Loss of exercise induced cardioprotection

Loss of exercise-induced cardioprotection after
cessation of exercise

Shannon L. Lennon, John Quindry, Karyn L. Hamilton, Joel French, Jessica
Staib, Jawahar L. Mehta and Scott K. Powers
J Appl Physiol 96:1299-1305, 2004. First published 12 December 2003;
doi:10.1152/japplphysiol.00920.2003
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Exercise-induced cardiac preconditioning: how exercise protects your achy-breaky heart
Chad R. Frasier, Russell L. Moore and David A. Brown
J Appl Physiol, September , 2011; 111 (3): 905-915.

J Appl Physiol 96: 1299–1305, 2004.
First published December 12, 2003; 10.1152/japplphysiol.00920.2003.

Loss of exercise-induced cardioprotection
after cessation of exercise
Shannon L. Lennon,1 John Quindry,1 Karyn L. Hamilton,1 Joel French,1
Jessica Staib,1 Jawahar L. Mehta,2 and Scott K. Powers1,3
1

Department of Exercise and Sport Sciences, Center for Exercise Science, University of Florida,
Gainesville, Florida 32611; 2Division of Cardiovascular Medicine, University of Arkansas for
Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205;
and 3Department of Physiology, University of Florida, Gainesville, Florida 32611
Submitted 27 August 2003; accepted in final form 8 December 2003

heart; reactive oxygen species; heat shock proteins; antioxidant enzymes; ischemia-reperfusion

5–20 min resulting in myocardial stunning) and long-duration
(i.e., Ͼ20 min resulting in MI) myocardial ischemia. Although
the mechanism(s) responsible for exercise-mediated cardioprotection remain unknown, it has been postulated that exerciseinduced increases in myocardial levels of heat shock protein
(HSP) 72 and/or antioxidant defenses contribute to this cardioprotection (9, 28, 38, 48, 49). In regard to HSP72 and cardioprotection, transgenic mice that overexpress HSP72 provide
convincing evidence that HSP72 imparts cardioprotection. Indeed, compared with wild type, these transgenic animals possess a cardioprotective phenotype as illustrated by improved
postischemic contractile function and decreased infarction size
(21, 40). Furthermore, it has been shown that enhanced endogenous antioxidants could also be an important component of
exercise-induced cardioprotection (49). Therefore, current evidence indicates that, collectively or independently, exerciseinduced increases in myocardial levels of HSP72 or antioxidants could lead to improved protection during I/R insults (9,
14, 15, 28, 38, 39).
Although it is clear that exercise provides cardioprotection,
it is currently unknown how long the heart retains a cardioprotective phenotype after the cessation of exercise. Therefore,
this study investigated the time course of the loss of cardioprotection after cessation of exercise. On the basis of the
half-life of putative cardioprotective mediators such as HSP72
(35) that are elevated in response to exercise, we hypothesized
that exercise-induced cardioprotection against I/R injury would
disappear within 9 days after the cessation of exercise training.
To test this postulate, cardioprotection against I/R injury was
evaluated at selected time periods after cessation of exercise
training by using an isolated working heart model of global
ischemia and reperfusion.
METHODS

REGULAR PHYSICAL ACTIVITY is associated with multiple cardiovascular benefits, including a decreased incidence of myocardial infarction (MI) and improved survival rate after MI (18).
These epidemiological findings have been confirmed by numerous experimental studies indicating that both acute and
chronic exercise provides protection against myocardial ischemia-reperfusion (I/R) injury in animals (4, 5, 9, 14–17, 28, 36,
38, 39, 43, 46, 48). Specifically, both short-term (i.e., 1–5 days)
and long-term (i.e., weeks to months) exercise decreases I/Rinduced cardiac injury resulting from both short-duration (i.e.,

Address for reprint requests and other correspondence: S. Lennon, Boston
University Medical Center, Boston, MA 02118 (E-mail: slennon@bu.edu).
http://www.jap.org

Animals and Experimental Design
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. Male Sprague-Dawley rats (ϳ4 mo old; ϳ370–
400 g) were randomly assigned to one of five experimental groups: 1)
sedentary “control” group, 2) exercise followed by 1 day of rest, 3)
exercise followed by 3 days of rest, 4) exercise followed by 9 days of
rest, and 5) exercise followed by 18 days of rest. These experimental
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.

