J Appl Physiol 109: 702–709, 2010.
First published July 1, 2010; doi:10.1152/japplphysiol.00281.2010.
Aerobic exercise training improves skeletal muscle function and Ca2⫹
handling-related protein expression in sympathetic hyperactivity-induced
heart failure
C. R. Bueno Jr., J. C. B. Ferreira, M. G. Pereira, A. V. N. Bacurau, and P. C. Brum
School of Physical Education and Sport, University of São Paulo, São Paulo, Brazil
Submitted 17 March 2010; accepted in final form 30 June 2010
cardiac diseases; muscular abnormalities; treadmill running; Ca2⫹
transient
HEART FAILURE (HF) is a clinical syndrome characterized by a
marked decrease in exercise tolerance and a distinct muscular
weakness. HF patients are commonly limited by exertional
fatigue during both normal daily activities and maximal exercise testing (23). Over the last decades, skeletal muscle fatigue
in HF patients has been presumed to be a consequence of
reduced blood flow secondary to decreased cardiac output (31).
However, severity of exercise intolerance has been poorly
correlated with some cardiovascular indexes such as stroke
Address for reprint requests and other correspondence: P. C. Brum, Escola
de Educação Física e Esporte da Universidade de São Paulo, Departamento de
Biodinâmica do Movimento do Corpo Humano, Av. Professor Mello Moraes,
65-Butantã, São Paulo, SP, CEP 05508-900 Brazil (e-mail: [email protected]).
702
volume, ejection fraction, and maximal cardiac output in HF
(10, 30). Additionally, increases in cardiac function and oxygen delivery to skeletal muscle by oxygen supplementation
failed to increase exercise tolerance and fatigue resistance in
HF (29). These observations suggest that muscle fatigue, at
least in some HF patients, may be due to intrinsic skeletal
muscle changes rather than insufficient skeletal muscle blood
flow.
Among several skeletal muscle abnormalities detected in
HF patients, such as muscle atrophy, impaired metabolic
response, and reduced mitochondrial function, alterations in
excitation-contraction coupling have been proposed to explain the muscle fatigue process seen during HF. Previous
studies have suggested that depressed sarcoplasmic Ca2⫹
levels and diminished rate of sarcoplasmic reticulum Ca2⫹
release and reuptake contribute to increased fatigue in HF.
In fact, reduced sarcoplasmic reticulum Ca2⫹ release (19)
and reuptake (12) were observed in fiber bundles and intact
fibers from fast-twitch muscle in severe HF rats. Skeletal
muscle of post-myocardial infarction rats displayed ryanodine receptor (RyR) hyperphosphorylation and impaired
Ca2⫹ release. In contrast, sarcoplasmic vesicles isolated
from skeletal muscle of moderate HF rats displayed accelerated Ca2⫹ release and reuptake (22, 28).
Aerobic exercise training is considered an efficient adjuvant therapy for HF, and its impact on skeletal muscle is
remarkable. In fact, exercise training was able to reverse
skeletal muscle myopathy by preventing muscle atrophy,
capillary rarefaction associated with improved skeletal muscle oxidative capacity, and decreased oxidative stress in HF
rodents (1, 11). We previously demonstrated (6) that aerobic
exercise training in nonfailing mice improves the Ca2⫹
handling of skeletal muscles comprising different fiber compositions. Similar results were recently demonstrated in
biopsied vastus lateralis of healthy humans (16) submitted
to leg extension. However, the effect of aerobic exercise
training on skeletal muscle Ca2⫹ handling in HF still needs
to be better understood.
We previously reported (1, 2, 7) that mice lacking ␣2A and
␣2C adrenoceptors (␣2A/␣2CARKO) develop sympathetic hyperactivity-induced HF related to exercise intolerance, established skeletal muscle myopathy, and cardiac dysfunction with
clinical signs of HF at 7 mo of age. The present study was
undertaken to evaluate the effect of aerobic exercise training on
expression of proteins involved in Ca2⫹ release and reuptake
by sarcoplasmic reticulum of skeletal muscles comprising
different fiber compositions in ␣2A/␣2CARKO mice. We tested
the hypothesis that exercise training would improve exercise
tolerance and skeletal muscle force in HF mice associated with
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Bueno CR Jr, Ferreira JC, Pereira MG, Bacurau AV, Brum
PC. Aerobic exercise training improves skeletal muscle function and
Ca2⫹ handling-related protein expression in sympathetic hyperactivity-induced heart failure. J Appl Physiol 109: 702–709, 2010. First
published July 1, 2010; doi:10.1152/japplphysiol.00281.2010.—The
cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart
failure (HF) remain unclear. We investigated potential Ca2⫹ abnormalities in skeletal muscles comprising different fiber compositions
and investigated whether aerobic exercise training would improve
muscle function in a genetic model of sympathetic hyperactivityinduced HF. A cohort of male 5-mo-old wild-type (WT) and congenic
␣2A/␣2C adrenoceptor knockout (ARKO) mice in a C57BL/6J genetic
background were randomly assigned into untrained and trained
groups. Exercise training consisted of a 8-wk running session of 60
min, 5 days/wk (from 5 to 7 mo of age). After completion of the
exercise training protocol, exercise tolerance was determined by
graded treadmill exercise test, muscle function test by Rotarod,
ambulation and resistance to inclination tests, cardiac function by
echocardiography, and Ca2⫹ handling-related protein expression by
Western blot. ␣2A/␣2CARKO mice displayed decreased ventricular
function, exercise intolerance, and muscle weakness paralleled by
decreased expression of sarcoplasmic Ca2⫹ release-related proteins
[␣1-, ␣2-, and ␤1-subunits of dihydropyridine receptor (DHPR) and
ryanodine receptor (RyR)] and Ca2⫹ reuptake-related proteins [sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA)1/2 and Na⫹/
Ca2⫹ exchanger (NCX)] in soleus and plantaris. Aerobic exercise
training significantly improved exercise tolerance and muscle function
and reestablished the expression of proteins involved in sarcoplasmic
Ca2⫹ handling toward WT levels. We provide evidence that Ca2⫹
handling-related protein expression is decreased in this HF model and
that exercise training improves skeletal muscle function associated
with changes in the net balance of skeletal muscle Ca2⫹ handling
proteins.
