue-stained 0.5-pmthick sections taken a t the muscle’s widest
girth with adjacent ultrathin sections viewed
with the electron microscope were used to
determine which of the myofibers were associated with myosatellite cells. Diameters of
myosatellite-cell-associated fibers were digitized into the computer from light micrographs ( x 1,675)as described above. Separate
files were maintained for normal and for dystrophic animals of each age group. The number of measured fibers varied from 50 to 200,
with the fewest fibers being measured in the
older animals where myosatellite cells were
less frequent.
Myonuclei and Satellite Cells
Diameter of the Myofibers
The diameters of 800-1,350 myofibers of
control and dystrophic muscles for each age
group were measured from light micrographs ( x 1,675) of randomly chosen muscle
fascicles seen at the muscles’ maximal girths.
(Calibration of the photographic enlarger was
made a t each printing.) Recognition of small
myofibers in dystrophic muscle was ensured
by comparison of light micrographs with serial ultrathin sections. Micrographs were
placed on a GRAFPEN sonic digitizer containing two linear microphones (one for each
axis). A spark-emitting stylus furnished direct binary input to a Digital LSI 11/03 processor with 24K memory and a Tetronix
Model 4010-1 terminal. When four points
along the circumference of each fiber, which
defined two perpendicular diameters that intersected a t the fiber’s center, were touched
by the sonic digitizer, the computer was programmed (Dunne et al., 1975; O’Leary et al.,
1976) to calculate the average diameter, display a histogram of the fiber diameter distribution for each group, and compute the mean
diameters for all of the fibers in a given
group.
Using transverse, ultrathin section, taken
a t the muscles’ maximal girths, 1,500-2,000
cross-sectioned fiber profiles from four dystrophic and control muscles of each age group
were examined, and the number of myonuclei andor myosatellite cells was recorded.
This permitted determination of changes in
the myonuclear and myosatellite cell population with age and dystrophy. The myonuclear and myosatellite cell populations were
also determined in ultrathin section taken
near the proximal and distal tendons, in order to determine whether the myonuclear
and myosatellite cell populations observed at
the widest girth were representative of that
found throughout the muscles.
Labeling Index of Satellite Cells
Diameter of Myosatellite-Cell-Associated
Myofi bers
In order to determine whether myosatellite
cells were preferentially associated with myofibers in a particular size range, montages of
micrographs of toluidine blue-stained 0.5-pmthick sections passing through the muscle’s
maximal girth were taken to the electron
microscope. An ultrathin section, serial to
the 0.5-pm section used for the montage, was
used to determine which of the fibers was
associated with a myosatellite cell.
Fifteen-micrometer-thick section from the
region of the muscles’ maximal girths were
remounted and sectioned into alternate semithin and ultrathin sections. Ultrathin sections were stained for electron microscopy.
Semithin sections were stained in Regaud’s
hematoxylin (Masson, 19291, a stain insoluble in the emulsion and the chemicals used
for autoradiographic studies, prior to dipping
them into melted Kodak NTB-2 emulsion.
Emulsion-coated slides were stored in a light
tight box for 8 weeks and then developed in
Kodak Dolmi (D-170) (Kopriwa and Leblond,
1962). Since background was quite low, a
nucleus that was associated with four or more
grains was considered to be labeled. Light
micrographs of the labeled cells were compared to adjacent ultrathin sections viewed
with the electron microscope, where the labeled cell could be “typed” with greater accuracy. The labeling index of the myosatellite
cells was determined.
162
M. ONTELL ET AL
Serial semithin and ultrathin sections
taken from tendinous ends of the muscle were
treated similarly in order to determine
whether the myosatellite cells in this region
showed different mitotic activity.
A similar procedure was carried out on the
muscles of animals killed 24 hours after injection with r3H]thymidine, in order to determine whether the myosatellite cells found in
dystrophic muscle were fusion competent.