8750-7587/04 $5.00 Copyright © 2004 the American Physiological Society

1299


Lennon, Shannon L., John Quindry, Karyn L. Hamilton, Joel
French, Jessica Staib, Jawahar L. Mehta, and Scott K. Powers.
Loss of exercise-induced cardioprotection after cessation of exercise.
J Appl Physiol 96: 1299–1305, 2004. First published December 12,
2003; 10.1152/japplphysiol.00920.2003.—Endurance exercise provides cardioprotection against ischemia-reperfusion (I/R) injury. Exercise-induced cardioprotection is associated with increases in cytoprotective proteins, including heat shock protein 72 (HSP72) and
increases in antioxidant enzyme activity. On the basis of the reported
half-life of these putative cardioprotective proteins, we hypothesized
that exercise-induced cardioprotection against I/R injury would be lost
within days after cessation of exercise. To test this, male rats (4 mo)
were randomly assigned to one of five experimental groups: 1)
sedentary control, 2) exercise followed by 1 day of rest, 3) exercise
followed by 3 days of rest, 4) exercise followed by 9 days of rest, and
5) exercise followed by 18 days of rest. Exercise-induced increases
(P Ͻ 0.05) in left ventricular catalase activity and HSP72 were
evident at 1 and 3 days postexercise. However, at 9 days postexercise,
myocardial HSP72 and catalase levels declined to sedentary control
values. To evaluate cardioprotection during recovery from I/R, hearts
were isolated, placed in working heart mode, and subjected to 20.5
min of global ischemia followed by 30 min of reperfusion. Compared
with sedentary controls, exercised animals sustained less I/R injury as
evidenced by maintenance of a higher (P Ͻ 0.05) percentage of
preischemia cardiac work during reperfusion at 1, 3, and 9 days
postexercise. The exercise-induced cardioprotection vanished by 18
days after exercise cessation. On the basis of the time course of the
loss of cardioprotection and the return of HSP72 and catalase to
preexercise levels, we conclude that HSP72 and catalase are not
essential for exercise-induced protection during myocardial stunning.
Therefore, other cytoprotective molecules are responsible for providing protection during I/R.

1300

LOSS OF EXERCISE-INDUCED CARDIOPROTECTION

groups were then subdivided into sham or in vitro I/R groups. Hearts
from the sham groups were used to assess exercise-induced changes in
antioxidant enzyme activities. During the study, all experimental
groups were maintained on a 12:12-h light-dark photoperiod and
provided food and water ad libitum. We selected the rat as an
experimental animal because this species is a widely accepted mammalian model for the study of myocardial function and adaptation.
Furthermore, choosing male animals avoided variances in blood
estrogen levels that occur in female animals. Finally, selection of
young adult animals removed confounding experimental variables
associated with both development and old age.
Exercise Protocol

