Satellite cell depletion prevents fiber hypertrophy in skeletal muscle

Abstract

The largest mammalian cells are the muscle fibers, and they have multiple nuclei to support their large cytoplasmic volumes. During hypertrophic growth, new myonuclei are recruited from satellite stem cells into the fiber syncytia, but it was recently suggested that such recruitment is not obligatory: overload hypertrophy after synergist ablation of the plantaris muscle appeared normal in transgenic mice in which most of the satellite cells were abolished. When we essentially repeated these experiments analyzing the muscles by immunohistochemistry and in vivo and ex vivo imaging, we found that overload hypertrophy was prevented in the satellite cell-deficient mice, in both the plantaris and the extensor digitorum longus muscles. We attribute the previous findings to a reliance on muscle mass as a proxy for fiber hypertrophy, and to the inclusion of a significant number of regenerating fibers in the analysis. We discuss that there is currently no model in which functional, sustainable hypertrophy has been unequivocally demonstrated in the absence of satellite cells; an exception is re-growth, which can occur using previously recruited myonuclei without addition of new myonuclei.

Full text

STEM CELLS AND REGENERATION RESEARCH ARTICLE
Satellite cell depletion prevents fiber hypertrophy in skeletal
muscle
Ingrid M. Egner
1
, Jo C. Bruusgaard
1,2
and Kristian Gundersen
1,
*
ABSTRACT
The largest mammalian cells are the muscle fibers, and they have
multiple nuclei to support their large cytoplasmic volumes. During
hypertrophic growth, new myonuclei are recruited from satellite stem
cells into the fiber syncytia, but it was recently suggested that such
recruitment is not obligatory: overload hypertrophy after synergist
ablation of the plantaris muscle appeared normal in transgenic mice
in which most of the satellite cells were abolished. When we
essentially repeated these experiments analyzing the muscles by
immunohistochemistry and in vivo and ex vivo imaging, we found that
overload hypertrophy was prevented in the satellite cell-deficient
mice, in both the plantaris and the extensor digitorum longus
muscles. We attribute the previous findings to a reliance on muscle
mass as a proxy for fiber hypertrophy, and to the inclusion of a
significant number of regenerating fibers in the analysis. We discuss
that there is currently no model in which functional, sustainable
hypertrophy has been unequivocally demonstrated in the absence of
satellite cells; an exception is re-growth, which can occur using
previously recruited myonuclei without addition of new myonuclei.
KEY WORDS: Hypertrophy, Plasticity, Satellite cells, Skeletal
muscle, Stem cells, Mouse
INTRODUCTION
Size matters in skeletal muscle because force is proportional to
cross-sectional area and mass to power. Muscle mass can vary
considerably, and is modified by hormones, disuse, strength
exercise or experimental overload (Egner et al., 2013; Eriksson
et al., 2005; Gundersen, 2011; Herbst and Bhasin, 2004). Muscle
mass is mainly altered by changing the size of pre-existing muscle
fibers, and fiber loss or de novo formation of fibers is believed to
play a much lesser role (Allen et al., 1999; Gollnick et al., 1981;
MacDougall et al., 1984; Taylor and Wilkinson, 1986; White et al.,
2010).
During hypertrophy each fiber displays radial growth, and it has
been believed that the number of myonuclei in each fiber syncytium
increases by satellite cells multiplying and fusing with the muscle
fibers in order to support the larger cytoplasmic volume. It is well-
documented that new myonuclei are recruited in this way during
many hypertrophic conditions (Allen et al., 1995, 1999; Aloisi et al.,
1973; Bruusgaard et al., 2010; Cabric et al., 1987; Cabrićand James,
1983; Cheek et al., 1971; Enesco and Puddy, 1964; Giddings and
Gonyea, 1992; Kadi et al., 1999; Lipton and Schultz, 1979; McCall
et al., 1998; Moss, 1968; Moss and Leblond, 1970; Roy et al., 1999;
Schiaffino et al., 1976; Seiden, 1976; Winchester and Gonyea,
1992), and recruitment of myonuclei seems to precede the radial
growth (Bruusgaard et al., 2010), thus a causal role is suggestive.
Attempts to prevent satellite cell activity by blocking DNA
synthesis by γ-irradiation (Adams et al., 2002; Barton-Davis et al.,
1999; Phelan and Gonyea, 1997; Rosenblatt and Parry, 1992;
Rosenblatt et al., 1994) have suggested that proliferation of satellite
cells is necessary for efficient hypertrophic growth. There have,
however, been some conflicting reports (Lowe and Alway, 1999;
Rosenblatt and Parry, 1993), and the specificity of the γ-irradiation
for cell proliferation has been questioned (McCarthy and Esser,
2007).
