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Am J Physiol Endocrinol Metab 305: E171–E182, 2013.
First published May 14, 2013; doi:10.1152/ajpendo.00065.2013.
Exercise restores decreased physical activity levels and increases markers
of autophagy and oxidative capacity in myostatin/activin-blocked mdx mice
Juha J. Hulmi,1 Bernardo M. Oliveira,1 Mika Silvennoinen,1 Willem M. H. Hoogaars,2 Arja Pasternack,3
Heikki Kainulainen,1 and Olli Ritvos3
1
Department of Biology of Physical Activity, Neuromuscular Research Center, University of Jyväskylä, Finland; 2Human
and Clinical Genetics/Leiden University Medical Center, Leiden, The Netherlands; and 3Department of Bacteriology
and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
Submitted 4 February 2013; accepted in final form 16 May 2013
myostatin; activin receptor IIB; exercise; skeletal muscle; hypertrophy
ADEQUATE SIZE AND FUNCTION of skeletal muscle are of paramount importance for health (3, 11, 49, 58, 60). Duchenne
muscular dystrophy (DMD) is a disease characterized by progressive wasting of skeletal muscle (28). Restoration of dystrophin expression in all muscles of the body is difficult, and
the effectiveness of such treatment likely depends on the
muscle quality of DMD patients. Therefore, approaches aimed
at stimulation of muscle growth and function are being developed that may complement dystrophin restoration approaches.
Type IIB activin receptors (ActRIIB) are expressed in skeletal muscle (21, 37) and mediate the signaling of myostatin and
Address for reprint requests and other correspondence: J. Hulmi, Dept. of
Biology of Physical Activity, Univ. of Jyväskylä, P.O. Box 35, 40014 Jyväskylä, Finland (e-mail: [email protected]).
http://www.ajpendo.org
activins (37, 59). Myostatin and activin are efficiently blocked
by systemic injection of a soluble ligand binding domain of
ActRIIB fused to the Fc domain of IgG (sActRIIB-Fc) (38).
sActRIIB-Fc rapidly increases muscle mass and function and
alleviates the disease phenotype in a number of mouse models
(1, 15, 22, 24, 46, 49, 60). Recent evidence showed that it also
increases muscle mass without reported major side-effects in
humans (5), supporting its possible therapeutic use in the future
in some neuromuscular diseases or in muscle wasting.
Booth and Laye conclude in their review (7) that studying
only sedentary animals and not allowing them to perform
normal wild-type physical activity levels may lead to inaccurate conclusions. Adequate level of physical activity and exercise has well-established beneficial effects on skeletal muscle
size and function as well as on overall health (32, 53). On the
other hand, low aerobic capacity is a risk factor for cardiovascular and metabolic diseases (57). Although as mentioned
above the lack or inhibition of myostatin seems to have various
positive effects on muscle, there is also some evidence that it
can decrease muscle oxidative capacity (2, 24, 41, 42, 50).
Thus, treatments to positively complement myostatin-blocked
or -deficient muscle are needed. Recent evidence indicates that
muscle endurance seems to be positively affected by exercise
when combined with myostatin blocking (35) and after myostatin deletion (41, 42). Because submaximal exercise seems to
offer some benefits in mdx mice, a relatively mild disease
model of DMD (9, 23, 34), exercise in combination with
myostatin and activin blocking may offer further benefits in
DMD treatment.
Maintaining autophagy at normal levels is important to
rejuvenate organelles and to prevent accumulation of dysfunctional proteins (39, 40, 43). Recent evidence also suggests that
autophagy may be impaired in muscle dystrophy (12) and that
increased autophagy by exercise accounts for exercise benefits
(18, 20). The effects of myostatin/activin blocking alone or in
combination with exercise on autophagy are not known.
Myostatin/activin blocking rapidly increases skeletal muscle
mass, but its effects on physical activity as well as the effects
of combinatorial exercise are unclear. Here, we demonstrate
the opposite and compensatory effects of exercise and myostatin blocking on the level of physical activity and skeletal
muscle autophagy and oxidative capacity.
MATERIALS AND METHODS
Animals. In the experiments we used 6- to 7-wk-old male mice
from the Jackson Laboratory (Bar Harbor, ME). The modestly dystrophic mdx mice were from a C57Bl/10ScSnJ background, and the
wild-type mice were from the C57Bl/10ScSnJ strain. The mice were
0193-1849/13 Copyright © 2013 the American Physiological Society
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Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on October 29, 2016
Hulmi JJ, Oliveira BM, Silvennoinen M, Hoogaars WM, Pasternack A, Kainulainen H, Ritvos O. Exercise restores decreased
physical activity levels and increases markers of autophagy and
oxidative capacity in myostatin/activin-blocked mdx mice. Am J
Physiol Endocrinol Metab 305: E171–E182, 2013. First published
May 14, 2013; doi:10.1152/ajpendo.00065.2013.—The importance of
adequate levels of muscle size and function and physical activity is
widely recognized. Myostatin/activin blocking increases skeletal muscle mass but may decrease muscle oxidative capacity and can thus be
hypothesized to affect voluntary physical activity. Soluble activin
receptor IIB (sActRIIB-Fc) was produced to block myostatin/activins.