SKELETAL MUSCLE Ca2⫹ HANDLING IN HEART FAILURE TRAINED MICE
an improved net balance of Ca2⫹ handling proteins in both
soleus and plantaris muscles.
MATERIALS AND METHODS
cardiography was performed with an Acuson Sequoia model 512
echocardiographer (Acuson, Mountain View, CA) equipped with a
14-MHz linear transducer. Left ventricle systolic function was estimated by fractional shortening as follows: fractional shortening (%) ⫽
[(LVEDD ⫺ LVESD)/LVEDD] ⫻ 100, where LVEDD is left ventricular end-diastolic dimension and LVESD is left ventricular endsystolic dimension.
Skeletal muscle functional assessment. To verify whether exercise
training would improve motor ability in HF mice, we performed motor
ability tests in trained and untrained 7-mo-old WT and ␣2A/␣2CARKO
mice. Mice were submitted to the following tests: 1) the inclined plane
test measured the maximal angle of a wood board on which the animal
was placed until it slipped; 2) the ambulation test determined the mean
length of a step, measured in hind foot ink prints while mice ran freely in
a corridor (length, 50 cm; width, 8 cm; height of lateral walls, 20 cm)
(27); and 3) Rotarod (IITC Life Science, Woodland Hills, CA), in which
the mice were placed on the rod, which was rotating at an initial speed of
1 rpm, the speed was gradually increased from 1 to 40 rpm over a period
of 5 min, and the time that the mice stayed on the rod was recorded. The
mice were subjected to three successive trials, and the performance of
each animal was measured as its best individual performance over the
three trials (26).
Skeletal muscle protein expression. Immunoblots of untrained and
exercise-trained 7-mo-old WT and ␣2A/␣2CARKO mouse soleus and
plantaris muscle homogenates were performed according to Towbin et
al. (25). Briefly, liquid nitrogen-frozen muscles were homogenized in
a buffer containing (in mM) 1 EDTA, 1 EGTA, 2 MgCl2, 5 KCl, 25
HEPES (pH 7.5), and 2 DTT, with 100 ␮M PMSF, 1% Triton X-100,
and protease inhibitor cocktail (1:100; Sigma-Aldrich, St. Louis,
MO). Samples were loaded and subjected to SDS-PAGE in polyacrylamide gels (10%). After electrophoresis, proteins were electrotransferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Equal loading of samples (25 ␮g) and even transfer
efficiency were monitored with the use of 0.5% Ponceau S staining of
the blotted membrane. The blotted membrane was then incubated in a
blocking buffer (5% nonfat dry milk, 10 mM Tris·HCl, pH 7.6, 150
mM NaCl, and 0.1% Tween 20) for 2 h at room temperature and then
incubated with a specific antibody overnight at 4°C. Mouse monoclonal antibodies to sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA)1
(1:2,500), SERCA2 (1:2,500), Na⫹/Ca2⫹ exchanger (NCX) (1:1,000),
dihydropyridine receptor (DHPR)␣1 subunit (1:500), DHPR␣2 subunit
(1:500), DHPR␤1 subunit (1:500), and RyR (1:500) were obtained from
Affinity BioReagents (Golden, CO) and rabbit polyclonal parvalbumin
(1:2,000) from Sigma-Aldrich. Binding of the primary antibody was
detected with the use of peroxidase-conjugated secondary antibodies
(anti-rabbit, 1:3,000, for 1.5 h at room temperature) and developed with
enhanced chemiluminescence (Amersham Biosciences) detected by autoradiography. Quantification analysis of blots was performed with the
use of Scion Image software (Scion based on NIH Image). Targeted
bands were normalized to ␣-tubulin antibody (1:1,000; Santa Cruz
Biotechnology).
Statistical analysis. Data are presented as means ⫾ SE. Two-way
ANOVA with post hoc testing by Tukey (Statistica software, StatSoft,
Tulsa, OK) was used to compare the effect of training (untrained and
exercise trained) and genotype (WT and ␣2A/␣2CARKO) on fractional
shortening, HR, BP, body weight, lung wet-to-dry ratio, distance run,
step length, fall angle, Rotarod, and protein expression levels. Statistical significance was considered achieved when the value of P was
⬍0.05.