The necessity of recruiting new myonuclei was directly
challenged by McCarthy et al. (2011) who utilized a mouse strain
with an IRES cassette in which Pax7 promoter elements drive
expression of Cre-recombinase in the presence of tamoxifen crossed
to a strain with a floxed diphtheria toxin A expression vector. This
model yields mice in which most of the satellite cells are ablated
when tamoxifen is administered.
In such animals, they subjected the plantaris muscle to overload
(OL+) by ablation of the synergistic soleus and gastrocnemius
muscles. They reported that there was little difference in the
hypertrophic response in the satellite-deficient mice (SC−)
compared with controls with intact satellite cells (SC+). Based on
these findings, they conclude that they ‘provide convincing
evidence that skeletal muscle fibers are capable of mounting a
robust hypertrophic response to mechanical overload that is not
dependent on satellite cells’.
In the present study, we essentially repeated the experiments of
McCarthy et al. (2011), but our data failed to support their
conclusions. We observed a robust overload hypertrophy in the
plantaris in SC+ mice, whereas in SC−mice hypertrophy was
prevented. Similar experiments on the extensor digitorum longus
(EDL) gave essentially the same result. Based on our data, we
conclude that the hypertrophic response to mechanical overload is
dependent on satellite cells.
RESULTS
Tamoxifen efficiently ablated satellite cells in overloaded
muscles
In order to investigate the efficiency of the satellite cell ablation after
administration of tamoxifen, the number of Pax7
+
cells was counted
on whole mid-belly cross-sections from plantaris muscles (Fig. 1A).
The crucial observation was that the absolute number of Pax7
+
cells in the tamoxifen-treated SC−OL+ mice remained low
(Fig. 1A), as the average number of Pax7
+
cells per cross-section
(5.2) was similar to that found in normal SC+OL−mice (5.0;
Fig. 1B). This finding was similar to the observations of McCarthy
et al. (2011).
Received 23 December 2015; Accepted 28 June 2016
1
Department of Biosciences, University of Oslo, Blindern, Oslo N-0316, Norway.
2
Department of Health Sciences, Kristiania University College, P.O. Box 1190,
Sentrum, Oslo N-0107, Norway.
*Author for correspondence (kgunder@ibv.uio.no)
J.C.B., 0000-0001-8163-5849; K.G., 0000-0001-9040-3126
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The number of Pax7
+
cells in SC−mice was 24% of that of SC+
controls without overload (Fig. 1B). After overload, the number of
Pax7
+
cells in SC+ control mice increased by 650%, which was a
stronger activation than that observed by McCarthy et al. (2011). In
our SC−mice, overload led to a 420% increase, indicating that
satellite activation took place, but resulting in a much smaller absolute
number of Pax7
+
cells in the tamoxifen-treated mice than inSC+ mice.
Functional overload of the plantaris but not EDL can lead to
significant muscle damage
Overload by synergist ablation is a rather invasive procedure,
particularly for the plantaris. The sudden load put on the muscle is
high, as it has to bear the load of the large triceps surae complex.
Also, the blood supply to the plantaris that passes through the
gastrocnemius is prone to damage during the ablation. In our
material, five out of the 30 muscles that were ablated were almost
completely degenerated, and were excluded from further analysis.
All individual muscles were investigated for central nuclei and
labeling for embryonic myosin (Myh3) as markers of regenerating
fibers (Fig. 2). In non-overloaded (OL−) plantaris muscles, only 0.7
and 1.4% of the fibers showed one or both of these signs in the SC−
and SC+ group, respectively (Fig. 2D, left-hand graph). This
increased to 4% and 13% after overload (OL+). For the latter group,
the variability was high, ranging from 2 to 23%.
In the EDL, damage and regeneration seemed to be less of a
problem (Fig. 2, right panels). The average number of fibers with
embryonic myosin and/or central nuclei was below 1.5% in all
experimental groups and in no single muscle did more than 5% of
the fibers display such signs. Nonetheless, these fibers were
excluded from our hypertrophy analysis.
Since we suggest below that the inclusion of regenerating fibers
might be a confounding factor if included in the analysis of
hypertrophy such as by McCarthy et al. (2011), it was of interest to
Fig. 2. The effect of overload on damage and/or regeneration markers.
(A) Micrographs of cryosections from overloaded plantaris (left) and EDL (right)
muscles stained for DNA (blue), laminin (red) and embryonic myosin (green).
Scale bar: 50 μm. White arrows indicate fibers positive for MYH3 staining;
arrowheads indicate fibers with central nuclei. (B-D) Quantification of fibers
with central nuclei (B), embryonic myosin (C) or at least one of these
characteristics (D) in plantaris (left panels) and EDL (right panels). Data are
given as mean±s.e.m., and the values from individual animals (circles).