Modestly dystrophic mdx mice were injected with sActRIIB-Fc or
PBS with or without voluntary wheel running exercise for 7 wk.
Healthy mice served as controls. Running for 7 wk attenuated the
sActRIIB-Fc-induced increase in body mass by decreasing fat mass.
Running also enhanced/restored the markers of muscle oxidative
capacity and autophagy in mdx mice to or above the levels of healthy
mice. Voluntary running activity was decreased by sActRIIB-Fc
during the first 3– 4 wk correlating with increased body mass. Home
cage physical activity of mice, quantified from the force plate signal,
was decreased by sActRIIB-Fc the whole 7-wk treatment in sedentary mice. To understand what happens during the first weeks
after sActRIIB-Fc administration, when mice are less active,
healthy mice were injected with sActRIIB-Fc or PBS for 2 wk.
During the sActRIIB-Fc-induced rapid 2-wk muscle growth period, oxidative capacity and autophagy were reduced, which may
possibly explain the decreased running activity. These results show
that increased muscle size and decreased markers of oxidative
capacity and autophagy during the first weeks of myostatin/activin
blocking are associated with decreased voluntary activity levels.
Voluntary exercise in dystrophic mice enhances the markers of oxidative capacity and autophagy to or above the levels of healthy mice.
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MYOSTATIN/ACTIVIN BLOCKING AND EXERCISE
measurement range of the strain gauge was ⫾ 250 g, while the
digitization precision was ⬍0.02 g. The outcome variable distance
was calculated similarly as previously (51). The force plate recording
has been validated against video recording and found to be perfectly
valid (51).
After the animal housing, the cage without the animal was positioned on the glass plate, and the force levels of all gauges were
adjusted to zero. Twenty-four-hour measurements were conducted for
the mice without running wheels. The three time points selected were
1–5 days before the first treatment injections, during the third week,
and the end of the sixth week. The measurement was conducted in the
animal’s own cage and in the same room with the other mice in this
experiment.
Muscle sampling. Lower leg muscles soleus, gastrocnemius, musculus quadriceps femoris (MQF), extensor digitorum longus (EDL),
and tibialis anterior were immediately removed, weighed, and frozen
in liquid nitrogen. The muscle weights reported are always average
weights of the left and right leg. The proximal part of gastrocnemius
muscle was mounted in an O.C.T. embedding medium (Tissue Tek,
Sakura Finetek Europe) with vertical orientation of muscle fibers and
snap-frozen in isopentane cooled with liquid nitrogen. Hematoxylin
and eosin (H&E) and succinate dehydrogenase (SDH) staining were
conducted from 8- to 10-␮m cross-sections cut with a cryomicrotome.
SDH analysis was conducted as earlier (52).
Tissue processing for protein analysis. The gastrocnemius muscle
was pulverized and homogenized in liquid nitrogen and then homogenized in ice-cold buffer with proper inhibitors and further treated as
previously reported (24, 25). The muscle homogenate was centrifuged
at 10,000 g for 10 min at 4°C. Total protein content was determined
using the bicinchoninic acid protein assay (Pierce Biotechnology,
Rockford, IL) with an automated KoneLab device (Thermo Scientific,
Vantaa, Finland).
Western immunoblot analyses. Muscle homogenates were solubilized in Laemmli sample buffer and heated at 95°C to denature proteins.
Samples containing 30 ␮g of protein were separated by SDS-PAGE and
transferred to PVDF membrane. The membrane was blocked in TBS
with 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk and
incubated overnight at 4°C with primary antibodies. The membrane was
then washed in TBS-T and incubated with horseradish peroxidaseconjugated secondary antibodies (Jackson ImmunoResearch Europe) for
1 h followed by washing in TBS-T. Proteins were visualized by ECL
(SuperSignal west femto maximum sensitivity substrate; Pierce Biotechnology) and quantified (band intensity ⫻ volume) using a ChemiDoc
XRS device in combination with Quantity One software (version 4.6.3,
Bio-Rad Laboratories).
