Exercise Training Prevents Oxidative Stress and
Ubiquitin-Proteasome System Overactivity and Reverse
Skeletal Muscle Atrophy in Heart Failure
Telma F. Cunha1, Aline V. N. Bacurau1, Jose B. N. Moreira1, Nathalie A. Paixa ˜o1, Juliane C. Campos1,
Julio C. B. Ferreira1, Marcelo L. Leal2, Carlos E. Negra ˜o1,3, Anselmo S. Moriscot2, Ulrik Wisløff4,
Patricia C. Brum1*
1School of Physical Education and Sport, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 2Biomedical Sciences Institute, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 3Heart
Institute (InCor), University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 4K. G. Jebsen Center of Exercise in Medicine, Norwegian University of Science and Technology, Trondheim,
Background: Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular
mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative
stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We
also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF.
Methods/Principal Findings: Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation,
protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic
hyperactivity-induced HF. At the 7thmonth of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress
and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by
reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human
HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome
activity was also increased and AET restored it to healthy control subjects’ levels.
Conclusions: Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS
overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of
particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF
patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in
the treatment of HF.
Citation: Cunha TF, Bacurau AVN, Moreira JBN, Paixa ˜o NA, Campos JC, et al. (2012) Exercise Training Prevents Oxidative Stress and Ubiquitin-Proteasome System
Overactivity and Reverse Skeletal Muscle Atrophy in Heart Failure. PLoS ONE 7(8): e41701. doi:10.1371/journal.pone.0041701
Editor: Antonio Musaro, University of Rome La Sapienza, Italy
Received April 10, 2012; Accepted June 25, 2012; Published August 3, 2012
Copyright: ? 2012 Cunha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo (FAPESP #2006/61523-7) and Conselho Nacional de Pesquisa e
Desenvolvimento (CNPq #473251/2009-4). PCB and CEN hold scholarships from CNPq (#301519/2008-0 and #301867/2010-0]. TFC held a scholarship from
FAPESP (#2006/58460-4). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
HF is a syndrome of poor prognosis characterized by exercise
intolerance, early fatigue and skeletal myopathy marked by
atrophy and shift toward fast twitch fibers [1,2], which may
culminate in cardiac cachexia, an underestimated problem for HF
prognosis and healthcare expenditure . Pathophysiological
determinants of skeletal myopathy in HF have begun to be
elucidated and a dynamic imbalance of anabolic and catabolic
processes has been proposed . In fact, increased protein
degradation, circulating proinflammatory cytokines and oxidative
stress are common features of systemic diseases-induced skeletal
muscle wasting, including HF [5–8].
UPS is a major proteolytic pathway responsible for disposal of
damaged proteins, which accumulate in skeletal myopathies .
Indeed, aggravation of skeletal muscle atrophy is associated with
UPS overactivation . Atrogin-1 and MuRF1, E3 ligases driving
conjugation of ubiquitin chains to proteasome substrates, are not
only directly associated with but required for skeletal muscle
atrophy [10,11], highlighting the importance of UPS beyond
associative findings. Despite the important role played by UPS in
atrophying states, little is known about its involvement in HF-
induced muscle atrophy.
The mechanisms underlying UPS overactivation in skeletal
myopathies have not been clarified. However, attention should be
driven to oxidative stress due to its differential modulation UPS
activation [12,13]. Even mild disturbance of redox balance causes
protein oxidation, leading to proteasomal overactivation for
maintenance of cell viability . Furthermore, increased
oxidative stress in HF has been associated with early fatigue and
PLoS ONE | www.plosone.org1 August 2012 | Volume 7 | Issue 8 | e41701
skeletal myopathy [15,16]. However, the association among
oxidative stress, UPS activation and skeletal muscle atrophy in
HF has been poorly addressed.
Even though muscle wasting is considered an independent
predictor of mortality in human HF , no available medication
is effective in counteracting HF skeletal myopathy. Therefore,
alternative therapies are of clinical relevance. AET has been
established as an adjuvant therapy for HF, counteracting exercise
intolerance and improving quality of life [18,19]. Additionally,
studies demonstrate beneficial effects of AET on skeletal muscle
structure and function in chronic diseases [20,21], however, its
impact on skeletal muscle UPS activation remains poorly un-
Using a mice model of sympathetic hyperactivity-induced HF
through disruption of a2Aand a2Cadrenergic receptor genes (a2A/
a2CARKO mice) [22,23], we hypothesized that: (a) UPS would be
up-regulated in plantaris muscle of a2A/a2CARKO mice and
associated with increased oxidative stress and muscle atrophy; (b)
AET would counteract HF-induced skeletal muscle oxidative
damage, UPS overactivation and atrophy. In addition, using
vastus lateralis muscle biopsies from HF patients and age-matched
healthy individuals, we tested the hypothesis that: (c) Proteasome
activity would also be increased in HF patients and (d) AET would
re-establish proteasome activity to healthy control levels, providing
novel insights into molecular mechanisms controlling skeletal
muscle phenotype in human HF and reinforcing the clinical
relevance of AET as an adjuvant therapy for HF.