Myocardial Biochemical Measurements
Measurement of myocardial antioxidant capacity. Because exposure of the heart to I/R can decrease antioxidant enzyme activity, left
ventricular tissue from sham groups was used to investigate the effects
of exercise training on antioxidant capacity. The antioxidants selected
were chosen because each plays an important role in regulation of
myocardial redox balance and has a key responsibility in providing
myocardial protection against oxidative injury. The hearts from sham
animals were rapidly removed, rinsed in antioxidant buffer (50 mM
NaHPO4, 0.1 mM butylated hydroxytoluene, 0.1 mM EDTA; pH 7.4),
and quickly frozen in liquid nitrogen. Sections of the left ventricular
free wall were individually minced and homogenized with ϳ40 passes
of a tight-fitting conical glass tissue homogenizer in cold 100 mM
phosphate buffer with 0.05% bovine serum albumin (1:20 wt/vol; pH
7.4). Homogenates were then centrifuged (4°C) for 10 min at 400 g.
The supernatant was decanted and assayed to determine total protein
content along with the activities of superoxide dismutase (SOD; EC
1.15.1.1), catalase (Cat; EC 1.11.1.6), and glutathione peroxidase
(GPX; EC 1.11.1.9). Protein content was determined by using methods described by Bradford (6). SOD and GPX activities were determined in the left ventricle by using a modification of the procedures
described by McCord and Fridovich (31) and Flohe and Gunzler (11),
respectively. Exercise-induced changes in SOD isoforms [i.e., manganese SOD (MnSOD) and copper-zinc (CuZnSOD)] were determined by KCN inhibition of the CuZnSOD isoform. Cat activity was
determined by using techniques described by Aebi (1). The coefficients of variation for SOD, GPX, and Cat assays ranged between 2
and 5%. Reduced glutathione levels are important in the regulation of
both cellular redox status and antioxidant capacity. Left ventricular
total glutathione was assayed to evaluate training-induced changes in
this important nonprotein thiol (Cayman Chemical, Ann Arbor, MI).
Note that because myocardial antioxidants and HSP72 returned to the
levels of sedentary control animals by 9 days of cessation of exercise,
J Appl Physiol • VOL

In Vitro I/R Experiments
To evaluate the time course of exercise-associated protection
against I/R contractile dysfunction, we used an isolated working heart
preparation. At the appropriate time span after cessation of exercise
training, animals were anesthetized with 100 mg/kg pentobarbital
sodium (ip). To facilitate removal of blood from the heart and to
prevent clot formation, 100 IU of heparin were injected directly into
the hepatic vein. Hearts were then rapidly excised, placed in ice-cold
saline, and weighed on an electronic scale (gross wet weight). The
aorta was quickly secured on a stainless steel catheter, and the heart
was retrograde perfused by using a modified Krebs Henseleit buffer
(1.5 mM CaCl2, 130 mM NaCl, 5.4 mM KCl, 11 mM glucose, 2 mM
pyruvate, 0.5 mM MgCl2, 0.5 mM NaH2PO4, and 25 mM NaHCO3,
pH 7.4) aerated with 95% O2-5% CO2 at a constant perfusion pressure
of 80 cmH2O and a temperature of 37 Ϯ 0.3°C. Excess tissue was
trimmed, weighed, and subtracted from the gross weight to get a final
wet weight of the heart. All subsequent values were normalized to this
final heart weight.
After 15 min of retrograde perfusion, the hearts were switched to
the working heart mode and preischemic heart function was evaluated
at 13 Ϯ 0.5 cmH2O (atrial filling pressure) with an 80-cm-high aortic
column. Coronary and aortic flows were determined by timed collections of the coronary effluent and aortic column overflow, respectively. Cardiac output was determined as the sum of coronary and
aortic flow.
Global, normothermic ischemia was then induced by simultaneously cross-clamping the atrial inflow and the aortic outflow for
20.5 min. During ischemia, hearts remained in a water-jacketed
chamber maintained at 37°C. After ischemia, hearts were initially
perfused in the retrograde mode for 15 min at a perfusion pressure of
80 cmH2O and then switched to the working heart mode for 15 min
for a total of 30 min of reperfusion. The initial reperfusion period in
the retrograde mode is necessary to stabilize the hearts before working
heart reperfusion. Coronary effluent was collected between minutes 5
and 7 of reperfusion, and lactate dehydrogenase (LDH; EC 1.1.1.27)
activity in the effluent was assayed as a marker of myocardial injury
(2). Furthermore, cardiac output was monitored at selected time
periods during this final 15 min of postischemia recovery in the
working heart mode.
We exposed these hearts to 20.5 min of global ischemia because
this level of myocardial injury results in a “stunned myocardium” with
a ϳ40% decrease in cardiac working capacity. Myocardial stunning is
defined as a nonlethal form of I/R injury that results in impaired
cardiac function for 24–48 h after I/R without death of cardiac
myocytes. We selected the isolated working heart model in these
experiments for several reasons. First, this preparation closely resembles the in vivo working heart, and isolated working heart experiments
are highly reproducible (44). Second, when contrasted to in vivo
models, an advantage of the isolated heart preparation is the ability to