Statistical Analyses
Statistical pairwise comparisons of fiber diameter distributions of normal and dystrophic muscle of various ages and statistical
analyses comparing the fiber diameter distributions of myosatellite-cell-associatedmyofibers with the diameter distributions of the
total myofiber population were carried out
using the University of Pittsburgh DEC-KL10, employing the nonparametric KruskalWallis test (Hollander and Wolfe, 1973). All
other statistical analyses (i.e., including
muscle weight, areas, fiber number, myonuclei, myosatellite cells, etc.) were carried out
on the DEC-KL-10 utilizing two-way analyses of variance. Appropriate transformation
of data (log or square root) were made t o
make the data compatible with the assumptions of the analysis of variance. Depending
on the significance of F ratios, appropriate
means were compared using the Student
Newman Keuls test (Sokal and Rohlf, 1969).
Values of P 2 0.05 were considered significant. All references to signfcant or insignificant changes, given in Results or Discussion,
refer to statistically determined differences.
All statements derived from statistical analysis are followed by a n asterisk (*).
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Morphology
The histologic changes that occur in murine dystrophic muscle have been described
using both the light microscope (Michelson
et al., 1955; West and Murphy, 1960; Platzer
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RESULTS
Gross Morphometry
At the time of onset of clinical symptoms (2
weeks postnatal) the weight of the extensor
digitorum longus muscle of the dystrophic
mouse was similar to that of age-matched
controls.* At all other ages studied, the
weight of the dystrophic muscles were significantly less than that of normal muscles,*
the ratio of dystrophic to control muscle
weight decreasing with age (Table 1).
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MURINE DYSTROPHY
and Chase, 1964)and the electron microscope
(Platzer and Chase, 1964; Banker, 1967; Bray
and Banker, 1970; Ontell and Haller, 1980;
Ontell, 1981; Ontell and Feng, 1981). Therefore, only those factors pertinent to the morphometric analyses will be discussed.
Alterations in the structure of the dystrophic extensor digitorum longus muscles
were present at 2 weeks postnatal (Fig. 1).
Three types of fibers were identified, based
on the extent and nature of their reactions to
the disease: relatively “healthy” fibers, necrotic fibers, and regenerating fibers. The
healthy fibers retained their polygonal shape
and peripheral nuclei, and many were indistinguishable from myofibers found in control
muscles. The necrotic fibers varied in appearance along their lengths, displaying swollen,
rounded profiles in regions characterized by
coagulation necrosis and being little more
than empty tubes enclosed by basal lamina
in other regions. Where the fiber consisted of
little more than a n irregular basal lamina1
tube, the fiber was not usually identifiable
in the light microscope. The regenerating
myofiber was,small in diameter, contained a
prominent, centrally placed or eccentric nuclei, and displayed few myofibrils. The ratio
of “healthy,” necrotic, and regenerating fibers varied between muscles of different animals of the same age and even between the
muscles of the two hindlimbs of a given dystrophic animal. Healthy fibers predominated
in all muscles studied. Necrotic fibers accounted for no more than 10% of the total
fiber population in any muscle. In some muscles as many as 25% of the fibers could be
classified as regenerating fibers.
The fascicular arrangement of the myofibers in dystrophic muscle was lost (Fig. 2)
with age. The variation in fiber diameters
within a given muscle became progressively
greater, and connective tissue infiltrated the
muscle, particularly in those regions where
extensive regeneration had occurred.
163
were marked reductions in the numbers of
fibers found in dystrophic muscles as compared to that found in age-matched controls
(Table l).*By 17 weeks, dystrophic muscles
had -59% of the fibers found in muscles of
age-matched controls.
Diameter of Myofibers at the Muscle’s
Widest Girth
In control animals, a continuous shift to
the right of fiber diameter distributions occurred with age, with a rather mature distribution pattern being established in 8-weekold mice (Figs. 3,4). The mean fiber diameter
increased nearly twofold during the period
studied (Figs. 3,4), as did the maximum-sized
fiber found in the muscle (Figs. 3, 4). There
was a progressive decrease in the number of
small diameter fibers (Figs. 3,4).