The animal care and protocols in this study were reviewed and
approved by the Ethical Committee of the University of Sa ˜o Paulo
The human study was performed according to the Helsinki
declaration and was approved by the Regional Committee for
Medical Research Ethics in Norway (Regionale Komiteer for Medisinsk
og Helsefaglig Forskningsetikk, REK midt) (clinical trial identifier
NCT00218933). The CONSORT (Consolidated Standards of
Reporting Trials) checklist for the study is available in Information
S1. Written consent was obtained from all patients.
and a2Cadrenergic receptors (a2A/a2CARKO mice) were used in
the present study. The absence of these receptors leads to
substantial increase in sympathetic tone, since they are presynaptic
receptors regulating noradrenaline release in sympathetic nerve
terminals . Previous studies of our group demonstrated that
those mice provide a physiologically relevant HF animal model
[23–27]. Male a2A/a2CARKO mice in a C57Bl6/J genetic
background and their wild type controls (WT) were studied at 3,
5 or 7 months of age. Subsets of animals were allocated into time-
course evaluation of skeletal muscle atrophy (WT and a2A/
a2CARKO at 3, 5 or 7 months of age, n=6 per group) or evaluation
of UPS activation and AET effects (7 month-old untrained WT
and a2A/a2CARKO [ARKO], and trained a2A/a2CARKO [AR-
KOT], n=6 per group). This time point was chosen because a2A/
a2CARKO mice display severe HF and established skeletal muscle
myopathy at 7 months of age [21,24,28]. Additionally, a2Aand
a2Cadrenoceptors have never been reported as modulators of
skeletal muscle function or structure. Mice were maintained in
a light (12–h light cycle) and temperature (22uC) controlled
environment and with free access to standard laboratory chow
Mice with genetic disruption of both a2A
(Nuvital Nutrientes, Brazil) and tap water. This study was
conducted in accordance with the ethical principles in animal
research adopted by the Brazilian College of Animal Experimen-
tation (www.cobea.org.br). The animal care and protocols in this
study were reviewed and approved by the Ethical Committee of
the University of Sa ˜o Paulo (CEP #2007/28).
was assessed by M-mode echocardiography in halothane-anesthe-
tized WT and a2A/a2CARKO mice. Mice were positioned in supine
position and ultrasound transmission gel was applied to the
precordium. Echocardiography was performed using an Acuson
Sequoia model 512 echocardiographer (Siemens, USA) equipped
with a 14-MHz linear transducer. LV systolic function was
means left ventricular end-diastolic dimension, and LVESD
means left ventricular end systolic dimension.
Graded treadmill tests until exhaustion
were performed as previously described by our group .
Running performance, here assessed by total distance run, was
used to verify exercise intolerance in HF animals. Exercise
tolerance was evaluated by graded treadmill running tests after
adaptation to treadmill exercises over a week (10 min/day).
Treadmill speed started at 6 m/min and was increased by 3 m/
min every 3 minutes until exhaustion, when mice were no longer
able to run. Tests were carried out by a single observer (TFC),
blinded to mice’s identity. Total distance run (meters) and peak
workload (m/min) were recorded.
Aerobic exercise training.
ate-intensity treadmill running during eight weeks (from 5 to
7 months of age), five days/week. This age was chosen due to our
previous findings demonstrating substantial cardiovascular im-
provements by AET at a time point (7mo-old) when a2A/a2CARKO
mice display severe cardiac dysfunction [30,31]. Each session
consisted of 60-minute running at 60% of maximal workload
achieved in a graded treadmill running test (protocol is described
above), corresponding to maximal lactate steady state (MLSS), as
we have previously described for the same animal model . At
the end of the fourth training week, animals were reevaluated for
running performance in order to adjust AET intensity. Untrained
mice were exposed to treadmill exercise (5 min at 40% of maximal
workload, 3 days/week) in order to maintain running skills .
