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

Staib, Jawahar L. Mehta and Scott K. PowersShannon L. Lennon, John Quindry, Karyn L. Hamilton, Joel French, Jessicacessation of exerciseLoss of exercise-induced cardioprotection after

You might find this additional info useful...

48 articles, 28 of which can be accessed free at:This article cites http://jap.physiology.org/content/96/4/1299.full.html#ref-list-1

19 other HighWire hosted articles, the first 5 are:This article has been cited by

  [PDF] [Full Text] [Abstract]

, September 27, 2010; .Cardiovasc ResJohn W. CalvertCardioprotective effects of nitrite during exercise 

[PDF] [Full Text] [Abstract], February 15, 2011; 89 (3): 499-506.Cardiovasc Res

John W. CalvertCardioprotective effects of nitrite during exercise 

[PDF] [Full Text] [Abstract], June 10, 2011; 108 (12): 1448-1458.Circulation Research

David J. LeferRebecca L. Hood, Amy L. Sindler, Susheel Gundewar, Douglas R. Seals, Lili A. Barouch and John W. Calvert, Marah E. Condit, Juan Pablo Aragón, Chad K. Nicholson, Bridgette F. Moody,Nitrosothiols-Adrenergic Receptors and Increased Nitric Oxide Signaling: Role of Nitrite and

3βReperfusion Injury via Stimulation of −Exercise Protects Against Myocardial Ischemia 

[PDF] [Full Text] [Abstract], September , 2011; 111 (3): 905-915.J Appl Physiol

Chad R. Frasier, Russell L. Moore and David A. BrownExercise-induced cardiac preconditioning: how exercise protects your achy-breaky heart 

[PDF] [Full Text] [Abstract], November , 2011; 301 (5): R1250-R1258.Am J Physiol Regul Integr Comp Physiol

Christopher P. BainesKyle S. McCommis, Allison M. McGee, M. Harold Laughlin, Douglas K. Bowles andresponse in the porcine myocardium: beneficial effects of chronic exerciseHypercholesterolemia increases mitochondrial oxidative stress and enhances the MPT

including high resolution figures, can be found at:Updated information and services http://jap.physiology.org/content/96/4/1299.full.html

can be found at:Journal of Applied Physiologyabout Additional material and information http://www.the-aps.org/publications/jappl

This information is current as of June 17, 2012. 

ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2004 by the American Physiological Society.those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American

publishes original papers that deal with diverse areas of research in applied physiology, especiallyJournal of Applied Physiology

on June 17, 2012jap.physiology.org

Dow

nloaded from

Loss of exercise-induced cardioprotectionafter 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

1Department of Exercise and Sport Sciences, Center for Exercise Science, University of Florida,Gainesville, Florida 32611; 2Division of Cardiovascular Medicine, University of Arkansas forMedical 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

Lennon, Shannon L., John Quindry, Karyn L. Hamilton, JoelFrench, 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 pro-vides cardioprotection against ischemia-reperfusion (I/R) injury. Ex-ercise-induced cardioprotection is associated with increases in cyto-protective proteins, including heat shock protein 72 (HSP72) andincreases in antioxidant enzyme activity. On the basis of the reportedhalf-life of these putative cardioprotective proteins, we hypothesizedthat exercise-induced cardioprotection against I/R injury would be lostwithin 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) exercisefollowed by 3 days of rest, 4) exercise followed by 9 days of rest, and5) exercise followed by 18 days of rest. Exercise-induced increases(P � 0.05) in left ventricular catalase activity and HSP72 wereevident at 1 and 3 days postexercise. However, at 9 days postexercise,myocardial HSP72 and catalase levels declined to sedentary controlvalues. To evaluate cardioprotection during recovery from I/R, heartswere isolated, placed in working heart mode, and subjected to 20.5min of global ischemia followed by 30 min of reperfusion. Comparedwith sedentary controls, exercised animals sustained less I/R injury asevidenced by maintenance of a higher (P � 0.05) percentage ofpreischemia cardiac work during reperfusion at 1, 3, and 9 dayspostexercise. The exercise-induced cardioprotection vanished by 18days after exercise cessation. On the basis of the time course of theloss of cardioprotection and the return of HSP72 and catalase topreexercise levels, we conclude that HSP72 and catalase are notessential for exercise-induced protection during myocardial stunning.Therefore, other cytoprotective molecules are responsible for provid-ing protection during I/R.