A different pattern was seen when the agerelated changes in the diameter distribution
of dystrophic muscle fibers were analyzed
(Figs. 5, 6). First, while there is evidence of
growth, with mean fiber diameter increasing
54% between the second and eighth week of
postnatal development (Fig. 5), the distribution of fiber diameters was much wider at
each age studied (Figs. 7-10). This wider distribution was the result of two factors: the
presence of very large fibers as well a s the
persistence of a substantial population of
small myotubes (< 6 pm in diameter). The
predominant type of large myofibers found
in the 2- and 4-week-old dystrophic muscle
was the “healthy”-looking, hypertrophied f i ber. There was also a small population of
swollen necrotic fibers. The population of
necrotic fibers was decreased in the older
muscles. While the maximal fiber diameter
was substantially greater in the dystrophic
muscle a t 8 weeks than it was in the control
muscle, the percentage of fibers > 30 pm in
diameter was less than that in the corresponding age-matched controls (Fig. 9). At 17
weeks, the population of fibers with diameters > 30 pm was further reduced (Figs. 6,
Number of Myofibers at the Muscle’s
10). Statistical comparisons (using the KrusWidest Girth
kal-Wallis test, Hollander and Wolfe, 1973)
The number of myofibers found at the wid- of diameter distributions of myofibers in dysest girth of the control muscles ranged from trophic and control muscles showed signifi900 to 1,250 myofibers, with muscles from cant differences (P < 0.01) in each age group.
the two oldest groups having a slightly deThe smallest-diameter fibers found in dyscreased number of myofibers as compared to trophic muscle, both by size and by ultrastructhe younger muscles (Table l).* At the onset tural criteria (central nuclei, glycogen accuof clinical symptoms the dystrophic muscle mulations, and myofibrillar arrangement),
had -77% of the fibers found in 2-week-old could be regarded as regenerating fibers. It
control muscle, and at all ages studied there should be noted that even at the earliest age
-
164
M. ONTELL ET AL
165
MURINE DYSTROPHY
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Figs. 3, 4. Comparison of the diameter distributions of the myofibers found in 2-, 4-, 8-, and
17-week-oldextensor digitorum longus muscles of the normal mouse. Mean diameters of myofibers at 2 , 4 , 8 , and 17 weeks are indicated by m ,o,x, and v, respectively.
Figs. 5, 6. Comparison of the diameter distributions of the myofibers found in 2-, 4-,8-, and
17-week-oldextensor digitorum longus muscles of the dystrophic mouse. Mean diameters of
myofibers at 2 , 4 , 8, and 17 weeks are indicated by m,0, x, and v, respectively.
Fig. 1. Semithin section through the widest girth of
an extensor digitorum longus muscle taken from a 2week-old dystrophic mouse. The myofibers are closely
spaced and arranged into fascicles (cf. Fig. 2). Based on
morphologic criteria three types of myofibers are identified polygonally shaped, “healthy” fibers (F) with peripherally located nuclei; palely stained, rounded necrotic
fibers (arrowheads); and small-diameter regenerating fibers (arrows) with centrally located nuclei. Toluidine
blue. Magnification, X500.
Fig. 2. Semithin section through the widest girth of
an extensor digitorum longus muscle taken from a 17week-old dystrophic mouse. The myofibers show a greater
variation in diameter as compared with muscle found in
younger dystrophic mice (Fig. 11, and they are separated
from each other by a significant amount of connective
tissue. Regionally, the fascicular arrangement of the myofibers is lost. Small-diameter, regenerating myofibers
(arrow) persist, but, as shown in this micrograph, the
frequency of finding necrotic fibers is greatly reduced.
Toluidine blue. Magnification, ~ 5 0 0 .
166
M. ONTELL ET AL.
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Fig. 7. Comparison of the diameter distributions of normal and dystrophic muscle fibers of
the extensor digitorum longus muscles of 2-week-old mice.
Fig. 8. Comparison of the diameter distributions of normal and dystrophic muscle fibers of
the extensor digitorum longus muscles of 4-week-old mice.
Fig. 9. Comparison of the diameter distributions of normal and dystrophic muscle fibers of
the extensor digitorum longus muscles of 8-week-oldmice.