Skeletal muscle cross-sectional area.
after the last functional assessment mice were killed and plantaris
muscle was carefully harvested, snap-frozen in isopentane and
stored in liquid nitrogen or 280uC depending on intended
experiments. Plantaris muscle was used due to the high prevalence
of type II fibers, known to display greater damage in pathological
states and superior response to mechanical overload than type I
fibers. Muscles were cut into 10 mm-thick sections using a cryostat
(Criostat Micron HM505E, Walldorf, Germany) and incubated
for myofibrillar ATPase activity after alkali (pH 10.3) preincuba-
tion. Whole muscle cross-sectional area (CSA) was evaluated at
6200 magnification and further analyzed on a digitalizing unit
connected to a computer (Image Pro-plus, Media Cybernetic,
USA). All analyses were conducted by a single observer (AVNB),
blinded to the mice’s identity.
uated using the ferrous oxidation-xylenol (FOX) orange technique
. Plantaris samples were homogenized (1:20 wt/vol) in
phosphate buffered saline (PBS; 100mM, pH 7.4) and centrifuged
at 12000g for 20 min at 4uC. Pellet was discarded and supernatant
was precipitated with trichloroacetic acid (10% wt/vol) and
centrifuged at 12000 g for 20 min at 4uC. Supernatant was mixed
Left ventricular function
Mice were submitted to moder-
Lipid hydroperoxides were eval-
Exercise Training Reduces UPS Activation in HF
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with FOX reagent containing 250 mM ammonium ferrous sulfate,
100mM xylenol orange, 25 mM H2SO4, and 4 mM butylated
hydroxytoluene in 90% methanol and incubated at room
temperature for 30 min. Absorbance of samples was read at
Total RNA was isolated from plantaris
samples using Trizol (Invitrogen, Carlsbad, California). RNA
concentration and integrity were assessed. cDNA was synthesized
using reverse transcriptase at 70uC for 10 min, followed by
incubation at 42uC for 60 min and at 95uC for 10 min. The genes
analyzed were: atrogin-1/MAFbx, MuRF-1, E3-a, USP14,
USP19, USP28 and Cyclophilin (reference gene). All primers
were synthesized by Invitrogen (sequences available in Informa-
tion S2). Real time PCR for all genes were run separately and
amplifications were performed by ABI Prism 5700 Sequence
Detection System (Applied Biosystems, USA) by using SYBR
Green PCR Master Mix (Applied Biosystems, USA). Results were
quantified as Ct values, where Ct is defined as the threshold cycle
of the polymerase chain reaction at which the amplified product is
first detected. Expression was normalized by cyclofilin levels as an
endogenous reference. WT group levels were arbitrarily set to 1.
Skeletal muscle protein expression.
subunits (a5, a7, b1, b5, b7 subunits) (Abcam item #ab22673),
polyubiquitinated proteins (Biomol item #BML-PW0930) and
carbonylated protein abundance (Millipore item #S7150) were
evaluated by western blotting in total extracts of plantaris muscle
from WT, a2A/a2CARKO and trained a2A/a2CARKO. Frozen
muscles were homogenized in a buffer containing 1 mM EDTA,
1 mM EGTA, 2 mM MgCl2, 5 mM KCl, 25 mM HEPES,
pH 7.5, 100 mM PMSF, 2 mM DTT, 1% Triton X-100, and
protease inhibitor cocktail (1:100, from Sigma-Aldrich). Centrifu-
gation was performed for 15 minutes at 10000g and 4uC, pellet
was discarded and supernatant (cytosolic proteins) was used.
Samples were subjected to SDS-PAGE in polyacrylamide gels
(10%). Proteins were electrotransferred to nitrocellulose mem-
brane. Equal loading of samples and transfer efficiency were
monitored with the use of 0.5% Ponceau S staining of the blotted
membrane. Membrane was then incubated in a blocking buffer
(5% nonfat dry milk, 10 mM Tris-HCl, 150 mM NaCl, and 0.1%
Tween 20, pH 7.6) for 2 h and then incubated overnight at 4uC
with specific antibodies against 20S proteasome subunits (Biomol
International, USA. Bands quantified in the 55-130 kDa range)
and ubiquitinated proteins (Biomol International, USA. Bands
quantified in the 55–130 kDa range). Protein carbonylation was
assessed by measuring the levels of carbonyl groups using the
OxyBlot Protein Oxidation Detection Kit (Millipore, USA),
following manufacturer’s instruction. Binding of the primary
antibody was detected with the use of peroxidase-conjugated
secondary antibodies (rabbit or mouse, 2h) and developed using
enhanced chemiluminescence detected by autoradiography. Anal-
ysis of blots was performed with Image J software (NIH, USA).
Results are expressed as percentage of age-matched WT group.
Assay of 26 S proteasome activity.
activity of proteasome was assayed using the fluorogenic peptide
(LLVY-MCA, Enzo Life Sciences item #P802-0005). Assays were
carried out in a microtiter plate by diluting 25 mg of cytosolic
protein into 200 mL of 10 mM MOPS, pH 7.4 containing 25 mM
LLVY-MCA (substrate), 25 mM ATP and 5.0 mM Mg2+. Rate of
fluorescent product formation was measured with excitation and
emission wavelengths of 350 and 440 nm, respectively. Peptidase
activities were measured in the absence and presence (20 mM) of
the proteasome-specific inhibitor epoxomicin and the difference
between the two rates was attributed to the proteasome.
