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Exp Physiol 95.2 pp 331–350
Experimental Physiology – Research Paper
Is oxidative stress a cause or consequence of disuse
muscle atrophy in mice? A proteomic approach
in hindlimb-unloaded mice
Lorenza Brocca1,2 , Maria Antonietta Pellegrino1,2 , Jean-François Desaphy3 , Sabata Pierno1,2 ,
Diana Conte Camerino3 and Roberto Bottinelli1,2
1
3
Department of Physiology, Human Physiology Unit and 2 Interuniversity Institute of Myology, University of Pavia, Italy
Section of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Italy
Two-dimensional proteomic maps of soleus (Sol), a slow oxidative muscle, and gastrocnemius
(Gas), a fast glycolytic muscle of control mice (CTRL), of mice hindlimb unloaded for 14 days
(HU mice) and of HU mice treated with trolox (HU-TRO), a selective and potent antioxidant,
were compared. The proteomic analysis identified a large number of differentially expressed
proteins in a pool of ∼800 proteins in both muscles. The protein pattern of Sol and Gas adapted
very differently to hindlimb unloading. The most interesting adaptations related to the cellular
defense systems against oxidative stress and energy metabolism. In HU Sol, the antioxidant
defense systems and heat shock proteins were downregulated, and protein oxidation index and
lipid peroxidation were higher compared with CTRL Sol. In contrast, in HU Gas the antioxidant
defense systems were upregulated, and protein oxidation index and lipid peroxidation were
normal. Notably, both Sol and Gas muscles and their muscle fibres were atrophic. Antioxidant
administration prevented the impairment of the antioxidant defense systems in Sol and further
enhanced them in Gas. Accordingly, it restored normal levels of protein oxidation and lipid
peroxidation in Sol. However, muscle and muscle fibre atrophy was not prevented either in Sol
or in Gas. A general downsizing of all energy production systems in Sol and a shift towards
glycolytic metabolism in Gas were observed. Trolox administration did not prevent metabolic
adaptations in either Sol or Gas. The present findings suggest that oxidative stress is not a major
determinant of muscle atrophy in HU mice.
(Received 15 August 2009; accepted after revision 9 October 2009; first published online 9 October 2009)
Corresponding author R. Bottinelli: Department of Physiology, Human Physiology Unit, University of Pavia,
Via Forlanini 6, 27100 Pavia, Italy. Email: [email protected]
Decrease in neuromuscular activity is a major factor
that shapes structure and function of skeletal muscle,
determining a loss of mass and strength and a shift in
the contractile and metabolic properties towards a faster
phenotype (Baldwin, 1996a,b).
It is generally accepted that the loss in muscle mass
depends on an imbalance between protein synthesis
and degradation (Thomason & Booth, 1990; Gamrin
et al. 1998). Among the potential triggers of protein
degradation, recent works have highlighted the role of
oxidative stress due to an imbalance between the cellular
antioxidant systems and free radical (ROS) production
L.B. and M.A.P. contributed equally to this work.
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(Lawler et al. 2003, 2006b; Smith & Reid, 2006; Powers et al.
2007). Accordingly, amelioration of disuse muscle atrophy
following antioxidant administration has been observed
(Kondo et al. 1992; Appell et al. 1997; Arbogast et al.
2007). Therefore, a pivotal role is now widely recognized
for oxidative stress in the pathogenesis of muscle wasting in
disuse and in a variety of pathological conditions (Moylan
& Reid, 2007).
However, many unanswered questions still remain
regarding the mechanisms underlying muscle atrophy
following disuse. A major puzzle is whether oxidative
stress is a requirement or merely plays a regulatory role
(Lawler et al. 2003; Powers et al. 2007). In this respect, it is
noteworthy that contradictory results have been obtained
regarding the impact of antioxidants on muscle atrophy
DOI: 10.1113/expphysiol.2009.050245
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L. Brocca and others
(Koesterer et al. 2002). Moreover, Servais et al. (2007)
have shown that vitamin E ameliorated muscle atrophy,
but, surprisingly, its protective effect did not depend on its
antioxidant function, but probably on a direct modulation
of muscle proteolysis.
It is also unclear whether fast and slow muscles share the
same mechanisms of disuse atrophy. Most studies (Kondo
et al. 1992; Appell et al. 1997; Lawler et al. 2003, 2006b;
Arbogast et al. 2007; Servais et al. 2007) focused on soleus
muscle which, being a postural slow muscle, is severely
affected by muscle atrophy, whereas limited information
is available on fast muscles because they have been
considered to be little involved in the process (Thomason
& Booth, 1990). However, fast muscles have recently been
shown to go through significant atrophy in some animal
studies (Stelzer & Widrick, 2003; Ferreira et al. 2007).
Moreover, a major potential mechanism of disuse atrophy,
such as oxidative stress, might not be equally relevant
in slow and fast muscles. In fact, slow and fast muscles
could be exposed to ROS production to different extents,
owing to their widely different phenotype and oxidative
metabolism.
To clarify the mechanisms of disuse atrophy, this study
compared the adaptations to hindlimb unloading (HU)
of the protein pattern of the soleus (Sol), a slow oxidative
muscle, and of the gastrocnemius (Gas), a fast glycolitic
muscle, of the mouse.
The proteomic approach appeared particularly well
suited to address the complexity of muscle phenotypic
adaptations (Spangenburg & Booth, 2003), because it
can be used to compare the expression of several
hundreds proteins (∼800; Gelfi et al. 2006; Brocca
et al. 2008) and provides a very comprehensive picture
of the adaptations in the global protein pattern of a
disused muscle. Importantly, the observation that proteins
involved in the cellular defense systems against oxidative
stress adapted very differently to HU in Sol and Gas
prompted experiments to clarify the role of oxidative stress
in disuse atrophy. The comparison between Sol and Gas
was combined with the analysis of trolox administration,
an antioxidant compound.
Two-dimensional (2-D) proteome maps were obtained
from Sol and Gas muscles of the following groups of mice:
(i) control, ground-based mice (CTRL mice); (ii) mice
hindlimb unloaded for 14 days (HU mice); and (iii) HU
mice subjected to trolox treatment (HU-TRO mice) for
1 week before HU and during the 2 weeks of HU. The
protein pattern of Sol and Gas showed large differences
in adaptation to HU, which comprised the antioxidant
defense systems and stress proteins, myofibrillar proteins,
energy production systems, transport proteins and several
other proteins having a variety of functional roles. In
particular, the following points were noted: (i) antioxidant
defense systems were impaired in HU Sol and enhanced in
HU Gas; (ii) consistently, protein oxidation index and lipid
Exp Physiol 95.2 pp 331–350
peroxidation were higher in HU Sol, but normal in HU
Gas; and (iii) trolox administration did not prevent muscle
atrophy, notwithstanding normal defense systems against
oxidative stress and normal levels of protein oxidation
and lipid peroxidation in HU-TRO Sol and Gas muscles.
Thus, the results of this study indicate that oxidative stress
is unlikely to be a major cause of muscle atrophy in
hindlimb-unloaded mice.
Methods
Animal care and hindlimb unloading
Experiments were approved by the Italian Health
Department and complied with the Italian guidelines
for the use of laboratory animals, which conform with
the European Community Directive published in 1986
(86/609/ECC). Six-month-old male C57BL mice weighing
27–32 g (Charles River Laboratories, Calco, Italy) were
randomly assigned to four groups: a control group
(CTRL, n = 5); a hindlimb-suspended group (HU, n = 5);
a hindlimb-suspended group which was treated with
an antioxidant, trolox (HU-TRO, n = 5; Sigma, Milan,
Italy); and a ground-based group (TRO; n = 3) which
was treated with trolox in the exactly same way as the
HU-TRO group, but was not hindlimb suspended. The
CTRL and TRO mice were maintained free in single cages
for 14 days. The method to induce muscle unloading by
hindlimb suspension (HU) has been described in detail
before (Desaphy et al. 2005). Briefly, the animals of HU
group were suspended individually in special cages for
2 weeks by thin string tied at one end to the tail and
at the other end to the top of the cage. All mice had
water ad libitum and received 8 g a day of standard rodent
chow. The HU-TRO and TRO mice received daily an
intraperitoneal injection of 0.25 ml of a 1 M NaHCO 3
solution containing 5 g l−1 trolox, corresponding to
∼45 mg kg−1 day−1 for 3 weeks, commencing 1 week
before HU and continuing for the 2 weeks of HU.