SC+OL−n=6; SC+OL+ n=7; SC−OL−n=5; SC−OL+ n=6. (E) CSA
distribution of regenerating fibers in SC+OL+ plantaris muscles (n=103 fibers
from three animals).
Fig. 1. The effect of tamoxifen treatment and overload on the number of
Pax7
+
cells in plantaris muscles. (A) Micrographs of cryosections from SC+
OL+ (left) and SC−OL+ (right) plantaris muscles stained for DNA (blue) and
Pax7 (green). Arrowheads indicate Pax7
+
nuclei. Scale bar: 50 µm. (B) The
number of Pax7+ cells identified on whole mid-belly cross-sections of the
plantaris muscle is given as mean±s.e.m., and values from individual animals
(circles). SC+OL−n=7; SC+OL+ n=7; SC−OL−n=5; SC−OL+ n=6.
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study the cross-sectional area (CSA) of this population separately.
We had a low number of such fibers, but CSA was measured in 103
fibers from three plantaris muscles from SC+OL+ mice. These
fibers had an average CSA of 334 µm
2
and the complete size
distribution is given in Fig. 2E.
Satellite cell ablation prevents overload hypertrophy
Based on histological observations of cryosections (Fig. 3A), in
SC+ muscles overload led to 26% increase in CSA in both the
plantaris and the EDL. By contrast, overload had essentially no
effect on average CSA in SC−muscles in either of the two muscles
(Fig. 3B). There was no significant effect on average CSA of
satellite cell ablation as such, since SC−OL−muscles were similar
to SC+OL−muscles.
As expected from the CSA averages, the CSA frequency
distribution for the SC+OL+ group displayed a marked right shift
(Fig. 3C) both in the plantaris and EDL. In the plantaris there were
fewer fibers in the range 500-1800 µm
2
and generally more fibers
above ≈2000 µm
2
. The EDL had fewer fibers below ≈1200 µm
2
and more fibers in the range 1400-2600 µm
2
after overload.
For the EDL, the CSA distribution of all the groups other than the
SC+OL+ group, were similar (Fig. 3C, right panel), suggesting that
none of the other treatments had an effect; thus, there was apparently
no effect of overload in the absence of satellite cells (SC−OL+), and
SC ablation as such had no effect (SC−OL−).
In the plantaris, overload had an effect on the CSA distribution
even in SC−muscles (Fig. 3C, left panel ). Although there was no
significant change in average CSA in response to overload, CSAs
became more homogenous as overload led to fewer fibers below
≈1000 µm
2
and above ≈2000 µm
2
. Thus, overload led to a narrower
distribution of CSA size peaking at ≈1600 µm
2
in this muscle.
Muscle mass is dependent on factors other than fiber
hypertrophy
Although the definition of hypertrophy is an increase in the size of
pre-existing fibers, muscle mass is often used as a proxy for
hypertrophy. We found that for the plantaris the overload surgery
led to an increase in mass both with (112%) and without (55%)
satellite cells (Fig. 4A, left panel). The EDL showed the same
tendency, but the increase in the SC−OL+ group was not
significant. The mass increases in the SC−muscles were not
related to any increase of the CSA and for the SC+ plantaris the
mass increase was much larger than the 26% increase in CSA.
We attribute the discrepancies between mass and CSA in the
plantaris mainly to post-operative complications and difficulties in
defining this muscle anatomically at the proximal end, particularly
Fig. 3. The effect of overload on fiber CSA.
(A) Micrographs of cryosections from plantaris (left
panels) and EDL (right panels), stained for DNA (blue)
and dystrophin (green). Scale bar: 30 µm. (B) Average
CSA values from individual muscles (circles), and mean±
s.e.m. for each experimental group of plantaris (left
panels) and EDL (right panels). n=7 animals in each
group. (C) CSA distribution of all fibers measured for each
experimental group from plantaris (left panel) and
EDL (right panel). Plantaris: SC+OL−n=1004; SC+OL+
n=936; SC−OL−n=900; SC−OL+ n=911 fibers. EDL:
SC+OL−n=929; SC+OL+ n=976; SC−OL−n=1083;
SC−OL+ n=873 fibers.
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after synergist ablation. Microscopical analysis of sections
frequently showed adhering tissue from other muscles after the
excision. Thus, at the proximal end, plantaris fuses with the
gastrocnemius and after ablation surgery adherences make it even
more difficult to dissect out the plantaris in a precise manner.
Histology on cross-sections showed that parts of gastrocnemius
were frequently part of the ‘lump’when the plantaris was dissected
out from synergist-ablated animals. Thus, in our hands mass is an
unreliable measure of hypertrophy in the plantaris overload model.