Fig. 10. Comparison of the diameter distributions of normal and dystrophic muscle fibers of
the extensor digitorum longus muscles of 17-week-oldmice.
studied, there were no fibers < 6 pm in any
control muscles (Fig. 3). While there was a
decrease in the percentage of small-diameter
fibers (< 6 pm) in the dystrophic muscles
between the second and fourth week (Fig. 5),
the percentage of small-diameter fibers remained at the 4-week level throughout the
next 13 weeks (Figs. 5,6).
muscle's maximal girth, increased with age,
with the adult girth being achieved by 8
weeks (Table l).* The only period when the
dystrophic muscle underwent a significant
increase in cross-sectional area was between
the second and fourth week of postnatal development (Table l).* At all ages studied the
maximal girth of the dystrophic muscle was
significantly less than that of age-matched
Ratio of Muscle Fiber Cross-Sectional Area controls.* The mean cross-sectional areas octo Nonmuscle Regions of the Extensor
cupied by the muscle fibers found at the maxDigitorum Longus
imal girth in the normal and dystrophic
The cross-sectional area of the control ex- muscles at various ages was calculated (crosstensor digitorum longus, measured at the sectional area occupied by muscle fibers =
MURINE DYSTROPHY
number of muscle fibers x mean diameter of
the fibers) and expressed as a percentage of
the cross-sectional area of the muscle (Table
1).In the normal muscle this percentage increased with age. An increase in this ratio
also occurred in dystrophic muscle between
the second and fourth week;" however, subsequently this ratio declined.* Thus, not only
were the dystrophic muscles smaller in girth,
the proportion of their area occupied by myofibers was less than that found in normal
muscles.
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Alterations in Number of Satellite Cells andl
or Myonuclei
Electron microscopic identification and
quantitation of the size of the myonuclear
and myosatellite cell population was completed using sections taken a t the muscles'
maximal girths. Despite the large increase
in the mean fiber diameter of control muscle
with age, the number of myonuclei per myofiber remained relatively constant (Table 2).
Myonuclei per myofiber in dystrophic muscle
also remained relatively constant with age
(Table 2). Myosatellite cells per myofiber in
normal and dystrophic muscles exhibited a
similar pattern of decrease with age;* however, there was a difference in the mean of
the control and dystrophic-averaged overtime,* with a slightly higher value being
observed in dystrophic muscles (Table 2). A
similar pattern was found when the ratio of
myosatellite cells to total sublaminal nuclei
(myosatellite cells and myonuclei) was calculated (Table 2).*
Quantitative studies were also carried out
at regions close to the proximal or distal tendon, in order to determine whether there
might be regional variations in the distribution of myonuclei or myosatellite cells. In
both the dystrophic and the normal extensor
digitorum longus muscles, it was found that
the number of myonucleilmyofiber was
greater a t the tendonous ends, with 64% of
the fiber profiles in both control and dystrophic muscles displaying a myonucleus.
Myosatellite cells per myofiber at the proximal or distal ends of the muscle did not differ
from that found at the muscle's widest girth.
Diameter of Myosatellite-Cell-Associated
Myofibers
The distribution of the diameters of myofibers displaying myosatellite cells was determined in order to establish whether the
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168
M. ONTELL ET AL
population of myosatellite cells found in
either normal or dystrophic muscles was selectively distributed to myofibers of a particular diameter. In all of the control groups,
the mean fiber diameters of myosatellite-cellassociated myofibers was similar to that
found in the total myofiber population (not
shown), and the fiber diameter distributions
(not shown) displayed no statistically significant differences as analyzed with the Kruskal-Wallis test (Hollander and Wolfe, 1973).
Thus, in the control extensor digitorum longus muscle, myosatellite cells are not preferentially distributed to myofibers of any given
size. In comparing the mean diameters of
myosatellite-cell-associated fibers in dystrophic muscle with the mean diameters of
the total population of extrafusal myofibers,
no significant difference were seen in the two
youngest age groups; however, in 8- and 17week-old muscles the mean diameters of the
myosatellite cell associated fibers were elevated (Figs. 11-14). None of the necrotic fiber
profiles were associated with a myosatellite
cell. Comparisons of the histograms of the
diameter distributions of the myosatellitecell-associated fibers with histograms of the
total fiber population of 2- and 4-week-old
animals showed considerable overlap (Figs.
