Download Voluntary exercise protects against acute doxorubicin cardiotoxicity

Am J Physiol Regul Integr Comp Physiol 289: R424 –R431, 2005.
First published April 21, 2005; doi:10.1152/ajpregu.00636.2004.
Voluntary exercise protects against acute doxorubicin cardiotoxicity
in the isolated perfused rat heart
Adam J. Chicco, Carole M. Schneider, and Reid Hayward
School of Sport and Exercise Science and the Rocky Mountain Cancer
Rehabilitation Institute, University of Northern Colorado, Greeley, Colorado
Submitted 17 September 2004; accepted in final form 13 April 2005
Address for reprint requests and other correspondence: R. Hayward, School
of Sport and Exercise Science, Univ. of Northern Colorado, Greeley, CO
80639 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
adriamycin; cardioprotection; heart; physical activity; cardiac function
R424
0363-6119/05 $8.00 Copyright © 2005 the American Physiological Society
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DOXORUBICIN (DOX) IS A HIGHLY effective antineoplastic antibiotic commonly used to treat a variety of solid tumors and
leukemias. Unfortunately, the clinical use of doxorubicin is
limited by a dose-dependent cardiotoxicity that may progress
to chronic cardiomyopathy and congestive heart failure (CHF)
(50). Characterization and prevention of DOX cardiotoxicity
(DCT) have been the subjects of intense research for decades,
but no single intervention has proven to be completely successful in ameliorating the damaging effects of DOX on the
heart, while maintaining its antineoplastic efficacy.
DOX cardiomyopathy and CHF typically develop after multiple intravenous DOX treatments over a period of several
months in humans. Incidence of chronic cardiomyopathy appears to be highly dependent on the cumulative DOX dose
administered (50) and has been recently reported to be as high
as 26% in patients receiving 550 mg/m2 (54). Evidence from
human (37) and animal (30) studies suggests that acute myocardial dysfunction and/or injury occurring early during the
course of DOX treatment is highly predictive of later cardiomyopathy. Furthermore, it appears that the chronic manifestations of DCT may result from myocardial injuries associated
with acute DOX exposure (40, 58). This has led to the use of
a wide variety of experimental models aimed at exploring the
mechanisms and circumvention of acute DCT in vivo and in
vitro. Several investigators have used the isolated perfused rat
heart model for this purpose, which has provided a reliable and
reproducible means of assessing the biochemical and functional aspects of acute DCT (19, 40, 42).
Free radical formation has been consistently reported to play
a pivotal role in several of the acute and chronic manifestations
of DCT (23, 27, 30, 34). DOX is believed to interact with
intracellular redox enzymes such as nicotinamide adenine
dinucleotide dehydrogenase to produce superoxide radicals and
increase the levels of cytotoxic reactive oxygen species (ROS)
in cardiomyocytes (10). The resulting oxidative stress is known
to initiate a myriad of destructive events in cardiomyocytes and
has been specifically linked to membrane lipid peroxidation
(27, 34, 39), impaired Ca2⫹ handling (15), mitochondrial
dysfunction (13), apoptosis (23), and DNA injury (30). Superoxide dismutase (SOD) is an endogenous antioxidant enzyme
that protects the heart from superoxide by catalyzing its dismutation into hydrogen peroxide (H2O2), which is then converted to oxygen and water by catalase (CAT) and the glutathione peroxidase (GPx) system. Acute DCT has been shown
to be enhanced when SOD activity is compromised (49) and is
attenuated in transgenic mice overexpressing the mitochondrial
isoform of SOD (MnSOD) (62). These studies further support
the role of superoxide in DCT and suggest that any treatment
that increases myocardial SOD activity may protect the heart
from DOX-induced injury.
Heat shock proteins of the 70-kDa family (particularly the
inducible isoform, HSP72) are believed to protect cardiomyocytes against injury during states of cellular stress (5, 21).
HSP72 induction has been specifically associated with the
prevention of apoptosis, necrosis, and oxidative injury in the
myocardium following exposure to ischemia-reperfusion (I/R)
(8, 45, 47), hydrogen peroxide (55), and DOX (17). HSP72
most likely protects cardiomyocytes by preserving protein
structure and function during states of stress (21) and may
selectively protect mitochondria from the damaging effects of
ROS (41).
Chicco, Adam J., Carole M. Schneider, and Reid Hayward.
