J Appl Physiol 110: 1638–1645, 2011.
First published April 7, 2011; doi:10.1152/japplphysiol.00020.2011.
Effects of endurance training on apoptotic susceptibility in striated muscle
Anna Vainshtein, Lawrence Kazak, and David A. Hood
School of Kinesiology and Health Science and the Muscle Health Research Center, York University, Toronto,
Ontario, Canada
Submitted 6 January 2011; accepted in final form 1 April 2011
exercise; cell death; mitochondria; heart; skeletal muscle
to adapt to environmental
demands. It has been well documented that under chronic
disuse conditions, such as prolonged bed rest, limb immobilization (e.g., casting), denervation, and microgravity (e.g.,
space flight), skeletal muscle atrophies and loses strength and
function. This type of muscle wasting is also observed with
aging. Similarly, cardiac muscle is also forced to adapt to
adverse conditions such as ischemia, resulting from atherosclerosis. Conversely, endurance training results in adaptations that
are beneficial in promoting healthier phenotypes in both heart
and skeletal muscle. With exercise training, skeletal muscle
becomes more oxidative and is better protected from oxidative
stress. This is also true for cardiac muscle, as the heart becomes
more resilient and less susceptible to ischemic insults (19).
Commonalities between cardiac and skeletal muscle adaptations have also been noted. Both muscle types experience an
elevation in reactive oxygen species (ROS) with progressive
aging (18). ROS production from mitochondria is also accelerated in muscle subject to chronic disuse and in cardiac
muscle ischemia. When the production of ROS exceeds cellular antioxidant capability, oxidative stress results and apoptosis
MUSCLE IS UNIQUE IN ITS ABILITY
Address for reprint requests and other correspondence: D. A. Hood, School
of Kinesiology and Health Science, York Univ., Toronto, Ontario M3J 1P3,
Canada (e-mail: [email protected]).
1638
is triggered (40). Apoptosis is a tightly regulated form of
programmed cell death, the aberrant regulation of which may
lead to pathological conditions including tumorigenesis, autoimmune, and neurodegenerative diseases (5) to name a few.
Apoptosis has also been implicated in ischemic heart disease
and sarcopenia (29), both of which emerge with advanced age.
Thus the increases in ROS observed with aging predispose
cardiac and skeletal muscle myofibers to mitochondrially mediated apoptosis. To date, although these apoptotic signaling
cascades have been studied extensively, some of the functions
and interactions of the major participants in the apoptotic
machinery remain poorly characterized. ROS-induced apoptotic events have been linked to the phosphorylation (P) of
c-Jun-N-terminal kinase (JNK) and the recruitment of the
structurally related Bcl-2 family of proteins to the mitochondria. These proteins function collectively to regulate organelle
membrane integrity (4). Bcl-2-associated X protein (Bax) is a
proapoptotic member of the Bcl-2 family that exists as a
monomer in the cytosol. Upon an apoptotic stimulus, Bax
undergoes a conformational change that allows it to homo- or
hetero-oligomerize and subsequently translocate and insert into
the mitochondrial membrane (2, 8, 10). This step is believed to
be critical, but not exclusive, for mitochondrial premeabilization and the formation of the mitochondrial permeability transition pore (mtPTP; Refs. 1, 2, 5, 8). In addition, the actin
binding protein cofilin-2 was recently found to translocate into
the mitochondria and help facilitate membrane permeabilization (21). The pathway by which this takes place, however,
remains unknown. The opening of the mtPTP is rapidly followed by an efflux of apoptogenic factors such as cytochrome
c and apoptosis inducing factor (AIF). These proteins can
either directly or indirectly cause DNA fragmentation (2, 4, 5),
resulting in the decay of the myonucleus, and ultimately in the
demise of the myonuclear domain that it governs. In contrast to
this, endurance-type physical activity has been associated with
both the reduced accumulation and production of ROS, as well
as with decreased levels of apoptosis (4). However, the mechanisms by which regular exercise mediates the interactions
between ROS and apoptosis remain to be elucidated.