11,12),with no statistically significant differences being found. At 8 (Fig. 13)and 17 weeks
(Fig. 14) the myosatellite cells were preferentially associated with large diameter fibers with few, if any, of the small-diameter
fibers displaying myosatellite cells. Comparisons of the diameter distributions of the total fiber population with that of the
myosatellite-cell-associated fiber population
for 8- and 17-week-oldanimals showed significant differences a t P < 0.05 and P < 0.2,
respectively. Interestingly, there was no preferential association of myosatellite cells with
smaller-diameter (regenerating) fibers at any
ages studied.
cation of all labeled cells found in the 5-pmthick sections was made in serial ultrathin
sections (Figs. 15, 16). In muscles removed
from mice 2 hours after 13H]thymidine injection, labeled nuclei could be found in endothelial cells, pericytes, connective tissue cells,
and myosatellite cells. The number of labeled
myosatellite cells found a t the muscles’ maximal girths was recorded. Having determined the mean number of fibers found in
normal and dystrophic muscles during each
age period and the number of myosatellite
cells per myofiber profile, it was possible to
estimate the total number of myosatellite
cells present in each autoradiograph. From
this informtion, the labeling index was calculated. Five percent of the myosatellite cells
found in a 2-week-old control extensor digitorum longus muscle were labeled. The labeling index dropped to 1% by 4 weeks
postnatal, and no labeled myosatellite cells
were found in older control muscles. In comparison, 23%, 7%, 5%, and 2% of the myosatellite cells of dystrophic muscles were
labeled 2,4, 8, and 17 weeks, respectively.
Examination of transverse sections taken
near the proximal and distal ends of normal
and dystrophic muscles revealed that there
were fewer fibers found in these regions than
a t the muscles’ widest girths ( - 10-15% of
the fibers found at the muscles’ widest girths
extended into these sections). Therefore, although the proximal and distal regions of
each of four muscles in the youngest two age
groups were studied, the number of myosatellite cells that were present was relatively
small and deemed insufficient for providing
exact information relative to the labeling index of myosatellite cells located a t the ends
of the muscles. However, in both control and
dystrophic 2- and 4-week-old animals the labeling index never exceeded that found a t
the muscles’ widest girths.
No attempt was made to determine quantitatively the fusion capabilities of myosatelLabeling Index of Satellite Cells
lite cells. However, labeled myonuclei were
The total number of labeled nuclei found found in muscles of 2-week-old control mice
in autoradiographs of 0.5-pm-thick cross sec- and 2-, 4-, 8-, and 17-week-olddystrophic mice
tions, taken a t the widest girths of normal that were injected with [3H]thymidine 24
muscles, was highest a t 2 weeks and rapidly hours prior to removal of the extensor digideclined with age. In 2-week-old dystrophic torum longus muscles.
muscle approximately three times as many
DISCUSSION
nuclei were labeled as in age-matched controls. While the number of labeled nuclei deThe three factors altering the pattern of
creased with age, the ratio of labeled nuclei growth of dystrophic muscle, as compared to
in dystrophic muscles to that found in con- normal murine muscle, are necrosis with retrols showed substantial increases. Identifi- sulting myofiber loss, regeneration, and hy-
169
MURINE DYSTROPHY
4 WEEK
2 WEEK
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DIAMETER
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DlAMETER(rm)
TOTAL FIBER POPULATION
-
M'Y 0 S A T E L L I T E C E L L A S S 0 C I A T E D F I B E R S
'...."."
Fig. 11. Comparison of the diameter distribution of the myosatellite-cell associated fibers
with the total myofiber population of the extensor digitorum longus muscles found in 2-weekold dystrophic mice. Mean diameter of total myofiber population indicated by x. Mean diameter
of myosatellite-cell-associated fibers indicated by 0.
Fig. 12. Comparison of the diameter distribution of the myosatellite-cell-associatedfibers
with the total myofiber population of the extensor digitorum longus muscles found in 4-weekold dystrophic mice. Mean diameter of total myofiber population indicated by x. Mean diameter
of myosatellite-cell-associatedfibers indicated by 0.