The uniformity of the protein loading was confirmed by staining
the membrane with Ponceau S and by reprobing the membrane with
an antibody against GAPDH (Abcam, Cambridge, MA). Our results
were not markedly affected whether the results were normalized to
GAPDH, to Ponceau S, or to total protein.
Autophagy. LC3 immunoblotting is an often-used marker for autophagy (20, 39). LC3 I is the cytosolic form of the protein, and LC3
II is conjugated to lipid phosphatidylethanolamine and is present in
autophagosomes (26). We carefully analyzed the LC3 I and II isoforms by specific antibody and with gel run, separating protein bands
at 16 and 14 kDA, respectively, as recommended (26, 44) (see below).
Antibodies. The antibodies recognized PGC-1␣ from Calbiochem
(Merck, Darmstadt, Germany), estrogen-related receptor-␣ (ERR␣)
from Novus Biologicals (Littleton, CO), cytochrome c from Santa
Cruz Biotechnology (Santa Cruz, CA), p-ULK1 (UNC-51-like kinase
1) at mTOR-stimulated site Ser757, FoxO1 at Thr24, and p-AS160 at
Thr642 from Cell Signaling Technology and LC3B from SigmaAldrich. For ubiquitinated proteins, horseradish peroxidase-conjugated anti-ubiquitin antibody was used (Santa Cruz Biotechnology).
Total FoxO1 was analyzed using specific antibody (Cell Signaling
Technology) by reprobing the membrane after careful stripping of the
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00065.2013 • www.ajpendo.org
housed in standard conditions (temperature 22°C, light from 8:00 AM
to 8:00 PM) and had free access to tap water and food pellets (R36,
4% fat, 55.7% carbohydrate, 18.5% protein, 3 kcal/g; Labfor, Stockholm Sweden).
Ethics statement. The treatment of the animals was in strict accordance with the European Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific Purposes. The
protocol was approved by the National Animal Experiment Board
(Permit No. ESLH-2009-08528/Ym-23).
Experimental design. In the long, 7-wk experiment, the mdx mice
were randomly divided into four groups: 1) PBS control, 2) PBS
running, 3) sActRIIB-Fc, and 4) sActRIIB-Fc running (n ⫽ 8 in each).
Wild-type mice served as healthy controls (n ⫽ 5). All animals were
housed in their individual cages. sActRIIB-Fc or PBS was injected
intraperitoneally once a week with a 5 mg/kg dose of sActRIIB-Fc.
The chosen exercise modality was voluntary wheel running. To allow
the treatment to take effect, the running wheels were locked during the
first injection day and the following day, preventing mice from
exercising. On the last 2 days, the mice did not have access to running
wheels, so that outcome effects would not reflect the acute effects of
exercise.
In the short, 2-wk experiment, the C57Bl/10ScSnJ mice were
randomly divided into three groups: 1) PBS, 2) sActRIIB-Fc once per
week, and 3) sActRIIB-Fc twice per week (n ⫽ 6 in each). The 5 and
10 mg/kg subgroups in groups 2 and 3 were pooled for the further
analysis as no differences were found between the 5 and 10 mg/kg
doses (see Ref. 24).
In the third experiment, the C57Bl/10ScSnJ mice were divided into
three groups (n ⫽ 6 in each group): 1) euthanized 1 or 2 days after a
single injection of PBS, 2) euthanized 1 day after, and 3) euthanized
2 days after sActRIIB-Fc.
During the experiments, all the conditions were standardized. The
mice were euthanized after the experiments by cervical dislocation,
and tissue and blood samples were collected.
sActRIIB-Fc production. The recombinant fusion protein effectively blocking myostatin and activins (24) was produced and purified
in house as described in detail earlier (4, 22, 24). In brief, the fusion
protein contains the ectodomain (ecd) of human sActRIIB and a
human IgG1 Fc domain. The protein was expressed in Chinese
hamster ovary (CHO) cells grown in suspension culture.
Voluntary wheel running, feed intake, and muscle function. Voluntary wheel running was selected as the exercise modality in this
study, as it may offer benefits in mdx mice (9, 34), whereas forced
exercise, such as running, may sometimes even be detrimental to
dystrophic mice or humans (16). Moreover, with voluntary monitored
running we were able to determine whether the blocking of myostatin/
activins affects running activity during the treatment period.
The mice were housed individually in cages where they had free
access to custom-made running wheels (diameter 24 cm, width 8 cm)
24 h/day. Total running distance was recorded 24 h daily. Sedentary
animals were housed individually in similar cages without the running
wheel. Body mass and feed consumption were measured every week
throughout the study.