Thus the purposes of this study were 1) to utilize a whole
muscle preparation for the investigation of apoptotic susceptibility of both skeletal and cardiac muscle in response to acute
oxidative stress, and 2) to evaluate the effect of endurance
training on apoptotic signaling in both of these muscle types.
We hypothesized that training would produce phenotypic adaptations that would confer protection of these striated muscles
against oxidative stress.
METHODS
Animals. Male Sprague-Dawley rats (n ⫽ 40; Charles River, St.
Constant, QC, Canada) weighing between 130 and 160 g were
individually housed and given food and water ad libitum. All proce-
8750-7587/11 Copyright © 2011 the American Physiological Society
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Vainshtein A, Kazak L, Hood DA. Effects of endurance
training on apoptotic susceptibility in striated muscle. J Appl
Physiol 110: 1638 –1645, 2011. First published April 7, 2011;
doi:10.1152/japplphysiol.00020.2011.—An increase in the production of reactive oxygen species occurs with muscle disuse, ischemic
cardiomyopathy, and conditions that arise with senescence. The resulting oxidative stress is associated with apoptosis-related myopathies. Recent research has suggested that chronic exercise is protective
against mitochondrially mediated programmed cell death. To further
investigate this, we compared soleus (Sol) and cardiac muscles of
voluntary wheel-trained (T; 10 wk) and untrained (C) animals. Training produced a 52% increase in muscle cytochrome c oxidase (COX)
activity. Sol and left ventricle (LV) strips were isolated and incubated
in vitro with H2O2 for 4 h. Strips were then fractionated into cytosolic
and mitochondrial fractions. Whole muscle apoptosis-inducing factor
(AIF) and Bax/Bcl-2 levels were reduced in both the Sol and LV from
T animals. H2O2 treatment induced increases in JNK phosphorylation,
cofilin-2 localization to the mitochondria, as well as cytosolic AIF in
both Sol and LV of T and C animals, respectively. Mitochondrial Bax
and cytosolic cytochrome c were augmented under oxidative stress in
the LV only. The H2O2-induced increases in P-JNK, mitochondrial
Bax, and cytosolic AIF were ablated in the LV of T animals. These
data suggest that short-term oxidative stress can induce apoptotic
signaling in striated muscles in vitro. In addition, training can attenuate oxidative stress-induced apoptotic signaling in a tissue-specific
manner, with an effect that is most prominent in cardiac muscle.
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EXERCISE AND APOPTOSIS
dures involving animals were approved by the York University
Animal Care Committee in accordance with the Canadian Council on
Animal Care.
Animal training. The animals were randomly divided into trained
(n ⫽ 20) or untrained (control, n ⫽ 20) groups. The trained animals
had access to loaded voluntary running wheels. Animals were
trained for 8 –10 wk. For the first 2 wk, the wheels remained
unloaded. Thereafter, 50 g of resistance was added on a weekly
basis until week 5, where a maximal load of 200 g was placed on
the wheels (weeks 6-10). This load was maintained until week 10
of training, as done previously (9). This was done to obtain
continuous training adaptations as the animals habituated to the
running. A training effect was determined by measuring the epi-
Table 1. Characteristics of control and trained animals
Measure
Control
Trained
Body weight, g (n ⫽ 19/20)
Heart weight-to-body weight ratio,
mg/g (n ⫽ 19/20)
Soleus weight-to-body weight ratio,
mg/g (n ⫽ 19/20)
Epididymal fat-to-body weight ratio,
mg/g (n ⫽ 8)
Food intake, g/day (n ⫽ 39/35)
Water intake, ml/day (n ⫽ 37/29)
540.50 ⫾ 16.86
527.60 ⫾ 14.39
2.34 ⫾ 0.04
2.60 ⫾ 0.04*
0.42 ⫾ 0.01
0.47 ⫾ 0.02*
15.19 ⫾ 1.52
25.89 ⫾ 0.718
35.32 ⫾ 1.835
10.40 ⫾ 1.54*
30.10 ⫾ 0.84*
60.52 ⫾ 4.65*
All values are means ⫾ SE; n, no. of experiments. *P ⬍ 0.05, control vs.
trained animals.