Voluntary exercise protects against acute doxorubicin cardiotoxicity
in the isolated perfused rat heart. Am J Physiol Regul Integr Comp
Physiol 289: R424 –R431, 2005. First published April 21, 2005;
doi:10.1152/ajpregu.00636.2004.—The clinical use of doxorubicin
(DOX) is limited by a dose-dependent cardiotoxicity. The purpose of
this study was to determine whether voluntary exercise training would
confer protection against DOX cardiotoxicity in the isolated perfused
rat heart. Female Sprague-Dawley rats were randomly assigned to
standard holding cages or cages with running wheels for 8 wk.
Twenty-four hours after the sedentary (SED) or voluntary exercise
(VEX) running period, rats were anesthetized with pentobarbital
sodium, and hearts were isolated and perfused with oxygenated
Krebs-Henseleit (KH) buffer at a constant flow of 15 ml/min. After a
20-min stabilization period, hearts were paced at 300 beats per minute
and perfused with KH buffer containing 10 ␮M DOX for 60 min. A
set of control hearts from SED and VEX rats were perfused under
identical conditions without DOX for the same period. DOX perfusion
led to significant decreases in left ventricular developed pressure,
⫹dP/dt, and ⫺dP/dt, and significant increases in LV lipid peroxidation in sedentary rats compared with non-DOX controls (P ⬍ 0.05).
Prior voluntary exercise training attenuated these DOX-induced effects and was associated with a significant increase (78%, P ⬍ 0.05)
in heat shock protein (HSP72), but not mitochondrial isoform of SOD
(MnSOD) or CuZnSOD protein expression in the hearts of wheel-run
animals. These data indicate that chronic physical activity may provide resistance against the cardiac dysfunction and oxidative damage
associated with DOX exposure and provide novel evidence of HSP72
induction in the heart after voluntary exercise.
VOLUNTARY EXERCISE ATTENUATES DOX CARDIOTOXICITY
METHODS
Animals and experimental design. All experimental procedures
were approved by the University of Northern Colorado Institutional
Animal Care and Use Committee and were in compliance with
guidelines established by the American Physiological Society. Female
Sprague-Dawley rats (200 –225 g) were randomly assigned to a
sedentary or voluntary exercise group. Female rats were selected
because of their greater propensity for voluntary running compared
with males (60). All animals were housed in a temperature-controlled
facility (19 –21°C) maintained on a 12:12-h light-dark cycle with food
and water provided ad libitum. Rats in the voluntary exercise group
were housed individually in cages with stainless-steel running wheels
(1.06 m circumference, Mini-Mitter, Bend, OR) and were allowed
free access to the wheel 24 h per day for 8 wk. Sedentary rats were
housed in standard holding cages without running wheels for the same
period. The voluntary wheel-running model was selected in an effort
to isolate the effects of habitual physical activity from the systemic
stress response observed in rats following forced (e.g., treadmill)
exercise training (32, 33). After the 8-wk sedentary or voluntary
running period, rats were randomly subdivided into four experimental
groups: sedentary control (SED; n ⫽ 6), SED⫹DOX (n ⫽ 7),
voluntary exercise (VEX; n ⫽ 8), or VEX⫹DOX (n ⫽ 7). Hearts
from SED and VEX rats were perfused with Krebs-Henseleit (KH)
buffer for the entire perfusion period. SED⫹DOX and VEX⫹DOX
hearts were perfused under identical conditions with KH buffer
containing 10-␮M DOX (Bedford Laboratories, Bedford, PA).
Isolated heart perfusion protocol. All rats were anesthetized with
heparinized (1,000 U/kg) pentobarbital sodium (40 mg ip), and hearts
were rapidly excised and placed in ice-cold KH buffer consisting of
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(in mM): 120 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl, 25 NaHCO3, 17
glucose, and 0.5 EDTA, (Sigma, St. Louis, MO). The aorta was then
immediately cannulated for retrograde cardiac perfusion at 37°C with
oxygenated (95% O2-5% CO2) KH buffer at a constant perfusion rate
of 15 ml/min. After a 15-min stabilization period, hearts were electrically paced at 300 beats per minute (bpm; 5 Hz) using a stimulus
isolator (AD Instruments, Colorado Springs, CO) and allowed to
stabilize for an additional 5 min before being perfused with KH buffer
(SED and VEX) or KH buffer containing 10 ␮M DOX (SED⫹DOX
and VEX⫹DOX) for an additional 60 min. This DOX perfusion
protocol has been previously shown to provide a reproducible
model of acute DOX cardiotoxicity in the isolated perfused rat heart
(19, 42, 43).