96 • APRIL 2004 •

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Animals assigned to exercise groups performed a total of 8 days of
exercise. Treadmill running was selected as our exercise mode to
avoid the physiological and psychological issues (e.g., hypoxic exercise and/or fear) associated with swim training in rodents. Exercise
training included 5 days of habituation to the treadmill beginning with
10 min of running at 30 m/min, 0% grade, with daily increases of 10
min until 50 min/day were achieved. Animals then performed 3
consecutive days of 60 min running/day (30 m/min, 0% grade) at an
estimated work rate of ϳ70% maximal O2 consumption (26). Note,
that during the 60 min of daily exercise, the animals were provided
three equally spaced 2- to 3-min rest periods. Mild electrical shocks
were used sparingly to motivate animals to run. The intensity and
duration of the exercise training protocol were selected because this
training program has been shown to promote myocardial adaptations
and provide cardioprotection against myocardial stunning (9, 14, 15,
37, 38). Exercised animals were killed with a lethal injection of
pentobarbital sodium at 1, 3, 9, or 18 days after their last exercise
session.

biochemical measurements were not performed on hearts removed
from animals in the 18 days postexercise group.
Measurement of myocardial HSPs. To determine the effects of
exercise on induction of myocardial HSPs, we performed polyacrylamide gel electrophoresis and immunoblotting by using the techniques as previously described (14, 28, 38). Briefly, left ventricular
samples from I/R animals were homogenized, and one-dimensional
sodium dodecyl sulfate-polyacrylamide (12%) gel electrophoresis was
performed to separate proteins by molecular weight. After separation,
proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Nitrocellulose membranes were blocked for 2 h with
(0.5%) bovine serum albumin. Blots were incubated for 2 h with
monoclonal antibodies for HSP72 (SPA-810, StressGen, Victoria,
Canada). After chemiluminescence detection (Amersham Biosciences, Piscataway, NJ) and computerized densitometry, results
were expressed as a percentage of sedentary controls.

1301


Table 1. Postexercise training animal body and heart weights for animals used in working heart experiments
Parameter

Control
(n ϭ 11)

1 Day
(n ϭ 10)

3 Days
(n ϭ 9)

9 Days
(n ϭ 9)

18 Days
(n ϭ 8)

Body weight, g
Heart weight, g
Heart-to-body weight ratio, mg/g

382.5Ϯ2.44
1.13Ϯ0.01
2.96Ϯ0.03

382.5Ϯ2.94
1.12Ϯ0.02
2.92Ϯ0.06

381Ϯ2.74
1.1Ϯ0.02
2.89Ϯ0.07

392.8Ϯ3.74
1.15Ϯ0.02
2.93Ϯ0.05

372.6Ϯ2.6
1.11Ϯ0.02
2.98Ϯ0.03

Values are means Ϯ SE; n, no. of animals. No differences existed in any measurement between experimental groups.

control afterload and preload of the heart. Third, this model is capable
of detecting small changes in cardiac function (50). Finally, because
of the collective aforementioned advantages, it has been argued that
the working heart is the “model of choice” to study myocardial
stunning (50).

activity returned to untrained levels by 9 days after cessation of
exercise, biochemical measurements were not performed on
hearts obtained from animals subjected to 18 days of detraining.

Statistical Analyses

Exercise Training and Cardiac Function During Recovery
From Ischemia

RESULTS

Animal Characteristics
Animal characteristics are presented in Table 1. No differences existed in animal body weights between the experimental
groups. Furthermore, no differences existed in heart weights or
the ratio of body weight to heart weight between the five
experimental groups.
Exercise-Induced Changes in Myocardial Antioxidants
and HSP72
Exercise did not significantly alter myocardial GPX or SOD
activities or cardiac levels of glutathione. The observation that
exercise-related increases in MnSOD activity did not reach
statistical significance is not consistent with our previous work
and appears to be due to large interanimal variability. In
contrast, exercise significantly elevated myocardial Cat activity
at both 1 and 3 days postexercise. By day 9, myocardial Cat
activity returned to a level not different from sedentary control
animals (Table 2).
Compared with the sedentary control animals, exercise resulted in significant increases in myocardial HSP72 levels at 1
and 3 days postexercise (Fig. 1). Similar to exercise-induced
changes in Cat activity, however, these training-induced
changes were not evident at 9 days after cessation of exercise.
Finally, because myocardial levels of HSP72 and antioxidant