Fig. 1. Running performance and training effects. Trained animals were
exercised with no load for the first 2 wk and then loaded incrementally by 50
g (week 2), 100 g (week 3), and 200 g (weeks 5–10), as indicated by the arrows.
Average daily running distance for each week of running (A); work done,
average per day at each week of training (B; n ⫽ 20); and cytochrome c
oxidase (COX) activity (C) in soleus muscle in trained and untrained animals.
Values are means ⫾ SE (n ⫽ 23, control; n ⫽ 24, trained). *P ⬍ 0.05, trained
vs. control.
J Appl Physiol • VOL
Fig. 2. Effects of training on soleus muscle apoptotic protein expression.
Typical Western blot and quantification of multiple samples of cytochrome c
(A; Cyto C), cofilin-2 (B), Bax-to-Bcl-2 ratio (C), and apoptosis-inducing
factor (AIF) protein levels (D) between control (C) and trained (T) animals.
E: typical Western blot for manganese superoxide dismutase (MnSOD) protein
expression. Aciculin was used as a loading control. Values are means ⫾ SE
(n ⫽ 6 –10). *P ⬍ 0.05, trained vs. control.
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didymal fat weight, heart weight-to-body weight ratios, as well as
cytochrome c oxidase (COX) activity.
Tissue extraction. Animals were taken 24 h past the immobilization
of the running wheel to avoid any acute exercise effects. The animals
were anesthetized with an intraperitoneal injection of ketamine and
xylazine (40 mg/ml ketamine; 5 mg/ml xylazine) at a dose of 0.2
ml/100 g body weight. Both epididymal fat pads, one soleus (Sol)
muscle and a portion of the left ventricle (LV), were removed and
immediately frozen in liquid nitrogen. The Sol and LV sections were
later used to determine COX activity and whole muscle apoptotic
protein expression.
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Bradford method. The fractions were frozen at ⫺20°C until further
use.
Immunoblotting. Whole muscle protein extracts or isolated mitochondrial and cytosolic fractions were subjected to SDS-PAGE.
Proteins were then transferred onto nitrocellulose membranes using a
wet electrotransfer technique. Nitrocellulose membranes were
blocked in a 5% skim milk solution containing 1 ⫻ TBST (Trisbuffered saline/Tween-20, 25 mM Tris, 1 mM NaCl, and 0.1%
Tween-20, pH 7.5). Membranes were then incubated overnight at 4°C
with the appropriate primary antibody [1:500 Bax; 1:5,000 AIF; 1:100
Bcl-2; 1:500 P-JNK; 1:1,000 cofilin-2; 1:750 cytochrome c; 1:4,000
COX IV; 1:1,000 manganese superoxide dismutase (MnSOD); 1:250
aciculin; or 1:20,000 GAPDH]. Membranes were subsequently
washed in TBST and then incubated with the appropriate secondary
antibody coupled to horseradish peroxidase at room temperature for
60 min. Membranes were washed once more and finally visualized
using an enhanced chemiluminescence kit. Films were scanned and
quantified using SigmaScan Pro (version 5) software (Jandel Scientific, San Rafael, CA).
Mitochondrial content. COX enzyme activity was used as a measure of mitochondrial content. Whole muscle tissue was pulverized
into fine powder in the presence of liquid nitrogen. This powder
(10 –20 mg) was then diluted 20-fold in muscle extraction buffer
(0.1 M KH2PO4 and 2 mM EDTA pH 7.2) and sonicated on ice (3
cycles of 3 s at 30% intensity each) to disrupt cellular membranes.
After centrifugation, the supernatant fractions were recovered as
previously described (10). Enzyme activity was determined by the
maximal oxidation rate of completely reduced cytochrome c,
evaluated as a change in absorbance at 550 nm using a Synergy HT
microplate reader. The data were compiled using KC4 software.
