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Am J Physiol Heart Circ Physiol
281: H1346–H1352, 2001.
Short-term exercise training can improve myocardial
tolerance to I/R without elevation in heat shock proteins
Received 14 August 2000; accepted in final form 18 June 2001
Hamilton, Karyn L., Scott K. Powers, Takao Sugiura,
Sunjoo Kim, Shannon Lennon, Nihal Tumer, and
Jawahar L. Mehta. Short-term exercise training can improve myocardial tolerance to I/R without elevation in heat
shock proteins. Am J Physiol Heart Circ Physiol 281:
H1346–H1352, 2001.—We examined the effects of 3 days of
exercise in a cold environment on the expression of left
ventricular (LV) heat shock proteins (HSPs) and contractile
performance during in vivo ischemia-reperfusion (I/R).
Sprague-Dawley rats were divided into the following three
groups (n ⫽ 12/group): 1) control, 2) exercise (60 min/day) at
4°C (E-Cold), and 3) exercise (60 min/day) at 25°C (E-Warm).
Left anterior descending coronary occlusion was maintained
for 20 min, followed by 30 min of reperfusion. Compared with
the control group, both the E-Cold and E-Warm groups maintained higher (P ⬍ 0.05) LV developed pressure, first derivative of pressure development over time (⫹dP/dt), and pressure relaxation over time (⫺dP/dt) throughout I/R. Relative
levels of HSP90, HSP72, and HSP40 were higher (P ⬍ 0.05)
in E-Warm animals compared with both control and E-Cold.
HSP10, HSP60, and HSP73 did not differ between groups.
Exercise increased manganese superoxide dismutase (MnSOD) activity in both E-Warm and E-Cold hearts (P ⬍ 0.05).
Protection against I/R-induced lipid peroxidation in the LV
paralleled the increase in MnSOD activity whereas lower
levels of lipid peroxidation were observed in both E-Warm
and E-Cold groups compared with control. We conclude that
exercise-induced myocardial protection against a moderate
duration I/R insult is not dependent on increases in myocardial HSPs. We postulate that exercise-associated cardioprotection may depend, in part, on increases in myocardial
antioxidant defenses.
exercise training provides
myocardial protection against ischemia-reperfusion
(I/R) injury (2, 4, 5, 9, 15, 19, 25, 27). Indeed, both
short-term (i.e., days) and long-term (i.e., months) exercise training enhances myocardial recovery after an
I/R insult as evidenced by an improved recovery of
cardiac contractile performance and reduced oxidative
damage to the myocardium (9, 25). The exact biochemical mechanisms responsible for this protection continue to be debated. Nonetheless, it has been argued
that elevated myocardial levels of heat shock protein
(HSP) 72, and perhaps other HSPs (e.g., HSP10,
HSP40, HSP60, and HSP90), play a significant role in
the exercise-induced improvement in myocardial protection during I/R (for reviews, see Refs. 20 and 21).
Although an exercise-induced increase in myocardial
HSP72 is a potential mechanism to explain the cardioprotection associated with exercise, recent work by Taylor et al. (27) indicates that exercise training in a cold
environment does not elevate myocardial levels of HSP72
but improves myocardial performance postischemia in an
isolated working heart model. Taylor et al. concluded
that whereas increases in myocardial HSP72 can contribute to improved postischemic function, other mechanisms
must be responsible for the exercise-induced cardioprotection in their experiments.
With the use of an in vivo model of I/R, the current
experiments expand on the work of Taylor et al. (27) by
exploring other potential mechanisms that could contribute to the exercise-induced myocardial protection
during an I/R insult. In particular, we hypothesized
that whereas exercise in a cold environment does not
upregulate the expression of myocardial HSP72, this
type of exercise could increase other important stress
proteins in the heart and/or enhance myocardial antioxidant defenses. To test this hypothesis, animals were
exercise trained in both warm and cold environments.
Myocardial responses to in vivo I/R were then exam-
Address for reprint requests and other correspondence: S. K.