Successful I/R protocols were completed on 11 animals from
the control group along with 10, 9, and 9 animals from the 1,
3, and 9 days postexercise groups, respectively. Animals were
excluded from data analysis if they did not complete the full
exercise protocol or if their hearts did not demonstrate stable
baseline performance during preischemia data collection. The
effects of exercise training on pre- and postischemic cardiac
function at each time point postexercise are presented in Table
3 and Fig. 2. Note that, before ischemia, no differences existed
between experimental groups in heart rate, coronary flow,
cardiac output, or cardiac work. Furthermore, no differences
existed between any of the experimental groups in heart rate or
coronary flow at 30 min of reperfusion. Also, no group differences existed in aortic diastolic pressure at 30 min of reperfusion (data not shown). Reperfusion after 20.5 min of global
ischemia resulted in a significant contractile dysfunction in
sedentary control animals. For example, compared with preischemic values, percent recovery of cardiac work was depressed
by Ͼ30% in sedentary control animals at 30 min of reperfusion. In contrast to control, exercise-trained animals studied at
1, 3, and 9 days postexercise maintained significantly higher
relative cardiac outputs and cardiac work at 30 min of reperfusion (Table 3, Fig. 2). Note that cardiac work increased
rapidly during reperfusion and that a steady state was achieved
within 5 min after reintroduction of the working heart mode.
Therefore, only values from minute 30 of reperfusion are
reported.
Absolute LDH activity in the coronary effluent (preischemia) ranged from 12 to 41 mU⅐minϪ1 ⅐g wet weightϪ1 across
the five experimental groups. Postischemic LDH activity in the

Table 2. Effects of short-term exercise training on left ventricular antioxidants
Enzyme Activity
(per mg protein)

Control
(n ϭ 10)

1 Day
(n ϭ 10)

3 Days
(n ϭ 10)

9 Days
(n ϭ 10)

GPX, ␮mol/min
Cat, units
Total SOD, units
MnSOD, units
CuZnSOD, units
Total glutathione, mmol

0.21Ϯ0.01
1.04Ϯ0.03
42.77Ϯ1.79
18.17Ϯ1.63
24.60Ϯ1.79
1.36Ϯ0.08

0.244Ϯ0.009
1.21Ϯ0.03*†
44.84Ϯ2.38
20.82Ϯ1.26
23.72Ϯ1.82
1.38Ϯ0.10

0.235Ϯ0.006
1.17Ϯ0.02*†
48.03Ϯ2.52
22.12Ϯ1.73
25.91Ϯ2.05
1.44Ϯ0.12

0.221Ϯ0.007
1.06Ϯ0.03
46.19Ϯ3.17
19.64Ϯ1.99
26.54Ϯ2.65
1.47Ϯ0.11

Values are means Ϯ SE; n, no. of animals. GPX, glutathione peroxidase; Cat, catalase; SOD, superoxide dismutase; MnSOD, manganese SOD; CuZnSOD,
copper-zinc SOD. Cat units were calculated by using the rate constant of a first-order reaction (K). K ϭ (2.3/t)⅐[log10 (A1/A2)], where t is time in min, A1 is
initial absorbance reading Ϫ blank absorbance, and A2 is absorbance reading at 1 min Ϫ blank absorbance. One unit of SOD activity is defined as the amount
of SOD required for a 50% decrease in cytochrome c reduction rate. *Different from control, P Ͻ 0.05. †Different from 9 days, P Ͻ 0.05.

96 • APRIL 2004 •

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Myocardial contractility and biochemical measurements were analyzed by using a one-way analysis of variance. When appropriate, a
Tukey’s post hoc test was applied. Significance was established a
priori at P Ͻ 0.05.