Cell death ELISA. DNA fragmentation levels were determined in
cytosolic extracts from fractionated muscle strips, using ELISA kit
(Cell Death Detection ELISA kit; Roche Diagnostics, Manheim,
Germany) modified from the manufacturer’s protocol. Briefly,
cytosolic fractions (20 ␮g protein) were diluted 4-fold (vol/vol) in
Immunoreagent as recommended by the manufacturer. The samples were subsequently pipetted on to streptavidin-coated microplates for 2 h at room temperature under vigorous shaking (300
rpm). After being washed, the nonbound antibody was removed
and 2,2=-azino-di-(3-ethylbenzthiazoline sulfonate) was added to
Fig. 3. Effects of training on H2O2-induced
apoptotic signaling in soleus muscle. Apoptotic signaling was determined by protein
expression in soleus muscle treated with either vehicle (V) or 3 mM H2O2 (H). Typical
Western blot and quantification of multiple
samples of cytosolic P-JNK (A), mitochondrial cofilin-2 (B), mitochondrial Bax (C),
cytosolic AIF (D), cytosolic cytochrome c
(E) between control (C) and trained (T) animals. Aciculin and GAPDH served as cytosolic fraction loading controls, while COXIV
was used as mitochondrial fraction loading
control. Values are means ⫾ SE (n ⫽ 4 –10).
*P ⬍ 0.05, H2O2 vs. vehicle treated.
J Appl Physiol • VOL
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Isolated muscle preparation. Muscle strips (⬃20 mg each) were
removed from the Sol and LV. Strips of this size can be adequately
oxygenated and have consistently been shown to remain viable when
incubated in vitro (6). The Sol and LV muscles were strategically
chosen to represent muscle of varying oxidative capacity. After
removal, the strips were incubated with either vehicle (double distilled
water) or hydrogen peroxide (H2O2; 3 mM) for a 4-h period. H2O2 is
a common ROS used to mimic oxidative stress conditions in muscle.
The incubation was performed in vitro using glass scintillation vials
containing 5 ml of warmed (30°C), pregassed (⬃20 min with 95%
O2:5% CO2) Krebs-Henseleit buffer (118.5 mM NaCl, 4.7 mM KCl,
1.2 mM KH2PO4, 25 mM NaHCO3, 1.2 mM MgSO4, and 3.4 mM
CaCl2 pH 7.4) supplemented with 5 mM D-glucose, 2 mM sodium
pyruvate, and 2 mU of insulin. The strips were continuously oxygenated with 95% O2-5% CO2 and kept at a constant temperature of 30°C
for the duration of the incubation period.
Tissue fractionation. Following incubation, muscle strips were
separated into mitochondrial and cytoslic fractions by way of
differential centrifugation (36), with modifications. Briefly, after 4
h of incubation the muscle strips were minced, homogenized using
a Teflon pestle and mortar, and suspended in mitochondrial isolation buffer (MIB; 250 mM sucrose, 20 mM HEPES, 10 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA pH 7.4) supplemented with a cocktail of protease inhibitors (1 mM PMSF, 10 ␮M
leupeptin, 1.5 ␮M aprotinin, 1.5 ␮M pepstatin A, and 1 mM
sodium orthovanadate). The homogenate was then centrifuged at
1,000 g for 10 min at 4°C to pellet the nuclei. The supernate was
carefully removed and centrifuged at 16,000 g for 20 min at 4°C to
pellet the mitochondria. The supernate was removed and spun
again at 16,000 g for 20 min at 4°C to pellet any residual
mitochondria; the resulting supernate was considered to be enriched cytosolic fraction. The mitochondrial pellet was resuspended twice in a fourfold dilution of MIB and centrifuged at
16,000 g for 20 min at 4°C after each resuspension. The supernate
was finally removed and the mitochondrial pellet was resuspended
in a onefold dilution of MIB. Mitochondria were subsequently
sonicated to yield the enriched mitochondrial fraction. Based on
this separation, we inferred the translocation of various apoptogenic proteins from one cellular location to another. Protein
concentrations within the samples were determined using the
EXERCISE AND APOPTOSIS
produce a color change, dependent on the amount of fragmented
DNA present. The extent of DNA fragmentation was quantified
photometrically at a light density of 405 nm, using a Synergy HT
microplate reader.