Trolox [(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid, Sigma, Milan, Italy] is a water-soluble
vitamin E analogue that freely crosses cell membranes.
The drug displays a very high antioxidant capacity and
may lack antioxidant-unrelated side-effects, so that it
is widely used as a standard compound in antioxidant
capacity assays. At the end of suspension, the mice
were unfastened from the string and killed by cervical
dislocation. The ground-based (TRO) or hindlimbunloaded (HU-TRO) mice receiving trolox injection
daily showed no difference in body weight and food
consumption compared with CTRL and HU non-treated
animals, respectively. Soleus (Sol) and gastrocnemius
(Gas) muscles were dissected and accurately weighed.
Muscles from one leg were immediately frozen in liquid
nitrogen and used for proteomic and oxyblot analysis,
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Proteomic analysis of disused mouse muscles
while muscles from the other leg were placed into OCT
(Tissue-Tek, Sakura Finetek Europe, Zoeterwoude, The
Netherlands) embedding medium, immediately frozen in
liquid nitrogen and thereafter used for cross-sectional area
analysis (CSA). All the muscle samples were stored at
−80◦ C.
Cross-sectional area analysis
The CSA of individual muscle fibres was determined
both in the midbelly region of Sol (n = 5) and in
the superficial portion of the Gas muscles (n = 5)
for all experimental groups. Serial transverse sections
(10 μm thick) of the muscle samples mounted in OCT
embedding medium were cut in a cryostat at −20◦ C
and collected on coated slides. The cross-cryosections
were immunostained using two monoclonal antibodies
against myosin heavy chain (MHC) isoforms (BA-F8
against MHC-I and SC-71 against MHC-IIA) as previously
described in detail (Bottinelli et al. 1991). A secondary
rabbit antimouse immunoglobulin G (IgG) antibody
conjugated with peroxidase (P-0260, DAKO, Glostrup,
Denmark) was used to reveal the binding of the primary
antibodies. The antibodies enabled identification of type I
and type IIA muscle fibres. In Sol muscles, most fibres were
either stained by BA-F8 (type I) or by SC-71 (type IIA)
or by both antibodies (hybrid fibres). However, several
fibres were not stained and were very probably type IIX
fibres, given the very low content in MHC-IIB of Sol even
following HU. In Sol, the CSA was determined only for
pure type I and type IIA fibres. The superficial Gas did not
show any positively stained fibres for either the antibody
against MHC-I or the antibody against MHC-IIA. Given
the lack of staining with such antibodies and the almost
homogeneous MHC-IIB content (∼90% in CTRL and
100% in HU and HU-TRO) of the Gas muscles in the
population of mice used, all fibres in the cross-sections of
the Gas samples were very probably type IIB. Therefore,
in Gas, CSA was determined in all fibres of the sections
and fibres were considered type IIB. Images of the stained
sections were captured from a light microscope (Leica
DMLS) and transferred to a personal computer using a
video camera (Leica DFC 280). Cross-sectional areas of
identified fibres was measured with Scion Image analysis
software (NIH, Bethesda, MD, USA) and expressed in
micrometres squared.
Analysis of MHC isoform content
The MHC isoform content was determined by an
approach previously described in detail (Pellegrino et al.
2003). Briefly, about 6 μg of each muscle sample were
dissolved in lysis buffer containing 8 M urea, 2 M thiourea,
4% Chaps, 65 mM dithiothreitol (DTT) and 40 mM Tris
base. The lysates were loaded onto 8% polyacrylamide
SDS-PAGE gels. Electrophoresis was run for 2 h at 200 V
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and then for 24 h at 250 V; the gels were stained with
Coomassie Blue staining. Four bands could be separated in
the region of MHC isoforms. These bands corresponded,
in order of migration from the fastest to the slowest,
to MHC-I or slow, MHC-IIB, MHC-IIX and MHC-IIA.
Densitometric analysis of MHC bands was performed to
assess the relative proportion of the four MHC isoforms
in the samples (Pellegrino et al. 2003). It should be noted
that the same samples, made from the whole Sol or Gas
muscles, were used for both analysis of MHC isoform
distribution and proteomic analysis, whereas CSA of Gas
fibres was determined in the superficial portion of the
muscle only where MHC-I and MHC-IIA were absent.
Oxyblot analysis
Muscle homogenates. Muscle samples previously stored
at −80◦ C were homogenized at 4◦ C in an antioxidant
buffer containing protease inhibitors, 25 mM imidazole
and 5 mM EDTA, pH 7.2 adjusted with NaOH. The protein
suspension was centrifuged at 12 000g for 10 min and the
pellet resuspended in 0.01% tetrafluoroacetic acid solution
(200 μl of TFA for 0.1 g of tissue). Following a second
centrifugation for 5 min at 12 000g, the supernatant was
collected and the protein concentration determined (RC
DC protein assay, Bio-Rad Laboratories, Hercules, CA,
USA).
Oxyblot procedure. The principle of oxyblot procedure
is to detect carbonyl groups deriving from protein
oxidation in the protein side-chains. The Oxyblot
Oxidized Protein Detection Kit was purchased from
Millipore (Vimodrone, Italy). The same experimental
approach has been used previously (Barreiro et al. 2003,
2005; Coirault et al. 2007) and was applied here with
minor modifications. Protein carbonyls were derivatized
to dinitrophenylhydrazone (DNP) by reaction with 2,4dinitrophenylhydrazine (DNPH). The DNP-derivatized
protein samples were separated by polyacrylamide gel
electrophoresis followed by Western blotting. In detail,
6 μg of proteins for each muscle sample were denatured
with SDS solution at a final concentration of 6%. The
DNPH solution was added to obtain the derivation;
the reaction was stopped after 15 min of incubation at
room temperature. One-dimensional electrophoresis was
carried out on 15% SDS-polyacrylamide gels loaded with
6 μg of derivatized proteins. A mixture of proteins of
known molecular weight, provided with the kit, was also
loaded to provide molecular weight markers. Proteins were
transferred to nitrocellulose membranes at 100 V for 2 h,
stained with Ponceau Red (Sigma) and then scanned. The
membranes were blocked by incubation with 3% bovine
serum albumin for 1 h and then incubated with rabbit
anti-DNP antibody (at 1:150 dilution) for 1 h at room
temperature. Blots were washed three times for 10 min
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and were then incubated with peroxidase-labelled antirabbit IgG for 1 h.
Blots were developed by using an enhanced
chemiluminescence method in which luminol was excited
by peroxidase in the presence of H 2 O 2 (ECL advance,
Healthcare, Chalfont St. Giles, UK). To assess the
selectivity of carbonyl measurements, a negative control
was always loaded (muscle protein sample without the
derivatization step). Autoradiographic films were imaged
using a high-resolution scanner (Epson V750 PRO image
system), and densitometric analysis was performed using
the software Adobe Photoshop 5.5 (Adobe; D’Antona et al.
2003). Protein oxidation was quantified by defining the
oxidative index (OI), i.e. the ratio between densitometric
values of the oxyblot bands and those stained with Ponceau
Red (Oxy/PR). To compare different experiments, the OI
was expressed relative to control samples.