Powers, Dept. of Exercise and Sport Sciences and Physiology, Center
for Exercise Science, Univ. of Florida, Gainesville, FL 32611 (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.
endurance exercise; heart; radicals; lipid peroxidation; antioxidant enzymes
IT IS CLEAR THAT ENDURANCE
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KARYN L. HAMILTON,1 SCOTT K. POWERS,2 TAKAO SUGIURA,5 SUNJOO KIM,6
SHANNON LENNON,2 NIHAL TUMER,3 AND JAWAHAR L. MEHTA4
1
Baylor College of Medicine, Cardiology Research, Veterans Affairs Medical Center,
Houston, Texas 77030; 2Department of Exercise and Sport Sciences and Physiology and the Center
for Exercise Science, University of Florida, Gainesville, 32611; 3Geriatric Research, Education
and Clinical Center, Department of Veterans Affairs Medical Center, and 4Division of Cardiology,
University of Florida College of Medicine, Gainesville, Florida 32610; 5Department of Exercise
and Health Science, Yamaguchi University, Yamaguchi, Japan 753; and 6Department of
Clinical Pathology, Gyeongsang National University School of Medicine, Chinju, Korea 660-702
EXERCISE TRAINING IMPROVES I/R TOLERANCE
ined and myocardial levels of stress proteins and the
activities of primary antioxidant enzymes were assessed.
METHODS
Animals and Experimental Design
Exercise Training Protocol
The animals selected to engage in exercise training were
habituated to treadmill exercise by daily treadmill running
for 3–5 consecutive days. Ambient temperature in which
training occurred was either 25°C or 4°C. The first day of
exercise began with a 5-min exercise bout; this duration was
increased by 10–15 min per day during the next 4 days.
Control animals were placed in a nonmoving treadmill. After
this habituation to exercise training, the exercise-trained
animals were then exercised for 3–5 consecutive days of
treadmill exercise, 60 min/day, 30 m/min, 0% grade, at ⬃70%
maximal O2 consumption (18). Mild electrical shocks were
used sparingly to motivate animals to run. Colonic temperature was measured before and after each 60-min exercise
bout. The decision to exercise train animals for 3–5 days at
this work rate was based on previous work from our laboratory (unpublished observations), indicating that this training
protocol promotes myocardial expression of HSP72 and provides cardioprotection during an in vivo I/R insult.
In Vivo Protocol for Studying Myocardial Responses
During I/R
In vivo I/R was performed 24 h after the last exercise
training session. Animals were anesthetized with 30 mg/kg of
pentobarbital sodium and ventilated with room air with the
use of a small animal ventilator (Kent Scientific; Litchfield,
CT). Throughout the surgery, body temperature was monitored via a rectal thermistor probe. Body temperature was
maintained at ⬃37 ⫾ 1°C with the use of a heated operating
platform and appropriate heating lamps. Cardiac rhythm
was monitored continuously via a standard electrocardiogram (lead II).
The chest was opened by a left thoracotomy and a ligature
was placed around the left anterior descending coronary
artery (LCA) close to its origin (ligature ends were exteriorized). At this point, any animals exhibiting significant ventricular arrhythmias were eliminated from the study. Coronary occlusion was achieved by passing both ends of the
ligature through a small plastic tube, which was then
pressed against the surface of the heart directly above the
LCA. The resulting arterial occlusion was maintained for 20
min by clamping the plastic tube and ligature with a small
hemostat. This duration of ischemia results in myocardial
stunning without significant infarction (3). Reperfusion duAJP-Heart Circ Physiol • VOL
ration was 30 min and was achieved by removing the clamp
and the tube. When necessary, gentle massage of the nonischemic portion of the heart was used to convert ventricular
fibrillation into a normal sinus rhythm. Animals exhibiting
⬎3 episodes of ventricular fibrillation were eliminated from
the study. Sham surgery included all surgical interventions
with the exception of coronary occlusion.
Validation of Coronary Occlusion and Reperfusion
The aforementioned technique of coronary occlusion has
been shown to be effective by other investigators (2, 15). More
importantly, we have validated that complete coronary occlusion, consistently resulting in ischemia in ⬃60% of the myocardium, is achieved in our hands (author’s unpublished
observations). Furthermore, we (25) have also performed
experiments to ensure that reperfusion is adequately
achieved in this model.
Measurement of Left Ventricular Developed Pressure
and dP/dt
To monitor cardiovascular function during the I/R protocol,
an arterial cannula was introduced via the carotid artery into
the left ventricle. Left ventricular developed pressure
(LVDP), first derivative of pressure development over time
(⫹dP/dt), and first derivative of pressure relaxation over time
(⫺dP/dt) were measured by using a pressure transducer
interfaced with a computerized heart performance analysis
system (Digi-Med; Louisville, KY).