Cardiology, St. Olav’s Hospital, Trondheim, Norway, and agreed
to participate in the study. The protocol started in October 2001
and ended in September 2005 due to study completion. None of
the HF patients had myocardial infarction in the 12 months
preceding the study. All HF patients exhibited LV ejection fraction
,40% (functional class II-III, NYHA), were clinically stable and
had received b-blockers, ACE inhibitors and statins for .12
months. HF patients were randomly assigned to either sedentary
(HF-S) or exercise training protocol (HF-T). Subjects were
randomized and stratified (by age) to HF-S or HF-T. Flowchart
of patient allocation is shown in Figure 1. The randomization code
was developed with a computer random-number generator to
select random permuted blocks. Participants were blinded to
assigned intervention. Physiological parameters such as cardiac
structure and function, aerobic capacity, height and body weigh
were similar between HF groups before experimental protocol.
Exclusion criteria were unstable angina pectoris, uncompensated
heart failure, myocardial infarction during the past 4 weeks,
complex ventricular arrhythmias, no use of b-blockers and ACE
inhibitors, and orthopedic or neurological limitations to exercise.
Medications did not change during the 12-week study period. The
study was performed according to the Helsinki declaration and
was approved by the regional medical research ethics committee
(clinical trial identifier NCT00218933). Written consent was
obtained from all patients.
After a 10-minute warm-up, a VO2peak
test (MetaMax II, Cortex, Germany) was performed with an
individualized treadmill ramp protocol and increased inclination
by 2% when oxygen uptake stabilized at each workload until
VO2peak was reached. Leveling off of oxygen uptake despite
increased workload and respiratory exchange ratio above 1.05
were used as criteria for maximal oxygen uptake. Immediately
after this workload, blood was drawn from a fingertip for
measurement of lactate concentration.
Aerobic exercise training.
training twice weekly and performed 1 weekly session at home.
HF-S and Control groups met for supervised exercise once every
three weeks. Training sessions consisted of ‘‘uphill’’ continuous
walking at 60% of VO2peak (60% to 70% of peak heart rate)
during 50 minutes. All subjects used a heart rate monitor (Polar
Electro, Finland) to obtain the assigned exercise intensity. Borg 6-
to-20 scale was used to assess the rate of perceived exertion during
and after each training session. Treadmill speed and inclination
were adjusted continuously to ensure that every training session
was carried out at the assigned heart rate. Home-based training
intensity was recorded twice by heart rate monitors, placed so that
the patients were unable to see their heart rate during the exercise.
Recordings confirmed correct exercise intensity during home
training. Patients were instructed to immediately stop home-based
training if they had chest pain or any other distressing symptoms
and contact the emergency department at the hospital. The
control group was told to follow advice from their family doctor
with regard to physical activity. In addition, they met for 47
minutes of continuous treadmill walking at 70% of peak heart rate
every 3 weeks.
Skeletal muscle biopsies.
were obtained from the vastus lateralis with a sterile 5 mm-
diameter biopsy needle (Bergstrom) under local anesthesia. Sample
preparation, proteasome activity and protein ubiquitination and
carbonylation measurements were carried out as described above
for mice samples.
Subjects were recruited from the Department of
HF-T group met for supervised
Skeletal muscle biopsy samples
Exercise Training Reduces UPS Activation in HF
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All values are presented as means 6 standard error from mean.
Data were tested for normal distribution and one-way analysis of
variance (ANOVA) followed by Duncan post hoc testing was used
to compare all variables. Statistical significance was considered
achieved when P value was set as ,0.05.
Physiological Parameters in a2A/a2CARKO Mice
Corroborating our previous findings [21,26,30], Table 1 shows
cardiac function deterioration in a2A/a2CARKO mice from 3 to 7
months of age, latter associated with signs of HF as cardiac
enlargement and severe dysfunction, lung edema and exercise
intolerance. It was also shown by our group that a2A/a2CARKO
mice display severe pathological cardiac remodeling, activation of
renin-angiotensinsystem andimpairedcalcium handling
[24,27,28,33], which are paralleled by a 30% mortality rate
observed at the 7thmonth of age (Information S3), supporting the
rationale of using a2A/a2CARKO mice as a model of severe HF.
Time-course of Skeletal Muscle Trophicity, Oxidative
Stress and Proteasome Activation in a2A/a2CARKO Mice
Severity of cardiac dysfunction was paralleled by progressive
reduction of plantaris muscle CSA from 3 to 7 months of age in
a2A/a2CARKO mice in comparison with age-matched WT
(Figure 2A). Plantaris muscle hypertrophy was observed in a2A/
a2CARKO mice at 3 months of age, which was no longer observed
at the 5thmonth, when a2A/a2CARKO plantaris CSA was similar
to age-matched WT mice. Finally, 7 month-old a2A/a2CARKO
mice displayed plantaris muscle atrophy. Representative histolog-
ical images are shown in Information S4. Lipid hydroperoxidation
in plantaris muscle were similar between groups at the 3rdmonth,
progressed to a strong trend toward elevation in 5 month-old a2A/
Figure 1. Flowchart of intervention assignment of HF patients. *Skeletal muscle biopsies were not taken from these patients, therefore, they
were excluded from analysis.