The EDL muscle was easier to excise after synergist ablation than
the plantaris, but post-operative adherences were a problem even for
this muscle. The increase in mass after overload in the SC+ group
was 23%, similar to the increase in CSA (Fig. 4, right panel). The
SC−group also had a slightly higher mass (13%) after overload, but
this was not statistically significant. Thus, in the EDL there was a
reasonably good agreement between the changes in mass and CSA.
In our study, tamoxifen-treated mice had on average a 13% lower
body weight than sham-treated animals. When correcting for body
weight, this would tend to increase the relative mass values for the
tamoxifen groups (Fig. 4B). These body weight corrections might,
however, represent an overcorrection, and hence a false hypertrophy
as a major effect of this estrogen antagonist is to reduce body fat
(Liu et al., 2015). McCarthy et al. reported only muscle mass
corrected for body weight, not absolute muscle mass.
Overload hypertrophy is related to an increase in the number
of myonuclei
Activation of SCs is thought to be important during hypertrophy in
order to increase the number of myonuclei to match the increase in
cytoplasmic volume. Thus, we investigated the number of
myonuclei per fiber on histological cross-sections co-labeled with
anti-dystrophin. Nuclei with their geometric center within the inner
rim of the dystrophin ring were defined as myonuclei (Fig. 5A,
arrows), and the number of these nuclei was divided by the number
of fibers analyzed on the same section.
Overload of SC+ muscles led to a 51% increase in the number of
myonuclei in the plantaris and 30% in the EDL (Fig. 5B). Thus, for
the plantaris the increase was twice what would be expected from
the 26% increase in cytoplasmic volume, whereas in the EDL the
increase was more similar to the increase in cytoplasmic volume. In
Fig. 4. The effect of overload on muscle mass. (A,B) Absolute muscle mass
(A) or muscle mass relative to body weight (B) given as mean±s.e.m., and
values from individual (circles) animals for plantaris (left panels) and EDL
(right panels) muscles. Plantaris: SC+OL−n=17; SC+OL+ n=8; SC−OL−
n=18; SC−OL+ n=10 animals. EDL: SC+OL−n=11; SC+OL+ n=8; SC−OL−
n=17; SC−OL+ n=7 animals.
Fig. 5. The effect of overload on number of
myonuclei determined from cross-sections.
(A) Micrographs of cryosections from plantaris (left)
and EDL (right), stained for DNA (blue) and dystrophin
(green). Arrows indicate myonuclei. Scale bars:
50 µm. (B) Quantification of myonuclei per fiber from
the plantaris (left) and EDL (right). Data are given as
mean±s.e.m., and values from individual muscles
(circles). n=7 animals in each group.
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the SC−group, overload led to no significant change in the number
of myonuclei in plantaris or EDL (Fig. 5B).
Identification of myonuclei on cross-sections can be unreliable
(Bruusgaard et al., 2012; Bruusgaard and Gundersen, 2008;
Gundersen and Bruusgaard, 2008). We therefore went on to study
myonuclei in single fibers. For the plantaris, single fibers were
teased out after maceration of fixed muscles (Fig. 6A). As in
previous papers using this technique, there were differences
between experimental groups in the degree of stretch (Bruusgaard
et al., 2010); thus, number of nuclei is given per sarcomere length.
The only group that showed an increase in this variable was the
SC+OL+ group (Fig. 6B).
The most precise technique to observe the myonuclei belonging
to a single fiber syncytium is in vivo imaging after intracellular
injection of a nuclear dye. Although this is not feasible in the
plantaris, surface fibers on the lateral side of EDL are well-suited for
such analysis (Fig. 7A). When such fibers were analyzed in vivo, it
was observed that overload led to a 60% increase in myonuclear
number in the SC+ group (Fig. 7B), and the increase in CSA
calculated from the en face diameter increased 34% (Fig. 7C). There
was also on average a 19% increase in the number of myonuclei in
the SC−group. This small increase could be related to residual SCs
(McCarthy et al., 2011), but was apparently not sufficient to support
any hypertrophy in our experiments (Fig. 7C).
The increase in the number of myonuclei was larger than the
increase in cytoplasmic volume, thus in SC+OL+ muscles the
myonuclear domain volume decreased by 22% (Fig. 7D). This
might be related to the myonuclear recruitment preceding the radial
growth as shown previously (Bruusgaard et al., 2010).