Strength endurance and body coordination were tested by a hanging wire test according to published methods (56). Mice were suspended above a metal wire secured 30 cm above a padded cell and
released. Depending on the functional ability of the mouse, use of all
limbs and the tail were accepted during a 10-min hanging session.
Three trials were given and the longest hanging time (up to the
maximum of 10 min) was used for the further analysis.
Home cage activity by force plate recording. Vertical ground
reaction forces were measured using a custom-made force plate
(linearity 99%, cross-talk ⬍2%). Forces were measured by four
uniaxial strain gauges which were rectangularly arranged under the
corners of a glass plate on which the animal housing cage was located.
A 14-bit A/D converter (DI-710; DATAQ Instruments, Akron, OH)
was used for digitizing the force data at a sampling rate of 80 Hz. The
RESULTS
Exercise attenuates further increase in body mass due to
myostatin and activin blocking by decreasing fat mass. The
effects of sActRIIB-Fc administration with or without 7 wk
of voluntary running were investigated in modestly dystrophic mdx muscle. Body mass increased rapidly during the
first 2–3 wk after sActRIIB-Fc but thereafter tended to
stabilize to the rate of growth in the PBS group (Fig. 1A).
Voluntary running for 7 wk attenuated the sActRIIB-Fcinduced increase in body mass compared with PBS controls,
mainly by decreasing fat mass (Fig. 1, A and B). Running
also decreased the sActRIIB-Fc-induced increase in absolute muscle mass, but significantly only in gastrocnemius
muscle, not in other muscles (Fig. 1C). Relative to body
mass, running in combination with sActRIIB-Fc actually
increased soleus and EDL muscle mass compared with
sActRIIB-Fc treatment alone (not shown). The mdx mice
had bigger muscles and lower brown and white fat mass than
the healthy mice (Fig. 1). ANOVA (2 ⫻ 2) also revealed a
trend for running to decrease brown fat mass (P ⫽ 0.06). No
effects of sActRIIB-Fc on absolute white or brown fat mass
were observed (Fig. 1B). But since sActRIIB-Fc increased
muscle and body mass, we found decreased epididymal
body fat and a trend (P ⫽ 0.10) for a reduced retroperitoneal
fat by sActRIIB-Fc when these variables were normalized to
body weight (not shown). This was so even though the mice
administered sActRIIB-Fc consumed more feed than their
controls (Fig. 1A).
sActRIIB-Fc treatment for 7 wk did not increase liver size
(not shown), in contrast to the previously published shorter
2-wk experiment (24). ANOVA (2 ⫻ 2) revealed a trend for
sActRIIB-Fc treatment to increase heart mass (P ⫽ 0.06), but
the effects of sActRIIB-Fc were not significant in the individual running or non-running groups (not shown).
sActRIIB-Fc and exercise: effect on the markers of muscle
oxidative capacity. To estimate muscle oxidative capacity, both
the number and capacity markers and regulators of mitochondria were examined in gastrocnemius muscle. Dystrophic mdx
mice had lower mtDNA and PGC1-␣ protein content and
tended to have decreased CS activity (P ⫽ 0.10) compared
with control mice (Fig. 2). All these variables in addition to
cytochrome c remained unchanged after 7 wk of sActRIIB-Fc
treatment but were normalized in mdx mice to control levels or
higher by running, showing the beneficial effects of exercise
(Fig. 2). Increase in oxidative capacity by running compared
with sedentary mice was also confirmed by more strongly
stained mitochondrial enzyme SDH (Fig. 2D).
sActRIIB-Fc administration delays voluntary running activity in mdx mice. During the first 2–3 wk, when the mice were
rapidly growing in the sActRIIB-Fc running group, their daily
running distance was ⬃50% lower than that of the PBS running
mice (Fig. 3A). However, during weeks 4 –7, the difference in
running distance disappeared. Therefore, the sActRIIB-Fctreated mice did not reach their peak in daily voluntary
running distance until ⬃week 6 compared with week 2 in the
PBS group. More detailed analysis of the running data
showed that during the first 4 wk the average velocity and
running distance of each individual running bout were lower
in the sActRIIB-Fc-treated mice than in the PBS mice (not
shown, P ⬍ 0.05). In contrast, the number of running bouts
did not differ between the groups (P ⫽ 0.32), and there was
only a trend for longer time spent on the running wheels in
the PBS running group compared with sActRIIB-Fc running
group (P ⫽ 0.07). As expected, during the last 3 wk, no
differences existed between the groups in the number, velocity, distance, or time of running bouts (P ⬎ 0.67).