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

REGULAR PHYSICAL ACTIVITY is associated with multiple cardio-vascular benefits, including a decreased incidence of myocar-dial infarction (MI) and improved survival rate after MI (18).These epidemiological findings have been confirmed by nu-merous experimental studies indicating that both acute andchronic exercise provides protection against myocardial ische-mia-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/R-induced cardiac injury resulting from both short-duration (i.e.,

5–20 min resulting in myocardial stunning) and long-duration(i.e., �20 min resulting in MI) myocardial ischemia. Althoughthe mechanism(s) responsible for exercise-mediated cardiopro-tection remain unknown, it has been postulated that exercise-induced increases in myocardial levels of heat shock protein(HSP) 72 and/or antioxidant defenses contribute to this cardio-protection (9, 28, 38, 48, 49). In regard to HSP72 and cardio-protection, transgenic mice that overexpress HSP72 provideconvincing evidence that HSP72 imparts cardioprotection. In-deed, compared with wild type, these transgenic animals pos-sess a cardioprotective phenotype as illustrated by improvedpostischemic contractile function and decreased infarction size(21, 40). Furthermore, it has been shown that enhanced endog-enous antioxidants could also be an important component ofexercise-induced cardioprotection (49). Therefore, current ev-idence indicates that, collectively or independently, exercise-induced increases in myocardial levels of HSP72 or antioxi-dants 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 cardio-protective phenotype after the cessation of exercise. Therefore,this study investigated the time course of the loss of cardio-protection after cessation of exercise. On the basis of thehalf-life of putative cardioprotective mediators such as HSP72(35) that are elevated in response to exercise, we hypothesizedthat exercise-induced cardioprotection against I/R injury woulddisappear within 9 days after the cessation of exercise training.To test this postulate, cardioprotection against I/R injury wasevaluated at selected time periods after cessation of exercisetraining by using an isolated working heart model of globalischemia and reperfusion.

METHODS

Animals and Experimental Design

This experimental protocol was approved by the University ofFlorida Animal Care and Use Committee and followed the guidelinesestablished by the American Physiological Society for the use ofanimals in research. Male Sprague-Dawley rats (�4 mo old; �370–400 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 ofrest, and 5) exercise followed by 18 days of rest. These experimental

Address for reprint requests and other correspondence: S. Lennon, BostonUniversity Medical Center, Boston, MA 02118 (E-mail: slennon@bu.edu).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

8750-7587/04 $5.00 Copyright © 2004 the American Physiological Societyhttp://www.jap.org 1299

on June 17, 2012jap.physiology.org

Dow

nloaded from

groups were then subdivided into sham or in vitro I/R groups. Heartsfrom the sham groups were used to assess exercise-induced changes inantioxidant enzyme activities. During the study, all experimentalgroups were maintained on a 12:12-h light-dark photoperiod andprovided food and water ad libitum. We selected the rat as anexperimental animal because this species is a widely accepted mam-malian model for the study of myocardial function and adaptation.Furthermore, choosing male animals avoided variances in bloodestrogen levels that occur in female animals. Finally, selection ofyoung adult animals removed confounding experimental variablesassociated with both development and old age.

Exercise Protocol

Animals assigned to exercise groups performed a total of 8 days ofexercise. Treadmill running was selected as our exercise mode toavoid the physiological and psychological issues (e.g., hypoxic exer-cise and/or fear) associated with swim training in rodents. Exercisetraining included 5 days of habituation to the treadmill beginning with10 min of running at 30 m/min, 0% grade, with daily increases of 10min until 50 min/day were achieved. Animals then performed 3consecutive days of 60 min running/day (30 m/min, 0% grade) at anestimated work rate of �70% maximal O2 consumption (26). Note,that during the 60 min of daily exercise, the animals were providedthree equally spaced 2- to 3-min rest periods. Mild electrical shockswere used sparingly to motivate animals to run. The intensity andduration of the exercise training protocol were selected because thistraining program has been shown to promote myocardial adaptationsand provide cardioprotection against myocardial stunning (9, 14, 15,37, 38). Exercised animals were killed with a lethal injection ofpentobarbital sodium at 1, 3, 9, or 18 days after their last exercisesession.