Tissue Preparation
Selected biochemical properties of the ventricular myocardium were studied in all experimental groups. To determine
the antioxidant capacity in the hearts of all experimental
groups, small samples of the LV were removed, rinsed free of
blood in ice-cold antioxidant buffer (100 ␮M EDTA, 50 mM
NaHPO4, and 1 mM BHT; pH 7.4), and quickly frozen in
liquid nitrogen. These samples were later assayed to determine the concentration of protein thiols as well as the activities of superoxide dismutase (SOD), glutathione peroxidase
(GPx), and catalase (CAT).
Furthermore, LV tissue that was inferior to the LCA
occlusion site was quickly removed, divided into three sections, and frozen in liquid nitrogen for subsequent biochemical analysis of lipid peroxidation and measurement of the
relative levels of HSP10, HSP40, HSP60, HSP72, HSP73,
and HSP90.
Biochemical Assays
To assess the effects of both exercise training and I/R on
the myocardium, we measured tissue lipid peroxidation, protein thiols, and the activities of antioxidant enzymes. Sections of the LV myocardium were minced and homogenized in
100 mM cold phosphate buffer with 0.05% bovine serum
albumin (1:20 wt/vol; pH 7.4). Homogenization was achieved
by using 40 passes of the homogenate in a tight-fitting Potter-Elvehjem homogenizer. Homogenates were then centrifuged (3°C) for 10 min at 400 g. The supernatant was decanted and assayed to determine total protein content along
with the activities of SOD (EC1.15.1.1), CAT (EC1.11.1.6),
and GPx (EC1.11.1.9). Protein content was determined using
methods described by Bradford (6). In sham surgery groups,
the supernatant was then assayed to determine the activities
of both manganese SOD (MnSOD) and copper-zinc SOD
(Cu/ZnSOD) using the cytochrome c reduction technique of
McCord and Fridovich (23). GPx and CAT activity were
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This experimental protocol was approved by the University of Florida Animal Care and Use Committee and followed
the guidelines established by the American Physiological
Society for the use of animals in research. Female SpragueDawley rats (4 mo old) were randomly assigned to one of
three experimental groups (n ⫽ 12/group): 1) control, 2) 3–5
consecutive days of treadmill exercise in a cold (4°C) environment (E-Cold), and 3) 3–5 consecutive days of treadmill
exercise in a warm (25°C) environment (E-Warm). Each of
these three groups was further divided into two surgical
groups: sham surgery and I/R surgery. During the experimental period, all groups were maintained on a 12:12-h
light-dark cycle and provided rat chow and water ad libitum.
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EXERCISE TRAINING IMPROVES I/R TOLERANCE
determined in the LV as described by Flohe and Gunzler (11)
and Aebi (1), respectively. In our laboratory, the coefficients
of variation for SOD, GPx, and CAT were ⬃2, 3, and 5%,
respectively. These and all other biochemical assays were
performed in triplicate at 25°C and samples from all experimental groups were assayed on the same day to avoid interassay variation.
Protein Thiol Measurements
Lipid Peroxidation Measurements
To determine the amount of oxidative damage in the heart,
LV levels of two by-products of lipid peroxidation were measured. Lipid hydroperoxides were quantified using the ferrous oxidation-xylenol orange technique reported by HermesLima et al. (13). Briefly, after the tissue was homogenized in
methanol, the membrane peroxides were mixed in solution
with an iron source (FeSO4), an acid (H2SO4), and a reactive
dye (xylenol orange). In this mixture, the membrane peroxides oxidize Fe2⫹ to Fe3⫹ and the peroxides are reduced. The
ionized acid assists in the reduction. The Fe3⫹ then reacts
with the xylenol orange to form a Fe3⫹-xylenol orange complex. Originally yellow in dye form, the Fe3⫹-xylenol complex
changes to a purplish color that can be detected spectrophotometrically at 580 nm. Cumene hydroperoxide was used to
generate a standard curve for this assay.
