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Maintenance of myonuclear domain size in rat soleus
after overload and growth hormone/IGF-I treatment
G. E. MCCALL,1 D. L. ALLEN,1 J. K. LINDERMAN,3 R. E. GRINDELAND,3
R. R. ROY,2 V. R. MUKKU,4 AND V. R. EDGERTON1,2
1Department of Physiological Science and 2Brain Research Institute, University of California,
Los Angeles 90095; 3National Aeronautics and Space Administration Ames Research Center,
Moffett Field 94035; and 4Genentech, South San Francisco, California 94080
compensatory hypertrophy; satellite cells; synergist ablation;
growth factors; myonuclear number; functional overload
ADULT SKELETAL MUSCLE is capable of significant growth
during periods of increased loading. During functional
overload (FO) induced by surgical ablation of synergist
muscles, muscle mass and fiber cross-sectional area
(CSA) increase in the remaining muscle(s) (28, 29, 33).
This increase in fiber size is thought to occur via several
mechanisms, including increased gene transcription
and protein synthesis rate (7). The signals that initiate
these intracellular changes are not well defined but are
believed to include alterations in the cellular mechanical forces (37), as well as local and systemic release of
growth factors (9, 37, 38).
As a result of FO, the number of myonuclei within a
single fiber increases (3). These newly formed myonuclei are believed to arise when satellite cells, quiescent
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muscle stem cells that lay adjacent to the adult muscle
fibers, are activated to proliferate during the early
stages of FO and are subsequently incorporated into
the muscle fiber (31, 32). Because an increase in
myonuclear number expands the quantity of DNA
available for protein production, the additional myonuclei may facilitate skeletal muscle hypertrophy. Two
lines of evidence suggest a mechanistic role for the
addition of myonuclei in the hypertrophic process.
First, satellite cells have been reported to proliferate
before muscle fiber hypertrophy in rats during FO of
the soleus (33) or extensor digitorum longus muscle (8).
The satellite cell and myonuclear numbers increase
sequentially (32, 33) in a pattern consistent with the
hypothesis that the new myonuclei arise from the
incorporation of proliferating satellite cells. Second,
inhibition of satellite cell activation and/or proliferation by g-irradiation prevents hypertrophy of the rat
extensor digitorum longus (27) and soleus (24) during
FO, strongly implying that satellite cell activation
and/or proliferation is a prerequisite for muscle hypertrophy.
Rosenblatt et al. (27) also reported a constant ratio of
‘‘myonucleus to myoplasmic volume’’ across a range of
muscle fiber sizes after irradiation, FO, or FO plus
irradiation in rats. This supports the proposition that
increases in myonuclear number and cellular volume
are proportional, such that the myonuclear domain size
(defined as the volume of cytoplasm per myonucleus) of
the muscle fiber remains constant. However, since
Allen et al. reported modulation of myonuclear domain
size of single muscle fibers after chronically increased
or decreased loading in cats (3) and unloading in rats
(4), there appear to be some conditions or stages of
adaptation when the modulation in myonuclear domain size may not be tightly controlled.
In addition to mechanical loading, hormonal factors
also influence cellular adaptations in muscle. Certain
growth factors, particularly insulin-like growth factors
(IGFs), are mitogens for myoblasts (12, 38) and satellite
cells (5, 10) in vitro. Daily injections of growth hormone
(GH) caused significant increases in fiber size and
satellite cell number and a nonsignificant increase in
myonuclear number in the extensor digitorum longus
and soleus muscles of normal adult rats (36). When
serum GH and IGF-I levels were elevated in vivo by
GH-secreting tumors, satellite cell proliferation and
hypertrophy of the soleus muscle were observed in
growing young, but not mature, rats (22). These results
demonstrate that GH and/or IGF-I elevation may enhance developmental muscle hypertrophy and further
8750-7587/98 $5.00 Copyright r 1998 the American Physiological Society
1407
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McCall, G. E., D. L. Allen, J. K. Linderman, R. E.
Grindeland, R. R. Roy, V. R. Mukku, and V. R. Edgerton.
Maintenance of myonuclear domain size in rat soleus after
overload and growth hormone/IGF-I treatment. J. Appl.
Physiol. 84(4): 1407–1412, 1998.—The purpose of this study
was to determine the effects of functional overload (FO)
combined with growth hormone/insulin-like growth factor I
(GH/IGF-I) administration on myonuclear number and domain size in rat soleus muscle fibers. Adult female rats
underwent bilateral ablation of the plantaris and gastrocnemius muscles and, after 7 days of recovery, were injected
three times daily for 14 days with GH/IGF-I (1 mg/kg each;
FO 1 GH/IGF-I group) or saline vehicle (FO group). Intact
rats receiving saline vehicle served as controls (Con group).