RESULTS
Fig. 1. Experimental design. WT, wild-type mice; KO, ␣2A/␣2C adrenoceptor
knockout (ARKO) mice.
J Appl Physiol • VOL
Exercise training reverses skeletal muscle dysfunction in
heart failure mice. Physiological parameters of untrained and
exercise-trained WT and ␣2A/␣2CARKO mice are presented
in Table 1. Untrained ␣2A/␣2CARKO mice displayed lower
fractional shortening, tachycardia, and increased lung wet-
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Study population. A cohort of male congenic ␣2A/␣2CARKO mice
in a C57BL/6J genetic background and their wild-type (WT) controls
aged 5–7 mo were studied. At 5 mo of age, ␣2A/␣2CARKO mice
display dysfunction associated with exercise intolerance (15). At 7 mo
of age, they present severe cardiac dysfunction associated with exercise intolerance, established skeletal muscle myopathy, and increased
mortality rate (1, 2, 7). Mice were maintained in a 12:12-h light-dark
cycle and a temperature-controlled environment (22°C) with free
access to standard laboratory chow (Nuvital Nutrientes, Curitiba, PR,
Brazil) and tap water. This study was in accordance with Ethical
Principles in animal research adopted by the Brazilian College of
Animal Experimentation (www.cobea.org.br.). In addition, this study
was approved by the University of São Paulo Ethical Committee (CEP
no. 2007/028). The experimental design is shown in Fig. 1.
Exercise training protocol. To verify whether exercise training
could improve skeletal muscle Ca2⫹ handling in 7-mo-old ␣2A/
␣2CARKO mice, we performed exercise training in ␣2A/␣2CARKO
and WT mice aging from 5 to 7 mo of age. Exercise training
consisted of 8 wk of running on a motor treadmill (ESD model 01
FUNBEC), 5 days/wk, for 60 min at maximal lactate steady-state
workload, as described elsewhere (8). All untrained mice were
exposed to treadmill exercise (5 min) three times a week to become
accustomed to the exercise protocol and handling.
Graded treadmill exercise test. Exercise capacity, estimated by
total distance run, correlates with skeletal muscle work capacity, and
it is a method used for detecting exercise intolerance in HF. Exercise
tolerance was evaluated with a graded treadmill exercise protocol for
mice as previously described (8). Briefly, after being adapted to
treadmill exercises over a week (10 min of exercise session), mice
were placed in the treadmill streak and allowed to acclimatize for at
least 30 min. Intensity of exercise was increased by 3 m/min (6 –33
m/min) every 3 min at 0% grade until exhaustion. The graded
treadmill exercise test was performed in WT and ␣2A/␣2CARKO mice
after completion of exercise training protocol at 7 mo of age.
Cardiovascular measurements. Resting blood pressure (BP) and
heart rate (HR) were determined noninvasively with a computerized
tail-cuff system (BP 2000 Visitech Systems, Apex, NC) described
elsewhere (4). Mice were acclimatized to the apparatus during daily
sessions over 4 days, 1 wk before starting the experimental period.
Noninvasive ventricular function was assessed by two-dimensional
guided M-mode echocardiography in halothane-anesthetized WT and
␣2A/␣2CARKO mice at 7 mo of age. Briefly, mice were positioned in
the supine position with front paws wide open, and an ultrasound
transmission gel was applied to the precordium. Transthoracic echo-
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SKELETAL MUSCLE Ca2⫹ HANDLING IN HEART FAILURE TRAINED MICE
Table 1. Physiological parameters in untrained and
exercise-trained wild-type and ␣2A/␣2CARKO mice
Parameter
WTun
(n ⫽ 6)
FS, %
HR, beats/min
BP, mmHg
BW, g
Lung wet-to-dry ratio
31.2 ⫾ 1.4
585 ⫾ 6
112 ⫾ 6
28.6 ⫾ 0.9
5.5 ⫾ 0.1
WTtr
(n ⫽ 5)
KOun
(n ⫽ 5)
31.3 ⫾ 2.1 24 ⫾ 1.3*‡
518 ⫾ 5* 687 ⫾ 5*‡
113 ⫾ 5
113 ⫾ 5
27.2 ⫾ 0.4 26.4 ⫾ 1.1
5.8 ⫾ 0.1 6.6 ⫾ 0.5*‡
KOtr
(n ⫽ 6)
30.7 ⫾ 1.3†
598 ⫾ 6†‡
108 ⫾ 6
25.9 ⫾ 0.5
5.8 ⫾ 0.2†
Data are presented as means ⫾ SE for n mice. WT, wild-type mice; KO,
␣2A/␣2C adrenoceptor knockout (ARKO) mice; un, untrained; tr, trained; FS,
fractional shortening; HR, heart rate; BP, blood pressure; BW, body weight. *P ⬍
0.05 vs. WTun; †P ⬍ 0.05 vs. KOun; ‡P ⬍ 0.05 vs. WTtr.