In addition to lipid hydroperoxides, total 8-isoprostane was
quantified by competitive enzyme immunoassay (EIA) as per
the manufacturer’s instruction (Cayman Chemical; Ann Arbor, MI). Measurement of 8-isoprostane, a member of the
eicosanoid family, is based on the competition between 8-isoprostane and an 8-isoprostane-acetylcholinesterase conjugate for a limited amount of 8-isoprostane polyclonal antiserum. Briefly, after homogenization in methanol, samples
are extracted and hydrolyzed via incubations in 100% ethanol and 15% potassium hydroxide. Resultant samples are
then purified on a methanol-activated Sep-Pak C18 column
and evaporated to dryness using a Supelco vacuum centrifugation system. The remaining residue is then redissolved in
EIA buffer, placed in microplates coated with antibody, and
mixed with antiserum to 8-isoprostane and 8-isoprostane
linked to acetylcholinesterase. After a 24-h incubation, wells
are washed free of unbound reagents, and Ellman’s reagent
is added to facilitate spectrophotometric measurement at 412
nm. Standard curves constructed using known concentrations of 8-isoprostane are used to quantify total 8-isoprostane
concentration in the samples.
Immunoblotting
To determine the effects of training on induction of myocardial HSPs, we performed polyacrylamide gel electrophoresis and immunoblotting using a modification of techniques
described by Locke et al. (21). Briefly, LV samples from I/R
animals were homogenized and one-dimensional sodium doAJP-Heart Circ Physiol • VOL
Data Analysis
Significance was established a priori at P ⬍ 0.05. All
dependent variables were analyzed using analysis of variance software (Systat, SPSS). Where appropriate, Tukey’s
honestly significant difference test was applied post hoc.
RESULTS
Colonic Temperature During Exercise
Sixty minutes of exercise in the cold (4°C) environment did not result in a significant increase (P ⬎ 0.05)
in colonic temperature (preexercise 38.2 ⫾ 0.1°C; postexercise 38.1 ⫾ 0.2°C). Hence, it seems unlikely that
myocardial temperature increased significantly in
these conditions. In contrast, 60 min of exercise in the
thermoneutral (25°C) environment resulted in a significant rise (P ⬍ 0.05) in colonic temperature from 37.6 ⫾
0.2°C (preexercise) to 41.1 ⫾ 0.2°C (postexercise).
Myocardial Performance During I/R
Successful I/R protocols were performed on 12 animals from each experimental group. Ventricular fibrillation mandated exclusion of five control animals, two
E-Warm animals, and two E-Cold animals. Figure 1
contains the mean values for LVDP during preischemia, ischemia, and after reperfusion in animals from
all experimental groups. No differences existed (P ⬎
0.05) between groups in these measures before ischemia. However, compared with control, both E-Cold and
E-Warm animals maintained higher (P ⬍ 0.05) LVDP
throughout both ischemia and reperfusion. Identical
results were found for LV ⫹dP/dt and ⫺dP/dt during
I/R (data not shown).
Biochemical Measurements
Myocardial HSP content. To determine if the relative
levels of HSP10, 40, 60, 72, 73, and 90 were elevated in
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Protein oxidation was assessed by spectrophotometric
measurement of LV protein thiols, as described by Jocelyn
(16). This assay measures the quantity and proportion of
sulfhydryl (SH) groups within tissue. Briefly, total cellular
thiols were determined by mixing 5,5⬘-dithiobis(2-nitrobenzoic acid) (DTNB; a SH reagent) with tissue homogenate and
quantifying spectrophotometrically at 412 nm. DTNB was
then reacted with homogenate after acid precipitation of
proteins. Protein-bound thiol content was obtained mathematically by subtracting the nonprotein-bound thiol concentration from the total thiol concentration.
decyl sulfate polyacrylamide gel electrophoresis was performed to separate proteins by molecular weight. The percentage of polyacrylamide used in gels varied as a function of
the molecular weight of the individual HSP as follows:
HSP90 (10%), HSP73 (12.5%), HSP72 (12.5%), HSP60
(12.5%), HSP40 (12.5%), and HSP10 (15%). After separation,
proteins were transferred to nitrocellulose membranes (0.45
mm thick, Bio-Rad; Hercules, CA) using the Bio-Rad transblot electrophoretic transfer cell at a constant voltage of 100
V for 1 h. After protein transfer, the nitrocellulose membranes were blocked for 2 h using either 1% bovine serum
albumin or 5% nonfat dry milk. Blots were incubated for 2 h
with the following antibodies: rabbit polyclonal anti-HSP40,
mouse monoclonal anti-HSP72, rabbit polyclonal antiCPN10, mouse monoclonal anti-HSP60, rat monoclonal antiHSP90, or rat monoclonal anti-HSP70 (Stress Gen; Victoria,
Canada). Antibodies were then reacted with the appropriate
secondary antibody conjugated to alkaline phosphatase before incubation with bromochloroindolyl phosphate-nitro
blue tetrazolium substrate (Sigma; St. Louis, MO). Quantification of the bands from the immunoblots was performed
using computerized densitometry and NIH Image Analysis
software. Standard curves were constructed during preliminary experiments to assure linearity.