Muscle wet weight was 32% greater in the FO than in the Con
group: 162 6 8 vs. 123 6 16 mg. Muscle weight in the FO 1
GH/IGF-I group (196 6 14 mg) was 59 and 21% larger than in
the Con and FO groups, respectively. Mean soleus fiber
cross-sectional area of the FO 1 GH/IGF-I group (2,826 6 445
µm2 ) was increased compared with the Con (2,044 6 108 µm2 )
and FO (2,267 6 301 µm2 ) groups. The difference in fiber size
between the FO and Con groups was not significant. Mean
myonuclear number increased in FO (187 6 15 myonuclei/
mm) and FO 1 GH/IGF-I (217 6 23 myonuclei/mm) rats
compared with Con (155 6 12 myonuclei/mm) rats, although
the difference between FO and FO 1 GH/IGF-I animals was
not significant. The mean cytoplasmic volume per myonucleus (myonuclear domain) was similar across groups.
These results demonstrate that the larger mean muscle
weight and fiber cross-sectional area occurred when FO was
combined with GH/IGF-I administration and that myonuclear number increased concomitantly with fiber volume.
Thus there appears to be some mechanism(s) that maintains
the myonuclear domain when a fiber hypertrophies.
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MYONUCLEAR DOMAIN AND HYPERTROPHY
METHODS
Experimental design. Adult female Sprague-Dawley rats
(,250 g body wt) were used in these experiments. Animal
care and use protocols were in accord with the Ames Research
Center Animal User’s Guide (AHB 7180) and the guidelines of
the National Institutes of Health and were approved by the
Institutional Animal Care and Use Committee of Ames
Research Center and the Animal Research Committee at the
University of California, Los Angeles. All animals were
housed in pairs and maintained in a room at 24 6 1°C on a
12:12-h light-dark cycle and allowed normal cage activity.
Standard rat chow (Purina) and water were provided ad
libitum.
Rats were assigned randomly and equally to one of three
conditions (n 5 5/group): 1) control, saline injected (Con); 2)
synergist ablated, saline injected (FO); or 3) synergist ablated, GH/IGF-I injected (FO 1 GH/IGF-I). FO rats were
anesthetized with a combination of ketamine and xylazine,
and a skin incision was made along the calf. The gastrocnemius and plantaris muscles were excised bilaterally, with
care taken not to damage the neural or vascular connections
of the soleus. After 7 days of recovery from the initial
inflammatory phase (6), FO 1 GH/IGF-I rats were injected
subcutaneously three times daily (at 0800, 1200, and 1600)
for 14 days with recombinant human GH/IGF-I (Genentech,
San Francisco, CA) at a total daily dosage of 1 mg/kg body wt
each dissolved in 0.85% saline at concentration of 250 µg/ml.
The Con and FO rats received a similar volume of saline
injected at the same times. Combined GH/IGF-I injections
were employed, because previous studies demonstrated that
optimal elevation of serum IGF-I and minimal effects on
insulin secretion are achieved with coinjection compared with
injection of either growth factor alone (17, 19). Combined
GH/IGF-I injections also increase anabolism (17, 19) and
result in optimal secretion of IGF-I-binding proteins from the
liver, thus increasing the half-life and potential tissue exposure of injected IGF-I (17). After 3 wk of FO the rats were
killed, and the soleus muscles were dissected, cleaned of
excess fat and connective tissue, wet weighed, frozen in Freon
cooled in liquid nitrogen, and stored at 270°C until use.
Analysis of myonuclear number and fiber size. Single
muscle fiber segments were microdissected and analyzed
using confocal microscopy, as described previously (4). Briefly,
the muscle was progressively thawed from 270 to 220°C,
then placed in a 220°C 50% glycerol-50% low-calcium relaxing solution (11) and held overnight at 25°C. The microdissection procedure involved placing the intact muscle in a 100%
relaxing solution to maintain the fibers in a pliant condition
during the mechanical isolation. Care was taken to sample
fibers from all regions of the muscle. Prior studies have shown
that the basal lamina and any adhering mononucleated
nonmuscle and satellite cells are stripped off during similar
isolation procedures (16). We also previously showed by
electron microscopy that our isolation procedure removes any
extracellular nuclei adhering to the plasma membrane (34).