Fig. 2. Exercise training increases skeletal muscle
function of heart failure mice. Exercise capacity
represented by maximal distance run (A), step
length (B), fall angle (C), and Rotarod test (D),
used as indexes of skeletal muscle function, were
evaluated at 7 mo of age in untrained and exercisetrained WT and ␣2A/␣2CARKO mice. Sample size
is indicated in the columns. Note that exercise
training significantly improved distance run, step
length, and fall angle in ␣2A/␣2CARKO mice. Data
are presented as means ⫹ SE. *P ⬍ 0.05 vs.
untrained WT mice; ‡P ⬍ 0.05 vs. untrained ␣2A/
␣2CARKO mice.
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to-dry ratio compared with WT mice. No changes in BP and
body weight were observed among groups. Exercise training
increased cardiac function, decreased resting HR, and reduced lung wet-to-dry ratio in ␣2A/␣2CARKO mice toward
the values in WT mice. Of interest, untrained ␣ 2A /
␣2CARKO mice also displayed exercise intolerance, shorter
step length, reduced fall angle, and diminished time in the
Rotarod test, which are good markers of skeletal muscle
dysfunction, compared with WT mice (Fig. 2). As expected,
exercise training improved exercise capacity and skeletal
muscle function in ␣2A/␣2CARKO mice (Fig. 2). Trained
WT mice improved exercise tolerance compared with the
untrained WT group.
Exercise training restores expression level of proteins involved in sarcoplasmic Ca2⫹ release in skeletal muscle from
heart failure mice. Since impaired Ca2⫹ release from the
sarcoplasmic reticulum has been identified as a contributor to
skeletal muscle fatigue in HF (24), we investigated whether the
expression of different DHPR subunits and RyR are altered in
soleus and plantaris of our ␣2A/␣2CARKO mice and whether
exercise training would change their expression profile.
DHPR␣1 subunit expression levels were decreased in
soleus of untrained ␣2A/␣2CARKO mice compared with WT
mice (Fig. 3B), while no changes in plantaris were observed
among groups (Fig. 4B). DHPR␣2 levels were significantly
reduced in plantaris of untrained ␣2A/␣2CARKO mice (Fig.
4C). DHPR␤1 subunit expression levels were significantly
reduced in both soleus and plantaris of untrained ␣2A/
␣2CARKO compared with WT control mice (Figs. 3D and
4D). RyR expression levels were significantly decreased in
soleus of untrained ␣2A/␣2CARKO mice compared with WT
control mice (Fig. 3E). No changes in plantaris RyR levels
were observed between untrained ␣2A/␣2CARKO and WT
groups (Fig. 4E). Of interest, exercise training reestablished
the expression of altered Ca2⫹ handling proteins in soleus
and plantaris of ␣2A/␣2CARKO mice toward untrained WT
values (Figs. 3 and 4). Exercise training also increased
DHPR␤1 levels in plantaris (Fig. 4D) and RyR levels in both
soleus and plantaris (Figs. 3E and 4E) of trained WT mice.
These data suggest that exercise training efficiently reverses
changes in expression level of proteins involved in sarco-
SKELETAL MUSCLE Ca2⫹ HANDLING IN HEART FAILURE TRAINED MICE
705
plasmic Ca2⫹ release in both soleus and plantaris of HF
mice.
Exercise training improves expression level of proteins involved in sarcoplasmic Ca2⫹ reuptake in skeletal muscle from
heart failure mice. Considering that a reduction in magnitude
and a slowing of sarcoplasmic reticulum Ca2⫹ reuptake are
associated with reductions in force and skeletal muscle
fatigue in HF (19), we investigated whether the expression
of SERCA1, SERCA2, NCX, and parvalbumin are altered in
soleus and plantaris of our ␣2A/␣2CARKO mice and whether
exercise training would change their expression profile.
As depicted in Fig. 5, untrained ␣2A/␣2CARKO mice
displayed a prominent reduction in SERCA2 and SERCA1
expression levels in soleus (Fig. 5B) and plantaris (Fig. 5E),
respectively, compared with WT control mice. NCX levels
were reduced in both soleus and plantaris of untrained
␣2A/␣2CARKO mice compared with the WT group (Fig. 5,
J Appl Physiol • VOL
C and F). In addition, the expression levels of parvalbumin,
a well-known cytosolic Ca2⫹ buffer, were significantly
elevated in untrained ␣2A/␣2CARKO mice compared with
the untrained WT group (Fig. 5G). Interestingly, exercise
training notably increased SERCA2 and SERCA1 expression levels in soleus and plantaris of ␣2A/␣2CARKO mice,
respectively, toward untrained WT levels (Fig. 5, B and E).
Exercise training also elevated SERCA2 and SERCA1 levels in the trained WT group. NCX expression levels were
significantly improved in trained ␣2A/␣2CARKO and WT
groups compared with genotype-matched untrained mice
(Fig. 5, C and F). Exercise training had a significant effect
in parvalbumin expression levels in plantaris of trained WT
mice (Fig. 5G). These results suggest that HF-induced
changes in skeletal muscle expression levels of proteins
involved in sarcoplasmic Ca2⫹ reuptake are reversed by
exercise training in soleus and plantaris muscles.