Home cage activity. We also studied the level of habitual
home cage physical activity in mdx mice without running
wheels. Physical activity was measured by the distance of
movement per day. The results obtained using the force plate
system developed by our group (51) showed that also the
sActRIIB-Fc-treated mdx mice living without access to running wheels were habitually less active than the PBS-injected
control mice. Interestingly in the 3-wk time point the magnitude of the effect was similar to running distance in the running
group, e.g., ⬃50% less (Fig. 3B). However, unlike the mice
with access to running wheels seemingly getting accustomed
with running, these sedentary mice with sActRIIB-Fc remained
less active than PBS mice throughout the 7-wk period, although the difference between the groups was much smaller at
the later time point (Fig. 3B).
membrane with Restore Western Blot Stripping Buffer (Pierce Biotechnology).
Serum creatine kinase, TNF-␣, and blood hemoglobin and hematocrit. Blood samples were collected after the 7-wk experiment during
euthanization of the animals. Blood hematocrit and hemoglobin were
analyzed by microcuvette HemoCue (EKF-diagnostic, Barleben, Germany). Serum creatine kinase (CK) was analyzed by a kinetic photometric method with KoneLab (Thermo Scientific, Vantaa, Finland).
Serum TNF-␣ was analyzed by an immunometric chemiluminescence
method with an Immulite 1000 (DPC, Los Angeles, CA).
DNA isolation and mtDNA. Gastrocnemius muscle was pulverized
and homogenized in liquid nitrogen and was weighed into TRIzol
reagent (Invitrogen, Carlsbad, CA) and further mixed and homogenized utilizing a FastPrep FP120 apparatus (MP Biomedicals, Illkrich,
France). Total DNA was extracted according to the manufacturer’s
guidelines. The DNA concentrations were analyzed with Nanodrop
ND-1000 (Thermo Fisher Scientific, Waltham MA) in duplicate.
mtDNA/gDNA levels were quantified with a real-time qPCR assay
using an Abi 7300 Real-Time PCR System (Applied Biosystems,
Foster City, CA) in triplicates. The probes and primers used were
predesigned transcripts validated by Applied Biosystems bioinformatics design pipelines. The PCR cycle parameters used were: 50°C for
2 min ⫹ 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for
1 min. The mtDNA copy number was calculated based on the
multiplex amplification of both the mitochondrial NADH dehydrogenase subunit 2 (ND2, mtDNA, ABI Assay ID Mm04225288_s1, VIC
with limited primers) and the nuclear gDNA marker melanomaassociated antigen (mutated) 1-like 1 (Mum1l1, gDNA, ABI Assay ID
Mm00615902_s1, FAM). PCR amplification was performed using 25
ng of DNA. In both assays, all primers and probes map within a single
exon. The results are expressed as the ratio of mtDNA to gDNA in
each specimen with the ⌬CT method. Quantification was performed in
the exponential amplification phase.
Citrate synthase activity. The activity of citrate synthase (CS) in
gastrocnemius muscle homogenate was measured using a kit (SigmaAldrich, CS0720) with an automated KoneLab device (Thermo Scientific).
Statistical methods. Differences between groups were evaluated by
analysis of variance. Fisher’s least significant difference test was used
for post hoc analysis. PASW statistics version 18.0 was used for the
statistical analyses (SPSS, Chicago, IL). The level of significance was
set at P ⱕ 0.05. Data are expressed as means ⫾ SE. Correlations were
analyzed using Pearson’s product moment coefficient.
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Fig. 1. Changes in masses of body (A), fat (B), and muscle (C), and feed consumption (A) after 7 wk of administration of cActRIIB-Fc (soluble ligand binding
domain of activin receptor IIB fused to the Fc domain of IgG) with or without physical exercise (Run). MQF, quadriceps; EDL, extensor digitorum longus.
*Difference vs. PBS; #difference between sActRIIB-Fc and sActRIIB-Fc run.
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Strength endurance. To roughly estimate functional strength
endurance, a hanging wire test (56) was conducted during the
last week of the study. As expected, at the age of ⬃13 wk,
healthy control mice were able to hang for a significantly
longer time than the mdx mice (Fig. 3D). No consistent
differences existed between the treated or nontreated groups in
hanging wire performance (Fig. 3D). Supporting these results,
we recently reported that treatment for 5 wk of mdx mice with
our sActRIIB-Fc increased muscle mass and myofiber size but
had little effect on muscle function (22).
sActRIIB-Fc administration with or without exercise: no
effect on blood inflammation or damage markers, hemoglobin,
or hematocrit. The muscles of the mdx mice were more
fibrotic, had centralized nuclei, and showed inflammatory
cell invasion (Fig. 3E). No apparent visible differences,
such as cytoplasmic inclusions, previously published for
myostatin KO mice (2), or the amount of centralized nuclei
were noticed in the H&E-stained muscle samples after 2 or
7 wk of treatments (Figs. 3E and Fig. 4D). Furthermore,
serum CK was higher (Fig. 3F) and blood hematocrit/
hemoglobin slightly lower in the mdx than in healthy control
mice (not shown). Serum CK and TNF-␣ were not significantly affected by either sActRIIB-Fc or exercise (Fig. 3F).