1302


Fig. 1. Left ventricular heat shock protein 72 in control and exercise groups
(exercise ϩ 1 day rest, exercise ϩ 3 days rest, and exercise ϩ 9 days rest)
obtained from scanning of Western blots reacted with antibody for heat shock
protein 72. Values are means Ϯ SE. *Different from control, P Ͻ 0.05.
†Different from exercise ϩ 9 days rest, P Ͻ 0.05.

induced cardioprotection against myocardial stunning remains
for at least 9 days after cessation of exercise training.
DISCUSSION

Overview of Principal Findings
To our knowledge, this is the first investigation of the
relationship between the loss of protection against myocardial
stunning after cessation of exercise and the reduction in putative cardioprotective proteins in the heart. Our data indicate
that exercise-induced cardioprotection against myocardial
stunning remains for at least 9 days after cessation of exercise
training. Importantly, we observed that cardioprotection persists in the absence of elevated myocardial levels of HSP72 and
antioxidants (e.g., Cat) at 9 days postexercise. These findings
indicate that exercise-associated cardioprotection is not critically dependent on increases in cardiac levels of these cytoprotective proteins.

Table 3. Effects of exercise training intensity on pre- and postischemic cardiac function
Control
(n ϭ 11)

Preischemia
HR, beats/min
CF, ml⅐minϪ1 ⅐g heart weightϪ1
CO, ml⅐minϪ1 ⅐g heart weightϪ1
SP, mmHg
Cardiac work, SP ϫ CO
RPP, HR ϫ SP
Postischemia
HR, beats/min
CF, ml⅐minϪ1 ⅐g heart weightϪ1
CO, ml⅐minϪ1 ⅐g heart weightϪ1
SP, mmHg
Cardiac work, SP ϫ CO
Cardiac work, %preischemia
RPP, HR ϫ SP

1 Day
(n ϭ 10)

3 Days
(n ϭ 9)

9 Days
(n ϭ 9)

18 Days
(n ϭ 8)

342.6Ϯ7.9
14.9Ϯ0.47
43Ϯ0.45
90.7Ϯ1.53†
3,900Ϯ75
30,953Ϯ256

347.2Ϯ7.9
16.5Ϯ0.55
43.6Ϯ1.07
89.4Ϯ1.4†
3,900Ϯ124
30,946Ϯ314

336.5Ϯ10.4
17.9Ϯ0.92†
42.7Ϯ1.1
91.3Ϯ1.9†
3,889Ϯ81
30,585Ϯ563

337.6Ϯ5.7
15.5Ϯ0.44
43Ϯ0.9
91.9Ϯ1.6†
3,948Ϯ87
30,974Ϯ338

311.2Ϯ16.3
14.3Ϯ0.5
41.5Ϯ1.6
100.4Ϯ3.1
4,158Ϯ178
30,976Ϯ1,228

328.7Ϯ10.5
14.5Ϯ0.99
33.6Ϯ1.6
82.9Ϯ2.7
2,818Ϯ212
68Ϯ4
27,006Ϯ477

328Ϯ8.9
16.5Ϯ0.97
39.3Ϯ1.5*
85.3Ϯ1.4
3,354Ϯ146
86Ϯ3*
27,881Ϯ395

341Ϯ13.8
16.1Ϯ1.1
41Ϯ1.2*
85Ϯ2.4
3,462Ϯ71
89Ϯ2*
28,699Ϯ551

336Ϯ5.8
15.2Ϯ0.78
38.5Ϯ1.4*
84.7Ϯ2.3
3,255Ϯ134
83Ϯ4*
28,394Ϯ510

310Ϯ16
12.2Ϯ0.75
33.2Ϯ2.9
87.3Ϯ3.4
2,916Ϯ278
74Ϯ3
26,806Ϯ112

Values are means Ϯ SE; n, no. of animals. Postischemia values represent 30 min of reperfusion measured in the working heart mode. HR, heart rate; CF,
coronary flow; CO, cardiac output; SP, systolic pressure; RPP, rate-pressure product. *Significantly different from control, P Ͻ 0.05. †Significantly different from
18 days, P Ͻ 0.05.