Myocardial Biochemical Measurements

Measurement of myocardial antioxidant capacity. Because expo-sure of the heart to I/R can decrease antioxidant enzyme activity, leftventricular tissue from sham groups was used to investigate the effectsof exercise training on antioxidant capacity. The antioxidants selectedwere chosen because each plays an important role in regulation ofmyocardial redox balance and has a key responsibility in providingmyocardial protection against oxidative injury. The hearts from shamanimals were rapidly removed, rinsed in antioxidant buffer (50 mMNaHPO4, 0.1 mM butylated hydroxytoluene, 0.1 mM EDTA; pH 7.4),and quickly frozen in liquid nitrogen. Sections of the left ventricularfree wall were individually minced and homogenized with �40 passesof a tight-fitting conical glass tissue homogenizer in cold 100 mMphosphate buffer with 0.05% bovine serum albumin (1:20 wt/vol; pH7.4). Homogenates were then centrifuged (4°C) for 10 min at 400 g.The supernatant was decanted and assayed to determine total proteincontent along with the activities of superoxide dismutase (SOD; EC1.15.1.1), catalase (Cat; EC 1.11.1.6), and glutathione peroxidase(GPX; EC 1.11.1.9). Protein content was determined by using meth-ods described by Bradford (6). SOD and GPX activities were deter-mined in the left ventricle by using a modification of the proceduresdescribed by McCord and Fridovich (31) and Flohe and Gunzler (11),respectively. Exercise-induced changes in SOD isoforms [i.e., man-ganese SOD (MnSOD) and copper-zinc (CuZnSOD)] were deter-mined by KCN inhibition of the CuZnSOD isoform. Cat activity wasdetermined by using techniques described by Aebi (1). The coeffi-cients of variation for SOD, GPX, and Cat assays ranged between 2and 5%. Reduced glutathione levels are important in the regulation ofboth cellular redox status and antioxidant capacity. Left ventriculartotal glutathione was assayed to evaluate training-induced changes inthis important nonprotein thiol (Cayman Chemical, Ann Arbor, MI).Note that because myocardial antioxidants and HSP72 returned to thelevels of sedentary control animals by 9 days of cessation of exercise,

biochemical measurements were not performed on hearts removedfrom animals in the 18 days postexercise group.

Measurement of myocardial HSPs. To determine the effects ofexercise on induction of myocardial HSPs, we performed polyacryl-amide gel electrophoresis and immunoblotting by using the tech-niques as previously described (14, 28, 38). Briefly, left ventricularsamples from I/R animals were homogenized, and one-dimensionalsodium dodecyl sulfate-polyacrylamide (12%) gel electrophoresis wasperformed to separate proteins by molecular weight. After separation,proteins were transferred to nitrocellulose membranes (Bio-Rad, Her-cules, CA). Nitrocellulose membranes were blocked for 2 h with(0.5%) bovine serum albumin. Blots were incubated for 2 h withmonoclonal antibodies for HSP72 (SPA-810, StressGen, Victoria,Canada). After chemiluminescence detection (Amersham Bio-sciences, Piscataway, NJ) and computerized densitometry, resultswere expressed as a percentage of sedentary controls.

In Vitro I/R Experiments

To evaluate the time course of exercise-associated protectionagainst I/R contractile dysfunction, we used an isolated working heartpreparation. At the appropriate time span after cessation of exercisetraining, animals were anesthetized with 100 mg/kg pentobarbitalsodium (ip). To facilitate removal of blood from the heart and toprevent clot formation, 100 IU of heparin were injected directly intothe hepatic vein. Hearts were then rapidly excised, placed in ice-coldsaline, and weighed on an electronic scale (gross wet weight). Theaorta was quickly secured on a stainless steel catheter, and the heartwas retrograde perfused by using a modified Krebs Henseleit buffer(1.5 mM CaCl2, 130 mM NaCl, 5.4 mM KCl, 11 mM glucose, 2 mMpyruvate, 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 pressureof 80 cmH2O and a temperature of 37 � 0.3°C. Excess tissue wastrimmed, weighed, and subtracted from the gross weight to get a finalwet weight of the heart. All subsequent values were normalized to thisfinal heart weight.

After 15 min of retrograde perfusion, the hearts were switched tothe working heart mode and preischemic heart function was evaluatedat 13 � 0.5 cmH2O (atrial filling pressure) with an 80-cm-high aorticcolumn. Coronary and aortic flows were determined by timed collec-tions of the coronary effluent and aortic column overflow, respec-tively. Cardiac output was determined as the sum of coronary andaortic flow.