Exercise Training Reduces UPS Activation in HF
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a2CARKO mice and culminated in significantly increased lipid
hydroperoxidation in 7 mo-old a2A/a2CARKO mice when
compared with age-matched WT (Figure 2B). Likewise, protein
carbonylation was elevated only in 7 mo-old a2A/a2CARKO
mice, when HF was present (Figure 2C). Skeletal muscle
proteasomal activity was significantly decreased in 3 mo-old
a2A/a2CARKO mice, progressing to unchanged levels at the 5th
month, ultimately reaching significant elevation in 7 mo-old a2A/
a2CARKO mice compared with age-matched WT (Figure 2D). As
plantaris atrophy and oxidative damage were observed only at 7
months of age, further experiments were carried out at this time
Effects of AET on Cardiac Function and Lung Water
Content in a2A/a2CARKO Mice
As shown in Table 2, AET restored FS of a2A/a2CARKO to
WT’s values. Lung edema was also prevented by AET in a2A/
a2CARKO mice (Table 2), which corroborates our previous
findings addressing cardiac function in this model [26,30,33].
Improved cardiac function after AET was associated with reduced
mortality rate (Information S3) at the 7thmonth of age.
mRNA Levels of Skeletal Muscle UPS Components in
a2A/a2CARKO Mice and Effects of AET
To further investigate the contribution of UPS components for
plantaris atrophy, we evaluated E3 ligases mRNA expression in 7
mo-old a2A/a2CARKO mice (Figure 3A). a2A/a2CARKO mice
displayed higher Atrogin-1/MAFbx and E3a mRNA levels than
age-matched WT (dashed line), which were effectively reduced by
AET (Figure 3A). Deubiquitinating enzymes may also modulate
protein tagging for proteasomal degradation, therefore, we
evaluated USP28, USP19 and USP14 mRNA levels. Figure 3B
shows that HF mice displayed increased USP28 and 14 mRNA
levels. USP28 mRNA levels were reestablished to WT levels while
USP14 mRNA levels were reduced to values below WT’s
Skeletal Muscle UPS Activation in a2A/a2CARKO Mice
and Effects of AET
In order to verify whether AET would affect skeletal UPS
activation in our model, we measured protein ubiquitination,
chymotrypsin-like proteasome activity and expression of protea-
some subunits in WT, untrained and aerobic exercise-trained a2A/
a2CARKO mice. Ubiquitinated proteins expression was signifi-
cantly increased in a2A/a2CARKO mice and AET effectively
reduced it to WT levels (Figure 4A). As mentioned above,
proteasome activity was increased in 7 month-old a2A/a2CARKO
mice (Figure 4B) even though no changes were observed in
proteasome subunits expression (data not shown). AET signifi-
cantly reduced proteasomal activity, restoring the WT pattern
(Figure 4B). Importantly, normal levels of protein carbonylation
accompanied restoration of ubiquitinated protein levels and
proteasome activity in trained mice (Figure 4).
AET Effects on Skeletal Muscle Trophicity and Function in
To test whether afore-mentioned effects of AET on UPS
components were associated with improved skeletal muscle
phenotype, plantaris CSA, running capacity and rotarod perfor-
mance were assessed. Plantaris atrophy and skeletal muscle global
dysfunction were completely prevented by AET in our HF model
(Figure 5), confirming improved skeletal muscle phenotype by the
intervention. Representative histological images are shown in
Information S4. Additionally, our group demonstrated in previous
publications that AET was able to prevent exercise intolerance,
metabolic impairment (e.g. maximal activity of citrate synthase
and hexokinase), fiber type shift toward type II fibers and calcium
handling disturbances on skeletal muscle in the same animal
model used in the present investigation [21,25].
Physiological AET Effects in Human HF
HF patients submitted to AET showed increased peak oxygen
uptake when compared with untrained HF patients (Table 3).
Likewise, work economy at submaximal intensity was also
improved in trained patients (9.460.7 vs. 7.360.4 mlO2.min-
1.kg-1in sedentary and trained HF patients, respectively, during
exercise at a fixed submaximal intensity; p,0.05).
Skeletal Muscle Proteasome Activation in Human HF and
Effects of AET
Similarly to observed in HF mice, chymotrypsin-like protea-
some activity in skeletal muscle biopsies from HF patients was
increased in comparison with healthy individuals (Figure 6A).