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coronary effluent ranged from 20 to 264 mU⅐minϪ1 ⅐g wet
weightϪ1 across the experimental groups; these values are
consistent with I/R insults that result in myocardial stunning.
For clarity, LDH activities were normalized (percent) to preischemic values. Consistent with the cardiac performance measures, analyses revealed LDH activity in the coronary effluent
collected during reperfusion was significantly higher from
control hearts compared with exercise trained hearts at 1, 3,
and 9 days postexercise (Fig. 3).
Because we observed protection against I/R-induced cardiac
dysfunction and LDH release that persisted through 9 days
postexercise, we further assessed the time course of exerciseinduced cardioprotection by adding another exercise-trained
group that was evaluated at 18 days postexercise. As illustrated
in Table 3 and Fig. 2, exercise-induced preservation of postischemic cardiac output and cardiac work was lost at 18 days
postexercise. Similarly, I/R-induced LDH release from the
heart at 18 days postexercise was not different from controls
(Fig. 3). Collectively, these findings indicate that exercise-

Fig. 2. Time course of exercise-induced protection on recovery of cardiac
function after 20.5 min of global ischemia. Percent recovery is calculated as
(preischemia cardiac output/cardiac output at minute 30 of reperfusion) ϫ 100.
Values are means Ϯ SE. *Different from control, P Ͻ 0.05.


diac Cat activity in both short- and long-duration exercise
training (16, 19, 23, 25, 41, 42, 47). In contrast, myocardial
glutathione levels are typically not elevated after short-term
exercise, but most long-term training studies report that several
weeks of training elevates cardiac glutathione levels (9, 19, 20,
27, 34). On the basis of these observations, it is tempting to
speculate that the myocardial strategy to eliminate hydrogen
peroxide changes over time in response to continuous exercise
training. That is, the increased ability to remove hydrogen
peroxide early during training adaptation is achieved by elevating Cat activity alone, whereas as exercise training continues, there is a gradual increase in glutathione levels. Increasing
cardiac levels of glutathione with chronic exercise training
would improve the ability of the heart to eliminate hydrogen
peroxide because glutathione works in tandem with GPX to
remove hydrogen peroxide and other organic hydroperoxides.
The biological rationale for this changing adaptive strategy is
unclear but could be linked to the fact that exercise-induced
increases in Cat activity can be achieved rapidly via gene
expression, whereas myocardial glutathione synthesis is a relatively slow process limited by the availability of cellular
levels of cysteine (13).
Postexercise Loss of Cardioprotection

Exercise-Induced Myocardial Adaptation
Our results confirm previous data that short-term endurance
exercise training results in cardioprotection against an in vitro
I/R insult that produces severe myocardial stunning. This
agrees with previous reports in which both in vivo and in vitro
I/R models were used to study exercise-induced cardioprotection (14–17, 28, 38, 46). Furthermore, our data show that acute
exercise training is associated with threefold increases in myocardial levels of HSP72. Exercise-induced increases in myocardial levels of HSP72 are widely reported in the literature,
and an increase in this stress protein has been postulated to play
a role in exercise-induced cardioprotection (14–16, 28, 46).
It is well established that both ischemia and reperfusion
promote the formation of radicals/oxidants and that oxidative
injury contributes to myocardial stunning (3). It follows that
exercise-induced increases in myocardial antioxidants could
provide cardioprotection against this type of I/R injury. Therefore, to determine the effects of exercise on myocardial antioxidant capacity, we measured glutathione levels along with
the activities of key antioxidant enzymes (Table 2). Our results
indicate that short-term exercise training does not elevate
myocardial glutathione or the activities of GPX or two intracellular isoforms of SOD (i.e., CuZnSOD or MnSOD). The
finding that acute exercise does not elevate myocardial levels
of glutathione, GPX, and CuZnSOD agrees with previous
reports (9, 14, 15, 37, 38). In contrast, several studies, including work in our laboratory, have reported that exercise is
associated with small (e.g., 10–20%) but significant increases
in myocardial MnSOD activity (9, 14, 15, 17, 38). Among this
work are studies indicating that exercise-induced increases in
MnSOD may be critical for protection against MI (17, 49).
Nonetheless, in the present study, although the mean MnSOD
activity tended to be higher in exercise-trained hearts compared
with control, these levels did not reach significance.
In contrast to other antioxidant enzymes, exercise training
elevated myocardial Cat activity. Although some controversy
exists, others have reported exercise-induced increases in carJ Appl Physiol • VOL