A significant correlation between CSA and nuclei per fiber length
was observed in all the four experimental groups (Fig. 7E). The
correlation was shifted to higher number of nuclei for the same CSA
in SC+OL+ muscles. For the other groups, experimental conditions
Fig. 6. The effect of overload on number of myonuclei in plantaris
muscles determined from single macerated fibers. (A) Micrographs of
macerated fibers from the plantaris muscle stained for DNA (blue). Scale bar:
50 μm. (B) Quantification of nuclei per sarcomere length. In the left panel,
each circle represents average values for an individual muscle based on a
sample of 8-32 fibers from each muscle. Columns denote mean±s.e.m. of five
animals each. In the right panel, each circle represents the individual fibers in
the same material. Columns denote mean±s.e.m. for all the fibers from five
animals in each group. SC+OL−n=84; SC+OL+ n=89; SC−OL−n=76;
SC−OL+ n=81.
Fig. 7. The effect of overloadon number of myonucleiin EDL determined by
in vivoimaging of single fibers.(A) In vivo images of fibersinjected with TRITC-
labeled oligonucleotides displaying the myonuclei. In situ sarcomere length is
≈3.1 µm. Scale bar: 30 µm. (B-D) Quantification of myonucleiper fiber length (B),
CSA (C) and myonuclear domain volume (MND) (D). Each circle represents the
value from one fiber, SC+OL−n=31(4); SC+OL+ n=39(5); SC−OL−n=45(7);
SC−OL+ n=37(7) (number of animals in parenthesis). Each column denotes the
mean±s.e.m. of 31-45 fibers from four to seven animals. (E) Correlation between
myonucleiper fiber length and CSA; each circle represents one fiber.
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had little effect, except that the variability in the number of nuclei
was larger in the OL+ groups (Fig. 7E, right-hand panels).
DISCUSSION
We show that ablation of satellite cells prevents overload
hypertrophy and suggest that SC activation and subsequent fusion
to muscle fibers resulting in an increase in the number of myonuclei
is obligatory for normal hypertrophy.
The finding seems to support the long-standing notion of nuclear
domains: that proteins are expressed locally in the vicinity of each
nucleus (Gundersen et al., 1993; Merlie and Sanes, 1985; Ralston
and Hall, 1992; Ralston et al., 1997; Sanes et al., 1991), and that
each nucleus can support only a limited volume of cytoplasm
(Gregory, 2001; Hall and Ralston, 1989; Strassburger, 1893). The
relevant bottleneck relating the number of nuclei to cytoplasmic
volume is not known, although it has been speculated that capacity
for shuttling of macromolecules over the nuclear membrane could
act as a limiting factor if the number of nuclei is not sufficiently high
(Gundersen, 2016; Hall et al., 2011).
Our findings are in conflict with McCarthy et al. (2011) who used
essentially the same approach as us. For both studies, the remaining
satellite cell pool in overloaded SC−muscles was the same relative
to normal muscles (SC+OL−), and was apparently sufficient to
prevent an increase in the number of myonuclei when these muscles
were overloaded. Therefore, we attribute the differences in the
results to differences in the way hypertrophy was analyzed, such as
the reliance on relative muscle mass as a proxy for fiber hypertrophy
and the inclusion of regenerating fibers in the McCarthy study.
Muscle mass as a proxy for hypertrophy
McCarthy et al. (2011) based their conclusions mainly on changes
in relative muscle mass as a proxy for hypertrophy. To some extent,
we reproduced their findings using this variable, but because CSA
did not increase along with the mass, we attribute this finding to
confounding factors related to adherences and effects of the surgery
rather than fiber hypertrophy (see Results).
McCarthy et al. (2011) did not report CSA averages, but from
their CSA histogram we calculated that the average overload
hypertrophy was ≈11% in SC+ muscles, and similarly so also in a
more recent paper (Kirby et al., 2016). This is a rather narrow scope,
considering it was compared to a ≈10% increase in the average CSA
in their SC−muscles. We suggest that the apparent unblunted
hypertrophy in the satellite cell-deficient group only appears robust
because there was so little hypertrophy in the control muscles. In our
overloaded controls (SC+OL+), by contrast, we observed an average
increase in CSA of 26%, and this magnitude is in agreement with
previous literature on short-term effects of plantaris overload by
synergist ablation (e.g. White et al., 2009; Zhang et al., 2014). This
robust SC+ hypertrophy gave us a sufficiently large scope for
observing the attenuation of hypertrophy in SC−muscles.
Although subtle differences in genetic background or overload
conditions might have played a role, for example we observed a
higher number of satellite cells in our SC+OL+ muscles, we suggest
that the differences in the CSA data of the SC+ plantaris muscles
between the present study and that of McCarthy et al. (2011) can be
attributed mainly to the degree of muscle damage and the inclusion
criteria for the fibers analyzed.
Muscle damage and the inclusion criteria for the fibers
analyzed
The plantaris overload model is rather extreme, as the relatively
small plantaris muscle suddenly has to bear the load that was
previously supported by both the soleus and the much larger
gastrocnemius muscle, and this could lead to rupture damage of
muscle fibers. In addition, the blood supply to the plantaris that
comes through the gastrocnemius is easily damaged during a radical
ablation of the gastrocnemius.