Statistical analyses. Data are expressed as means ⫾ SE. Comparisons between trained and untrained animals in two panel
figures were done using unpaired student’s t-test. Comparisons
between trained and untrained animals, and between Vehicle and
H2O2 treatments in RESULTS (see Figs. 2–5) were made using paired
(Veh and H2O2) two-way analyses of variance followed by Bonferroni post hoc test. Statistical differences were considered significant at P ⬍ 0.05. All statistical analyses were carried out using
Graph Pad Prism 4.0. Number of experiments (n ⫽ x) varies from
experiment to experiment due to the small amount of protein
available per sample.
RESULTS
J Appl Physiol • VOL
Training resulted in a decrease in both basal apoptotic
protein expression and stress-induced apoptotic signaling in
cardiac muscle. Endurance training had no effect on cytochrome c or cofilin-2 expression in cardiac muscle (Fig. 4, A
and B). However, a 50% decrease (P ⬍ 0.05; Fig. 4C) in the
Bax-to-Bcl-2 ratio, as well as a 32% decrease in AIF levels (P ⬍
0.05; Fig. 4D) were observed following training. In contrast, no
change in the expression of MnSOD was found following
exercise training (Fig. 4E). Upon the induction of apoptotic
signaling with H2O2 treatment, JNK phosphorylation rose by
76% (P ⬍ 0.05) in control animals and only by 58% (P ⬍ 0.05)
in trained animals (Fig. 5A). JNK phosphorylation was significantly attenuated with exercise training both basally, and in
response to oxidative stress (P ⬍ 0.05). Moreover, treatment
with H2O2 resulted in 128% and 68% increases in cofilin-2
translocation to the mitochondria of control and trained animals, respectively (P ⬍ 0.05; Fig. 5B). H2O2 treatment also
provoked a 59% increase in mitochondrial Bax and a 39%
increase in cytosolic AIF in control animals only (P ⬍ 0.05;
Fig. 5, C and D). Thus exercise training successfully attenuated
both Bax translocation and AIF release in cardiac muscle (Fig.
5, C and D). Interestingly, the H2O2-induced increase in
cytosolic cytochrome c was not reduced by training and was
42–50% higher in cardiac muscle from both trained and control
animals in response to H2O2 (P ⬍ 0.05; Fig. 5E). Thus training
Fig. 4. Effects of voluntary running on cardiac muscle apoptotic protein
expression. Western blot and quantification of multiple samples of cytochrome
c (A), cofilin-2 (B), Bax-to-Bcl-2 ratio (C), and AIF protein levels (D) between
control (C) and trained (T) animals. E: typical Western blot for MnSOD
protein expression. Aciculin was used as a loading control. Values are
means ⫾ SE (n ⫽ 5–9). *P ⬍ 0.05, trained vs. control.
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Animal running behavior, COX activity, and general
characteristics. Animals progressively increased their running distance, and work performed, over the first 3 wk of
training. Following the third week, the work plateaued and
then declined, as the loads increased and the distance run
decreased (Fig. 1, A and B). This behavior appears to be
typical during loaded wheel running, as shown previously
(9). A training effect was determined using three indices.
First, epididymal fat weight-to-body weight ratio was 32%
lower (P ⬍ 0.05) in trained animals (Table 1). Second, the
heart weight-to-body weight ratio was 11% higher in the
trained animals (P ⬍ 0.05), typical of a modest traininginduced cardiac hypertrophy. Third, COX activity was also
measured, to provide an assessment of whole muscle mitochondrial content. Compared with the control animals, COX
activity was 52% higher in the Sol muscle of trained animals
(P ⬍ 0.05; Fig. 1C).
Training resulted in reduced basal apoptotic protein expression but not stress-induced apoptotic signaling in Sol.