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Fig. 3. Exercise training improves expression level
of proteins involved in sarcoplasmic Ca2⫹ release
in soleus from heart failure mice. A: representative
blots show the effect of exercise training on the
levels of proteins related to sarcoplasmic Ca2⫹
release. DHPR, dihydropyridine receptor; RyR, ryanodine receptor. B–E: soleus levels of DHPR␣1
subunit (B), DHPR␣2 subunit (C), DHPR␤1 subunit
(D), and RyR (E) in untrained and exercise-trained
WT and ␣2A/␣2CARKO mice. Sample size is indicated in the columns. Data are presented after
normalization against ␣-tubulin. *P ⬍ 0.05 vs.
untrained WT mice (WTun); ‡P ⬍ 0.05 vs. untrained KO mice.
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SKELETAL MUSCLE Ca2⫹ HANDLING IN HEART FAILURE TRAINED MICE
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Fig. 4. Exercise training augments expression level
of proteins involved in sarcoplasmic Ca2⫹ release
in plantaris from heart failure mice. A: representative blots show the effect of exercise training on the
levels of proteins related to sarcoplasmic Ca2⫹
release. B–E: plantaris levels of DHPR␣1 subunit
(B), DHPR␣2 subunit (C), DHPR␤1 subunit (D),
and RyR (E) in untrained and exercise-trained WT
and ␣2A/␣2C ARKO mice. Sample size is indicated
in the columns. Data are presented after normalization against ␣-tubulin. *P ⬍ 0.05 vs. untrained WT
mice; ‡P ⬍ 0.05 vs. untrained KO mice.
DISCUSSION
Our results, based on a well-established aerobic exercise
training protocol, show that exercise training-induced
changes in the net balance of skeletal muscle Ca2⫹ handling
proteins contribute to improved exercise tolerance and muscle performance in sympathetic hyperactivity-induced HF
mice. The main findings of the present study are that
exercise training reestablished the expression levels of proteins involved in sarcoplasmic Ca2⫹ release (DHPR␣1,
DHPR␣2, DHPR␤1, and RyR) and reuptake (SERCA1,
SERCA2, and NCX) in soleus and plantaris muscles of
␣2A/␣2CARKO mice toward untrained WT values (see Fig.
6, a schematic illustration of the main results of the present
study).
Large-scale epidemiologic studies demonstrated that low
aerobic exercise capacity in subjects with cardiovascular disease is a stronger predictor of mortality than other established
risk factors (3, 5). Therefore, the mechanisms underlying
J Appl Physiol • VOL
exercise intolerance in HF are of main interest. Accumulated
evidence indicates skeletal muscle myopathy as a main contributor to reduced exercise capacity in HF. Muscles are often
atrophied with reduced muscle oxidative capacity and impaired
contractile properties, which further culminates in muscle fatigue. We previously demonstrated (1) that sympathetic hyperactivity-induced HF in mice is associated with skeletal muscle
myopathy, and here we provide evidence for an imbalanced
expression of Ca2⫹ handling protein-related muscular dysfunction in HF mice.
The process involved in excitation-contraction coupling
in skeletal muscle is initiated when T tubules are depolarized, leading to conformational changes in the DHPR-RyR
complex, leading to Ca2⫹ ion flow to the sarcoplasm, which
triggers contraction. After contraction, SERCA performs the
critical function of promoting muscle relaxation by sequestering Ca2⫹ from the sarcoplasm at the expense of ATP
hydrolysis.
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SKELETAL MUSCLE Ca2⫹ HANDLING IN HEART FAILURE TRAINED MICE
707
We showed that sympathetic hyperactivity-induced HF
mice displayed significant decrease in expression profile of
proteins involved in both sarcoplasmic reticulum Ca2⫹ release (different DHPR subunits and RyR) and reuptake
(SERCA and NCX) in skeletal muscles comprising different
fiber type compositions. This phenomenon was associated
with muscle weakness and exercise intolerance. However,
other studies observed accelerated Ca2⫹ release and reuptake (22, 28) or even increased RyR and SERCA protein
levels that were not paralleled by changes in Ca2⫹ homeostasis and tetanic force (13) in moderate HF rats. These
apparent contrasting results might be related to HF etiology
J Appl Physiol • VOL
or severity. In fact, some studies reported that during the
time course of myocardial infarction-induced cardiac dysfunction in rats increased SERCA expression and activity in
the early stage was reversed to decreased SERCA expression and activity and impaired Ca2⫹ homeostasis in the late
stage associated with HF (13, 21, 28). Our data in severe HF
mice support, at least in part, the hypothesis that decreases
in expression of sarcoplasmic reticulum Ca2⫹-related proteins are associated with muscle weakness and fatigue in
chronic HF.
Aerobic exercise training is a potent adjuvant therapy for
HF, with positive impact in both cardiac and skeletal mus-
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Fig. 5. Exercise training improves expression
level of proteins involved in sarcoplasmic Ca2⫹
reuptake in soleus and plantaris from heart failure mice. A: representative blots show the effect
of exercise training on the levels of proteins
related to sarcoplasmic Ca2⫹ reuptake in soleus.