McCarthy et al. (2011) report that ∼30% of fibers in their SC+OL+
group expressed embryonic myosin and/or centrally located nuclei as
signs of damage or regeneration. Importantly, the authors used
automated CSA measurements and these regenerating/damaged
fibers were included in their analysis. We observed that such fibers
were generally smaller than fibers with normal morphology (compare
Fig. 2E and Fig. 3C). Inclusion of a substantial population of such
fibers would tend to shift the total CSA distribution to the left and
hence explain the low degree of hypertrophy in their SC+OL+. This
would apply to their SC+OL+ mice only, and not to the SC−OL+
mice. This would tend to mask a difference in the hypertrophy of pre-
existing fibers between the two groups.
In contrast to McCarthy et al. (2011), we excluded fibers with
signs of damage from our analysis. In any case, the number of such
fibers was lower in our plantaris material, and in the EDL such fibers
were rather rare so this confounding factor was not much of a
problem in this muscle. EDL was not analyzed by McCarthy et al.
(2011).
In a recent paper, the Peterson group (Fry et al., 2014) essentially
repeated their previous experiments on the plantaris, but extended
the overload time to 8 weeks. Their new data suggest a role for
satellite cells in long-term hypertrophy as SC ablation attenuated the
hypertrophy response from a 36% to a 26% increase in CSA. The
latter increase seemed to occur without an increase in the number of
myonuclei. This finding might be an example of a hypertrophy
without an increase in the number of myonuclei, but the extensive
initial tissue damage reported for the plantaris overload model by
McCarthy et al. (2011), probably also applies to the experiments of
Fry et al. (2014) since at 8 weeks 9% of the fibers still had central
nuclei. Thus, the muscle history could be a confounding factor for
interpretations related to hypertrophy of pre-existing fibers also for
the Fry et al. (2014) study.
Is recruitment of SCs obligatory for hypertrophy?
Our data confirms most of the studies in which hypertrophy was
prevented by γ-irradiation (Adams et al., 2002; Barton-Davis et al.,
1999; Phelan and Gonyea, 1997; Rosenblatt and Parry, 1992;
Rosenblatt et al., 1994). Are there then any remaining models
suggesting that recruitment of myonuclei are not obligatory for
functional hypertrophy?
Transgenic models related to myostatin inhibition display large
fibers without a corresponding increase in the number of myonuclei
(Amthor et al., 2009; Bruusgaard et al., 2005; Lee et al., 2012;
Raffaello et al., 2010), but the hypertrophy seems not to be fully
functional as the specific force is reduced in such muscles (Amthor
et al., 2007; Charge et al., 2002; Mendias et al., 2011).
It has been suggested that the beta-agonist clenbuterol induces
hypertrophy in rats without addition of myonuclei by satellite cell
proliferation (Rehfeldt et al., 1994), but the assessment of
myonuclei would have to be repeated with modern methods, and
the functionality of the hypertrophy was not determined.
Hypertrophy without activation of satellite cells or increase in the
number of myonuclei is induced by overexpression of the serine/
threonine kinase Akt/PKB. Such muscle fibers seemed to have
normal specific force after 3 weeks of overexpression (Blaauwet al.,
2009), but it remains unclear if this condition is sustainable over
longer periods (Blaauw and Reggiani, 2014).
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In conclusion, a fully functional, sustainable, de novo
hypertrophy without SC contribution remains to be unequivocally
demonstrated.
The demonstration of the muscle memory phenomenon
(Bruusgaard and Gundersen, 2008; Bruusgaard et al., 2010;
Egner et al., 2013; for a review, see Gundersen, 2016), for which
efficient re-growth relies on myonuclei recruited during a previous
hypertrophic episode, is an important example of functional radial
growth without the need for new myonuclei from satellite cells. This
has been directly demonstrated using the hind limb suspension
model, in which atrophy is induced by lifting up the hind part of
rodents by the tail and thereby unloading the hind leg. Myonuclei
are not lost during this atrophy. When the muscles were reloaded by
letting the animal back down again the fibers displayed a 60% radial
re-growth, and this re-growth was not accompanied by any increase
in the number of myonuclei (Bruusgaard et al., 2012). Moreover,
using a similar mouse model as used in the present study it was
demonstrated that the re-growth was not affected by satellite cell
ablation (Jackson et al., 2012). This notion is also supported by
previous suggestions that until a certain limit of hypertrophy is
reached, it can occur without recruiting new myonuclei (Kadi et al.,
2005; Petrella et al., 2006, 2008), and we have suggested a ‘peak
pegging’hypothesis in which the number of myonuclei reflects the
largest size a muscle fiber has had in its history (Gundersen, 2016).