Global, normothermic ischemia was then induced by simulta-neously cross-clamping the atrial inflow and the aortic outflow for20.5 min. During ischemia, hearts remained in a water-jacketedchamber maintained at 37°C. After ischemia, hearts were initiallyperfused in the retrograde mode for 15 min at a perfusion pressure of80 cmH2O and then switched to the working heart mode for 15 minfor a total of 30 min of reperfusion. The initial reperfusion period inthe retrograde mode is necessary to stabilize the hearts before workingheart reperfusion. Coronary effluent was collected between minutes 5and 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 timeperiods during this final 15 min of postischemia recovery in theworking heart mode.

We exposed these hearts to 20.5 min of global ischemia becausethis level of myocardial injury results in a “stunned myocardium” witha �40% decrease in cardiac working capacity. Myocardial stunning isdefined as a nonlethal form of I/R injury that results in impairedcardiac function for 24–48 h after I/R without death of cardiacmyocytes. We selected the isolated working heart model in theseexperiments for several reasons. First, this preparation closely resem-bles the in vivo working heart, and isolated working heart experimentsare highly reproducible (44). Second, when contrasted to in vivomodels, an advantage of the isolated heart preparation is the ability to

1300 LOSS OF EXERCISE-INDUCED CARDIOPROTECTION

J Appl Physiol • VOL 96 • APRIL 2004 • www.jap.org

on June 17, 2012jap.physiology.org

Dow

nloaded from

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

Statistical Analyses

Myocardial contractility and biochemical measurements were an-alyzed by using a one-way analysis of variance. When appropriate, aTukey’s post hoc test was applied. Significance was established apriori at P � 0.05.

RESULTS

Animal Characteristics

Animal characteristics are presented in Table 1. No differ-ences existed in animal body weights between the experimentalgroups. Furthermore, no differences existed in heart weights orthe ratio of body weight to heart weight between the fiveexperimental groups.

Exercise-Induced Changes in Myocardial Antioxidantsand HSP72

Exercise did not significantly alter myocardial GPX or SODactivities or cardiac levels of glutathione. The observation thatexercise-related increases in MnSOD activity did not reachstatistical significance is not consistent with our previous workand appears to be due to large interanimal variability. Incontrast, exercise significantly elevated myocardial Cat activityat both 1 and 3 days postexercise. By day 9, myocardial Catactivity returned to a level not different from sedentary controlanimals (Table 2).

Compared with the sedentary control animals, exercise re-sulted in significant increases in myocardial HSP72 levels at 1and 3 days postexercise (Fig. 1). Similar to exercise-inducedchanges in Cat activity, however, these training-inducedchanges were not evident at 9 days after cessation of exercise.Finally, because myocardial levels of HSP72 and antioxidant

activity returned to untrained levels by 9 days after cessation ofexercise, biochemical measurements were not performed onhearts obtained from animals subjected to 18 days of de-training.

Exercise Training and Cardiac Function During RecoveryFrom Ischemia

Successful I/R protocols were completed on 11 animals fromthe control group along with 10, 9, and 9 animals from the 1,3, and 9 days postexercise groups, respectively. Animals wereexcluded from data analysis if they did not complete the fullexercise protocol or if their hearts did not demonstrate stablebaseline performance during preischemia data collection. Theeffects of exercise training on pre- and postischemic cardiacfunction at each time point postexercise are presented in Table3 and Fig. 2. Note that, before ischemia, no differences existedbetween experimental groups in heart rate, coronary flow,cardiac output, or cardiac work. Furthermore, no differencesexisted between any of the experimental groups in heart rate orcoronary flow at 30 min of reperfusion. Also, no group differ-ences existed in aortic diastolic pressure at 30 min of reperfu-sion (data not shown). Reperfusion after 20.5 min of globalischemia resulted in a significant contractile dysfunction insedentary control animals. For example, compared with preis-chemic values, percent recovery of cardiac work was depressedby �30% in sedentary control animals at 30 min of reperfu-sion. In contrast to control, exercise-trained animals studied at1, 3, and 9 days postexercise maintained significantly higherrelative cardiac outputs and cardiac work at 30 min of reper-fusion (Table 3, Fig. 2). Note that cardiac work increasedrapidly during reperfusion and that a steady state was achievedwithin 5 min after reintroduction of the working heart mode.Therefore, only values from minute 30 of reperfusion arereported.