EXERCISE TRAINING IMPROVES I/R TOLERANCE
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4). More importantly, it should be noted that 8-isoprostane concentrations were also lower in both E-Warm
sham and E-Cold sham groups compared with control
sham (P ⬍ 0.05). These results indicate that exercise
training in both warm and cold environments provided
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Fig. 1. Left ventricular developed pressure (LVDP) in the three
experimental groups before ischemia (I), during ischemia, and during reperfusion (R). Data are means ⫾ SE and are expressed as a
percentage of initial (preischemia) values. #P ⬍ 0.05, both warm
environment (E-Warm) and cold environment (E-Cold) significantly
different from control (C). *P ⬍ 0.05, E-Warm significantly different
from control.
the hearts from exercise-trained animals, portions of
the LV from control, E-Cold, and E-Warm animals
were analyzed for the relative levels of HSP isoforms
by using Western blotting. The results revealed that
myocardial levels of HSP40, HSP72, and HSP90 were
significantly greater (P ⬍ 0.05) in the LV of E-Warm
animals compared with both control and E-Cold animals (Fig. 2, A–C). Whereas a trend toward increased
HSP90 content was apparent in E-Cold compared with
control, this difference did not reach statistical significance (P ⫽ 0.85). Exercise training (both environmental conditions) did not alter (P ⬎ 0.05) ventricular
levels of HSP10, HSP60, and HSP73 (Fig. 3, A–C).
Myocardial antioxidant enzyme activity. To determine if exercise training altered myocardial antioxidant capacity we measured the activities of MnSOD,
Cu/ZnSOD, CAT, and GPx in the LV of animals from
all experimental groups. These data are presented in
Table 1. The results revealed that compared with control, exercise training elevated (P ⬍ 0.05) myocardial
activity of MnSOD in both E-Cold and E-Warm animals. In contrast, exercise training (E-Cold and EWarm animals) did not alter the activities of Cu/ZnSOD or CAT in the LV. Also, myocardial GPx activity
was greater (P ⬍ 0.05) in E-Cold animals compared
with both control and E-Warm animals. Antioxidant
enzyme activities were assayed in LV tissues from
sham surgery animals and, therefore, do not reflect the
impact of ischemia or reperfusion on enzyme activities.
Myocardial lipid peroxidation and protein thiols. To
determine if the exercise-induced improvement in myocardial contractile performance after an I/R insult was
associated with improved protection against oxidative
stress, we measured two markers of lipid peroxidation.
Our results indicated that in hearts exposed to I/R,
myocardial levels of both lipid hydroperoxides, and
8-isoprostane were lower (P ⬍ 0.05) in both E-Cold and
E-Warm animals compared with control animals (Fig.
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Fig. 2. LV heat shock proteins HSP40 (A), HSP72 (B), and HSP90
(C) levels in C, E-Cold, and E-Warm animals obtained from densitometric scanning of Western blots reacted with an antibody for these
HSPs. Lanes 1 and 4, C; Lanes 2 and 5, E-Warm; Lanes 3 and 6,
E-Cold. Values are means ⫾ SE, expressed as a percentage of the
control group. *P ⬍ 0.05, different from C and E-Cold.
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EXERCISE TRAINING IMPROVES I/R TOLERANCE
Table 1. Activities of important antioxidant enzymes
in the left ventricle of sham surgery animals
Enzyme, activity per
mg protein
GPx, ␮mol/min
MnSOD, units
Cu/ZnSOD, units
CAT, units
Control
(n ⫽ 10)
0.335 ⫾ 0.014
87.13 ⫾ 5.18
108.05 ⫾ 7.63
2.46 ⫾ 0.30
E-Warm
(n ⫽ 10)
E-Cold (n ⫽ 12)
0.326 ⫾ 0.020 0.375 ⫾ 0.020*
103.80 ⫾ 7.72† 105.11 ⫾ 4.02†
109.81 ⫾ 9.88 104.71 ⫾ 8.54
2.86 ⫾ 0.28
3.01 ⫾ 0.24
Values are means ⫾ SE; n, no. of rats. E-Warm, 25°C warm
environment; E-Cold, 4°C cold environment; GPx, glutathione peroxidase; MnSOD, manganese superoxide dismutase; Cu/ZnSOD, copper-zinc; CAT, catalase. * P ⬍ 0.05, different from control and
E-Warm. † P ⬍ 0.05, different from control.