Measurement of LV function. To monitor left ventricular function
during the perfusion period, a microtip catheter pressure transducer
(Millar Instruments, Houston, TX) was inserted into the left ventricular (LV) cavity through the mitral valve for continuous measurement
of LV developed pressure (LVDP) and its maximal and minimal first
derivatives (⫹dP/dt and ⫺dP/dt, respectively). After the 20-min
stabilization period, LV pressure data were recorded under paced
conditions (300 bpm) at 10-min intervals using a PowerLab/8e data
acquisition system (ADInstruments). Immediately after the perfusion
period, hearts were trimmed free of surrounding connective tissue and
fat, blotted dry, and weighed. The LV was then isolated, frozen in
liquid nitrogen, and stored at ⫺80°C for future biochemical analyses.
Lipid peroxidation assay. To examine the effect of DOX perfusion
on myocardial lipid peroxidation in sedentary and wheel-run animals,
malondialdehyde and 4-hydroxy-alkenals (MDA⫹4-HAE) were analyzed in LV of all hearts after the perfusion experiments described
above using a spectrophotometric assay kit (Calbiochem, San Diego,
CA), according to the manufacturer’s instructions. Frozen LV tissue
was homogenized in ice-cold 20 mM Tris 䡠 HCl, pH 7.4, containing 5
mM butylated hydroxytoluene (to prevent lipid peroxidation during
homogenization) at a dilution of 1:5 (wt/vol) and centrifuged at 3,000
g for 10 min at 4°C. The supernatant was collected and used for future
spectrophotometic analyses. MDA ⫹ 4-HAE is expressed relative to
the protein content of sample supernatant determined by the method
of Bradford (1) using bovine serum albumin as the standard.
Left ventricular SOD and HSP72 content. To examine the effect of
voluntary exercise on potential candidates of exercise-induced cardioprotection, HSP72, CuZnSOD, and MnSOD contents were determined in LV samples obtained from SED and VEX animals following
the perfusion protocol described above using Western immunoblotting methods previously described (2, 28). Briefly, LV samples were
homogenized in ice-cold buffer containing (in mM) 100 KCl, 50
MOPS, 5 MgCl2, 1 ATP, 1 EGTA (pH 7.4), and protease inhibitor
cocktail (Roche Diagnostics, Mannheim, Germany). Homogenates
then were diluted 1:3 (vol/vol) in lysis buffer containing 20 mM
HEPES, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.1% SDS (pH
7.4); allowed to incubate at room temperature for 10 min; and then
centrifuged at 3,000 g for 5 min. The supernatant was then assayed for
total protein (1), and a volume of supernatant containing 50 ␮g
(HSP72) or 3 ␮g (SOD isoforms) of LV protein was electrophoresed
on polyacrylamide gels for separation of proteins by molecular
weight. Proteins were then transferred to membranes (Amersham,
Piscataway, NJ) and incubated with an alkaline phosophatase-conjugated polyclonal antibody specific for rat HSP72, or polyclonal
antibodies specific for CuZnSOD or MnSOD (Stressgen, Victoria,
British Columbia, Canada). HSP72 blots were then reacted with
5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium substrate (Sigma Chemical; HSP72) and scanned for band density analysis. SOD isoform blots were incubated with a secondary horseradish
peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a chemiluminescent substrate
(Western Lighting, Perkin-Elmer Life Sciences, Boston, MA), and
developed on film. Quantification of relative HSP72 and SOD isoforms band densities were performed on computerized scans of
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Exercise training has been reported to increase SOD (2, 44)
and HSP72 content (28, 45) in the myocardium. These adaptations have been associated with an exercise-induced preservation of cardiac function and attenuation of myocardial lipid
peroxidation observed following I/R (2, 45). Therefore, it is
plausible that exercise training may also confer protection
against the cardiac dysfunction and myocardial lipid peroxidation induced by DOX exposure. The authors are aware of only
one study that has examined the effect of prior exercise training
on DCT (20). These investigators reported that mice swimtrained before, during, and after DOX therapy in vivo had
fewer myocardial lesions than untrained DOX-treated mice,
but no assessment of cardiac function was conducted.
The vast majority of studies that have examined the cardioprotective effects of exercise training have utilized a forced
treadmill running or swimming model. Although these protocols permit strict control of the duration and intensity of
exercise training, animals appear to experience significant
systemic stress, which may have far-reaching physiological
implications (32, 48). Therefore, we have employed a voluntary running model in an effort to isolate the effects of habitual
physical activity from the additional stress associated with
forced-exercise protocols.