Absolute LDH activity in the coronary effluent (preische-mia) ranged from 12 to 41 mU�min�1�g wet weight�1 acrossthe five experimental groups. Postischemic LDH activity in the

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

ParameterControl

(n � 11)1 Day

(n � 10)3 Days(n � 9)

9 Days(n � 9)

18 Days(n � 8)

Body weight, g 382.5�2.44 382.5�2.94 381�2.74 392.8�3.74 372.6�2.6Heart weight, g 1.13�0.01 1.12�0.02 1.1�0.02 1.15�0.02 1.11�0.02Heart-to-body weight ratio, mg/g 2.96�0.03 2.92�0.06 2.89�0.07 2.93�0.05 2.98�0.03

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

Table 2. Effects of short-term exercise training on left ventricular antioxidants

Enzyme Activity(per mg protein)

Control(n � 10)

1 Day(n � 10)

3 Days(n � 10)

9 Days(n � 10)

GPX, �mol/min 0.21�0.01 0.244�0.009 0.235�0.006 0.221�0.007Cat, units 1.04�0.03 1.21�0.03*† 1.17�0.02*† 1.06�0.03Total SOD, units 42.77�1.79 44.84�2.38 48.03�2.52 46.19�3.17MnSOD, units 18.17�1.63 20.82�1.26 22.12�1.73 19.64�1.99CuZnSOD, units 24.60�1.79 23.72�1.82 25.91�2.05 26.54�2.65Total glutathione, mmol 1.36�0.08 1.38�0.10 1.44�0.12 1.47�0.11

Values are means � 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 � (2.3/t)�[log10 (A1/A2)], where t is time in min, A1 isinitial absorbance reading � blank absorbance, and A2 is absorbance reading at 1 min � blank absorbance. One unit of SOD activity is defined as the amountof SOD required for a 50% decrease in cytochrome c reduction rate. *Different from control, P � 0.05. †Different from 9 days, P � 0.05.

1301LOSS OF EXERCISE-INDUCED CARDIOPROTECTION

J Appl Physiol • VOL 96 • APRIL 2004 • www.jap.org

on June 17, 2012jap.physiology.org

Dow

nloaded from

coronary effluent ranged from 20 to 264 mU�min�1�g wetweight�1 across the experimental groups; these values areconsistent with I/R insults that result in myocardial stunning.For clarity, LDH activities were normalized (percent) to preis-chemic values. Consistent with the cardiac performance mea-sures, analyses revealed LDH activity in the coronary effluentcollected during reperfusion was significantly higher fromcontrol hearts compared with exercise trained hearts at 1, 3,and 9 days postexercise (Fig. 3).

Because we observed protection against I/R-induced cardiacdysfunction and LDH release that persisted through 9 dayspostexercise, we further assessed the time course of exercise-induced cardioprotection by adding another exercise-trainedgroup that was evaluated at 18 days postexercise. As illustratedin Table 3 and Fig. 2, exercise-induced preservation of postis-chemic cardiac output and cardiac work was lost at 18 dayspostexercise. Similarly, I/R-induced LDH release from theheart at 18 days postexercise was not different from controls(Fig. 3). Collectively, these findings indicate that exercise-

induced cardioprotection against myocardial stunning remainsfor at least 9 days after cessation of exercise training.

DISCUSSION

Overview of Principal Findings

To our knowledge, this is the first investigation of therelationship between the loss of protection against myocardialstunning after cessation of exercise and the reduction in puta-tive cardioprotective proteins in the heart. Our data indicatethat exercise-induced cardioprotection against myocardialstunning remains for at least 9 days after cessation of exercisetraining. Importantly, we observed that cardioprotection per-sists in the absence of elevated myocardial levels of HSP72 andantioxidants (e.g., Cat) at 9 days postexercise. These findingsindicate that exercise-associated cardioprotection is not criti-cally dependent on increases in cardiac levels of these cyto-protective proteins.

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

Table 3. Effects of exercise training intensity on pre- and postischemic cardiac function

Control(n � 11)

1 Day(n � 10)

3 Days(n � 9)

9 Days(n � 9)

18 Days(n � 8)

PreischemiaHR, beats/min 342.6�7.9 347.2�7.9 336.5�10.4 337.6�5.7 311.2�16.3CF, ml�min�1�g heart weight�1 14.9�0.47 16.5�0.55 17.9�0.92† 15.5�0.44 14.3�0.5CO, ml�min�1�g heart weight�1 43�0.45 43.6�1.07 42.7�1.1 43�0.9 41.5�1.6SP, mmHg 90.7�1.53† 89.4�1.4† 91.3�1.9† 91.9�1.6† 100.4�3.1Cardiac work, SP CO 3,900�75 3,900�124 3,889�81 3,948�87 4,158�178RPP, HR SP 30,953�256 30,946�314 30,585�563 30,974�338 30,976�1,228