Fig. 13. Comparison of the diameter distribution of the myosatellite-cell-associatedfibers
with the total myofiber population of the extensor digitorum longus muscles found in 8-weekold dystrophic mice. Mean diameter of total myofiber population indicated by x. Mean diameter
of myosatellite-cell-associated fibers indicated by 0 .
Fig. 14. Comparison of the diameter distribution of the myosatellite-cell-associated fibers
with the total myofiber population of the extensor digitorum longus muscles found in 17-weekold dystrophic mice. Mean diameter of total myofiber population indicated by x. Mean diameter
of myosatellite-cell-associated fibers indicated by 0.
pertrophy. These result in dystrophic muscles
having a reduced population of myofibers
with greater diameter range. Interestingly,
despite the marked decrease in the number
of myofibers, necrotic muscle fibers account
for only a small percentage of the total fiber
population of dystrophic muscle. This may
indicate that once a muscle fiber shows evidence of necrotic changes, necrosis and subsequent elimination of the myofiber proceeds
rather rapidly. Hypertrophy, reportedly a
distinguishing characteristic of dystrophic
170
M. ONTELL ET AL.
Fig. 15. Light microscopic autoradiograph of a labeled nucleus (arrow) found in a 2-week-old
dystrophic muscle. In a serial ultrathin section, this cell is identified as a myosatellite cell (Fig.
16).Hematoxylin. x 1,650.
Fig. 16. Serial ultrathin section shows that the labeled cell (Fig. 15) lies under the basal
lamina of a muscle fiber. An electron-lucent space (arrows) separates this myosatellite cell (S)
from the underlying muscle fiber (MI. Magnification, x 9,750.
murine muscle (West and Murphy, 1960),actually involves relatively few of the myofibers. No more than 10% of the fibers found
in dystrophic murine muscle, at any age
studied, have diameters exceeding those
found in age-matched controls. The most significant difference between control and dystrophic muscles, in terms of the number of
myofibers involved, is the presence of segments of the dystrophic fiber population
which are significantly smaller than the myofibers found in control muscle. Based on their
size and their ultrastructural characteristics,
these fibers can clearly be identified as regenerating myotubes.
There is an apparent paradox in finding
substantial populations of regenerating myofibers in muscle whose ultimate fate is progressive degeneration. It can be suggested
that there may be some innate morphologic
or biochemical defect in the regenerating
myotubes found in dystrophic muscle that
inhibits them from fully maturing or which
is incompatible with their survival. Alternatively it may be suggested that the dys-
trophic environment (i.e., the presence of
dystrophic nerve) may be a limiting factor in
effective regeneration. Finally, it can be suggested that the magnitude of the regenerative response is insufficient to keep pace with
degenerative changes. In the present study,
only the magnitude of the regenerative response has been examined.
The magnitude of the regenerative response has been evaluated in two ways. First,
the frequency of finding small-diameter myofibers whose ultrastructural characteristics
appear similar to regenerating fibers in other
systems, has been determined. The frequency of finding small-diameter fibers (< 6
pm) is increased in 2-week-old dystrophic
muscle as compared to normal muscle; and,
although there is a decline in the percentage
of these fibers during the next 2 weeks, it
remains at a markedly elevated level even in
17-week-oldmice whose muscles are in a severely degenerated state. However, judging
the magnitude of the regenerative response
entirely on the basis of the frequency of finding regenerating fibers is not entirely satis-
MURINE DYSTROPHY
factory. While it is possible that the de novo
formation of myotubes may persist in older,
more severely affected animals, the possibility also exists that the regenerating myotubes found in older dystrophic mice may
have been formed during a n earlier, more
moderate stage of the disease and that they
are unable to undergo normal growth and
maturation to form mature myofibers. A second method, which has permitted assessment of the regenerative capacity of the
muscle, involves determination of the number, distribution, and labeling index of myosatellite cells.
Myosatellite cells are believed to be responsible for both the addition of myonuclei to
existing fibers in normal growing muscle
(Moss and Leblond, 1970, 1971) and for the
regenerative response of muscle to disease
and trauma (Church et al., 1966; Shafiq et
al., 1967; Teravainen, 1970). It is generally
believed that trauma or disease causes the
myosatellite cell population to increase and
that fusion of myosatellite cells results in de
novo formation of regenerating myotubes (cf.