The mechanism(s) responsible for exercise-induced cardioprotection remains controversial. It has been suggested that
exercise-induced increases in cardiac levels of HSP72 and/or
key antioxidants are potential mechanisms to explain the cardioprotection associated with physical activity (8, 17, 22,
28–30, 32, 49). Our hypothesis that exercise-induced cardioprotection against myocardial stunning would disappear within
9 days after the cessation of exercise training was formulated
by reports indicating that the half-life of putative cardioprotective proteins (e.g., HSP72) is ϳ2–3 days (35). The half-life of
circulating levels of some of the isoforms of antioxidant
enzymes, however, can be as short as a few hours (12).
Therefore, after cessation of exercise training, the postexercise
decline in HSP72 would result in an ϳ88% reduction in
myocardial levels within 9 days. It follows that, if HSP72
and/or Cat is essential for exercise-induced cardioprotection,
the protection would disappear after myocardial levels of these
protective proteins decline to sedentary control levels. Our
results reveal that cardiac levels of both HSP72 and Cat decline
over time and reach sedentary control values at day 9 postexercise. Nonetheless, the finding that cardioprotection remains
at 9 days postexercise indicates that mechanisms other than
HSP72 and Cat are involved in exercise-related protection
against I/R-induced myocardial stunning.
Potential Mechanisms of Exercise-Induced Protection
Against Myocardial Stunning
If exercise-induced increases in myocardial Cat and HSP72
are not essential to achieve exercise-mediated protection
against myocardial stunning, other mechanism(s) must be responsible for this form of cardioprotection. The two primary
theories to explain the pathogenesis of myocardial stunning are
the radical hypothesis and the calcium hypothesis (3). The
radical hypothesis postulates that myocardial stunning results
from oxidative injury by radicals and other reactive species
produced during ischemia and reperfusion. This oxidative

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Fig. 3. Effects of ischemia-reperfusion injury on the release of lactate dehydrogenase from isolated rat hearts from control and exercise groups (exercise ϩ 1 day rest, exercise ϩ 3 days rest, exercise ϩ 9 days rest, and
exercise ϩ 18 days rest). Lactate dehydrogenase activity was measured in
coronary effluent before and after ischemia-reperfusion. Lactate dehydrogenase activity was normalized to wet weight of the heart and expressed as the
percent difference between preischemia and postischemic values. Values are
means Ϯ SE. *Different from control, P Ͻ 0.05. †Different from exercise ϩ
18 days rest, P Ͻ 0.05.

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Summary and Conclusions
These experiments investigated the rate of decline of exercise-induced cardioprotection after the cessation of exercise
training. Our data indicate that exercise-induced cardioprotection remains for 9 days after cessation of exercise training but
that this protection is absent at 18 days postexercise. The fact
that cardioprotection persists in the absence of elevated myocardial levels of HSP72 and Cat at day 9 postexercise implies
that cytoprotective molecules other than HSP72 and Cat
provide cardioprotection against myocardial stunning. Although the mechanism(s) responsible for exercise-induced cardioprotection remains elusive, continuing advances in biotechnology (i.e., microarray analysis) will permit the measurement of transcription in a large number of genes and will
greatly accelerate research progress in our laboratory and
others (7, 10).

GRANTS
This work was supported by a grant-in-aid from the Department of Defense
(to J. L. Mehta) and National Heart, Lung, and Blood Institute Grant R01
HL-067855 (to S. K. Powers).
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injury could impair myocardial contractility due to a reduced
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