Running in combination with sActRIIB-Fc increased blood
hemoglobin and hematocrit, but running or sActRIIB-Fc
treatment alone did not have an effect (not shown).
Decrease in voluntary running activity is accompanied by an
increase in body mass and decrease in markers of muscle
oxidative capacity. To understand the possible reasons for the
observed running activity results, a correlation analysis was
Fig. 2. A and B: markers and regulators (B) of oxidative capacity in muscle after 7-wk sActRIIB-Fc administration with or without physical exercise (Run).
C: representative blot. D: oxidative properties of adult gastrocnemius muscle stained using SDH. Fibers stained dark contain high SDH activity. CytC,
cytochrome c; PGC-1␣, PPAR␥ coactivator 1␣. *Significant difference vs. PBS. In mtDNA figure, #difference in sedentary mice vs. healthy controls.
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Fig. 3. Weekly voluntary running (A) and home cage physical activity (B) after 7-wk sActRIIB-Fc administration with or without physical exercise (Run).
C: correlation between running activity and increase in body mass, an example from week 2. Dark symbols refer to sActRIIB-Fc and gray to PBS. D: longest
hanging time in hanging wire test. E: representative hematoxylin and eosin-stained cryosections from gastrocnemius muscle. F: serum markers of muscle damage
(creatine kinase, CK) and inflammation marker tumor necrosis factor-␣ (TNF-␣). *Significance vs. PBS.
first conducted. Running distance correlated negatively with
the increase in body mass gained during the first 2–3 wk.
Figure 3C shows the correlation (r ⫽ ⫺0.75, P ⫽ 0.001)
between running activity at weeks 0 –2 and the increase in body
mass during that time.
In order to further understand what happened during the first
weeks, mice were euthanized after a 2-wk myostatin and
activin blocking period. Similarly to capillary density, which
was reported earlier (24), the concentrations of proteins important for muscle oxidative capacity, PGC1-␣ and cytochrome c,
decreased after 2 wk of sActRIIB-Fc treatment, with a similar
trend in ERR-␣ (Fig. 4). No changes were yet seen acutely 1 or
2 days after a single injection. AS160 is a critical factor for
skeletal muscle glucose uptake (31). No changes were observed in the phosphorylation of p-AS160 (not shown). Therefore, a lower capillary density (see Ref. 24), and trends for
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lower markers of aerobic capacity were observed after 2 wk of
sActRIIB-Fc injections, suggesting that some proteins or organelles such as mitochondria or tissue structures such as
capillaries do not seem to be in balance with the rapid muscle
growth.
A further correlation analysis supported the hypothesis that
the rapid increase in muscle size during the first weeks of
myostatin and activin blocking was associated with decreased
oxidative capacity. It was found that, after 2 wk of myostatin
blocking, muscle fiber size correlated negatively with capillary
density (r ⫽ ⫺0.87, P ⬍ 0.001) [fiber size and capillary
density changes were reported earlier (24)] and CS activity
(r ⫽ ⫺0.61, P ⫽ 0.02).
Autophagy. LC3 immunoblotting was examined as marker
for autophagy (20, 39). Both cytosolic (LC3 I) and autophagosomal (LC3 II) forms of LC3 decreased after the 2-wk
sActRIIB-Fc administration (Fig. 5). In particular, the content
of LC3 II, not, e.g., the LC3 II/LC3 I ratio, has been suggested
to be the best indicator of autophagy (44). Supporting the 2-wk
results in healthy mice, also 7 wk of sActRIIB-Fc in mdx mice
decreased LC3 II content (2 ⫻ 2 ANOVA sActRIIB-Fc treatment effect P ⬍ 0.05), even though in post hoc analysis the
effect of sActRIIB-Fc alone only tended to be (P ⫽ 0.1)
significant (Fig. 5A). In contrast, running for 7 wk increased
LC3 II (2 ⫻ 2 ANOVA running effect P ⬍ 0.05; Fig. 5A). Post
hoc analysis revealed that LC3 II also increased significantly
compared with the non-running group in the exercise-only
mice (PBS running). Our preliminary microarray data did not,
however, reveal major changes in most of the autophagy genes
(e.g., Atg5, Atg7, LC3A, LC3B, p62, Beclin 1, Bnip1–5) due
to 7 wk of exercise or myostatin blocking. However, various
anti- or proautophagy BCL2 and BCL2-like genes either in-
Fig. 4. In gastrocnemius muscle, altered markers (A) and regulators (B) of oxidative capacity after short-term sActRIIB-Fc administration. C: representative blot.