Carlson, 1973). In the present investigation,
the myosatellite cell population has been
studied relative to the total sublaminal nuclear population, the more commonly used
method in studies of muscle growth and regeneration, and also in terms of myosatellite
cells and myonuclei per-myofiber profile. The
latter method is necessary since there have
been studies that report a n increased number of myonuclei per myofiber cross-sectional
profile with age (Kelly, 1978) and also because it has been suggested that dystrophic
murine muscle (Michaelson et al., 1955; West
and Murphy, 1960) and Duchenne muscle
(Wakayama et al., 1979) have elevated numbers of myonuclei. If only the first method
(myosatellite cells/total sublaminal nuclei) is
used, changes in the myonuclear population
may obscure alterations in the myosatellite
cell population.
The validity of using sections through the
muscle’s widest girth for quantitating the
myosatellite cell population of normal mouse
muscle has been demonstrated by Snow
(1981),who showed that in the normal mouse
the myosatellite population is uniformly distributed along the length of the muscle fibers. Since, in the present study, a uniform
pattern of myosatellite cell distribution also
has been demonstrated in the dystrophic
mouse muscle, comparisons of age-matched
dystrophic and control muscles are possible.
171
In both normal and dystrophic muscle the
myonuclei are uniformly distributed over
most of the muscle’s length, with a n increased number of myonuclei occurring at
the tendonous regions. This similarity in the
distribution patterns allows valid comparisons of the size of the myonuclear population
of normal and dystrophic muscles.
In the present study, no biologically significant age-related increase in myonucleilmyofiber profile has been found in the mouse, in
contrast to previous studies using the normal
growing rat, where there is a n approximately 100% increase between 14 and 100
days after birth (Kelly, 1978), and human
children, where there is a four-fold increase
between 4 and 15 years (Wakayama et al.,
1979). Since the present study has examined
1,500-2,000 profiles in each age group, the
explanation may lie in species differences.
While it has been suggested that the number
of myonuclei is elevated in the dystrophic
mouse (Michaelson et al., 1955; West and
Murphy, 1960), there are no published quantitative reports of the number of myonuclei
in murine dystrophic muscle. In the present
study, no elevation in this population has
been found, and it may be that the illusion of
myonuclei being more prevalent in dystrophic muscle is fostered by the presence in
this muscle of a population of small fibers
with very prominent nuclei.
The size of the myosatellite cell population
of the control muscle, both in terms of the
sublaminal nuclear population and their frequency per myofiber profile, decreases with
age. This is consistent with the pattern usually described in growing muscle (Allbrook
et al., 1971; Ontell, 1974; Schultz, 1974,1976).
Wakayama et al. (1979), however, examining
normal human muscle, has found a n agerelated decrease in the percentage of myosatellite cell nuclei relative to total sublaminal
nuclei but found no effect of age on the number of myosatellite cells per myofiber profile. .
Thus, it appears that a n age-related increase
in myonuclei in human muscle (Wayakama,
et al., 1979), rather than a decrease in the
number of myosatellite cells, is responsible
for the altered ratio of myosatellite cells to
the total sublaminal nuclear population.
Again, this difference may be species specific.
At the time of the onset of clinical symptoms in the mouse, a period when regeneration has already begun, there is no difference
in the size of the myosatellite cell population
of dystrophic mice as compared to age-
172
M. ONTELL ET AL.
matched controls. The myosatellite cell population of murine dystrophic muscle decreases with age as it does in normal
muscle.* In a preliminary study, using only
one dystrophic mouse in each age group, no
consistent age-related changes in the myosatellite cell population have been demonstrated (Summers and Parsons, 1981). The
present results are in marked contrast with
what apparently occurs in Duchenne dystrophy, where there is a two- to fourfold increase
in the myosatellite cell population and where,
throughout the first 9 years of life, this population fails to decrease with age (Wakayama et al., 1979). The absence of equivalent
responses of myosatellite cells to the dystrophic process may indicate another significant difference between Duchenne dystrophy
and the mouse dystrophy model.