The purpose of this study was to determine whether chronic
voluntary exercise before DOX exposure protects the heart
from the acute manifestations of DCT. We have used an
established model of acute DCT (19, 42, 43) to examine the
direct effects of DOX exposure on intrinsic cardiac function
and lipid peroxidation in isolated perfused hearts from sedentary and physically active rats. Given prior evidence of their
role in exercise-induced cardioprotection against oxidative
stress, MnSOD, CuZnSOD, and HSP72 content were also
examined in hearts of sedentary and physically active animals.
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VOLUNTARY EXERCISE ATTENUATES DOX CARDIOTOXICITY
Fig. 1. Weekly running distances for rats in running wheel cages. Data are
presented as means ⫾ SE.
RESULTS
Voluntary exercise and animal characteristics. Animals in
the running-wheel cages voluntarily ran an average of 6,880 ⫾
348 m/day. Fig. 1 illustrates the average distance run per week
over the 8-wk period. The effects of voluntary exercise on
animal characteristics are presented in Table 1. No significant
differences in body weight were observed between the sedentary and wheel-run rats at the end of the 8-wk study. However,
wheel running led to significant cardiac hypertrophy, indicated
by greater heart weight and heart-to-body weight ratio in the
VEX compared with SED animals (P ⬍ 0.001). In addition, a
skeletal muscle training effect was observed in the VEX
animals, indicated by significantly higher soleus weight and
citrate synthase activity compared with SED (P ⬍ 0.05).
Effect of voluntary exercise and DOX perfusion on LV
function. At the end of the stabilization period, there were no
differences in any of the selected parameters of LV function
observed between the sedentary or wheel-run animals, indicating that voluntary exercise did not significantly alter basal
cardiac function (Table 2). Fig. 2 illustrates the effect of VEX
and DOX perfusion on LV function relative to baseline levels
throughout the 60-min perfusion period. Perfusion with KH
buffer alone had no significant effect on LV function in the
SED or VEX hearts relative to baseline values, and there were
no significant functional differences observed between these
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DISCUSSION
The results of this study indicate that prior voluntary exercise training attenuates the cardiac dysfunction and lipid peroxidation induced by acute DOX exposure. To our knowledge,
these data are the first to provide direct evidence of exerciseinduced protection against the acute functional manifestations
of DCT. In addition, we provide novel evidence of HSP72
induction in the rat heart by chronic voluntary exercise.
Effect of DOX perfusion on LV function and lipid peroxidation. To determine the effects of DOX exposure on intrinsic
cardiac function in previously sedentary or physically active
Table 1. Effects of voluntary wheel running
on animal characteristics
Final body weight, g
Heart weight, g
Heart/body weight ratio, mg/g
Soleus weight, mg/g body wt
Soleus citrate synthase activity,
␮mol䡠g wet wt⫺1䡠min⫺1
SED
VEX
P Value
266⫾12
1.09⫾0.04
4.12⫾0.10
0.39⫾0.01
287⫾4
1.36⫾0.04
4.72⫾0.11
0.44⫾0.02
0.12
⬍ 0.001
⬍ 0.001
0.03
25.2⫾2.3
30.2⫾1.6
0.04
Values are presented as means ⫾ SE. SED, sedentary (n ⫽ 15); VEX,
voluntary exercise (n ⫽ 13).
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immunoblots using ImageJ densitometry software (National Institutes
of Health, Bethesda, MD). VEX band density is expressed as a
percentage of the mean SED band density from the same membrane.
Soleus citrate synthase activity. To examine any training effect
resulting from the voluntary running protocol on skeletal muscle, the
right soleus muscle was excised from sedentary and wheel-run rats
and analyzed for citrate synthase activity at 30°C by the method of
Srere (52).
Statistical analyses and protein assay. All data are expressed as
group means ⫾ SE. Cardiac function parameters were analyzed using
a two-way (VEX ⫻ DOX) ANOVA with repeated measures, followed
by independent sample t-tests to examine individual group differences. In addition, within-group differences between baseline and
time point measurements during perfusion were examined using
paired t-tests with the Bonferroni correction for multiple comparisons.
The MDA⫹4-HAE data were analyzed by a two-way ANOVA with
independent sample t-tests post hoc. HSP72, SOD isoforms, and
morphological data from SED and VEX animals were compared by
independent sample t-tests. Significance was established at P ⬍ 0.05
for all statistical analyses.
two groups. DOX perfusion led to a significant decline in all
parameters of LV function in the hearts from sedentary animals
throughout the perfusion period. LVDP decreased to 84% of
baseline values after 30 min of DOX perfusion (P ⬍ 0.05) and
steadily declined thereafter to 38% of baseline by 60 min.