Measurement of malondialdehyde (MDA) levels
To assess oxidative stress using a method independent
from oxyblot, we determined the levels of
malondialdehyde in three Sol and three Gas muscles
dissected from separate groups of three hindlimbsuspended mice and three hindlimb-suspended mice
subjected to trolox administration. Muscles were
dissected, weighed and frozen in liquid nitrogen. The
MDA level, which reflects the level of lipid peroxidation,
was measured by fluorescence assay of thiobarbituric
acid reactive substance (TBARS) formation, following
the indications of a commercial kit (OXItek, ZeptoMetrix
Corp., Buffalo, NY, USA). Fluorescence measurements
were performed in triplicate using a 96-well plate reader
(Victor3 V mutilabel counter, Perkin Elmer, Waltham,
MA, USA). The measured MDA levels were normalized
with respect to homogenization volume and muscle
weight.
Proteome analysis [two-dimensional (2-D) gel
electrophoresis, 2-DE]
Sample preparation. The methods of proteome analysis
are mostly the same as those previously used (Brocca et al.
2008). Muscle samples previously stored at −80◦ C were
pulverized in a steel mortar with liquid nitrogen to obtain a
powder that was immediately resuspended in a lysis buffer
(8 M urea, 2 M thiourea, 4% Chaps, 65 mM DTT and 40 mM
Tris base). The samples were vortexed, frozen with liquid
nitrogen and thawed at room temperature four times and
then incubated with DNase and RNase for 45 min at 4◦ C
to separate proteins from nucleic acids and then spun at
35 000g for 30 min. Protein concentration in the dissolved
samples was determined with a protein assay kit (2D quant
Kit, GE Healthcare).
In order to perform proteome analysis, a sample mix
was obtained for each experimental animal group (CTRL,
Exp Physiol 95.2 pp 331–350
HU and HU-TRO) for both soleus and gastrocnemius.
Sample mix contained an equal quantity of protein taken
from each muscle sample of CTRL, HU and HU-TRO.
As the TRO group did not differ from the CTRL group it
was not subjected to proteome analysis (see ‘Adaptation
to protein pattern’ for detail).
Two-dimensional gel electrophoresis. Isoelectrofocusing
was carried out using the IPGphor system (Ettan IPGphor
isoelectric focusing system, GE Healthcare). The IPG
gel strips, pH 3–11 NL (non-linear), 13 cm long, were
rehydrated for 14 h, at 30 V and 20◦ C, in 250 μl of
reswelling buffer [8 M urea, 2 M thiourea, 2% (w/v) Chaps,
0.1% (v/v) tergitol NP7 (Sigma), 65 mM DTT and 0.5%
(v/v) pH 3–11NL (GE Healthcare)] containing 100 μg
protein sample. Strips were focused at 20 000 V h–1 , at
a constant temperature of 20◦ C, and the current was
limited to 50 μA per IPG gel strip. After isoelectrofocusing,
the strips were stored at −80◦ C until use or equilibrated
immediately for 10–12 min in 5 ml of equilibration buffer
[50 mM Tris pH 6.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v)
SDS and 3% (w/v) iodoacetamide]. Then, the immobilized
IPG gel strips were applied to 15% total acrylamidebisacrylamide monomer concentration, 2.5% crosslinker
concentration polyacrylamide gels without a stacking gel.
The separation was performed at 80 V for 17 h at room
temperature.
The 2-D gels were fixed for 2 h in fixing solution [40%
(v/v) ethanol and 10% (v/v) acetic acid], stained with
fluorescent staining (FlamingoTM fluorescent gel stain,
Bio-Rad) for 3 h and destained with 0.1% (w/v) Tween 20
solution for 10 min.
Triplicate gels of each sample were visualized using
a Typhoon laser scanner (GE Healthcare) and analysed
with Platinum software (GE Healthcare). One gel was
chosen as the master gel, and used for the automatic
matching of spots in the other 2-D gels. Only spots
present in all gels used for the analysis were considered.
The software provided the normalized volume for each
spot (representing the amount of protein). A very good
reproducibility of the spots among the triplicate gels
of each experimental group was found. When the spot
volumes for matched spots were plotted on a linear
scale, regression analysis yielded correlation coefficients
in the range 0.89–0.94. The volumes of each spots in
the triplicate gels were averaged, and spots statistically
changed were obtained (P < 0.05). The average volumes of
each differentially expressed spot were used to determine
the volume ratios reported in the figures and Supplemental
tables. Supplemental Tables 1–4 also report the P values of
each differentially expressed spot.
Protein identification
Electrophoresis fractionation and in situ digestion. For
protein identification, 2-D gels were loaded with 300 μg
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Proteomic analysis of disused mouse muscles
of protein per strip; the electrophoretic conditions were
the same as those described in the ‘two-dimensional
gel electrophoresis’ section. After staining with Colloidal
Coomassie, spots were excised from the gel and washed
in 50 mM ammonium bicarbonate (pH 8.0) in 50%
acetonitrile to a complete destaining. The gel pieces were
resuspended in 50 mM ammonium bicarbonate (pH 8.0)
containing 100 ng of trypsin and incubated for 2 h at 4◦ C
and overnight at 37◦ C. The supernatant containing the
resulting peptide mixtures was removed and the gel pieces
were re-extracted with acetonitrile. The two fractions were
then collected and freeze dried.
Matrix-Assisted Laser Desorption/Ionization
Spectrometry analysis. The Matrix-Assisted
335
which definitive mass spectral data had previously been
acquired. Moreover, a permanent exclusion list of the
most frequent peptide contaminants (keratins and trypsin
peptides) was included in the acquisition method in order
to focus the analyses on significant data.
Statistical analysis
Data are expressed as means ± S.E.M. Statistical
significance of the differences between means was assessed
by Student’s t test or by one-way ANOVA followed by
Student–Newman–Keuls test (data in Figs 2 and 9). A
probability of less than 5% was considered significant
(P < 0.05).
Mass
Laser
Desorption/Ionization Mass Spectrometry mass spectra
(MALDI MS) were recorded on an Applied Biosystem
Voyager DE-PRO mass spectrometer equipped with a
reflectron analyser and used in delayed extraction mode.
One microlitre of peptide sample was mixed with an
equal volume of α-cyano-4-hydroxycynnamic acid as
matrix (10 mg ml−1 in 0.2% TFA in 70% acetonitrile),
applied to the metallic sample plate and air dried. Mass
calibration was performed by using the standard mixture
provided by the manufacturer. Mass signals were then
used for database searching using the MASCOT peptide
fingerprinting search program (Matrix Science, Boston,
MA, USA) available at http://www.matrixscience.com.
Liquid Chromatography Tandem Mass Spectrometry
analysis. When the identity of the proteins could
not be established by peptide mass fingerprinting,
the peptide mixtures were further analysed by Liquid
Chromatography Tandem Mass Spectrometry (LCMSMS)
using the LC/MSD Trap XCT Ultra (Agilent Technologies,
Palo Alto, CA, USA) equipped with a 1100 HPLC system
and a chip cube (Agilent Technologies). After loading, the
peptide mixture (7 μl in 0.5% TFA) was first concentrated
and washed either at 1 μl min−1 onto a C18 reverse-phase
precolumn (Waters, Milford, MA, USA) or at 4 μl min−1
in 40 nl enrichment column (Agilent Technologies chip),
with 0.1% formic acid as the eluent. The sample was
then fractionated on a C18 reverse-phase capillary column
(75 mm × 20 cm in the Waters system, 75 mm × 43 mm
in the Agilent Technologies chip) at a flow rate of
200 nl min−1 , with a linear gradient of eluent B (0.1%
formic acid in acetonitrile) in A (0.1% formic acid)
from 5 to 60% in 50 min. Elution was monitored on the
mass spectrometers without any splitting device. Peptide
analysis was performed using data-dependent acquisition
of one MS scan (m/z range from 400 to 2000 Da/e)
followed by MS/MS scans of the three most abundant ions
in each MS scan. Dynamic exclusion was used to acquire
a more complete survey of the peptides by automatic
recognition and temporary exclusion (2 min) of ions from
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Results
Muscle atrophy and MHC distribution
Muscle weight to body weight ratio was significantly lower
following 14 days HU in both Sol (CTRL, 0.38 ± 0.01,
n = 5; and HU, 0.30 ± 0.01, n = 5) and Gas (CTRL,
5.17 ± 0.07, n = 5; and HU, 4.67 ± 0.17, n = 5). Atrophy
was more pronounced in Sol (–22%) compared with Gas
(−10%).