Since it has been suggested, but not demonstrated, that myosatellite cells in diseased
muscle are more prevalent in foci of regeneration (Muir, 1970; Summers and Parsons,
1981), a n attempt has been made to determine whether these cells are preferentially
associated with regenerating (i.e., small) fibers in murine dystrophy. No preferential
association of myosatellite cells with small
fibers has been found either in control or
dystrophic muscles. In fact, in the two oldest
dystrophic groups, few of the small-diameter
fibers are associated with myosatellite cells
while in the oldest dystrophic group these
cells are preferentially associated with the
larger fibers. Hypertrophied fibers, in other
systems, show a n increased number of myosatellite cells (Schiaffho et al., 1972; Hanzlikova et al., 1975). A question is raised as to
why the frequency of myosatellite cells on
the small-diameter, regenerating fibers decreases as the disease progresses. While no
definitive information is available, it may be
that myosatellite cells are capable of a limited number of mitotic divisions subsequent
to which they either must remain as postmitotic cells or fuse with their associated myofiber. Some, if not all, muscle regeneration
subsequent to disease and trauma occurs in
the old basal laminal tubes of degenerating
myofibers. In other regenerating systems, the
myoblastic cells taking part in the regenerative effort are believed to be derived either
from myosatellite cells found associated with
the degenerating fibers or from myosatellite
cells recruited from surrounding intact fibers
(cf. Carlson, 1973). The absence of myosatellite cells beneath the basal lamina of the
necrotic fibers of the dy mouse is in agreement with a previous report in the dyZJ
mouse (Ontell, 1981) and suggests that the
myosatellite cells from neighboring fibers
may “seed” the empty basal laminal tubes
with myoblasts. The ability of myosatellite
cells to pass through muscle basal lamina
has been demonstrated (Lipton and Schultz,
1979).Whatever the source of the myoblastic
cells, the regenerative effort may exhaust
their mitotic capability, and the fusion of the
myosatellite cells with the myotubes could
result in the decreased frequency of these
cells on regenerating fibers in the present
study. The reduction in the number of myosatellite cells associated with the regenerating fibers may have had an effect on the
growth, maturation, or survival of the regenerating fibers.
That the labeling index of myosatellite cells
of normal muscle declined with age is similar
to what has been reported in the literature
(Schultz et al., 1978); however, the labeling
index of < 1%at 4 weeks of age appears to
be rather low. Since all of the animals in this
study have been injected a t 11A.M. and killed
at 1P.M., a n attempt has been made to determine whether a circadian rhythm (Laudert
and Venable, 1982) might make this time
period inappropriate for judging myosatellite
cell proliferative activity. Comparisons have
been made with a n autoradiographic study
of muscles from two 4-week-old normal mice
injected with a total of 8 pCVg body weight
of [3H]thymidine (at 4-hour intervals) for a
24-hour period prior to muscle removal. The
percentage of labeled myosatellite cells and
myonuclei in these animals (Klueber and
Ontell, unpublished observations) indicates
that the low labeling index found in the 4week-old normal animals is a reliable measurement of myosatellite cell proliferative
activity.
There are no previous studies of the labeling index of myosatellite cells in dystrophic
muscle. At all ages, even at the onset of dystrophy when the myosatellite cell population
is not yet elevated, the labeling index of
myosatellite cells is quite elevated compared
to that found in muscle from age-matched
control mice. While the dynamics of development and growth of normal muscle may explain the age-related decrease in the labeling
index of myosatellite cells, the reason the
labeling index of dystrophic myosatellite cells
decreases with age is not immediately apparent, since the degeneration of the muscle con-
MURINE DYSTROPHY
173
tinues throughout the period studied. Again, stitutes of Health grant NS 13688.
it may be related to the exhaustion of the
The competent technical assistance of Ms.
mitotic capabilities of the myosatellite cells. Gloria Diluiso and Ms. Janet Lieberman is
The age-related decline in the number and in gratefully acknowledged.
the labeling index of myosatellite cells in
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This study was supported by National In-
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muscle, growth, murine, stud, regenerative, myosatellite, quantitative, cells, dystrophy
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