Similar decrements were observed in LV ⫺dP/dt and ⫹dP/dt,
which decreased to 46% and 50% of baseline by 60 min,
respectively (P ⬍ 0.05). These DOX-induced decrements in
LV function were attenuated in the hearts of wheel-run rats.
LVDP remained at baseline levels for 40 min in the hearts of
VEX⫹DOX rats, then steadily declined to 60% of baseline
after 60 min (P ⫽ not significant). Although ⫺dP/dt and
⫹dP/dt also declined in hearts of wheel-run animals, after 60
min of DOX perfusion (decreased to 91% and 78% of baseline,
respectively), these decreases were not statistically significant.
Furthermore, relative values of LVDP, ⫺dP/dt and ⫹dP/dt
were significantly higher in the VEX⫹DOX than SED⫹DOX
hearts at several time points throughout the perfusion period
(see Fig. 2). These data suggest that VEX before DOX exposure results in an attenuation of DOX-induced LV dysfunction.
Lipid peroxidation. Two-way ANOVA analysis revealed a
significant effect of DOX perfusion on myocardial lipid peroxidation indexes. Although there was no effect of VEX alone
on MDA⫹4-HAE levels, there was a significant interaction
effect of VEX and DOX (P ⬍ 0.05). Compared with SED and
VEX hearts, MDA⫹4-HAE levels were significantly higher in
the SED⫹DOX hearts (P ⬍ 0.05), but not the VEX⫹DOX
hearts (Fig. 3). These data suggest that voluntary exercise
attenuates myocardial lipid peroxidation induced by DOX
exposure.
Left ventricular SOD and HSP72 content. No significant
differences in LV MnSOD or CuZnSOD content were observed between SED and VEX animals (Fig. 4). HSP72 content, however, was 78% higher in the hearts of VEX compared
with SED (Fig. 5), indicating that voluntary exercise induced
HSP72 protein expression in the heart.
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VOLUNTARY EXERCISE ATTENUATES DOX CARDIOTOXICITY
Table 2. Absolute values of left ventricular function in isolated hearts
ESP, mmHg
LVDP, mmHg
⫹dP/dt, mmHg/s
⫺dP/dt, mmHg/s
2.1⫾0.1
1.5⫾0.1
2.3⫾0.2
2.0⫾0.1
86.6⫾2.8
88.4⫾3.7
85.4⫾2.9
86.8⫾3.1
84.5⫾3.2
86.9⫾6.7
83.1⫾6.4
84.8⫾4.7
2507⫾96
2497⫾73
2332⫾58
2445⫾124
⫺1610⫾60
⫺1698⫾42
⫺1573⫾40
⫺1627⫾61
2.2⫾0.1
1.5⫾0.1
14.6⫾4.1*
7.9⫾1.8
91.8⫾2.7
89.8⫾5.0
84.4⫾4.3
93.0⫾5.0
89.6⫾3.1
88.3⫾5.2
69.8⫾2.9*
85.1⫾4.7
2566⫾100
2822⫾351
1909⫾82*
2288⫾121
⫺1826⫾169
⫺1817⫾48
⫺1272⫾75*
⫺1665⫾93
2.3⫾0.1
1.4⫾0.1
59.6⫾49.1*
51.8⫾70.2
95.3⫾3.0
95.3⫾4.5
90.1⫾7.5
102.6⫾17.2
93.0⫾3.6
93.9⫾2.0
30.5⫾1.4*
50.1⫾7.7
2563⫾109
3080⫾518
1168⫾52*
1896⫾179
⫺1753⫾111
⫺1954⫾62
⫺720⫾44*
⫺1481⫾196
Values are means ⫾ SE. EDP, end-diastolic pressure; ESP, end-systolic pressure; LVDP, left ventricular developed pressure (ESP-EDP); ⫹dP/dt, maximum
rate of pressure development; ⫺dP/dt, maximum rate of pressure decline; SED, sedentary (n ⫽ 6); VEX, voluntary exercise (n ⫽ 8); SED ⫹ DOX, sedentary
perfused with DOX (n ⫽ 7); VEX ⫹ DOX, VEX perfused with DOX (n ⫽ 7). *Significantly different than baseline level in the same group (P ⬍ 0.05).
rats, we have monitored LV pressure characteristics of isolated
hearts perfused with 10-␮M DOX over a 60-min period. This
model provides a rapid and reproducible means of assessing
the cumulative effects of continuous DOX exposure at a
Fig. 2. Response of left ventricular developed pressure (A), maximum rate of left
ventricular pressure development (⫹dP/dt) (B), and maximum rate of left ventricular
pressure decline (⫺dP/dt) (C) during the 60-min perfusion period. SED, sedentary
hearts perfused with buffer only (n ⫽ 6); VEX, voluntary exercise hearts perfused with
buffer only (n ⫽ 7); SED⫹DOX, sedentary hearts perfused with buffer ⫹ 10 ␮M
DOX (n ⫽ 8); and VEX, voluntary exercise hearts perfused with buffer ⫹ 10 ␮M
DOX (n ⫽ 7). All data are expressed as mean % of baseline (0 min) values ⫾ SE.