The CSA of individual muscle fibres was determined in
the midbelly region of Sol and in the superficial portion
of the proximal Gas in CTRL and HU mice. Crosscryosections were immunostained with monoclonal
antibodies against MHC-I and MHC-IIA (Fig. 1). In Sol,
type I fibres went through a lower degree of atrophy (16%)
than type IIA fibres (30%; Fig. 2). Muscle fibres from Gas
also went through a significant degree of atrophy (25%;
Fig. 2). All Gas fibres were very likely to be type IIB given
the almost homogeneous MHC-IIB composition of Gas
(see below) and the lack of staining with anti-MHC-I and
anti-MHC-IIA antibodies (Fig. 1).
A slower to faster shift in MHC isoform distribution
was observed in both Sol and Gas muscle following
HU in all five animals studied, although the shift
in Gas was small owing to the almost homogeneous
MHC-IIB content in CTRL Gas. In Sol, although the
MHC-I relative content remained unchanged (CTRL Sol,
38.5 ± 2.1%; and HU Sol, 37.9 ± 2.5%), the MHC-IIA
relative content was significantly (P < 0.05) lower in
HU Sol (30.4 ± 3.7%) than in CTRL Sol (50.5 ± 2.6%),
the MHC-IIX content was significantly higher in HU
Sol (28.1 ± 3.6%) than in CTRL Sol (10.9 ± 2.0%), and
MHC-IIB, which was absent in CTRL Sol, was present
in HU Sol (3.8 ± 1.2%). The CTRL Gas was mostly
composed of MHC-IIB (87.1 ± 2.0%), but also showed
some MHC-I (2.8 ± 1.1%), MHC-IIA (4.0 ± 0.9%) and
MHC-IIX content (5.9 ± 1.4%). In contrast, HU Gas was
homogeneously composed of MHC-IIB (100%) in all five
muscles analysed.
A group of five animals was treated for 1 week before
HU and for the 2 weeks of HU with a specific and highly
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Exp Physiol 95.2 pp 331–350
Figure 1. Cross-cryosections of soleus muscles and superficial proximal gastrocnemius muscles used
for determination of cross-sectional area of individual muscle fibres in control conditions (CTRL Sol and
CTRL Gas), following HU (HU Sol and HU Gas) and following trolox treatment before and during HU
(HU-TRO Sol and HU-TRO Gas)
Cross-sections were immunostained using anti-MHC-I (BA-F8) and anti-MHC-IIA (SC-71) monoclonal antibodies
as indicated on the left. Since all muscle fibres in cross-sections of gastrocnemius muscles were negative with
both anti-MHC-I and anti MHC-IIA antibodies in CTRL, HU and HU-TRO samples, only cross-sections stained by
anti-MHC-IIA antibody were analysed.
efficient antioxidant, trolox, to counteract oxidative stress
and clarify its role in disuse atrophy. Trolox did not
counteract muscle atrophy. The muscle weight to body
weight ratio of both Sol (0.30 ± 0.03, n = 5) and Gas
(4.54 ± 0.15) were not different in HU-TRO mice and
in HU mice. Figure 2 shows that trolox treatment did not
prevent muscle fibre atrophy either in Sol or in Gas, in
any fibre type. Trolox did not affect MHC distribution
in Gas and partly affected it in Sol; MHC-IIA relative
content was higher (43.0 ± 4.6%) and MHC-IIX content
was lower (18.2 ± 2.0%) than in HU Sol; MHC-IIB was
absent.
Figure 2. Mean values (+ S.E.M.) of cross-sectional areas of individual muscle fibres from soleus (Sol)
and gastrocnemius (Gas) muscles in control conditions (CTRL), following hindlimb suspension (HU) and
following trolox treatment before and during HU (HU-TRO)
Cross-sectional areas were determined on immunostained cross-cryosections. ∗ Significantly different from CTRL
(P < 0.05). The number of fibres analysed is reported above each bar.
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Proteomic analysis of disused mouse muscles
To ensure that trolox treatment per se did not determine
muscle atrophy or relevant changes in muscle phenotype,
a ground-based group of mice (n = 3) was treated with
trolox. The muscle weight to body weight ratio of
Sol (0.42 ± 0.04, n = 3) and Gas muscles (5.45 ± 0.31,
n = 3) was not different from CTRL. The myosin heavy
chain isoform distribution was also not different from
control values either in Sol (MHC-I, 37.4 ± 3.1%; MHCIIA, 51.55 ± 4.63%; MHC-IIX, 10.91 ± 3.5%; and MHCIIB, 0%) or in Gas (MHC-I, 1.51 ± 0.31%; MHC-IIA,
2.49 ± 0.81%; MHC-IIX, 6.01 ± 0.49%; and MHC-IIB,
90.0 ± 2.2%).
Adaptations of protein pattern
Figure 3 shows examples of proteomic maps obtained
from Sol and Gas muscles of CTRL, HU and HUTRO mice. The following comparisons were performed:
(i) between muscles from HU and CTRL mice to identify
the proteins differentially expressed as a result of disuse;
and (ii) between muscles from HU-TRO and HU mice
to assess whether and to what extent the antioxidant
treatment could counteract the disuse-induced alteration
in the protein pattern. Proteomic analysis was not
performed on the ground-based trolox-treated group
(TRO) because neither the whole animals (body weight
and food consumption) nor their Sol and Gas muscles
(muscle weight to body weight ratio and MHC isoform
distribution) showed any difference from ground-based
untreated mice (CTRL).
The differentially expressed proteins were grouped
on the basis of their functional role into the following
categories: myofibrillar proteins; antioxidant defense
systems and heat shock proteins; energy production
systems (glycolytic metabolism, oxidative metabolism and
creatine kinase); and transport proteins. The remainding
proteins, with variable functional roles, were pooled into
a single group (other proteins).
The circles and numbers in the control maps of Sol
(Fig. 4A) and Gas (Fig. 4B) indicate all proteins found to
be differentially expressed and which could be identified
by MALDI-Tof. The numbers enable identification of the
protein and all related information in Figs 5 and 7 for Sol
and in Figs 6 and 8 for Gas. The full set of information
regarding the differentially expressed proteins is reported
in Supplemental Tables 1–4.
In Sol, 774 spots were found to be present in both
CTRL and HU maps and were compared. The percentage
of differentially expressed spots was 8.26% (n = 64). Of
the 64 spots found to be differentially expressed, 19
Figure 3. Representative two-dimensional gels of Sol and Gas from the CTRL, HU and HU-TRO groups
Thirteen centimetre IPG gel strips, pH 3–11 NL (non-linear), were used in the first dimension, and SDS gels (15%
T, 2.5% C) were used in the second dimension.
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Figure 4. Representative two-dimensional gels of control soleus (A) and gastrocnemius muscles (B)
Thirteen centimetre IPG gel strips, pH 3–11 NL (non-linear), were used in the first dimension, and SDS gels (15% T,
2.5% C) were used in the second dimension. In A (CTRL Sol), the protein spots found to be differentially expressed
either in HU Sol versus CTRL Sol or in HU-TRO Sol versus HU Sol are circled and numbered. The numbers enable
identification of the circled spots using Figs 5 and 7. The same applies to B (CTRL Gas), in which the numbers
enable identification of the circled spots using Figs 6 and 8.