D: representative hematoxylin and eosin-stained gastrocnemius cryosections from PBS and sActRIIB-Fc after 2 wk of treatment. Western blot results are shown
as relative to control (PBS) ⫽ 1. ERR␣, estrogen-related receptor-␣. *Difference vs. PBS (P ⬍ 0.05); in A.
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Fig. 5. Changes in LC3 II and LC3 I protein (A) and phosphorylated ULK1 at Ser757 levels (B) after short- and long-term sActRIIB-Fc (ActRIIB) administration
with or without physical exercise (Run) in gastrocnemius muscle. In addition to the short-term effect, 7 wk of sActRIIB-Fc in mdx mice decreased LC3 II content
(2 ⫻ 2 ANOVA sActRIIB-Fc treatment effect P ⬍ 0.05), whereas running increased LC3 II (2 ⫻ 2 ANOVA running effect P ⬍ 0.05). Representative blots
of LC3 and p-ULK1 (C) and ubiquitinated proteins and p-FoxO1 and total FoxO1 (D). Results are depicted as relative to PBS ⫽ 1. *Difference vs. PBS (P ⬍
0.05). P ⫽ 0.06 depicts a trend of a difference in LC3 II at A2D vs. PBS.
creased or decreased due to the treatments (not shown). BCL2regulated autophagy has been recently shown to be required for
some of the positive responses of exercise (20).
Increased mTORC1 signaling by sActRIIB-Fc reported earlier by us (24) is in theory capable of directly inactivating
autophagy-initiating kinase Ulk1 by phosphorylating (p) it at
Ser757 (27). sActRIIB-Fc did not, however, affect p-ULK1 at
either 1 or 2 days, 2 wk, or 7 wk. In partial support of the LC3II
result, p-ULK increased in the combined sActRIIB-Fc and
running group. It was also noticed that the mdx mice had
elevated p-ULK1 levels. No clear and consistent effects of the
treatments were found, but protein ubiquitination tended to be
elevated in the muscles of the mdx mice. In support of that
result, FoxO1 (8) was dephosphorylated in the mdx mice
compared with the control group (see representative blots in
Fig. 5).
DISCUSSION
Further studies are needed to investigate the effects of different
modes, intensities, and doses of exercise in muscle dystrophy.
Low aerobic fitness and low level of physical activity are
associated with various health risks (32, 57). In this study, we
show that rapid muscle growth induced by sActRIIB-Fc decreased voluntary running during the first few weeks. Moreover, it delayed the time at which peak voluntary running
activity was reached. Our finding suggests that at least mdx
mice require extra time to adjust to the effects of the blocking
of myostatin and activins in order to tolerate a typical, fairly
large amount of voluntary running. We also showed that, in
normal cage conditions without access to running wheels, the
sActRIIB-Fc treated mdx mice were habitually less active than
the nontreated mice throughout the 7-wk intervention but
especially at first weeks. Thus, we postulate that 5– 6 wk of
exercise may be required for the previously sedentary dystrophic mice to tolerate increased levels of physical activity after
myostatin and activin blocking. Previously, postdevelopmental
myostatin depletion and thus increased muscle mass decreased
voluntary running in healthy mice, and this difference tended
to last throughout the 12-wk period (48). In contrast to this and
our results, myostatin blocking by neutralizing antibody has
been reported to increase habitual physical activity and performance in ob/ob mice (6). The difference between the studies
may be the experimental model used. Obese mice seem to
benefit functionally from the increased muscle mass and decreased fat mass caused by myostatin blocking (6, 29).
Possible explanations for the decreased physical activity
after sActRIIB-Fc treatment are intriguing. We found that
sActRIIB-Fc decreased running activity the more body mass
increased. Muscle growth and increased protein synthesis,
which were observed after blocking myostatin and activins
(24), is an energy-consuming process. We previously reported
a rapid sActRIIB-Fc-induced increase in the phosphorylation
of AMPK, a marker of energy stress (24). Large muscles and
high body mass are also not necessarily beneficial for efficient
running. For instance, selective breeding of mice for high
levels of voluntary running has favored a phenotype with
markedly reduced muscle mass (13). Other changes during the
rapid muscle growth were also observed. Previously, we observed decreased capillary density per muscle area after 2 wk
of sActRIIB-Fc treatment (24). Expanding on this finding, we
show here a decrease in the markers of oxidative energy
production capacity per muscle mass after 2 wk of myostatin
blocking. The result is supported by a previous microarray
study with sActRIIB-Fc (50) and studies using myostatin-null
mice (2, 41, 42), all of which showed a reduction in various
markers of aerobic capacity or metabolism. Thus, in addition to
increased body and muscle mass per se and energy stress,
decreased oxidative energy production capacity per muscle
mass may also explain, in part, the reduced amount of voluntary running during the first few weeks after the blocking of
myostatin and activins.