B and C: soleus levels of sarco(endo)plasmic
reticulum Ca2⫹-ATPase (SERCA)2 (B) and
Na⫹/Ca2⫹ exchanger (NCX) (C). D: representative blots show the effect of exercise training
on the levels of proteins related to sarcoplasmic
Ca2⫹ reuptake in plantaris. E–G: SERCA1 (E),
NCX (F), and parvalbumin (G) in untrained and
exercise-trained WT and ␣2A/␣2CARKO mice.
Sample size is indicated in the columns. Data
are presented after normalization against ␣-tubulin. *P ⬍ 0.05 vs. untrained WT mice; ‡P ⬍
0.05 vs. untrained KO mice.
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SKELETAL MUSCLE Ca2⫹ HANDLING IN HEART FAILURE TRAINED MICE
cles (1, 17). In cardiac muscle, we previously demonstrated
(15, 18, 20) that aerobic exercise training increases cardiac
contractility coupled with an improved Ca2⫹ homeostasis in
both mild and severe HF mice, which highlights the preventive and therapeutic impact of aerobic exercise training on
cardiac function in HF. Regarding skeletal muscles, it is
known that aerobic exercise training improves exercise
tolerance in both animal and human HF, mainly because of
improvements in skeletal muscle function (1, 5). However,
the mechanisms underlying the skeletal muscle gain of
function after exercise training in HF are still incipient.
Recently, we demonstrated (6) that aerobic exercise training
rearranged the network of proteins involved in skeletal
muscle Ca2⫹ handling, which culminated in an increased run
performance in WT mice. Improved skeletal muscle function and
increased sarcoplasmic Ca2⫹ release were reported in healthy
humans submitted to aerobic exercise training (16). Presently, we
extend this knowledge to mice with severe HF, in which aerobic
exercise training reestablished the levels of sarcoplasmic Ca2⫹
release (DHPR␣1, DHPR␣2, DHPR␤1, and RyR) and reuptake (SERCA1, SERCA2, and NCX) in soleus and plantaris muscles toward untrained WT values.
Ca2⫹ release from the sarcoplasmic reticulum is considerably suppressed because of DHPR-RyR complex uncoupling, which further culminates in diminished Ca2⫹ spark
amplitude in HF (24). Since the expression of DHPR␣1,
DHPR␣2, and DHPR␤1 subunits are closely related to
DHPR-RyR complex stability and excitation-contraction
coupling, we hypothesized that the reestablishment of
DHPR and RyR protein levels induced by aerobic exercise
training may strengthen and stabilize the direct coupling of
the DHPR and the RyR, leading to better amplitude of Ca2⫹
currents and release of Ca2⫹ from the sarcoplasmic reticulum. Additionally, the increased expression of NCX after
aerobic exercise training in HF mice may contribute to
better contractile activity of adult skeletal muscle (9, 32),
where NCX presumably works in a reverse mode, transporting Ca2⫹ into the sarcoplasm of skeletal muscle.
J Appl Physiol • VOL
GRANTS
The study was supported by grants from Fundação de Amparo a Pesquisa
do Estado de São Paolo-Brasil (FAPESP) (05/59740-7). C. R. Bueno, Jr. held
a master fellowship from FAPESP (FAPESP 06/57836-0R). P. C. Brum holds
a scholarship from Conselho Nacional de Pesquisa e Desenvolvimento-Brasil
(CNPq, BPQ 301519/2008-0). J. C. B. Ferreira holds a post doc fellowship
from FAPESP (2009/03143-1). A. V. N. Bacurau holds a doctoral fellowship
from FAPESP (2008/50777-3).
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Fig. 6. Schematic illustration with the main results of the present study.
Similar to increased levels of Ca2⫹ release-related protein, changes in Ca2⫹ reuptake-related protein levels were
also observed after aerobic exercise training in HF mice.
SERCA levels were downregulated in HF mice, and aerobic
exercise training reestablished SERCA expression to WT
levels. This is of particular interest since skeletal muscle is
highly dependent on sarcoplasmic reticulum Ca2⫹ stores for
a proper contraction.
To our knowledge, this study is the first to show that Ca2⫹
handling proteins are differentially regulated in muscles of
HF mice comprising different fiber types, and aerobic exercise training plays an interestingly homeostatic role by
reestablishing the protein expression to WT levels. Previous
studies have reported that in HF slow-twitch muscles (e.g.,
soleus) are more susceptible to fatigue and reduction in
muscle force than fast-twitch muscles (e.g., plantaris) (14).
These responses might be related to specific properties of
proteins embedded in sarcoplasmic reticulum of slow- and
fast-twitch muscles. In fact, we showed that soleus (but not
plantaris) of HF mice displayed a significant reduction in
DHPR␣1 (main subunit of DHPR pore) and RyR levels
compared with WT mice. These changes would contribute
to disrupted signal transduction through transverse tubule/
sarcoplasmic reticulum junction (e.g., DHPR-RyR complex)
and abrogated RyR opening during excitation-contraction
coupling in slow-twitch muscles, probably anticipating fatigue response. It worth mentioning that slow-twitch muscles are more susceptible to mitochondrial Ca2⫹ overload
since sarcoplasmic buffers, such as parvalbumin, are missing. In plantaris muscle, an upregulation of parvalbumin
was observed in HF mice, probably as a compensatory
response to increased sarcoplasmic Ca2⫹ levels, and aerobic
exercise training kept parvalbumin levels increased. Of
note, aerobic exercise training also increased parvalbumin
expression levels in WT mice, which may increase sarcoplasmic Ca2⫹ buffer capacity, rendering a better contraction-relaxation synchronism.