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Proteomic analysis of disused mouse muscles
were upregulated and 45 downregulated. Forty-three of
these spots could be identified by MALDI-Tof (Fig. 5 and
Supplemental Table 1).
In Gas, 802 spots were found to be present in both CTRL
and HU maps and were compared. The percentage of
differentially expressed proteins following HU was lower
in Gas (4.5%, n = 36) than in Sol, suggesting a lower
impact of HU. Of the 36 differentially expressed spots,
12 were upregulated and 24 downregulated. Twenty of
these spots could be identified by MALDI-Tof (Fig. 6 and
Supplemental Table 2).
Seven hundred and sixty-two protein spots in Sol and
732 protein spots in Gas were found to be present in
both HU and HU-TRO maps and were compared. The
percentage of differentially expressed spots was not large
either in Sol (4.46%, n = 34) or in Gas (4.09%, n = 30).
Figure 5. Histogram of volume ratios of differentially expressed proteins in HU Sol in comparison with
CTRL Sol
As shown on the right, proteins were grouped on the basis of their functional role as follows: myofibrillar proteins;
antioxidant defense systems and heat shock proteins; energy production systems (glycolytic metabolism, oxidative
metabolism and creatine kinase); transport proteins; and other proteins. The numbers on the x axis indicate the
ratio between the average volume of a given protein expressed in HU Sol and the average volume of the same
protein in CTRL Sol. Positive numbers (on the right) indicate upregulation of a protein in HU, whereas negative
numbers (on the left) indicate downregulation. On the left of the bars, the accession number of each protein
(Swiss-Prot accession number) and the numbers we attributed to the spots in the 2-D maps of Fig. 4A (spot
number) are shown. The full information on the proteins and the numerical ratios between volumes are reported
in Supplemental Table 1.
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Of the 34 spots found to be differentially expressed in Sol,
nine were upregulated and 25 downregulated. Nineteen
of these spots could be identified by MALDI-Tof (Fig. 7
and Supplemental Table 3). Of the 30 spots found to
be differentially expressed in Gas, 8 were upregulated
and 22 downregulated. Fifteen of such spots could
be identified by MALDI-Tof (Fig. 8 and Supplemental
Table 4).
Several of the differentially expressed proteins reported
in Figs 5–8 and Supplemental Tables 1–4 were found at
multiple spot locations (see, for example, myozenin in
Fig. 5 and Supplemental Table 1 and aconitase in Fig. 8 and
Supplemental Table 4) and therefore appear more than
once in the figures and Supplemental tables. The presence
Exp Physiol 95.2 pp 331–350
of multiple spots of a protein is frequent in 2-D gels and
is due to the existence of isoforms or to post-translational
modifications of a protein. Different isoforms of the
same protein might differ both in molecular weight
and in isoelectric point, whereas multiple spots due to
post-translational modifications usually have the same
molecular weight. Mass spectrometry was not able to
identify all isoforms of some proteins or the nature of
their post-translational modifications. However, in only
two cases, MLC-Is and MLC-If, the multiple spots of a
protein varied in opposite directions (some upregulated
and some downregulated). In such cases, to assess whether
the trend was towards up- or downregulation, the multiple
spots of MLC-Is and MLC-If were averaged.
Figure 6. Histogram of volume ratios of differentially expressed proteins in HU Gas in comparison with
CTRL Gas
The same approach to represent differentially expressed proteins was used as in Fig. 5. ‘Spot number’ refers to
the number of circled proteins in Fig. 4B. The full information on the proteins and the numerical ratios between
volumes are reported in Supplemental Table 2.
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Proteomic analysis of disused mouse muscles
Myofibrillar proteins. In HU Sol, several spots
corresponding to myosin light chain isoforms were
differentially expressed (Fig. 5). Myosin light chain-IIf
was upregulated. Myosin light chain-If was on average
upregulated, whereas MLC-Is content was on average
unchanged. The content of troponin I fast and troponin C
was higher, whereas actin content was lower in HU Sol
(Fig. 5).
Two load-bearing cytoskeleton proteins were also
downregulated in Sol: desmin, which plays a critical role in
the maintenance of structural and mechanical integrity of
the contractile apparatus, being involved in longitudinal
and especially lateral force bearing (Paulin & Li, 2004);
and myozenin, a member of a family of Z-disk proteins,
also called calsarcins or FATZ (Frey et al. 2000; Takada
et al. 2001), which mechanically link adjacent sarcomeres.
We identified a total of four spots for myozenin in Sol
and Gas 2-D maps, which we simply indicated as A, B,
C and D, because the correspondence between myozenin
and calsarcin isoforms (Frey et al. 2000) and between the
four spots and such isoforms is still unknown.
In HU Gas, fewer myofibrillar proteins were
differentially expressed compared with HU Sol (Fig. 6).
Troponin I fast and MLC-IIf content was higher in HU
Gas, consistent with what was observed in Sol and with
the slower to faster shift in MHC isoform content. Actin
was downregulated, as in soleus. In contrast to what
observed in Sol, three spots corresponding to myozenin (B,
C and D) were upregulated. Only one of the differentially
expressed myozenin spots (C) was common to Sol and
Gas, and was upregulated in Gas and downregulated in
Sol.
Figure 7. Histogram of volume ratios of differentially expressed proteins in hindlimb-suspended soleus
of mice treated with trolox (HU-TRO Sol) in comparison with HU Sol
The same approach to represent differentially expressed proteins was used as in Figs 5 and 6. ‘Spot number’ refers
to the number of circled proteins in Fig. 4A. The full information on the proteins and the numerical ratios between
volumes are reported in Supplemental Table 3.
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Trolox administration had little impact on myofibrillar
proteins. Troponin I fast and myozenin (spot C) were
downregulated in both Sol (Fig. 7) and Gas (Fig. 8), and
actin was downregulated in Gas.
Antioxidant defence systems and heat shock proteins. In
HU Sol, a major group of differentially expressed proteins
were those involved in cellular defence systems against
reactive oxygen species or free radicals (ROS). Superoxide
dismutase (Zn/Cu SOD) was upregulated, peroxiredoxin6 (PRDX6) and carbonic anhydrase 3 (CAH III) were
downregulated, suggesting an impairment of antioxidant
defence systems. Consistently, several heat shock proteins
(HSPs), which also protect cells against oxidative stress
(Lawler et al. 2006a), were downregulated (Fig. 5).
In contrast to what observed in HU Sol, all the
differentially expressed proteins involved in antioxidant
defense systems, Zn/Cu SOD, CAH III and PRDX6, were
upregulated in Gas following HU (Fig. 6). Interestingly,
the content of mitochondrial SOD (mn-SOD) was
Exp Physiol 95.2 pp 331–350
also higher in HU Gas. Therefore, no impairment of
antioxidant defense systems was observed in HU Gas.
Consistently, no differences were observed among HSPs.
Trolox administration resulted in an upregulation of
antioxidant defense systems in both Sol (Fig. 7) and Gas
(Fig. 8). The free radical scavenging properties of trolox,
therefore, prevented the HU-induced deterioration of the
antioxidant defense systems in Sol and enhanced them
above control levels in Gas, but no effect was observed on
muscle and muscle fibre atrophy (Fig. 2).
Energy production systems. In
HU Sol, all the
differentially expressed proteins involved in the
three cellular energy production systems (glycolytic
enzymes, oxidative enzymes and creatine kinase) were
downregulated, except for glyceraldehyde 3-phosphate
dehydrogenase (Fig. 5).