Importantly, skeletal muscle from trained patients presented
normal proteasome activity (Figure 6A). Protein ubiquitination
and carbonylation were unchanged among groups (Figures 6B and
A wealth of data suggests AET as a key intervention for
prevention and treatment in cardiology. Recent reports demon-
strate protection provided by AET against HF-induced skeletal
myopathy [20,21,25]. However, the molecular mechanisms by
which AET delay or reverse skeletal muscle myopathy in HF
Table 1. a2A/a2CARKO mice physiological parameters.
3 month-old 5 month-old7 month-old
WT ARKOWT ARKO WTARKO
Body mass (g)2660.5 2560.4 2760.52960.33060.32860.3
FS, %2160.51760.2 2260.4 1560.3*2260.71560.5*
LVEDD (mm) 0.3860.01 0.3860.010.3960.010.4060.0010.3860.01 0.4160.01*
Distance run (m)403628362619 397610 331613355618 235621*
Body mass, left ventricular fractional shortening (FS), left ventricular end-diastolic diameter (LVEDD) and running performance in 3, 5 and 7 month-old wild type (WT)
and a2A/a2CARKO mice. Data were analyzed by one-way ANOVA followed by Duncan post hoc test and are presented as mean 6 standard error from mean. *p,0.05 vs.
Exercise Training Reduces UPS Activation in HF
PLoS ONE | www.plosone.org5August 2012 | Volume 7 | Issue 8 | e41701
remain elusive. Several key findings emerge from this study, in
which we analyzed the contribution of AET in preventing
plantaris atrophy in sympathetic hyperactivity induced-HF mice:
(a) progressive skeletal muscle loss in a2A/a2CARKO mice was
associated with increasingly oxidative stress and proteasomal
activity; (b) UPS overactivation and oxidative damage were
detected when plantaris muscle atrophy was established in 7
month-old a2A/a2CARKO mice; (c) AET efficiently reestablished
plantaris phenotype, UPS and oxidative stress to WT levels. In
human HF: (d) increased skeletal muscle proteasome activity
suggests overactivation of UPS and (e) AET restored proteasome
activity to healthy control levels.
Overwhelming evidence demonstrates a striking association
between disease-induced skeletal muscle atrophy and UPS
activation , which also occurs in HF [34,35]. In fact,
ubiquitination of skeletal muscle contractile proteins has been
suggested in HF . In line with these findings, we demonstrate
that plantaris atrophy in HF mice is associated with UPS
overactivation and that these phenomena display interesting
relationship with severity of the disease, since skeletal myopathy
was associated with worsening cardiac function and clinical signs
At 3 months of age, when a2A/a2CARKO mice display
preserved cardiac function and exercise tolerance, plantaris
hypertrophy is observed in comparison with WT, which might
be explained by b2-adrenoceptor overactivation due to sympa-
thetic hyperactivity, as previously demonstrated . Moreover,
decreased proteasomal activity in 3 month-old a2A/a2CARKO
mice indicates reduced skeletal muscle proteolysis, favoring muscle
hypertrophy. This finding might also be related to b2-adrenocep-
tor overactivation due to sympathetic hyperactivity, which
activates hypertrophic signaling pathways besides inhibiting UPS
activation in skeletal muscle in atrophic states [37,38]. At 5 months
of age, skeletal muscle hypertrophy was no longer observed and
Figure 2. Plantaris trophicity, oxidative stress and chymotrypsin-like proteasomal activity in a2A/a2CARKO mice. Plantaris muscle
cross-sectional area (CSA) (A), lipid hydroperoxidation (B), protein carbonylation (C) and chymotrypsin-like proteasome activity (D) in 3, 5 and 7
month-old wild type (WT) and a2A/a2CARKO mice (ARKO). Immunobloting data are shown as percentage of age-matched WT group (set to 100%)).
Representative images of immunoblots are shown below respective charts. Data are presented as mean 6 standard error from mean. *p,0.05 vs.
Table 2. Cardiac function and lung water content.
FS, %2461 1461* 1961
Lung wet/dry ratio5,5760,136,5860,49* 5,8260,22#
Left ventricular fractional shortening (FS) and lung wet/dry ratio in 7 month-old
wild type (WT), untrained (ARKO) and trained a2A/a2CARKO mice ARKOT. Data
are presented as mean 6 standard error from mean.
*p,0.05 vs. age-matched WT.
#p,0,05 vs. age-matched ARKO.
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proteasomal activity was similar between a2A/a2CARKO and
WT. However, when severe HF was established in 7 month-old
a2A/a2CARKO mice, plantaris muscle atrophy and substantial
proteasomal overactivation were detected. Among the possible
mechanisms contributing to these observations, oxidative stress
should be highlighted, since increasing lipid hydroperoxidation
and protein carbonylation from 3 to 7 months of age were
observed in a2A/a2CARKO mice and redox unbalance is known
to modulate UPS activation and leads to skeletal muscle atrophy.