DISCUSSION
Our data indicate that exercise training in both a
cold and warm environment provides myocardial protection as indicated by improved cardiac contractile
performance during an in vivo I/R protocol designed to
result in myocardial stunning. Exercise in the warm
environment was associated with significant increases
in the relative levels of myocardial HSP40, HSP72, and
HSP90. However, exercise in the cold environment did
not alter myocardial HSP levels. Hence, we interpret
these results as evidence that the exercise training-
Fig. 3. LV HSP10 (A), HSP60 (B), and HSP73 (C) levels in C, E-cold,
and E-Warm animals obtained from densitometric scanning of Western blots reacted with an antibody for these HSPs. Lanes 1 and 4, C;
Lanes 2 and 5, E-Warm; Lanes 3 and 6, E-Cold. Values are means ⫾
SE expressed as a percentage of the C group.
myocardial protection against I/R-induced lipid peroxidation. This was the result of a lower sham or “unstressed” level of lipid peroxidation.
Finally, protein thiol concentrations were higher
(P ⬍ 0.05) in hearts exposed to I/R from both E-Cold
and E-Warm animals compared with control animals
(Fig. 5). These data indicate that exercise training
provides cardioprotection against I/R-induced protein
oxidation.
AJP-Heart Circ Physiol • VOL
Fig. 4. A: content of total 8-isoprostanes. B: lipid hydroperoxides in
the LV of both sham and ischemic-reperfused (I/R) animals from all
experimental groups. Values are means ⫾ SE. *P ⬍ 0.05, different
from C I/R; !P ⬍ 0.05, different from E-Warm and E-Cold I/R; ⫹P ⬍
0.05, different from C sham.
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Overview of Principal Findings
EXERCISE TRAINING IMPROVES I/R TOLERANCE
induced protection against in vivo I/R injury can be
achieved without an increase in myocardial HSPs.
Because exercise training (both cold and warm environments) was associated with elevated myocardial
antioxidant defenses and a decrease in I/R-induced
myocardial lipid peroxidation, we postulate exerciseinduced cardioprotection is due, in part, to an elevation
in cardiac antioxidant defenses.
Exercise Training Improves Myocardial Performance
During I/R Without Elevation in HSPs
Our data indicate that short-term (3–5 days) endurance exercise training results in myocardial protection
against I/R injury, as evidenced by the improved cardiac pressure generation during both ischemia and
reperfusion (Fig. 1). These results agree with previous
reports by others (21, 27).
Our results also indicate that exercise-induced cardioprotection against I/R-induced injury does not require an elevation in myocardial levels of HSPs. Nonetheless, there is convincing evidence that HSP72 as
well as other HSPs can contribute to myocardial protection against I/R injury. Indeed, recent studies (7, 22,
24) involving transfection of constructs into cultured
cells and transgenic animal models have provided direct evidence that HSP72 is a cytoprotective protein.
For example, studies (7, 22, 24) employing transgenic
mice overexpressing HSP72 transgene products in
their myocardium have provided strong evidence for
HSP72-mediated myocardial protection during an I/R
insult. Therefore, in the current experiments, the increases in myocardial levels of HSPs (i.e., HSP40,
HSP72, and HSP90) after exercise bouts at room temperature are a potential contributor to the improved
myocardial protection against I/R injury. However, our
results reveal that the exercise training in a cold environment can provide protection against I/R-induced
myocardial injury in vivo without increasing HSPs in
the heart (i.e., HSP10, HSP40, HSP60, HSP72, HSP73,
and HSP90). These data support and extend the findings of Taylor et al. (27), who reported that HSP72 is
AJP-Heart Circ Physiol • VOL
not the factor directly responsible for the exerciseinduced myocardial protection after an in vitro I/R
insult. Recently, however, Harris and Starnes (12) reported that preventing a rise in core temperature during chronic exercise, thus preventing accumulation of
myocardial HSP72, abolishes the cardioprotection observed when core temperature rises during training
resulting in increased myocardial HSP72. Collectively,
we interpret these results as evidence that exercise
training promotes complex biochemical alterations,
other than an increased expression of major HSPs,
capable of providing myocardial protection against an
I/R insult involving ischemia of short-to-moderate duration.