In HU Gas, four glycolytic enzymes and creatine
kinase were more abundant than in CTRL Gas and,
of the three differentially expressed enzymes involved
Figure 8. Histogram of volume ratios of differentially expressed proteins in hindlimb-suspended
gastrocnemius of mice treated with trolox (HU-TRO Gas) in comparison with HU Gas
The same approach to represent differentially expressed proteins was used as in Figs 5, 6 and 7. ‘Spot number’
refers to the number of circled proteins in Fig. 4B. The full information on the proteins and the numerical ratios
between volumes are reported in Supplemental Table 4.
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Proteomic analysis of disused mouse muscles
in oxidative metabolism, two were upregulated (NADH
dehydrogenase 1α complex 4 and α-electron transfer
flavoprotein,) and one (2-oxoglutarate dehydrogenase)
was downregulated (Fig. 6). Thus, in contrast to what
was observed in Sol, no evidence of a general impairment
of energy production systems was observed in Gas, but
merely a shift towards a more glycolytic metabolism.
Trolox administration did not prevent the effect of
disuse on energy production systems in either Sol (Fig. 7)
or Gas (Fig. 8).
In HU-TRO Sol, glycolytic enzymes and creatine
kinase were even further downregulated, whereas no
differentially expressed oxidative enzymes were found,
suggesting that trolox did not prevent the downregulation
of oxidative enzymes observed following HU.
In HU-TRO Gas, trolox administration affected
expression of the following three enzymes of the energy
production systems, which were all downregulated:
phosphoglucomutase (a glycolytic enzyme); aconitase (an
oxidative enzyme); and creatine kinase, reverting the
increase in the latter observed following HU (Fig. 8).
Transport proteins. In Sol, two downregulated spots
corresponded to different isoforms of myoglobin (Fig. 5).
Blood proteins found in our proteomic maps
(haemoglobin, serum albumin, serotransferrin and
apolipoprotein A-I) come from capillaries and very small
blood vessels in the muscle sample. The lower content
of most of them in HU Sol (Fig. 5) finds a possible
explanation in the lower muscle vascularization that
has been suggested to occur following HU (Desplanches
et al. 1990). Interestingly, apoliprotein A-I, a fat-binding
protein that represents the major component of the highdensity lipoproteins (HDL) in plasma, was upregulated.
In HU-TRO Gas, myoglobin, which was not affected in
HU Gas, was downregulated (Fig. 8).
Other proteins. In Sol, several proteins were not assigned
to a specific functional group and were pooled into the
category ‘other proteins’. Within this group, sarcalumenin
and parvalbumin play important roles in calcium handling
within muscles cells (Fig. 5). Sarcalumenin, which is a
calcium-binding protein in the sarcoplasmic reticulum
(Yoshida et al. 2005), was downregulated. In contrast,
parvalbumin, a calcium buffer in the cytosol, was
upregulated.
Two fatty acid-binding proteins (FABPH3s) were less
abundant in HU Sol than in CTRL Sol.
Aldose reductase, a putative antioxidant defense system
(Kang et al. 2007), was downregulated in HU Sol. Trolox
downregulated it even further in Sol and downregulated
it in Gas. Trolox downregulated parvalbumin in Sol.
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Protein oxidation and lipid peroxidation
Protein oxidation index was much higher in HU Sol than
in CTRL Sol, whereas it was not different in HU Gas
and CTRL Gas. These findings are consistent with the
proteomic analysis, which suggested an impairment of the
cellular defense systems against oxidative stress in HU Sol
(Fig. 5), but not in HU Gas (Fig. 6).
In Sol, trolox administration fully counteracted the
disuse-induced increase in oxidation index (Fig. 9). This
finding is consistent with the proteomic analysis, which
showed a reversal of the impairment of the antioxidant
defense systems in HU-TRO Sol. In HU-TRO Gas, the
oxidation index was slightly, but significantly, lower than
in HU Gas, consistent with the upregulation of the proteins
involved in antioxidant defense systems (Fig. 8).
Indeed, the oxidation index of both HU-TRO Sol and
HU-TRO Gas was slightly, but significantly, lower than in
the corresponding CTRL muscles (Fig. 9).
The redox balance was also assessed by an approach
independent from oxyblot, i.e. the analysis of lipid
peroxidation based on determination of the levels of
MDA. Malondialdehyde levels were significantly higher in
HU Sol (331 ± 51 nmol g−1 ) than in CTRL Sol (197 ±
11 nmol g−1 ), whereas the difference between HU Gas
(109 ± 21 nmol g−1 ) and CTRL Gas (149 ± 7 nmol g−1 )
did not reach statistical significance. Following trolox
administration, HU-TRO Sol (201 ± 11 nmol g−1 ) and
HU-TRO Gas (102 ± 18 nmol g−1 ) showed the same level
of MDA as the corresponding CTRL muscles.
The results of the analyses of protein oxidation and lipid
peroxidation were, therefore, consistent.
Discussion
In this study, the adaptations to muscle disuse of the
protein pattern of a slow oxidative muscle, the Sol, and a
fast glycolytic muscle, the Gas, were studied by proteomic
analysis. This analysis enabled the complexity of muscle
phenotype adaptations to be addressed (Spangenburg &
Booth, 2003) by identifying the proteins differentially
expressed following HU and antioxidant administration
among the very large number (∼800) of protein spots,
which could be separated in two-dimensional maps.
The differentially expressed proteins can be considered
the targets of the disuse process and of the antioxidant
treatment.
Notably, although a large number of proteins, belonging
to several functional categories, were differentially
expressed in the different conditions, their changes
were fully consistent with one another, building a very
comprehensive and solid picture of the adaptations of both
Sol and Gas to HU and antioxidant administration.
Soleus and Gas adapted to disuse very differently in
all the following protein categories identified: myofibrillar
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proteins; antioxidant defense systems; energy production
systems; and a group of proteins with variable functional
roles (‘other proteins’). The major findings related
to muscle atrophy and myofibrillar protein pattern,
antioxidant defense systems and oxidative stress, and
energy metabolism.
Hindlimb unloading and muscle mass and myofibrillar
protein pattern
Although it is generally believed that slow muscles are most
affected by disuse (Fitts et al. 2001), in agreement with
previous observations (Stelzer & Widrick, 2003; Ferreira
et al. 2007), the present findings indicate that 14 days HU
resulted in significant atrophy not only of Sol, a slow
muscle, but also of Gas, a fast muscle.
The greater atrophy of type IIA Sol and type IIB Gas
fibres compared with type I Sol fibres is surprising, because
it is not in agreement with the long-standing idea of a
Exp Physiol 95.2 pp 331–350
preferential atrophy of slow fibres in disuse (Baldwin,
1996b; di Prampero & Narici, 2003). However, similar
atrophy in slow and fast muscle fibres both in humans
(Hather et al. 1992; Fitts et al. 2001) and mice (Stelzer &
Widrick, 2003) has been previously observed. Collectively,
the results suggest that the variable sensitivity to disuse
atrophy does not depend on the different fibre-type
composition of slow and fast muscles, but more probably
on their function in maintaining posture.
In HU Sol, the downregulation of actin, desmin and
myozenin indicated by the proteome analysis (Fig. 5)
complements the picture of muscle atrophy. The lower
actin content is consistent with previous observations
(Chopard et al. 2001; Riley et al. 2002; Moriggi et al. 2008).
Since both desmin (Paulin & Li, 2004) and myozenin
(Frey et al. 2000; Takada et al. 2001) are load-bearing
cytoskeleton proteins, their downregulation is probably
an adaptation to the lower load applied to muscle fibres
during HU and is bound to result in a decrease in
Figure 9. Protein oxidation index (OI) in soleus (A) and gastrocnemius muscles (B) in control conditions
(CTRL, lane 1), following hindlimb unloading (HU, lane 2) and following trolox treatment before and
during HU (HU-TRO, lane 3)
The OI is determined as the ratio between the densitometric values of the oxyblot bands and those stained with
Ponceau Red in Western blots of muscle homogenates (see Methods). The height of each vertical bar represents
the mean + S.E.M. ∗ Significantly different from all other groups (P < 0.05). The band close to 43 kDa is actin.