As expected, training resulted in a 49% (P ⬍ 0.05) increase
in Sol cytochrome c expression (Fig. 2A), directly mirroring
the observed increase in COX activity (Fig. 1C). On the
other hand, no change in the expression of the actin-binding
protein cofilin-2 was observed (Fig. 2B). Endurance training
also resulted in a 40% decrease (P ⬍ 0.05) in the Bax-toBcl-2 ratio, an indicator of apoptotic susceptibility, as well
as a 34% reduction in whole muscle AIF level (P ⬍ 0.05;
Fig. 2, C and D). The protein expression of the antioxidant
enzyme MnSOD was not significantly altered in the Sol
muscle of trained, compared with control, animals (Fig. 2E).
In response to H2O2, JNK phosphorylation was elevated by
18 –30% (P ⬍ 0.05), and an approximate twofold increase in
cofilin-2 localization to the mitochondria (P ⬍ 0.05) was
observed in both control and trained animals (Fig. 3, A and B).
In contrast, H2O2 treatment did not cause measurable Bax
translocation nor did it increase cytosolic cytochrome c expression in either of the two groups (Fig. 3, C and E). However,
AIF localization to the cytosol was elevated by 58% (P ⬍ 0.05)
in controls, but only by 31% (P ⬍ 0.05) in trained animals
(Fig. 3D) as a result of oxidative stress. Thus, exercise training
resulted in some protein-specific adaptations favoring muscle
cell survival.
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resulted in cardiac muscle that is protected from basal and
oxidative stress-induced apoptotic signaling.
DNA fragmentation. Given the increases in apoptotic signaling observed with H2O2 treatment, and the attenuation of
these events with training, we further investigated whether
this protective effect was evident at the level of DNA
fragmentation, the most distal step in the apoptotic signaling
pathway. Our results revealed that the levels of DNA fragmentation were not different in either heart or Sol muscles
in response to training, or in response to 4 h of treatment
with H2O2 (Fig. 6, A and B).
DISCUSSION
The results of this study suggest that exercise training
evokes cardiac and skeletal muscle adaptations that favor
cellular survival over apoptotic cell death. Under basal conditions, voluntary wheel running resulted in decreased expression of proapototic factors in both muscle types, which is
indicative of reduced apoptotic potential. Training also resulted
in cardiac and skeletal muscles that were less susceptible to
oxidative stress-induced apoptotic signaling in the absence of
J Appl Physiol • VOL
notable changes in antioxidant enzyme protein levels. This
beneficial adaptation was most prominent in cardiac muscle.
There is an increasing body of evidence suggesting that
apoptosis may play a vital role in muscular atrophy, as well as
in geriatric-related disorders. This type of programmed cell
death has been implicated in disuse-related atrophy (3, 11, 36,
39), sarcopenia (7, 10, 13, 14), as well as in ischemic
cardiomyopathy (16, 17, 22, 24, 25, 30 –32). Moreover,
these conditions are often accompanied by increased levels
of oxidative stress. On the other hand, regular physical
activity has been demonstrated to suppress ROS production
(1, 35), increase antioxidant defense systems (20, 38), as well
as alleviate some of the asthenia and fatigability that often
accompany muscular atrophy (10, 15, 20). With this in mind,
we set out to investigate whether exercise training could
attenuate H2O2-induced apoptotic susceptibility in cardiac and
skeletal muscles.
Our progressively loaded voluntary wheel running protocol
produced typical muscle and whole body training effects
within 8 –10 wk, as is indicated by an increase in mitochondrial
content, reduction in epididymal fat stores, as well as a mild
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Fig. 5. Effects of training on H2O2-induced apoptotic signaling in cardiac muscle. Apoptotic signaling as determined by protein expression in cardiac muscle
treated with either vehicle (V) or 3 mM H2O2 (H). Typical Western blot and quantification of multiple samples of cytosolic phosphorylated (P)-JNK (A),
mitochondrial cofilin-2 (B), mitochondrial Bax (C), cytosolic AIF (D), cytosolic cytochrome c (E) between control (C) and trained (T) animals. Aciculin and
GAPDH served as cytosolic fraction loading controls while COX IV was used as mitochondrial fraction loading control. Values are means ⫾ SE (n ⫽ 5–10).
*P ⬍ 0.05. H2O2 vs. vehicle treated. ¶P ⬍ 0.05, trained vs. control.