Increased oxidative stress arises from imbalance between pro-
and antioxidant activity  and is depicted in skeletal muscle
under catabolic or dysfunctional states [6,7,40,41]. Importantly,
while increased oxidative stress through superoxide dismutase
(SOD) deletion accelerates aging-induced skeletal muscle atrophy
, antioxidant treatment effectively attenuates skeletal muscle
loss in a cancer model . Therefore, strong evidence of redox
imbalance-induced skeletal muscle atrophy supports our hypoth-
esis that oxidative damage is a major determinant of skeletal
muscle loss in HF.
UPS modulation by redox balance depends upon oxidative
damage extension. Protein damage by mild or moderate redox
imbalance increases UPS substrate availability, causing elevation
of proteasome activity . Conversely, severe oxidative damage
impairs substrate tagging by E3 ligases and causes proteasomal
dysfunction due to accumulation of non-degradable aggregates
, resulting in overall UPS inactivation. Therefore, we suggest
that skeletal muscle oxidative stress reaches moderate levels in 7
month-old a2A/a2CARKO mice, since accumulation of lipid
hydroperoxides and carbonylated proteins were observed con-
comitantly with UPS overactivation.
Studies suggest proteasome inhibition as a treatment against
skeletal muscle loss [43,44]. However, potentially dangerous effects
of such intervention must be considered, since the UPS is a major
effector of the protein quality control mechanism in all cells
[45,46]. In fact, cardiac dysfunction occurs when the proteasome
is inhibited in vivo . In contrast, AET undoubtedly promotes
beneficial effects in several tissues, including cardiac and skeletal
muscles in HF [18,48]. Thus, we have recently shown that AET
prevents skeletal muscle atrophy in our experimental model ,
and here we extend our findings to the preventive effect of AET on
oxidative stress and UPS overactivation.
Restoration of redox balance by AET is probably driven by
antioxidant defense, such as augmented activity of free radical
scavengers and reduced levels of inflammatory cytokines [49,50].
Figure 3. mRNA levels of UPS components. mRNA levels of E3 ligases E3-a, MuRF-1 and Atrogin-1 (A), and deubiquitinating enzymes USP28,
USP19 and USP14 (B). WT values were arbitrarily set to 1.0 and are represented by the dashed line. Data are shown as fold change over wild type
control group and presented as mean 6 standard error from mean. *p,0.05 vs. WT; #p,0.05 vs. age-matched ARKO.
Figure 4. UPS activation and effects of AET. Protein ubiquitination (A), chymotrypsin-like proteasome activity (B) and protein carbonylation (C)
in 7 month-old wild type (WT), untrained a2A/a2CARKO (ARKO) and trained a2A/a2CARKO mice (ARKOT). Representative images of immunoblots are
shown below respective charts. Data are shown as percentage of WT control group values (set to 100%) and presented as mean 6 standard error
from mean. *p,0.05 vs. age-matched WT; #p,0.05 vs. age-matched ARKO.
Exercise Training Reduces UPS Activation in HF
PLoS ONE | www.plosone.org7 August 2012 | Volume 7 | Issue 8 | e41701
Accordingly, we demonstrate here that AET reduced skeletal
muscle lipid hydroperoxidation and protein carbonylation,
accounting for reduced intracellular stress and relief of UPS
overload. Therefore, reduced UPS activation by AET possibly
occurred due to improvements in redox balance. These results
suggest that AET ultimately counteracts increased protein
degradation by the UPS.
Besides UPS overactivation by oxidative stress, it may also be
purposed that redox unbalance is involved in HF-induced skeletal
myopathy by reducing skeletal muscle regenerative capacity
through disturbance of satellite cell pool or differentiation rate
[51,52]. Indeed, direct negative effects of oxidative stress on
skeletal muscle satellite cells have been reported . Further-
more, it has been shown that HF patients present depressed IGF-1
signaling in skeletal muscle  and that anabolic effects of IGF-1
are partially attributed to satellite cells activation [55,56].
Following this rationale and considering that satellite cells
activation is the leading process mediating muscle regeneration,
it is also reasonable to speculate that anti-atrophic effects of AET
could be blunted in our model, even though we observed several
beneficial outcomes. In this sense, investigation of satellite cells
participation in cardiac cachexia is a promising topic for future
In human HF, AET also improved aerobic capacity (VO2peak)
and work economy, which were mainly due to skeletal muscle
improvements, since cardiac function did not differ between
sedentary and trained HF patients (2865 vs. 3562% are EF
values in sedentary and trained HF patients, respectively; p.0.05).
Importantly, skeletal muscle UPS overactivation is suggested by
increased proteasome activity in sedentary HF patients, corrob-
orating findings of a recent study that presented increased
abundance of MuRF1 in skeletal muscle in a larger population
of HF patients, regardless of age . This same study also showed
that MuRF1 expression is reduced by AET, which goes in line
with our finding that AET reverted 26 S Proteasome over-
activation in HF patients.