In addition, only extremely scant laminin staining was found
on occasional fiber segments isolated using the same technique (3). Thus we are confident that the overwhelming
majority of nuclei counted in the present study were true
myonuclei. Isolated fiber segments were placed on gelatincoated slides and stored at 240°C until use. After they were
thawed and air dried, fibers were stained for 5 min with 54
µM acridine orange and for 4 min with 1.5 3 1027 M
propidium iodide. PBS was used to rinse stains before the
phosphate-buffered saline samples were mounted with glycerol under ‘‘strutted’’ coverslips.
Three 173-µm regions along the proximal, middle, and
distal portions of each fiber segment were examined using
confocal microscopy (Sarasoto 2000, Molecular Dynamics,
Sunnyvale, CA) and averaged to determine the CSA (µm2 ),
myonuclear number (number of myonuclei/mm), and myonuclear domain size (cytoplasmic volume/myonucleus, µm3 )
of the fiber. First, the fibers were optically sectioned in steps
of 1.0–1.5 µm to create a stack of images encompassing the
entire fiber thickness. The number of myonuclei in each step
was counted and summed to determine the total number of
myonuclei in the viewing field. The stack images were
optically rotated orthogonal to the long axis of the fiber, and
the CSA was measured for two different regions within each
viewing field using calibrated measurement software (Silicon
Graphics, Salt Lake City, UT). Fiber CSA and myonuclear
number were adjusted (normalized) to a sarcomere length of
2.5 µm, as previously described (4), to correct for any stretching or shortening of the fiber during the mechanical isolation.
The cytoplasmic volume-to-myonucleus ratio was calculated
by multiplying the fiber CSA by the viewing field (173 µm)
and dividing by the number of myonuclei in that same field.
A total of 519 fibers were included in the statistical
analyses, representing 178 fibers from the Con, 186 fibers
from the FO, and 155 fibers from the FO 1 GH/IGF-I group.
To determine a mean value for each rat, 35 6 7 (SD) fibers
were analyzed per muscle and averaged. The mean values for
each rat within a group were averaged to determine the group
mean. Analyses were performed to evaluate the adequacy of
the fiber sample size of individual rats for detecting treatment group mean effects for fiber CSA. These analyses
involved calculating the rolling cumulative mean of each
additional five measurements. Beginning with the first measurement, sequential blocks of five measurements were drawn
from the total pool of fibers available for each rat. From each
rat’s block means, treatment group rolling cumulative aver-
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suggest a role of growth factors in increasing satellite
cell proliferation and myonuclear number.
A number of other studies have demonstrated an
interactive effect of increased mechanical loading and
administration of exogenous growth factors on skeletal
muscle mass. For example, GH or IGF-I administration
and brief bouts of daily resistance exercise resulted in a
greater prevention of the atrophy accompanying hindlimb unloading than either factor alone (15, 18, 30).
Additionally, a combination of exercise and GH/IGF-I
treatment of hindlimb-suspended rats was recently
shown to prevent most of the decrease in myonuclear
number, whereas either factor alone did not (2). Finally,
in vitro studies have demonstrated that passive mechanical stretch combined with IGF-I administration
resulted in greater myofiber hypertrophy than either
factor alone (38).
The purpose of the present study was to evaluate the
effects of FO, both alone and combined with GH/IGF-I
administration, on the myonuclear number and fiber
size of the rat soleus muscle. The combination of
GH/IGF-I administration and FO was expected to elicit
greater hypertrophy than FO alone. Furthermore, if an
increase in myonuclei is indeed a prerequisite for
muscle fiber hypertrophy, then the additional hypertrophy of GH/IGF-I-treated animals should be accompanied by an increase in myonuclear number and the
maintenance of myonuclear domain size.
MYONUCLEAR DOMAIN AND HYPERTROPHY
RESULTS
Muscle wet weight. Three weeks of overload increased muscle wet weight by 32% in the FO compared
with the Con rats (Fig. 1). Injections of GH/IGF-I
combined with FO (FO 1 GH/IGF-I) increased the
soleus wet weight by 21 and 59% compared with the FO
and Con groups, respectively.
Fiber CSA. Fiber CSA was 11% larger in FO than in
Con rats; however, this increase was not statistically
significant (Fig. 2). CSA was 25 and 38% larger in FO 1
GH/IGF-I than in FO and Con rats, respectively.
Myonuclear number and domain size. Myonuclear
number was 21 and 40% higher than control in the FO
and FO 1 GH/IGF-I groups, respectively (Fig. 3). The
16% higher mean myonuclear number in FO 1 GH/
IGF-I than in FO rats, however, was not statistically
significant (P 5 0.0582).