Whereas several mechanisms could contribute to the
training-induced cardioprotection, an exercise-induced
upregulation in cardiac antioxidant capacity is a potential contributor. In this regard, it is well known that
reactive oxygen species (ROS) are produced during I/R
and are important contributors to I/R-induced myocardial injury (for a review, see Ref. 10). Furthermore,
administration of ROS scavengers has been shown to
attenuate I/R-induced cardiac injury (3). In the current
experiments, exercise training promoted an upregulation of MnSOD activity in hearts from both E-Cold- and
E-Warm-trained animals. Hence, we postulate that the
training-induced increase in myocardial antioxidant
capacity is a contributor to the cardioprotection associated with exercise. Yamashita et al. (28) reached
similar conclusions. Interestingly, in a recent study by
Harris and Starnes (12), transient changes in the activities of Cu/ZnSOD and MnSOD, as well as CAT,
were reported after exercise training of various durations with or without changes in core temperature.
Changes in enzyme activities were noted only with
exercise training lasting 3 wk, and, because antioxidant activities returned to sedentary levels by 9 wk of
training, changes in antioxidant defenses were ruled
out as a contributing factor in the exercise-associated
cardioprotection. Nonetheless, it remains unclear
whether an increase in myocardial antioxidant capacity is the primary mechanism responsible for the cardioprotection associated with short-term exercise or
whether an augmented antioxidant capacity is simply
one of many redundant protective mechanisms affected
by exercise training.
Influence of Body Temperature on Exercise-Induced
HSP Expression
The component of exercise that is responsible for
increasing the expression of myocardial HSPs continues to be investigated. A variety of stresses associated
with exercise could contribute to the elevation in myocardial levels of HSPs. For example, heat stress, hypoxia, production of ROS, and stretching of cardiac myocytes are all potential contributors to HSP synthesis
(see Ref. 17 for a review). In our experiments, when the
exercise-induced rise in body temperature was prevented by exercise in the cold, myocardial levels of
HSPs in E-Cold animals were not elevated above con-
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Fig. 5. Protein thiol content in the LV of both sham and I/R animals
from all experimental groups. *P ⬍ 0.05, different from C I/R; !P ⬍
0.05, different from C sham.
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This work was supported by a grant-in-aid from the American
Heart Association, Florida Affiliate (to S. K. Powers) and by a
contract with the Department of Defense (to J. L. Mehta).
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trol. This finding indicates that the rise in body temperature during exercise is an essential component of
the exercise-induced expression of myocardial HSP72
as well as other HSPs. Others have reached a similar
conclusion (12, 27).
Finally, whereas our results and those of others (12,
27) indicate that an exercise-induced increase in body
temperature is required for an exercise-induced elevation in myocardial levels of HSPs, these findings differ
from the work of others (26). Skidmore et al. (26)
reported that when colonic temperature was maintained at resting temperature during exercise (i.e.,
exercise in a cool environment), small but significant
exercise-induced increases in myocardial levels of
HSP72 occurred. Close examination of these studies
does not provide an explanation for the divergent findings; additional research is required to resolve this
controversy.
In conclusion, to our knowledge, this is the first
experiment to examine the effects of exercise training
in a cold environment on cardioprotection during in
vivo I/R. Furthermore, we also examined metabolic
factors that might contribute to this exercise-induced
protection during I/R (i.e., myocardial HSPs and antioxidant enzymes). The major finding of this study was
that exercise-induced myocardial protection against an
I/R insult is not dependent on increases in the levels of
myocardial HSP10, HSP40, HSP60, HSP72, HSP73, or
HSP90. Whereas numerous mechanisms could contribute to the training-induced cardioprotection, it seems
likely that an exercise-induced upregulation in cardiac
antioxidant capacity is a contributor. It is unclear if the
exercise-induced increase in myocardial antioxidant
capacity is the primary mechanism to explain the myocardial protection associated with exercise or if the
improved antioxidant capacity is simply one of several
protective mechanisms influenced by exercise training.
Determining the mechanisms responsible for the exercise-induced cardioprotection is an important area for
future research.