Thus, during re-growth satellite cells are not required, not because a
large number of myonuclei is not needed in large fibers, but because
the nuclei are already there. The present data indicate that
recruitment of new myonuclei is obligatory for de novo
hypertrophy.
MATERIALS AND METHODS
Animal experiments
All animal experiments were approved by the Norwegian Animal Research
Authority and were conducted in accordance with the Norwegian Animal
Welfare Act of 20 December 1974. The Norwegian Animal Research
Authority provided governance to ensure that facilities and experiments
were in accordance with the Animal Welfare Act; the National Regulations
of 15 January 1996; and the European Convention for the Protection of
Vertebrate Animals used for Experimental and Other Scientific Purposes of
18 March 1986.
Inhalation of gas anesthesia with 2% (vol/vol) isoflurane in air was used
for all non-terminal experiments.
For terminal experiments, intraperitoneal injections at a dose of 10 μl/g
body weight of a mixture of 18.7 mg/ml Zoletil Forte (Virbac Laboratories,
France), 0.45 mg/ml Narcorxyl/Rompun (Bayer Animal Health, Germany)
and 2.62 μg/ml Fentanyl (Hameln Pharmaceuticals, Germany) was used.
All live animal imaging and surgery were performed under deep anesthesia
checked regularly by pinching the metatarsus region of the limb. Additional
doses were given if necessary.
A Pax7-DTA mouse strain that allows for conditional ablation of Pax7
+
cells was used for all experiments. This strain was generated by crossing the
previously described (Murphy et al., 2011) strain B6.Cg-Pax7tm1(cre/
ERT2)Gaka/J (Jackson Laboratory stock number: 017763) to the previously
described (Wu et al., 2006) strain B6;129-Gt(ROSA)26Sortm1(DTA)Mrc/J
(Jackson Laboratory stock number: 010527). Homozygous breeding pairs
were used and correct genotype was confirmed by PCR according to the
Jackson Laboratory protocol.
Mice were housed in a temperature- and humidity-controlled room and
maintained on a 12:12 h light-dark cycle with food and water ad libitum.
Adult (3-4 months) female Pax7-DTA mice were randomly assigned to
receive either an intraperitoneal injection of tamoxifen (Sigma-Aldrich,
T5648-56) at a dose of 2.0 mg/day, or vehicle containing 10% ethanol in
corn oil (Sigma-Aldrich, C8267) for 5 days, followed by a 2-week washout
period. Following the 2-week washout period, mice were divided into either
synergist ablation of the EDL or the plantaris muscle, or non-ablated control.
Overload of the plantaris muscle was induced by excising the distal half of
both the gastrocnemius and the soleus. Overload of the EDL muscle was
achieved by excising approximately two-thirds of the distal end of the
tibialis anterior. Ablations were performed unilaterally, and all animals were
subjected to 2 weeks of synergist ablation. Mice on which no surgery was
performed served as controls (OL−).
Immunohistochemistry
The muscles were dissected free from surrounding connective tissue,
weighed, and pinned to a rubber form at resting length, embedded in OCT
Tissue-Tek (Sakura Finetek Europe B.V.), quickly frozen in melting
isopentane, and stored at −80°C until sectioning. Muscles were
cryosectioned at 10 μm, air dried and stored at −80°C. Before
immunostaining, frozen sections were air-dried and subsequently blocked
in 1% bovine serum albumin and incubated with primary antibodies at 4°C
overnight and secondary antibodies for 1 h at room temperature. The
following primary antibodies were used: anti-dystrophin (1:200; Abcam,
ab15277), anti-MYH3 (1:50; Santa Cruz Biotechnology, sc53091) or anti-
laminin (1:200; Sigma-Aldrich, L9393). After three 10 min washes in PBS,
sections were incubated with secondary antibodies: goat anti-mouse IgG-
TRITC (1:200; Sigma-Aldrich, T7782) or goat anti-rabbit IgG-FITC (1:200;
Abcam, ab150077). Nuclei were co-stained using Hoechst dye 33342
(Invitrogen; 0.1 μg/ml in PBS).
Satellite cells were labeled by fixing in 2% paraformaldehyde for 5 min
followed by epitope retrieval using a sodium citrate/TWEEN buffer
(10 mM, pH 6.5, 0.05% TWEEN) at 95°C for 30 min. Sections were
stained with primary antibodies against Pax7 (1:5 dilution in PBS, 2% BSA,
0.1% Triton X-100; Developmental Studies Hybridoma Study Bank, AB
528428) at 4°C overnight, and subsequently stained with secondary
antibodies (1:200 dilution in PBS, 2% BSA; goat anti-mouse IgG Alexa
Fluor 488, Thermo Fisher Scientific, A27012). All sections were
counterstained with Hoechst 33342 to verify nuclear Pax7 staining.