Fig. 1. Soleus wet weight in control (Con), functional overload and
saline injected (FO), and functional overload and growth hormone/
insulin-like growth factor I (GH/IGF-I)-injected (FO 1 GH/IGF-I)
groups. Values are means 6 SD. * and † Significantly different from
Con and FO, respectively (P , 0.05).
Fig. 2. Soleus muscle fiber cross-sectional area (CSA) in Con, FO,
and FO 1 GH/IGF-I groups. Values are means 6 SD. * and
† Significantly different from Con and FO, respectively (P , 0.05).
Myonuclear domain size, expressed as the volume of
cytoplasm per myonucleus, was similar for all three
groups (Fig. 4), suggesting that increases in myonuclear number and fiber volume were coupled in both
FO groups. The correlation between myonuclear number and fiber CSA for the individual animals across all
groups was 10.77 (P , 0.001; Fig. 5). Within groups,
these correlations were 10.22, 10.40, and 10.48 (P .
0.05) for the Con, FO, and FO 1 GH/IGF-I groups,
respectively.
DISCUSSION
In the present study the greatest increase in soleus
muscle weight occurred when FO was combined with
GH/IGF-I administration. These results are consistent
with the enhanced muscle hypertrophy observed in rats
with GH-secreting tumors during postnatal growth
(22). Although another study by Riss et al. (25) found
comparable hypertrophy relative to body weight in the
plantaris after FO in rats with and without GHsecreting tumors, they suggested that GH-induced
muscle growth might occur by a mechanism different
from work-induced hypertrophy. Other studies have
reported an interactive effect of resistance exercise and
GH administration in attenuating muscle atrophy during hindlimb suspension (15, 18). For example, Linder-
Fig. 3. Soleus myonuclei number per millimeter myofiber length in
Con, FO, and FO 1 GH/IGF-I groups. Values are means 6 SD.
* Significantly different from Con (P , 0.05).
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ages were then computed for each additional block of five
measurements. After measurement of 20 fibers per rat, the
treatment group means were within 2.7–9.6% of the means
calculated using the total pool of measurements. As more
fibers per rat were included, this percentage regressed closer
to the mean calculated from all available measurements. The
coefficients of variation within a given treatment group also
had plateaued after measurement of 20–25 fibers. Moreover,
after 20 fibers were measured the correlation between any
subsequent means of five additional fibers and the means
from the total pool of fibers ranged from 0.86 to 0.99,
indicating that individual rat means were consistent after
measurement of 20 fibers. Therefore, an average of 35 fibers
per rat appears to be a more than adequate sample size to
determine fiber CSA, and the present analysis indicates that
the CSA means would not have changed substantially with
additional measurements.
Statistical analysis. Analysis of variance was used to
compare the group means for each of the dependent variables.
When the F ratio was significant, Scheffé’s post hoc test was
used to compare between treatment groups. Pearson’s productmoment correlation was computed to evaluate the relationship between myonuclear number and fiber CSA across or
within groups. For all tests, an a-level of 0.05 was chosen for
statistical significance.
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MYONUCLEAR DOMAIN AND HYPERTROPHY
Fig. 4. Soleus myonuclear domain size (cytoplasmic volume per
myonucleus) in Con, FO, and FO 1 GH/IGF-I groups. Values are
means 6 SD.
Fig. 5. Relationship between fiber CSA and myonuclei number per
millimeter for all treatment groups. Open symbols represent mean
value for each treatment group and correspond to filled symbols used
for individual data points. Regression line is plotted for individual
animal values across all treatment groups. r 5 10.77 (P , 0.001);
r2 5 0.59; f(x) 5 11.29 · x 1 275.57.
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man et al. (18) reported maintenance of gastrocnemius
myofibrillar protein content in hindlimb-suspended
rats that performed resistive exercise and received GH
injections but not in rats that received either intervention alone. Although the interaction effect was not
observed in the soleus muscle by Linderman et al.,
Allen et al. (2) recently reported that resistance exercise combined with GH/IGF-I injections maintained
soleus muscle mass, fiber CSA, and myonuclear number in hindlimb-suspended rats more effectively than
either intervention alone (2). The present study also
suggests that the effect of overloading the soleus is
potentiated by GH/IGF-I treatment.