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load-bearing capacity upon reloading. Contradictory
findings have previously been reported on the impact
of disuse on desmin content in both humans and rats
(Chopard et al. 2001, 2005; Moriggi et al. 2008).
In HU Gas, actin was downregulated, in agreement with
the observation of a disproportionate loss of thin filaments
in Gas (Riley et al. 2002). Myozenin was upregulated, in
contrast to what was observed in Sol (Fig. 6). This finding
suggests that there are different adaptations of the Z-disks
to HU in the two muscles. It should be noted that Zdisks are not only mere mechanical joints between adjacent
sarcomeres, but can also play a role in sensing the stress
applied to muscle cells (Hoshijima, 2006).
The variations of MHC isoform distribution and of
MLC and troponin I isoform content (Figs 5 and 6)
observed in both Sol and Gas confirm the slower to faster
shift in muscle phenotype generally observed in disuse
(Fitts et al. 2001) and are consistent with a very recent and
detailed analysis of myofibrillar protein isoforms in disuse
(Yu et al. 2007). In Gas, the almost homogeneous MHCIIB content of control muscles prevented a large slower to
faster shift in MHC content.
Interestingly, some adaptations of the Sol protein
pattern, i.e. the downregulation of desmin, various
oxidative enzymes and carbonic anhydrase and the
upregulation of parvalbumin, might depend on the disuseinduced activation of a gene expression programme
transforming Sol muscle fibres from slower to faster. Such
proteins are, in fact, differentially expressed in slow and
fast muscles or muscle fibres (Celio & Heizmann, 1982;
Schiaffino & Reggiani, 1996; Chopard et al. 2001). In
contrast, many adaptations observed either did not have
a known relation with a shift in muscle fibre types or
were in the opposite direction of what was expected,
among which were the adaptations of glycolytic enzymes
and creatine kinase. It is interesting to note that most
protein adaptations in Gas could not be related to a
shift in MHC isoform distribution because very limited
shift was observed. These observations are consistent with
the hypothesis that differences among muscle fibres are
controlled by several gene expression programmes, and
not by a single ‘master switch’, changing fibre type and all
other proteins in co-ordinated fashion (Spangenburg &
Booth, 2003)
Importantly, antioxidant administration did not
prevent muscle and muscle fibre atrophy either in Sol or in
Gas. The downregulation of myozenin in both HU-TRO
Sol and HU-TRO Gas and of actin in HU-TRO Gas (Figs 7
and 8) is consistent with persistent muscle atrophy.
Hindlimb unloading and oxidative stress
Profound alterations in antioxidant defense systems and
HSPs (Fig. 5), fully consistent with a higher protein
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oxidation index (Fig. 9) and lipid peroxidation, occurred
in HU Sol. Zinc/copper superoxide dismutase was
upregulated, whereas all other differentially expressed
proteins of this category were downregulated. Although
an increase in Zn/Cu SOD might at first appear protective
against oxidative stress, the decrease in the capacity to
convert hydrogen peroxide into water resulting from
a downregulation of enzymes downstream of Zn/Cu
SOD, including peroxiredoxin (PRDX6), can indeed result
in accumulation of hydrogen peroxide and promote
oxidative stress (Lawler et al. 2003). Carbonic anhydrase 3
(CAH III) protects against oxidative damage by binding
free radicals (Cabiscol & Levine, 1995; Raisanen et al.
1999). Its reduced content is in agreement with a
recent gene expression study in mouse HU Sol (Dapp
et al. 2004). Heat shock proteins work downstream
of the other antioxidant defense systems by removing
products caused by the formation of free radicals and
by protecting cells against oxidative stress (Lawler et al.
2006b). The downregulation of HSPs is consistent with
the lower content of Hsp70 and Hsp25 previously observed
following HU (Lawler et al. 2006b).
Notably, the findings in mouse Sol recapitulated what
was previously observed using different experimental
approaches in rat Sol and consequently could suggest a
major pathogenic role of oxidative stress in disuse atrophy,
in agreement with previous studies (Lawler et al. 2003,
2006b; Servais et al. 2007).
Surprisingly, the analysis of HU Gas which, being a
fast muscle, has been little studied, suggests the opposite
conclusion that oxidative stress, at least in mice, does
not play a major role in determining atrophy. In HU
Gas, Zn/Cu SOD, CAH III and PRDX6 were upregulated
(Fig. 6), HSPs were not differentially expressed, protein
oxidation index (Fig. 9) and lipid peroxidation were not
different from control Gas, but muscle and muscle fibre
(Fig. 2) atrophy occurred. Importantly, the effects of
antioxidant treatment on Sol and Gas strongly support
the lack of a major causal role of oxidative stress.
Trolox prevented the HU-induced deterioration of the
antioxidant defense systems in Sol (Fig. 7) and enhanced
them above control levels in Gas (Fig. 8). Consistently, it
counteracted the increase in oxidation index in HU Sol,
decreased it below normal in Gas (Fig. 9) and counteracted
the increase in lipid peroxidation in HU Sol. However, no
effect was observed on muscle and muscle fibre atrophy
(Fig. 2).
To reduce the risk of an unexpected non-specific effect,
the dose of trolox was chosen at an intermediate level
between the minimal (10 mg kg−1 day−1 , e.g. Sagach et al.
Pharmacol Res 2002) and maximal doses (92 mg kg−1 d−1 ;
e.g. McClung et al. 2007) reported in the literature. The
possibility that maximal doses of trolox could have affected
muscle atrophy cannot definitely be rule out, although it
appears very unlikely because lipid and protein oxidation
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were fully prevented and antioxidant defense systems were
upregulated by trolox treatment.
No information was available on antioxidant defense
systems in HU Gas until a very recent study in rats (Siu
et al. 2008). Interestingly, in substantial agreement with
the present work, Siu et al. (2008) showed no impairment
in antioxidant defense systems, in contrast to what was
previously observed in rat soleus (Lawler et al. 2003, 2006b;
Servais et al. 2007). However, the authors suggested a
pathogenic role of oxidative stress based on higher lipid
peroxidation, whereas we did not find a significant increase
in lipid peroxidation, in agreement with no increase
in protein oxidation index in Gas (Fig. 9). The slower
phenotype and higher oxidative metabolism of rat Gas
(Rijkelijkhuizen et al. 2003) compared with mouse Gas
could explain the somewhat different adaptation to disuse
reported by Siu et al. (2008) and by this work (Dapp et al.
2004).
The impact of antioxidant treatments on muscle
atrophy has been studied previously, and contradictory
results have been obtained. Amelioration of disuse atrophy
was observed in some studies (Kondo et al. 1992; Appell
et al. 1997; Arbogast et al. 2007; McClung et al. 2007;
Servais et al. 2007), but not in others (Koesterer et al.
2002; Matuszczak et al. 2004). The discrepancies could
depend on the disuse models and on the drugs employed,
which could have a more complex action than only
an antioxidant action. Disuse atrophy resulting from
mechanical ventilation (McClung et al. 2007) and oneleg immobilization (Kondo et al. 1992; Appell et al. 1997)
could be more sensitive to antioxidant treatment because
these models result in a much faster atrophy, which might
have pathogenic mechanisms somewhat different from
HU. Servais et al. (2007) suggested that the amelioration
of Sol atrophy following vitamin E administration did
not depend on its antioxidant properties, but on its
inhibition of proteolytic enzymes. The Bowman–Birk
inhibitor used by Arbogast et al. (2007) has multiple
actions, including the inhibition of serine proteases, and
not only an antioxidant action.