*Significantly lower than baseline in the SED⫹DOX group (P ⬍ 0.05). †Significant
difference between VEX⫹DOX and SED⫹DOX (P ⬍ 0.05).
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concentration similar to peak levels observed in plasma following single intravenous treatments in humans (7, 12). In
accordance with previous studies (19, 40, 42), we observed that
perfusion of hearts with 10-␮M DOX results in a progressive
decline in LVDP, with parallel decrements in dP/dt over a
60-min perfusion period.
The mechanisms by which DOX exposure induces cardiac
dysfunction have not been fully elucidated. Prevailing hypotheses suggest that myocardial oxidative stress is a primary event
in the etiology of DCT and is believed to initiate several of the
deleterious cellular events reported following DOX treatment
(6, 63). Oxidative destruction of fatty acids in cell and organelle membranes indicated by increased levels of lipid peroxidation products has been frequently reported following
DOX exposure (26, 39, 53) and has long been considered to be
a critical event in DCT (34). In accordance with these studies,
we observed a significant increase in MDA⫹4-HAE in the LV
from sedentary animals exposed to DOX, which supports the
association between lipid peroxidation and DOX-induced cardiac dysfunction.
Cardioprotective effects of voluntary wheel running. Voluntary exercise elicited significant cardiac hypertrophy and skel-
Fig. 3. Left ventricular malondialdehyde and 4-hydroxy-alkenals (MDA ⫹
4-HAE) content after the 60-min perfusion period. Data are means ⫾ SE.
Two-way ANOVA revealed a significant VEX⫻DOX interaction. *Significantly greater than SED and VEX (P ⬍ 0.05).
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Baseline (0 min)
SED
VEX
SED ⫹ DOX
VEX ⫹ DOX
30-min perfusion
SED
VEX
SED ⫹ DOX
VEX ⫹ DOX
60-min perfusion
SED
VEX
SED ⫹ DOX
VEX ⫹ DOX
EDP, mmHg
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VOLUNTARY EXERCISE ATTENUATES DOX CARDIOTOXICITY
etal muscle adaptations characteristic of wheel-run female rats
in previous studies (22, 61). Despite reports of favorable
adaptations in cardiac muscle after voluntary exercise training
(35, 59, 61), no differences in absolute parameters of LV
function were observed between the SED and VEX hearts in
the current study when perfused in the absence of DOX (see
Table 2). However, this finding is in agreement with previous
reports that exercise training does not alter baseline LV function in the isolated perfused rat heart under standard perfusion
conditions, despite eliciting significant cardiac hypertrophy
and cardioprotection against I/R stress and hydrogen peroxide
exposure (3, 45, 55). Similarly, no alteration in myocardial
lipid peroxidation was detected in the VEX vs. SED hearts
after KH buffer perfusion in the current study. This finding is
also in agreement with previous investigations that reported no
effect of 8 –10 wk of treadmill training on myocardial lipid
peroxidation in unchallenged hearts, despite observing a significant training-induced attenuation of lipid peroxidation following I/R or ethanol treatment (16, 45). Collectively, these
data suggest that the physiological benefits of these traininginduced adaptations may be evident only in the context of
myocardial stresses that exceed those encountered under normal resting conditions.
Despite the numerous reports of exercise-induced cardioprotection following treadmill exercise training, few studies have
explored the cardioprotective effects of voluntary exercise
training. Woodiwiss et al. (59) reported a preservation of
diastolic function after 16 wk of voluntary wheel running in
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Fig. 5. Left ventricular HSP72 content with representative blots (A) and SOD
activity (B) from sedentary (SED; n ⫽ 6) and wheel run (VEX; n ⫽ 8) rats.
*Significantly greater than SED (P ⬍ 0.001).
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Fig. 4. Left ventricular MnSOD (A) and CuZnSOD (B) content with representative blots from sedentary (SED; n ⫽ 6) and wheel run (VEX; n ⫽ 8) rats.