Autophagy is the major cellular route for the degradation of
long-lived proteins and cytoplasmic organelles (20, 45, 61).
After the first 2 wk of myostatin and activin blocking, concurrently with the decreased running after sActRIIB-Fc administration, we also observed a decreased level of LC3 II protein,
a marker of autophagy (44). Previously, autophagy has been
shown to be increased by treating cells with myostatin in vitro
(36). Maintaining autophagy at normal levels is important to
The importance of an adequate size and function of skeletal
muscle for health and well-being have been recognized during
the past few years (3, 49, 58, 60). In the present study, we
provide data that contribute to a better understanding of the
processes that occur during muscle growth when myostatin/
activin signaling is blocked in inactive, but also in active,
exercised muscles. This is important, as studying animals in a
restricted environment that does not allow their normal wildtype physical activity levels, may sometimes lead to inaccurate
conclusions (7).
In DMD, correction of the cause of the disease, i.e., restoration of dystrophin expression, may not be enough if muscle
wasting is in a progressed state. Therefore, novel treatment
modalities complementing the efficacy of gene defect correction approaches are being studied. Previously, exercise has
improved muscle function in myostatin-deficient (41, 42) and
in myostatin-blocked healthy mice (35). In the present study,
exercise decreased the myostatin/activin blocking-induced increase in body mass by reducing fat mass. In early DMD and
in mdx mice, in addition to possible hypertrophy of muscle
fibers, so called increase in nonfunctional muscle size or
“pseudohypertrophy” and thus increase in body mass is common (30). Thus, conceivably, the prevention of an excessive
increase in body mass is beneficial when treating muscle
dystrophies to optimize muscle mass to body mass ratio.
Some, but not all, markers of oxidative capacity were decreased in the muscle of mdx mice. This is supported by earlier
evidence of a decrease in genes important for oxidative energy
production in DMD (55) and decreased mitochondrial function
in mdx mice (33) in skeletal muscles. Importantly, exercise
enhanced or restored the oxidative phenotype of modestly
dystrophic muscle to or above the levels of healthy wild-type
mice. This improvement in oxidative phenotype occurred in
dystrophic muscles without any major effect of 7 wk of myostatin
and activin blocking. This is in contrast to the effects of the
short-term myostatin and activin blocking in healthy mice (see
below).
The protein content of PGC1-␣, one of the main regulators
of muscle oxidative capacity (10), was decreased in mdx mice.
However, voluntary running restored the decreased PGC1-␣
content in mdx mice to or above the levels of healthy mice. In
the long run, this may be important, because in addition to the
positive influence on oxidative capacity (10), overexpression
of PGC1-␣ in muscle has been also reported to ameliorate
DMD pathology (19). We could not, however, notice any
improvements for mdx muscle dystrophic phenotype and in a
muscle endurance test (hanging wire) after running. This is
even though mdx mice could increase their daily voluntary
running from the first days of the experiment. Increased
amount of voluntary running suggests improved ability in
addition to motivation for running (14). Previously, voluntary
running has been reported to be beneficial for mdx mice (9, 23,
34), but also some negative effects have been reported (23, 54).
E179
E180
ACKNOWLEDGMENTS
We thank Hongqiang Ma, Kaisa-Leena Tulla, Miro Nyqvist, Mervi Matero,
Michael Freeman, Simon Walker, Risto Puurtinen, Aila Ollikainen, Paavo
Rahkila, Eliisa Kiukkanen, and Mia Horttanainen for their help. We also thank
the students who assisted in the data collection.
GRANTS
This work was supported by Academy of Finland (Decision No. 137787 to
JJH) and Paulo Foundation (HK).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: J.J.H., W.M.H., H.K., and O.R. conception and design of
research; J.J.H., B.M.O., and A.P. performed experiments; J.J.H. and M.S. analyzed data; J.J.H., B.M.O., H.K., and O.R. interpreted results of experiments; J.J.H.
prepared figures; J.J.H. drafted manuscript; J.J.H., W.M.H., H.K., and O.R. edited
and revised manuscript; J.J.H., B.M.O., M.S., W.M.H., A.P., H.K., and O.R.
approved final version of manuscript.
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