PostischemiaHR, beats/min 328.7�10.5 328�8.9 341�13.8 336�5.8 310�16CF, ml�min�1�g heart weight�1 14.5�0.99 16.5�0.97 16.1�1.1 15.2�0.78 12.2�0.75CO, ml�min�1�g heart weight�1 33.6�1.6 39.3�1.5* 41�1.2* 38.5�1.4* 33.2�2.9SP, mmHg 82.9�2.7 85.3�1.4 85�2.4 84.7�2.3 87.3�3.4Cardiac work, SP CO 2,818�212 3,354�146 3,462�71 3,255�134 2,916�278Cardiac work, %preischemia 68�4 86�3* 89�2* 83�4* 74�3RPP, HR SP 27,006�477 27,881�395 28,699�551 28,394�510 26,806�112

Values are means � 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 � 0.05. †Significantly different from18 days, P � 0.05.

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

1302 LOSS OF EXERCISE-INDUCED CARDIOPROTECTION

J Appl Physiol • VOL 96 • APRIL 2004 • www.jap.org

on June 17, 2012jap.physiology.org

Dow

nloaded from

Exercise-Induced Myocardial Adaptation

Our results confirm previous data that short-term enduranceexercise training results in cardioprotection against an in vitroI/R insult that produces severe myocardial stunning. Thisagrees with previous reports in which both in vivo and in vitroI/R models were used to study exercise-induced cardioprotec-tion (14–17, 28, 38, 46). Furthermore, our data show that acuteexercise training is associated with threefold increases in myo-cardial levels of HSP72. Exercise-induced increases in myo-cardial levels of HSP72 are widely reported in the literature,and an increase in this stress protein has been postulated to playa role in exercise-induced cardioprotection (14–16, 28, 46).

It is well established that both ischemia and reperfusionpromote the formation of radicals/oxidants and that oxidativeinjury contributes to myocardial stunning (3). It follows thatexercise-induced increases in myocardial antioxidants couldprovide cardioprotection against this type of I/R injury. There-fore, to determine the effects of exercise on myocardial anti-oxidant capacity, we measured glutathione levels along withthe activities of key antioxidant enzymes (Table 2). Our resultsindicate that short-term exercise training does not elevatemyocardial glutathione or the activities of GPX or two intra-cellular isoforms of SOD (i.e., CuZnSOD or MnSOD). Thefinding that acute exercise does not elevate myocardial levelsof glutathione, GPX, and CuZnSOD agrees with previousreports (9, 14, 15, 37, 38). In contrast, several studies, includ-ing work in our laboratory, have reported that exercise isassociated with small (e.g., 10–20%) but significant increasesin myocardial MnSOD activity (9, 14, 15, 17, 38). Among thiswork are studies indicating that exercise-induced increases inMnSOD may be critical for protection against MI (17, 49).Nonetheless, in the present study, although the mean MnSODactivity tended to be higher in exercise-trained hearts comparedwith control, these levels did not reach significance.

In contrast to other antioxidant enzymes, exercise trainingelevated myocardial Cat activity. Although some controversyexists, others have reported exercise-induced increases in car-

diac Cat activity in both short- and long-duration exercisetraining (16, 19, 23, 25, 41, 42, 47). In contrast, myocardialglutathione levels are typically not elevated after short-termexercise, but most long-term training studies report that severalweeks of training elevates cardiac glutathione levels (9, 19, 20,27, 34). On the basis of these observations, it is tempting tospeculate that the myocardial strategy to eliminate hydrogenperoxide changes over time in response to continuous exercisetraining. That is, the increased ability to remove hydrogenperoxide early during training adaptation is achieved by ele-vating Cat activity alone, whereas as exercise training contin-ues, there is a gradual increase in glutathione levels. Increasingcardiac levels of glutathione with chronic exercise trainingwould improve the ability of the heart to eliminate hydrogenperoxide because glutathione works in tandem with GPX toremove hydrogen peroxide and other organic hydroperoxides.The biological rationale for this changing adaptive strategy isunclear but could be linked to the fact that exercise-inducedincreases in Cat activity can be achieved rapidly via geneexpression, whereas myocardial glutathione synthesis is a rel-atively slow process limited by the availability of cellularlevels of cysteine (13).