No significant differences were detected between SED and VEX.
rats with streptozocin-induced diabetes mellitus. Friberg et al.
(11) provided evidence that chronic wheel running enhances
cardiac contractile function in spontaneously hypertensive rats.
However, the current study is the first to examine the effects of
voluntary running on the deleterious effects of DOX exposure.
Our results indicate that hearts isolated from wheel-run animals
resist the DOX-induced LV dysfunction and lipid peroxidation
observed in the hearts of sedentary animals. Although this is
the first report of an exercise-induced cardioprotection against
DOX exposure, several previous investigations have demonstrated similar cardioprotection against other forms of oxidative stress after chronic exercise (2, 45, 55). However, the vast
majority of these studies used a forced treadmill exercise
training model rather than voluntary running. We have used a
voluntary running model in an effort to isolate the effects of
physical activity from the additional stress associated with
forced exercise protocols (32, 48). Therefore, our results provide evidence of exercise-induced cardioprotection in the absence of the coercive stress that may accompany forcedexercise training.
Mechanisms of exercise-induced cardioprotection. Myocardial antioxidant enzymes defend the heart against the damaging
effects of ROS and have been hypothesized to play an important role in exercise-induced resistance to oxidative stress (46)
and in the attenuation of DOX cardiotoxicity (51). In particular, several studies have indicated that the presence of myocardial SOD is critical for the prevention of DOX cardiotoxicity (18, 49, 62). This is reasonable, as SOD removes superoxide from cells, thereby providing the first line of defense
against DOX-induced oxidative stress. The activity and protein
expression of myocardial SOD isoforms has been reported to
be greater in the male and female rat heart following chronic
(ⱖ8 wk) forced treadmill running in some (2, 16, 44, 45), but
not all studies (8, 24). To our knowledge, the current study is
the first to examine myocardial SOD isoforms in the healthy rat
heart after chronic voluntary wheel running. We detected no
effect of voluntary exercise on the protein expression of MnSOD or CuZnSOD in the LV of rats in the current study,
suggesting that other mechanisms must be involved in the
observed cardioprotection against DOX exposure. A putative
explanation for the lack of SOD increase observed in our study
VOLUNTARY EXERCISE ATTENUATES DOX CARDIOTOXICITY
AJP-Regul Integr Comp Physiol • VOL
cardiac HSP72 induction following a 60-min treadmill running
bout occurred only when rats ran at speeds of 24 m/min or
greater. The intensity (i.e., speed) and duration of wheelrunning bouts were not measured in our study; however,
female rats have been reported to voluntarily attain running
speeds of 48 – 68 m/min for up to 5 min with heart rates
exceeding 500 bpm (38). It is not known whether brief episodes of high-intensity running occurring intermittently over a
period of several weeks provides sufficient stimulus for HSP72
induction in the heart. Further investigation is required to
elucidate the mechanisms by which exercise intensity, duration, or their interaction may influence HSP72 induction in the
rat heart.
Although our data suggest a potential role of cardiac HSP72
induction in the cardioprotection observed in the wheel-run rats
of the present study, evidence from prior studies suggest that
other mechanisms may be involved. Joyeux et al. (19) examined the effect of heat stress (42°C for 15 min) on acute DOX
cardiotoxicity in the isolated rat heart, similar to the model
used in the current study. They reported that although prior
heat stress protected the myocardium against electrophysiological injury, it did not attenuate DOX-induced decreases in
LVDP to the extent observed in our study. Although cardiac
HSP72 was not determined in this study, Demirel et al. (9)
reported a nearly sevenfold increase in cardiac HSP72 content
after an identical heat stress protocol. Furthermore, recent
evidence from Lennon et al. (25) suggests that sustained
increases in HSP72 are not essential for exercise-induced
protection during myocardial stunning. Therefore, it is plausible that mechanisms other than HSP72 may have contributed to
the observed exercise-induced cardioprotection against DOX
exposure.
Summary and Conclusions. In summary, our data provide
the first direct evidence of an exercise-induced protection
against the cardiac dysfunction induced by acute DOX exposure. This functional cardioprotection was associated with an
attenuation of DOX-induced lipid peroxidation and higher
levels of HSP72 protein in the hearts of wheel-run rats. Although these data suggest a possible role of HSP72 induction
in the cardioprotection conferred by voluntary exercise, the
precise mechanisms of protection remain to be elucidated. In
conclusion, our results indicate that chronic physical activity
provides resistance against DOX cardiotoxicity, thereby potentially improving the therapeutic value of DOX in individuals
who have been previously active.