In summary, the present findings provide evidence for the
first time that sympathetic hyperactivity-induced HF mice
display skeletal muscle dysfunction paralleled by impaired net
balance of proteins involved in sarcoplasmic Ca2⫹ release and
reuptake in muscles comprising different fiber types. Interestingly, aerobic exercise training, by rearranging the network of
Ca2⫹ handling proteins, improved exercise tolerance and muscle performance in HF mice. However, we may not exclude
that exercise training, by improving ventricular function and
muscle perfusion, could also collaborate in the increased muscle performance presently observed. Altogether these results
provide new insights on intracellular Ca2⫹ regulatory mechanisms underlying improved skeletal muscle contractility by
aerobic exercise training in HF. Further studies on other
intracellular targets are warranted.
SKELETAL MUSCLE Ca2⫹ HANDLING IN HEART FAILURE TRAINED MICE
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
16.
REFERENCES
J Appl Physiol • VOL
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
dling abnormalities in sympathetic hyperactivity-induced heart failure
mice. J Appl Physiol 104: 103–109, 2007.
Munkvik M, Rehn TA, Slettalokken G, Hasic A, Hallen J, Sjaastad I,
Sejersted OM, Lunde PK. Training effects on skeletal muscle calcium
handling in human chronic heart failure. Med Sci Sports Exerc 42:
847–855, 2009.
Oliveira RS, Ferreira JC, Gomes ER, Paixao NA, Rolim NP, Medeiros
A, Guatimosim S, Brum PC. Cardiac anti-remodelling effect of aerobic
training is associated with a reduction in the calcineurin/NFAT signalling
pathway in heart failure mice. J Physiol 587: 3899 –3910, 2009.
Pereira MG, Ferreira JC, Bueno CR Jr, Mattos KC, Rosa KT,
Irigoyen MC, Oliveira EM, Krieger JE, Brum PC. Exercise training
reduces cardiac angiotensin II levels and prevents cardiac dysfunction in
a genetic model of sympathetic hyperactivity-induced heart failure in
mice. Eur J Appl Physiol 105: 843–850, 2009.
Perreault CL, Gonzalez-Serratos H, Litwin SE, Sun X, FranziniArmstrong C, Morgan JP. Alterations in contractility and intracellular
Ca2⫹ transients in isolated bundles of skeletal muscle fibers from rats with
chronic heart failure. Circ Res 73: 405–412, 1993.
Rolim NP, Medeiros A, Rosa KT, Mattos KC, Irigoyen MC, Krieger
EM, Krieger JE, Negrao CE, Brum PC. Exercise training improves the
net balance of cardiac Ca2⫹ handling protein expression in heart failure.
Physiol Genomics 29: 246 –252, 2007.
Shah KR, Ganguly PK, Netticadan T, Arneja AS, Dhalla NS. Changes
in skeletal muscle SR Ca2⫹ pump in congestive heart failure due to
myocardial infarction are prevented by angiotensin II blockade. Can J
Physiol Pharmacol 82: 438 –447, 2004.
Spangenburg EE, Lees SJ, Otis JS, Musch TI, Talmadge RJ, Williams
JH. Effects of moderate heart failure and functional overload on rat
plantaris muscle. J Appl Physiol 92: 18 –24, 2002.
Sullivan MJ, Knight JD, Higginbotham MB, Cobb FR. Relation
between central and peripheral hemodynamics during exercise in patients
with chronic heart failure. Muscle blood flow is reduced with maintenance
of arterial perfusion pressure. Circulation 80: 769 –781, 1989.
Szigeti GP, Almassy J, Sztretye M, Dienes B, Szabo L, Szentesi P,
Vassort G, Sarkozi S, Csernoch L, Jona I. Alterations in the calcium
homeostasis of skeletal muscle from postmyocardial infarcted rats.
Pflügers Arch 455: 541–553, 2007.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proc Natl Acad Sci USA 76: 4350 –4354, 1979.
Turgeman T, Hagai Y, Huebner K, Jassal DS, Anderson JE, Genin O,
Nagler A, Halevy O, Pines M. Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone.
Neuromuscul Disord 18: 857–868, 2008.
Vieira NM, Bueno CR Jr, Brandalise V, Moraes LV, Zucconi E, Secco
M, Suzuki MF, Camargo MM, Bartolini P, Brum PC, Vainzof M, Zatz
M. SJL dystrophic mice express a significant amount of human muscle
proteins following systemic delivery of human adipose-derived stromal
cells without immunosuppression. Stem Cells 26: 2391–2398, 2008.
Williams JH, Ward CW. Changes in skeletal muscle sarcoplasmic
reticulum function and force production following myocardial infarction
in rats. Exp Physiol 83: 85–94, 1998.
Wilson JR, Ferraro N. Exercise intolerance in patients with chronic left
heart failure: relation to oxygen transport and ventilatory abnormalities.