In the present study the myonuclear domain size was
similar among groups, demonstrating that myonuclear
number and fiber volume increased concomitantly during muscle hypertrophy. In addition, there was a significant positive correlation between the number of myonuclei per millimeter and fiber CSA across all treatment
groups. Similar results were reported for fibers containing primarily slow myosin heavy chain in the cat
plantaris after 3 mo of FO (3). However, although the
mean myonuclear number was significantly increased
in both FO groups compared with the Con group, the
16% higher mean in the FO 1 GH/IGF-I group was
different from the FO group at the 0.0582 level of
probability, rather than the usually accepted 0.05 level.
Furthermore, although there was a significant increase
in muscle weight in FO compared with Con rats, the
11% higher mean fiber CSA in the FO group was not
significantly different from that in the Con group, as
indicated by Scheffé’s post hoc test.
From the present results we can only speculate as to
whether the fiber hypertrophy stimulated the increase
in myonuclear number or vice versa, although the
results of previous studies suggest that satellite cell
activation, proliferation, and fusion are prerequisites
for muscle fiber hypertrophy (8, 24, 26, 27, 33). The
results of these previous studies suggested that proliferating satellite cells were the source of the new myonuclei, and our correlative results indicate that the increases in myonuclei and cell volume are proportional
in the FO rat soleus. Together these findings suggest
that one consequence of satellite cell mitogenic activity
is the fusion of the progeny with existing myofibers,
resulting in an increased net nuclear capacity to synthesize contractile proteins required for cellular hypertrophy. However, the exact mechanism(s) by which proliferating satellite cells become incorporated into existing
myofibers as new myonuclei is not well defined.
The specific mechanisms by which GH/IGF-I treatment increased myonuclear number and fiber size to
maintain a constant myonuclear domain size during
FO cannot be determined from the present study. On
the basis of in vitro (5, 10) and in vivo (22) studies,
IGF-I could have stimulated the proliferation of satellite cells. Additionally, Vandenburgh et al. (38) found
that IGF-I stimulated hypertrophy and increased the
number of myonuclei per millimeter in myofibers differentiated in vitro from primary satellite cells. Although
the exogenous IGF-I may have acted directly on the
satellite cells, the exogenous GH could have exerted its
effects by stimulating systemic (i.e., hepatic) and/or
local autocrine/paracrine IGF-I expression in muscle
(20, 35). Moreover, mechanical work/overload of rat
hindlimb muscle increases IGF-I mRNA (9, 20) and
peptide (1) concentrations within the overloaded muscle.
Recently, Adams and Haddad (1) reported a positive
relationship between IGF-I protein and total DNA
content in rat plantaris muscle during FO and speculated that the elevated DNA content was due to satellite cell proliferation stimulated by the locally produced
IGF-I. In their study, similar responses also were
observed in FO hypophysectomized rats, indicating
that the hypertrophic mechanisms were independent of
systemic GH or IGF-I and apparently related to the
elevated muscle IGF-I (1). Thus the administration of
GH/IGF-I in the present study most likely augmented
the actions regulated by the work-induced elevation of
these growth factors.
In the present study, direct GH actions also may have
occurred given that the GH receptor and GH-binding
protein transcripts have been localized to myonuclei of
adult rat hindlimb muscle fibers (23). Additionally,
local infusion of GH increases muscle protein synthesis
MYONUCLEAR DOMAIN AND HYPERTROPHY
rates in humans (13, 14). Further investigation is
required to elucidate the mechanisms by which GH and
IGF-I could exert their effects on skeletal muscle independently and in conjunction with mechanical overload.
In conclusion, during FO with and without hormone
treatment, myonuclear number and fiber size were
coupled, such that the myonuclear domain size was
maintained. On the basis of the results of the present
and previous studies (8, 24, 26, 27, 33), we hypothesize
that an increase in myonuclear number may be a
prerequisite for prolonged and substantial workinduced skeletal muscle fiber hypertrophy. Implicit in
this hypothesis is the assertion that the increased
myonuclear number supplements the existing genetic
machinery in the synthesis of new contractile proteins
to meet the demands of the mechanical overload.
Received 4 June 1997; accepted in final form 10 December 1997.
13.
14.
15.
16.
17.
18.
19.
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This study was supported by National Institute of Neurological
Disorders and Stroke Grant NS-16333, National Aeronautics and
Space Administration Grant 199-26-12-09, National Academy of
Science Grant 9-18773, and National Institute of Dental Research
National Research Service Award DE-07212.
Part of this work has been published in abstract form (21).
Address for reprint requests: G. E. McCall, Dept. of Physiological
Science, 621 Circle Dr. South, 2301 Life Science, Los Angeles, CA
90095-1527.
12.
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