Hindlimb unloading and energy metabolism
In HU Sol, a general downsizing of all energy production
systems occurred for glycolytic enzymes, oxidative
enzymes and creatine kinase. While the downregulation
of oxidative enzymes is expected on the basis of the
slower to faster shift in muscle phenotype (Abadi et al.
2009), the downregulation of glycolytic enzymes is not in
agreement with their higher activity generally observed in
rats following HU or space flight (Fitts et al. 2001) and
with a very recent proteomic analysis of rat Sol muscle
following HU (Moriggi et al. 2008). However, Dapp
Exp Physiol 95.2 pp 331–350
et al. (2004) have shown a lower expression of glycolytic
enzymes following 7 days HU in mice and suggested that
the discrepancy with rat Sol could depend on the much
faster starting phenotype of mice and on the consequently
different shift in fibre types following HU. Notably, a
similar downregulation of glycolytic enzymes has been
observed following 4–11 days immobilization in humans
by microarray analysis (Chen et al. 2007).
Moreover, the downregulation of desmin, myoglobin
and FABPH3 in Sol are consistent with and could indeed
contribute to a major impairment of cell metabolism.
In fact, desmin is not only a load-bearing protein, but
is involved in mitochondrial positioning and respiratory
function (Milner et al. 2000). Myoglobin is not only a
short-term oxygen reservoir, but could facilitate oxygen
diffusion within the cell and stimulate mitochondrial
biogenesis by downregulating NO (Flogel et al. 2001).
It is noteworthy that desmin-null mice have structural
and functional alterations of the mitochondria (Goldfarb
et al. 2004). Moreover, FABPH3s could control lipid
metabolism and fuel usage (Makowski & Hotamisligil,
2004) and could play a central role in the control of lipid
signalling in the cells (Makowski & Hotamisligil, 2004),
which might be involved in modulating inflammation and
metabolism.
Interstingly, the downregulation of NADH-ubiquinone
oxidoreductase 49 kDa, cytochrome c1 and ATP synthase
(Fig. 5), belonging to mitochondrial complex I, III and V,
respectively, could not only impair energy metabolism,
but also contribute to ROS production in HU Sol. The
respiratory chain is in fact a major site of ROS production.
A slowing down of any of its steps (Powers et al. 2007)
and its asymmetric inhibition could increase its ROS
production.
In HU Gas, an upregulation of glycolytic enzymes and
no evidence of a general impairment of energy production
systems were observed. The higher content of glycolytic
enzymes in a fast muscle following disuse is in agreement
with previous observations (Thomason & Booth, 1990).
It is unlikely that this shift can be explained by the
small slower to faster shift in MHC isoform distribution
observed in HU Gas. It is more likely that unloading
determined an increase in glycolytic metabolism mostly
independent of fibre-type shift (Grichko et al. 2000). The
higher content of glycolytic enzymes following HU in Gas
is consistent with a shift in substrate usage away from
lipids and towards glucose and could determine metabolic
inflexibility, a phenomenon recently suggested to play a
role in disuse atrophy (Mazzatti et al. 2008).
Trolox administration did not counteract, but even
further decreased glycolytic metabolism, creatine kinase
and myoglobin in Sol and decreased oxidative and
glycolytic metabolism and myoglobin in Gas, suggesting
that the impairment of energy metabolism in disuse is not
due to free radical production.
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Proteomic analysis of disused mouse muscles
Several observations reported here suggest that
metabolic adaptations could concur to the pathogenesis
of disuse atrophy and not be a mere consequence of
it. Together with myofibrillar proteins and antioxidant
defense systems, the proteins directly (metabolic enzymes)
or indirectly involved (myoglobin, desmin, FHBS) in
energy metabolism were the most important targets of
HU. Their adaptations were not merely a consequence of
a slow to fast shift in muscle phenotype. The antioxidant
treatment, which restored apparently normal defense
systems against oxidative stress and counteracted protein
oxidation and lipid peroxidation, did not reverse both
metabolic adaptations and muscle atrophy.
The possible contribution of a metabolic programme
in modulating cell size is now widely recognized (Sandri,
2008). Indeed, a general impairment in energy production
has also been observed in a number of pathological
conditions in which muscle wasting occurs, such as
muscular dystrophy (Porter et al. 2004), cancer, fasting,
diabetes and uraemia (Lecker et al. 2004) and in burn
sepsis (Duan et al. 2006). Recently, metabolic inflexibility
has been stressed as a possible cause of disuse atrophy
(Mazzatti et al. 2008).
The possible link between metabolic adaptations and
structural and functional alterations in disused muscle is
still unclear. Interestingly, an impaired energy metabolism
per se could decrease protein synthesis, which is an
ATP-requiring process. Moreover, the downregulation of
creatine kinase might play a role in muscle atrophy. In
transgenic mice lacking either the cytoplasmic (M-CK) or
both the cytoplasmic and mitochondrial creatine kinase,
muscle atrophy and damage occur (Momken et al. 2005).
Interestingly, RING-finger protein-1 (MuRF1), a major
ubiquitin ligase involved in muscle proteolysis in disuse,
has very recently been found to ubiquinate creatine kinase
and to favour its catabolism (Koyama et al. 2008).
In Gas, metabolic inflexibility per se could play a role
in muscle deterioration following disuse (Mazzatti et al.
2008), although metabolic alterations appear much less
evident than in Sol.
Hindlimb unloading and calcium handling
The downregulation of sarcalumenin and the upregulation
of parvalbumin suggest a disuse-induced alteration of
calcium handling within the cell. The lower content
of sarcalumenin suggests a weakening of the calciumhandling capacity of the sarcoplasmic reticulum (Yoshida
et al. 2005), whereas the upregulation of parvalbumin
suggests an increase in the calcium-buffering capacity of
the cytosol. The higher content of parvalbumin following
HU could be related to the slow to fast shift in muscle
phenotype (Celio & Heizmann, 1982). The upregulation
of parvalbumin is in agreement with previous gene
C 2009 The Authors. Journal compilation C 2010 The Physiological Society
505
347
expression studies on immobilized human muscle (StAmand et al. 2001) and on muscles of small mammals in
a variety of conditions characterized by muscle wasting
(Lecker et al. 2004; Duan et al. 2006). In this respect, it
should be noted that disuse-induced alterations of resting
calcium concentration following HU have been observed,
although there is no agreement on whether resting calcium
concentration increases (Ingalls et al. 1999) or decreases
(Fraysse et al. 2003).
Conclusions
The present findings indicate, surprisingly, that oxidative
stress is more likely to be a consequence than a cause
of muscle atrophy following HU in mice, either in a
slow oxidative muscle, the soleus, or in a fast glycolitic
muscle, the gastrocnemius. Although the roles of different
pathogenic mechanisms in disuse muscle atrophy could
vary according to species and experimental models, our
findings in mice open the possibility that oxidative stress
might not be a major requirement of muscle atrophy
also in other conditions. Metabolic alterations emerge
as a possible component of the pathogenesis of disuse
atrophy in mice Sol, whereas in Gas their role is much
less clear. This study does not rule out the possibility
that oxidative stress can play a role in disuse-induced
alterations of muscle force production through oxidation
of myofibrillar proteins (Dalla Libera et al. 2005) and
effects on electrophysiological parameters (Desaphy et al.
2005; Powers & Jackson, 2008), independently from its
lack of effect on muscle atrophy.
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Acknowledgements
This work was supported by the Italian Space Agency grants
to R. Bottinelli and D. Conte Camerino (project OSMA
Exp Physiol 95.2 pp 331–350
‘Osteoporosis and Muscle Atrophy’). We thank CEINGE
Advanced Biotecnologies s.c.a.r.l. center, Naples, Italy, for help
in protein identification by MALDI-Tof analysis.
Supplemental material
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