Postexercise Loss of Cardioprotection

The mechanism(s) responsible for exercise-induced cardio-protection remains controversial. It has been suggested thatexercise-induced increases in cardiac levels of HSP72 and/orkey antioxidants are potential mechanisms to explain the car-dioprotection associated with physical activity (8, 17, 22,28–30, 32, 49). Our hypothesis that exercise-induced cardio-protection against myocardial stunning would disappear within9 days after the cessation of exercise training was formulatedby reports indicating that the half-life of putative cardioprotec-tive proteins (e.g., HSP72) is �2–3 days (35). The half-life ofcirculating levels of some of the isoforms of antioxidantenzymes, however, can be as short as a few hours (12).Therefore, after cessation of exercise training, the postexercisedecline in HSP72 would result in an �88% reduction inmyocardial levels within 9 days. It follows that, if HSP72and/or Cat is essential for exercise-induced cardioprotection,the protection would disappear after myocardial levels of theseprotective proteins decline to sedentary control levels. Ourresults reveal that cardiac levels of both HSP72 and Cat declineover time and reach sedentary control values at day 9 postex-ercise. Nonetheless, the finding that cardioprotection remainsat 9 days postexercise indicates that mechanisms other thanHSP72 and Cat are involved in exercise-related protectionagainst I/R-induced myocardial stunning.

Potential Mechanisms of Exercise-Induced ProtectionAgainst Myocardial Stunning

If exercise-induced increases in myocardial Cat and HSP72are not essential to achieve exercise-mediated protectionagainst myocardial stunning, other mechanism(s) must be re-sponsible for this form of cardioprotection. The two primarytheories to explain the pathogenesis of myocardial stunning arethe radical hypothesis and the calcium hypothesis (3). Theradical hypothesis postulates that myocardial stunning resultsfrom oxidative injury by radicals and other reactive speciesproduced during ischemia and reperfusion. This oxidative

Fig. 3. Effects of ischemia-reperfusion injury on the release of lactate dehy-drogenase from isolated rat hearts from control and exercise groups (exer-cise � 1 day rest, exercise � 3 days rest, exercise � 9 days rest, andexercise � 18 days rest). Lactate dehydrogenase activity was measured incoronary effluent before and after ischemia-reperfusion. Lactate dehydroge-nase activity was normalized to wet weight of the heart and expressed as thepercent difference between preischemia and postischemic values. Values aremeans � SE. *Different from control, P � 0.05. †Different from exercise �18 days rest, P � 0.05.

1303LOSS OF EXERCISE-INDUCED CARDIOPROTECTION

J Appl Physiol • VOL 96 • APRIL 2004 • www.jap.org

on June 17, 2012jap.physiology.org

Dow

nloaded from

injury could impair myocardial contractility due to a reducedresponsiveness of contractile filaments to calcium (3). Thecalcium hypothesis is based on evidence that I/R results in atransient calcium overload in the myocardium that triggerscalcium-activated proteases (i.e., calpains) to degrade keycytoskeletal proteins. The radical and calcium hypotheses arenot mutually exclusive because calcium overload in cells cancontribute to radical production and oxidation of calciumtransport proteins can promote disturbances in calcium ho-meostasis (3). Therefore, it is feasible that the mechanismresponsible for exercise-induced protection against myocardialstunning is linked to exercise provoked expression of antioxi-dants and/or calcium handling proteins.

As mentioned previously, others have concluded that exer-cise-induced increases in MnSOD may be critical for protec-tion against myocardial infarction (17, 49). Nonetheless, in thepresent study, although the mean MnSOD activity tended to behigher in exercise-trained hearts compared with control, theselevels did not reach significance. Furthermore, work in ourlaboratory using antisense oligonucleotides indicates that ex-ercise-induced increases in MnSOD are not essential for pro-tection against myocardial stunning (16, 24). However, itseems likely that other antioxidants may be involved in car-dioprotection. In this regard, preliminary experiments in ourlaboratory suggest that exercise elevates cardiac mRNA levelsof several other antioxidant proteins (e.g., thioredoxin reduc-tase, metallothionien) in the heart (unpublished observations).Future experiments are required to determine whether one ormore of these antioxidants are essential to achieve exercise-induced protection against myocardial stunning.