Am J Cardiol 51: 1358 –1363, 1983.
Wilson JR, Mancini DM, Dunkman WB. Exertional fatigue due to
skeletal muscle dysfunction in patients with heart failure. Circulation 87:
470 –475, 1993.
Wilson JR, Martin JL, Schwartz D, Ferraro N. Exercise intolerance in
patients with chronic heart failure: role of impaired nutritive flow to
skeletal muscle. Circulation 69: 1079 –1087, 1984.
Yamamoto S, Greeff K. Effect of intracellular sodium on calcium uptake
in isolated guinea-pig diaphragm and atria. Biochim Biophys Acta 646:
348 –352, 1981.
109 • SEPTEMBER 2010 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 18, 2017
1. Bacurau AV, Jardim MA, Ferreira JC, Bechara LR, Bueno CR Jr,
Alba-Loureiro TC, Negrao CE, Casarini DE, Curi R, Ramires PR,
Moriscot AS, Brum PC. Sympathetic hyperactivity differentially affects
skeletal muscle mass in developing heart failure: role of exercise training.
J Appl Physiol 106: 1631–1640, 2009.
2. Bartholomeu JB, Vanzelli AS, Rolim NP, Ferreira JC, Bechara LR,
Tanaka LY, Rosa KT, Alves MM, Medeiros A, Mattos KC, Coelho
MA, Irigoyen MC, Krieger EM, Krieger JE, Negrao CE, Ramires PR,
Guatimosim S, Brum PC. Intracellular mechanisms of specific betaadrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure. J Mol Cell Cardiol 45: 240 –249,
2008.
3. Belardinelli R, Georgiou D, Cianci G, Purcaro A. Randomized, controlled trial of long-term moderate exercise training in chronic heart
failure: effects on functional capacity, quality of life, and clinical outcome.
Circulation 99: 1173–1182, 1999.
4. Brum PC, Kosek J, Patterson A, Bernstein D, Kobilka B. Abnormal
cardiac function associated with sympathetic nervous system hyperactivity
in mice. Am J Physiol Heart Circ Physiol 283: H1838 –H1845, 2002.
5. Coats AJ, Adamopoulos S, Meyer TE, Conway J, Sleight P. Effects of
physical training in chronic heart failure. Lancet 335: 63–66, 1990.
6. Ferreira JC, Bacurau AV, Bueno CR Jr, Cunha TC, Tanaka LY,
Jardim MA, Ramires PR, Brum PC. Aerobic exercise training improves
Ca2⫹ handling and redox status of skeletal muscle in mice. Exp Biol Med
(Maywood) 235: 497–505, 2010.
7. Ferreira JC, Bacurau AV, Evangelista FS, Coelho MA, Oliveira EM,
Casarini DE, Krieger JE, Brum PC. The role of local and systemic renin
angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice. Am J Physiol Regul Integr Comp
Physiol 294: R26 –R32, 2008.
8. Ferreira JC, Rolim NP, Bartholomeu JB, Gobatto CA, Kokubun E,
Brum PC. Maximal lactate steady state in running mice: effect of exercise
training. Clin Exp Pharmacol Physiol 34: 760 –765, 2007.
9. Fraysse B, Rouaud T, Millour M, Fontaine-Perus J, Gardahaut MF,
Levitsky DO. Expression of the Na⫹/Ca2⫹ exchanger in skeletal muscle.
Am J Physiol Cell Physiol 280: C146 –C154, 2001.
10. Jondeau G, Katz SD, Zohman L, Goldberger M, McCarthy M,
Bourdarias JP, LeJemtel TH. Active skeletal muscle mass and cardiopulmonary reserve. Failure to attain peak aerobic capacity during maximal
bicycle exercise in patients with severe congestive heart failure. Circulation 86: 1351–1356, 1992.
11. Linke A, Adams V, Schulze PC, Erbs S, Gielen S, Fiehn E, MobiusWinkler S, Schubert A, Schuler G, Hambrecht R. Antioxidative effects
of exercise training in patients with chronic heart failure: increase in
radical scavenger enzyme activity in skeletal muscle. Circulation 111:
1763–1770, 2005.
12. Lunde PK, Dahlstedt AJ, Bruton JD, Lannergren J, Thoren P,
Sejersted OM, Westerblad H. Contraction and intracellular Ca2⫹ handling in isolated skeletal muscle of rats with congestive heart failure. Circ
Res 88: 1299 –1305, 2001.
13. Lunde PK, Sejersted OM, Thorud HM, Tonnessen T, Henriksen UL,
Christensen G, Westerblad H, Bruton J. Effects of congestive heart
failure on Ca2⫹ handling in skeletal muscle during fatigue. Circ Res 98:
1514 –1519, 2006.
14. Lunde PK, Verburg E, Eriksen M, Sejersted OM. Contractile properties of in situ perfused skeletal muscles from rats with congestive heart
failure. J Physiol 540: 571–580, 2002.
15. Medeiros A, Rolim NP, Oliveira RS, Rosa KT, Mattos KC, Casarini
DE, Irigoyen MC, Krieger EM, Krieger JE, Negrao CE, Brum PC.
Exercise training delays cardiac dysfunction and prevents calcium han-
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