Sections were imaged using an Olympus BX-50WI fluorescence
microscope with a 40×0.8 NA long working distance water immersion
objective and an Andor iXion+ camera, controlled by Andor SOLIS
software.
For sampling fibers for CSA quantification, a grid was placed over the
images and fibers that were located in the grid intersections from all parts of
the muscle were measured such that an average of 161 (range 101-205) were
sampled from each whole muscle cross-section. For the sampled fibers,
CSA was measured manually by circling the dystrophin ring. A set of
images of each muscle was captured using an Olympus BX-50WI
fluorescence microscope with a 40×0.8 NA long working distance water
immersion objective and a Canon 60D SLR camera. As discussed
previously (Bruusgaard et al., 2012; Gundersen and Bruusgaard, 2008),
to ensure that only nuclei inside the sarcolemma (the myonuclei proper)
were included in the analysis, myonuclei were defined as nuclei with their
geometrical center inside the inner rim of the dystrophin ring. Fibers with
centrally located nuclei and/or embryonic staining were excluded from the
analysis.
In vivo myonuclear imaging
For in vivo labeling of myonuclei, single fibers in the EDL were injected
with a solution containing a 5′-TRITC-labeled random 17-mer
oligonucleotide with a phosphorothioated backbone (Yorkshire
Biosciences) dissolved in an injection buffer (10 mM NaCl, 10 mM Tris,
pH 7.5, 0.1 mM EDTA and 100 mM potassium gluconate). The
oligonucleotides are taken up into the nuclei inside the injected fibers
apparently by active transport, and serve solely as an intravital nuclear dye in
our experiments. The methods have been described in detail previously
(Bruusgaard et al., 2003; Utvik et al., 1999).
The oligonucleotides contained the randomly selected sequence
TAGTCCTAAGTGGACGC, and a BLAST analysis confirmed that the
sequence was not represented in the mouse genome either in the sense or
antisense direction.
In vivo imaging was performed essentiallyas described previously (Balice-
Gordon and Lichtman, 1994; Bruusgaard et al., 2010, 2003; Utvik et al.,
1999). Fiber segments of 250-1000 μm were analyzed by acquiring images in
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DEVELOPMENT

different focal planes 5 μm apart on an Olympus BX-50WI fluorescence
microscope with a 20×0.5 NA long working distance water immersion
objective. All images were acquired with an Andor iXion+ camera, controlled
by Andor SOLIS software. By importing the images to a Macintosh computer
running Adobe Photoshop and NIH ImageJ software, a stack was generated
and used to count all the nuclei in the segment. The counting of nuclei was
performed by evaluating all of the images in each stack.
Ex vivo single fiber analysis
The plantaris muscle was fixed in 4% paraformaldehyde (wt/vol) at 4°C for
48 h. The muscles were subsequently incubated in a 40% (wt/vol) NaOH
solution for 2-3 h at room temperature, followed by shaking for 8 min in
20% (wt/vol) NaOH and three washes in dH
2
O. Fibers were then transferred
to a Petri dish with 5 ml dH
2
O and stained with two or three droplets of
Hematoxylin (Santa Cruz Biotechnology, sc-24973) for visualization of
fibers and fiber structure. Watchmaker’s forceps and a binocular microscope
were then used to disperse single fibers on microscope slides (Superfrost
Plus, Thermo Scientific, J1800AMNZ). For visualizing nuclei, fibers were
mounted with ProLong Diamond Antifade Mountant with DAPI (Molecular
Probes, P36962). Segments of 171-623 μm from 330 fibers were analyzed
by acquiring images in different focal planes 5 µm apart on an Olympus BX-
50WI fluorescence microscope with a 40×0.8-N.A. water immersion
objective. Using ImageJ software (National Institutes of Health), a z-stack of
the images were generated and Adobe Photoshop (Adobe Systems) were
used for further measurements.
Statistics
Differences between groups were analyzed using GraphPad Prism software
using one-way ANOVA statistical analysis with Holm–Sidak post-tests.
Acknowledgements
We are grateful to Dr Einar Eftestøl and Kenth-Arne Hansson for comments on the
manuscript.
Competing interests
The authors declare no competing or financial interests.
Author contributions
I.M.E. and J.C.B. performed the experiments. J.C.B. prepared the figures. I.M.E.,
J.C.B. and K.G. designed the experiments. K.G. wrote the paper with input from
I.M.E. and J.C.B.
Funding
This study was supported by the Research Council of Norway (Norges
Forskningsråd) [grant 240374].
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