Increased proteasomal activity was not accompanied by in-
creased protein carbonylation, indicating that our HF patients
displayed only mild skeletal muscle oxidative stress, but sufficient
to induce myofibrillar protein damage and proteasomal activation.
It is important to highlight that all HF patients were under b-
blockade, ACE inhibition and statins, which independently
provide antioxidant effects [58-60] and may have relieved skeletal
muscle oxidative stress. However, we reinforce the role of AET in
HF treatment by showing that even optimal drug treatment does
not improve aerobic capacity and could not maintain skeletal
muscle proteasomal activity, which is clearly achieved by AET.
Our study shows that AET reduced oxidative stress, UPS
overactivation and prevented skeletal muscle atrophy in HF mice,
however, it does not provide direct evidence of cause-effect among
these findings. However, our hypothesis that relieved oxidative
stress counteracts UPS overactivation is partly supported by the
literature . One might argue that our sympathetic hyperac-
Figure 5. Skeletal muscle structure and global function. Plantaris
cross-sectional area (CSA) (A), running performance (B) and time on
rotarod (C) in 7 month-old wild type (WT), untrained a2A/a2CARKO
(ARKO) and trained a2A/a2CARKO mice (ARKOT). Data are presented as
mean 6 standard error from mean. *p,0.05 vs. age-matched WT;
#p,0.05 vs. age-matched ARKO.
Table 3. Patient physiological parameters.
Age, years72.261.675.066.5 75.065.5
24.161.1 25.562.2 24.763.0
VO2peak, mLO2.kg-1.min-137.062.912.460.5* 14.560.4*#
RER at VO2peak 1.0560.01 1.1260.01 1.1060.02
b-Blockers0/6 5/5 6/6
Age, body-mass index (BMI), peak oxygen uptake (VO2peak), respiratory
exchange ratio (RER) at VO2peak and used medication in healthy individuals
(Control), sedentary heart failure patients (HF-S) and trained heart failure
patients (HF-T). Data are presented as mean 6 standard error from mean.
*p,0.05 vs. Control.
#p,0.05 vs. HF-S.
Exercise Training Reduces UPS Activation in HF
PLoS ONE | www.plosone.org8 August 2012 | Volume 7 | Issue 8 | e41701
tivity-induced HF model is not the most similar to human HF,
however, we provided strong evidence that the progression of HF
in our model recapitulates many aspects of human HF . Since
enrolled HF patients were under optimal pharmacological
therapy, we could not isolate the effects of AET. Additionally,
small biopsy fragments didn’t allow further exploration of UPS
modulation and evaluation of mild oxidative stress indicators, such
as lipid hydroperoxidation. Left ventricular FS values in WT mice
were lower than found in our previous work , which probably
occurred due to the anesthetic agent used in the present study
(halothane presently used vs. isoflurane previously used ). Even
though halothane depresses cardiac contractility in a higher degree
than isoflurane , we were able to reproduce the same pattern
of cardiac dysfunction in our HF model and the effects of exercise
In conclusion, we provide evidence that AET prevents skeletal
muscle oxidative stress and UPS activation in HF mice, which
probably contributes to prevention of skeletal myopathy. The
clinical relevance of the present investigation is demonstrated by
attenuation in skeletal muscle proteasome activity in exercise-
trained HF patients, which is not achieved by drug treatment itself.
Altogether these findings strengthen AET as an efficient non-
pharmacological tool for HF therapy.
CONSORT checklist for non-pharmaco-
Primer sequence used for real-time
untrained a2A/a2CARKO (ARKO) and trained a2A/
a2CARKO mice (ARKOT) after starting experimental
protocol, when mice were 5 month-old. ***p=0.002 vs.
Cumulative survival of wild type (WT),
untrained wild type (WT) and a2A/a2CARKO (ARKO)
mice at 3, 5 and 7 months of age and trained a2A/
a2CARKO mice (ARKOT) at 7 months of age. Muscle
sections were prepared and analyzed as described in the Methods
section of the manuscript. Dashed lines represent the location of
plantaris muscle. Same magnification (50x) was applied to all
Representative histological images of
We thank Alex Monteiro, Katt Mattos, Marcele Coelho and Carlos B. Jr
for technical assistance and Luiz Bechara for support in discussion
regarding oxidative stress.
Conceived and designed the experiments: TFC PCB JCBF ASM CEN.
Performed the experiments: TFC NAP JCC JBNM JCBF MLL AVNB.
Analyzed the data: TFC JBNM JCC PCB NAP JCBF AVNB MLL.
Contributed reagents/materials/analysis tools: ASM CEN UW. Wrote the
paper: TFC JBNM PCB JCBF.
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