Limitations and future study. We have used an established
model of acute DCT in the isolated rat heart to examine the
direct effects of DOX exposure on intrinsic cardiac function in
previously sedentary or physically active rats. Although our
data clearly indicate that prior physical activity is associated
with a reduction in lipid peroxidation and preservation of
cardiac function during acute DOX exposure, the protective
effect of exercise training against chronic DOX treatment in
vivo cannot be determined by our experiments. As previously
mentioned, clinical presentation of DOX cardiomyopathy typically occurs after completion of several intravenous DOX
injections over a period of several months. Therefore, further
investigation is needed to determine the effect of exercise
training before, during, or after chronic DOX treatment in vivo
to fully explore the potential benefits of physical activity on the
acute and chronic cardiotoxicity of DOX treatment.
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and the equivocal findings of prior studies that used treadmill
training may be differences in the duration and/or intensity of
the selected exercise training protocols. Moran et al. (31)
recently reported that although 24 wk of treadmill exercise
training elicited significant increases in myocardial SOD activity, no changes were detected after 12 wk. Powers et al. (44,
45) and Brown et al. (2) reported increases in myocardial SOD
activity and protein expression after high-intensity treadmill
running protocols (ⱖ30 m/min, ⱖ10% grade) lasting 10 to
⬎20 wk, whereas other studies using less intense treadmill
running protocols have reported no effect (8, 24). Although
female rats have been reported to voluntarily achieve brief
periods of high-intensity running in the wheels (⬎45 m/min),
the running is intermittent in nature (38). Therefore, is it
plausible that the voluntary wheel-running protocol used in the
current study did not provide the sustained high-intensity
running that may be required to elicit increases in myocardial
SOD protein.
Although no changes in SOD isoforms were observed in this
study, earlier investigations have indicated that an increase in
myocardial SOD is not required for exercise-induced cardioprotection (8, 14, 25). Although it is possible that increases in
other antioxidant enyzmes (e.g., CAT and GPx) may have
contributed to the cardioprotection observed in wheel-run rats,
exercise has been reported to attenuate lipid peroxidation and
LV dysfunction after I/R in the absence of any increase in CAT
or GPx (8, 14, 25). Therefore, it appears that whereas antioxidant enzymes clearly play a role in defending the heart against
ROS, an increase in their expression may not be required for
the enhanced cardioprotection conferred by exercise training.
Increasing evidence suggests that myocardial HSP72 induction plays a pivotal role in the exercise-induced cardioprotection against oxidative stress (14, 46, 47, 56). HSP72 induction
has been shown to attenuate apoptosis, necrosis, and oxidative
injury in the myocardium following I/R (14, 47) and DOX
exposure (17). Interestingly, HSP72 induction by exercise
training or heat stress has attenuated myocardial lipid peroxidation and preserved cardiac function following I/R without a
concomitant increase in cardiac SOD, CAT, or GPx activity (8,
14, 57). Although previous studies have reported 400 –500%
increases in cardiac HSP72 content after forced treadmill
training (36, 47), a similar effect has not been observed after
voluntary wheel running. To our knowledge, only two studies
have previously examined cardiac HSP72 content after voluntary wheel running, and both reported no effect (4, 36). In
contrast, we have observed a 78% higher HSP72 level in the
hearts of wheel-run rats compared with the hearts of sedentary
rats (P ⬍ 0.001). These discrepant findings may be explained
by the fact that the two aforementioned studies that reported no
effect of wheel running on cardiac HSP72 used male rats. The
present study is first to examine the effect of voluntary wheel
running on myocardial HSP72 protein in female rats, which
were selected because of their greater propensity for voluntary
exercise compared with males (60). Indeed, the females in the
present study averaged 9- to 15-fold longer running distances
per week than the males in the previous studies (4, 36).
Therefore, a substantially greater volume of physical activity
may account for the observed differences in HSP72 protein
expression. However, it has been suggested that intensity,
rather than duration of exercise, is the critical factor for cardiac
HSP72 induction (29, 36). Milne and Noble (29) reported that
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VOLUNTARY EXERCISE ATTENUATES DOX CARDIOTOXICITY
ACKNOWLEDGMENTS
We wish to acknowledge Russell Moore, David Brown, Korinne Jew, and
Micah Johnson at the University of Colorado, Boulder, for their technical
advice and assistance with Western blotting experiments.
20.
GRANTS
21.
This study was supported by grants from the American Cancer Society and
the University of Northern Colorado Sponsored Programs and Academic
Research Center to R. Hayward.
22.
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