EXERCISE AND APOPTOSIS
cardiac hypertrophy. Training also resulted in reduced basal
proapoptotic protein expression. Accordingly, AIF and Baxto-Bcl-2 ratios were both reduced in the Sol and cardiac
muscles. It is important to note that the execution of apoptosis
is dependent on the interplay between pro- and antiapoptotic
factors working simultaneously at any given time (27). Thus
the ratio of Bax to Bcl-2 is a good indicator of apoptotic
potential. This ratio was lower in trained animals, suggesting
that training resulted in a muscle phenotype possessing a
reduced apoptotic capacity. We then assessed some potential
signaling events involved in mediating apoptosis in both skeletal and cardiac muscle.
To test the response of intact striated muscle to a death
signal, we incubated muscle strips with supraphysiological
level of H2O2 for a period of 4 h to mimic the severe induction
of oxidative stress (1, 2, 20, 40, 41). Tissues were subsequently
separated into cytosolic and mitochondrial fractions so that
protein translocation from one cellular compartment to another
could be inferred.
Cardiac muscle appeared to undergo the most robust antiapoptotic training adaptations in accordance with previous
work (27, 37). Under basal conditions, both the Bax-to-Bcl-2
ratio as well as total AIF levels were reduced by training. In
response to oxidative stress, Bax localization to the mitochondria was increased. This may be due, in part, to the activation
of cytosolic JNK, which is phosphorylated in the presence of
cellular stressors, subsequently inhibits Bcl-2, and allows for
Bax translocation to the mitochondria. Once within the organelle, Bax participates in the opening of the mtPTP, which is
promptly followed by release of proapoptotic AIF and cytochrome c into the cytosol (12, 28, 32, 33, 40). Upon release,
J Appl Physiol • VOL
AIF and cytochrome c proceed to induce DNA fragmentation
either directly or via a caspase cascade (12, 28, 32, 33, 40).
Accordingly, AIF and cytochrome c localization to the cytosol
was evident with H2O2 treatment. In response to training, the
same level of oxidative stress resulted in lower JNK phosphorylation in the heart. This coincided with reduced Bax translocation to the mitochondria. In addition, AIF translocation
observed with oxidative stress was diminished in the trained
heart, further indicating suppressed apoptotic signaling. This
reduction in cytosolic AIF may have been mediated by reduced
levels of the AIF cleaving protease calpain, which has been
found to decrease with chronic contractile activity in previous
studies (15). Training-induced reductions in cell death signaling may be occurring as an attempt to spare the cardiomyocyte
in response to cellular stressors. This is a favorable adaptation,
as apoptotic environments have been strongly implicated in
age-related cardiac pathophysiology (20, 23).
In contrast to the adaptations evident in cardiac muscle,
endurance training appeared to have little effect on H2O2induced apoptotic signaling in the Sol muscle. This occurred
despite clear evidence of a mitochondrial augmentation to the
exercise training regimen, as well as reduced basal proapoptotic protein expression. Although we did observe increases in
JNK phosphorylation, cofilin-2 localization to the mitochondria, as well as increased cytosolic AIF with H2O2 treatment,
these were unaltered by exercise training in Sol muscle. This
may be partially explained by the strength of stimulus provided
by the oxidative stress treatment. Perhaps a lower concentration of H2O2, adequate to evoke apoptotic signaling, but
insufficient to mask the benefits of exercise observed in whole
muscle, could have been employed. Alternatively, since heart
and muscle adapt differentially to training, it is possible that
adaptations in skeletal muscle would have been evident at an
earlier time point within the training protocol. Nonetheless, it
is apparent that skeletal muscle is less receptive to exercise
training-induced apoptotic adaptations compared with the
heart. This is consistent with previous studies (27, 37) where
ventricular muscle adapted to a greater extent than skeletal
muscle. This discrepancy in adaptation between the two striated muscle types could be due to 1) the differential loading of
cardiac vs. skeletal muscle throughout the training period, and
2) the wide variation in inherent tissue mitochondrial content,
as mitochondria are prime producers of ROS and contain
antioxidant enzymes.
Curiously, cofilin-2, cytochrome c, and MnSOD behaved
differently than originally hypothesized. Cofilin-2, an actin
binding cytoskeletal protein, was recently associated with oxidative stress-induced apoptosis (21). It resides in the cytosol,
but upon oxidation it translocates into the mitochondria where
it is thought to help facilitate the opening of the mtPTP. In our
study, H2O2 treatment resulted in cofilin-2 translocation to the
mitochondria in both cardiac and skeletal muscles, supporting
a role for cofilin-2 in apoptosis. However, the level of cofilin-2
was unaltered by training, suggesting that it does not participate in exercise-induced adaptations. In addition, we did not
observe an increase in cytosolic cytochrome c with H2O2
treatment in the Sol muscle, but it was elevated in cardiac
muscle. The reason for this differential response is not yet
clear, but this may be due to an inherently greater mitochondrial content in cardiac muscle, thus leading to more detectable
cytochrome c efflux in response to H2O2. Moreover, exercise
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Fig. 6. Cell death. Cell death as measured by DNA fragmentation ELISA in the
cytosolic fraction of control (C) and trained (T) animals treated with either
vehicle (V) or 3 mM H2O2 (H) in soleus (A) and cardiac muscle (B). Values
are means ⫾ SE (n ⫽ 7–11). OD, optical density.
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GRANTS
This work was supported by funding from the Natural Sciences and
Engineering Research Council of Canada (NSERC) to D. A. Hood. A.
Vainshtein is a recipient of the Heart and Stroke Foundation Masters Studentship Award. D. A. Hood is the holder of a Canada Research Chair in Cell
Physiology.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
REFERENCES
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activity on SS and IMF mitochondrial apoptotic susceptibility in skeletal
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did not appear to alter cytochrome c localization in either
tissue. This may not have been detectable in our model due to
the increase in total cytochrome c expression observed with the
exercise training. Additionally, no differences in MnSOD levels were found in heart or skeletal muscle following training.
MnSOD is an enzyme responsible for catalyzing the dismutation of superoxide radicals into their less reactive form of
oxygen and hydrogen peroxide. This enzyme is found in the
mitochondria where it plays a key antioxidant role. The activity
of this enzyme has previously been shown to increase in the
heart following exercise (15), but we were unable to detect a
change using immunoblotting techniques with the wheel running training program employed in this study. Our data are in
accordance with previous work, where MnSOD protein levels
remained constant following 7 days of chronic contractile
activity (1, 26). Thus our results suggest that the reduced
apoptotic susceptibility observed in trained muscles occurs in
the absence of increases in antioxidant enzyme activity, assuming that MnSOD is representative of the expression of other
enzymes, such as glutathione peroxidase. However, this remains to be determined.
DNA fragmentation levels were extremely low in both heart
and skeletal muscle under steady-state conditions. Thus it was
difficult to observe a difference in this measure between trained
and control muscle. Furthermore, our incubation time with
H2O2 was only 4 h, and while this is sufficient to induce
apoptotic signaling, this treatment time was not long enough to
elicit DNA fragmentation. Our results are in accordance with
current literature, since 24 – 48 h of treatment with H2O2 are
required to induce DNA fragmentation in cell culture (34, 40).
This study has revealed that 1) treatment with a high concentration of H2O2 in a whole muscle ex vivo preparation is
sufficient for the activation of programmed cell death cascades,
and 2) endurance training can attenuate oxidative stress-induced apoptotic signaling in a tissue-specific manner. Moreover, skeletal muscle appears to be more resistant to change
than the continuously contracting cardiac muscle. It will be of
interest to continue comparing the apoptotic susceptibility and
differential adaptations of these two tissues. Nevertheless,
these data are consistent with the hypothesis that endurance
training results in a striated muscle phenotype that is resistant
to oxidative stress-induced apoptosis. Since there is evidence
to suggest that oxidative stress is elevated in atrophic muscle
conditions, as well as in heart disease and aging, our findings
suggest that endurance training and an active lifestyle may
provide some protection from apoptotic cell death in both
skeletal and cardiac muscle.
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