Related to the calcium hypothesis of myocardial stunning,several laboratories have investigated exercise-inducedchanges in myocardial calcium handling. For example, al-though exercise does not alter the number of L-type calciumchannels in the heart (33), exercise training does increase therate of calcium transport by calcium ATPtase in the sarcoplas-mic reticulum of rat hearts (45). At present, other than sarco-plasmic reticulum calcium ATPases, limited information existsregarding exercise-mediated changes in other calcium handlingproteins in the heart. It seems possible that exercise trainingmay alter calcium-handling proteins resulting in protectionagainst I/R-induced calcium overload. This could provide anexciting area for future research.

Summary and Conclusions

These experiments investigated the rate of decline of exer-cise-induced cardioprotection after the cessation of exercisetraining. Our data indicate that exercise-induced cardioprotec-tion remains for 9 days after cessation of exercise training butthat this protection is absent at 18 days postexercise. The factthat cardioprotection persists in the absence of elevated myo-cardial levels of HSP72 and Cat at day 9 postexercise impliesthat cytoprotective molecules other than HSP72 and Catprovide cardioprotection against myocardial stunning. Al-though the mechanism(s) responsible for exercise-induced car-dioprotection remains elusive, continuing advances in biotech-nology (i.e., microarray analysis) will permit the measure-ment of transcription in a large number of genes and willgreatly accelerate research progress in our laboratory andothers (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 R01HL-067855 (to S. K. Powers).

REFERENCES

1. Aebi H. Catalase in vitro. Methods Enzymol 105: 121–126, 1984.2. Bergmeyer H, Bernt E, and Hess B. Methods of Enzymatic Analysis.

New York: Academic, 1965.3. Bolli R and Marban E. Molecular and cellular mechanisms of myocar-

dial stunning. Physiol Rev 79: 609–634, 1999.4. Bowles DK, Farrar RP, and Starnes JW. Exercise training improves

cardiac function after ischemia in the isolated, working rat heart. Am JPhysiol Heart Circ Physiol 263: H804–H809, 1992.

5. Bowles DK and Starnes JW. Exercise training improves metabolicresponse after ischemia in isolated working rat heart. J Appl Physiol 76:1608–1614, 1994.

6. Bradford MM. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 72: 248–254, 1976.

7. Bronikowski AM, Carter PA, Morgan TJ, Garland T Jr, Ung N, PughTD, Weindruch R, and Prolla TA. Lifelong voluntary exercise in themouse prevents age-related alterations in gene expression in the heart.Physiol Genomics 12: 129–138, 2003.

8. Chen Z, Siu B, Ho YS, Vincent R, Chua CC, Hamdy RC, and ChuaBH. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J Mol Cell Cardiol 30: 2281–2289,1998.

9. Demirel HA, Powers SK, Zergeroglu MA, Shanely RA, Hamilton K,Coombes J, and Naito H. Short-term exercise improves myocardialtolerance to in vivo ischemia-reperfusion in the rat. J Appl Physiol 91:2205–2212, 2001.

10. Diffee GM, Seversen EA, Stein TD, and Johnson JA. Microarrayexpression analysis of effects of exercise training: increase in atrialMLC-1 in rat ventricles. Am J Physiol Heart Circ Physiol 284: H830–H837, 2003.

11. Flohe L and Gunzler WA. Assays of glutathione peroxidase. MethodsEnzymol 105: 114–121, 1984.

12. Greenwald RA. Superoxide dismutase and catalase as therapeutic agentsfor human diseases. A critical review. Free Radic Biol Med 8: 201–209,1990.

13. Halliwell B and Gutteridge J. Free Radicals in Biology and Medicine.New York: Oxford University Press, 1989.

14. Hamilton KL, Powers SK, Sugiura T, Kim S, Lennon S, Tumer N, andMehta JL. Short-term exercise training can improve myocardial toleranceto I/R without elevation in heat shock proteins. Am J Physiol Heart CircPhysiol 281: H1346–H1352, 2001.

15. Hamilton KL, Staib JL, Hess A, Phillips T, Lennon S, and Powers SK.Exercise, antioxidants, and protection against myocardial ische-mia-reperfusion. Free Radic Biol Med 34: 800–809, 2003.

16. Harris MB and Starnes JW. Effects of body temperature during exercisetraining on myocardial adaptations. Am J Physiol Heart Circ Physiol 280:H2271–H2280, 2001.

17. Hoshida S, Yamashita N, Otsu K, and Hori M. Repeated physiologicstresses provide persistent cardioprotection against ischemia-reperfusioninjury in rats. J Am Coll Cardiol 40: 826–831, 2002.

18. Hull SS Jr, Vanoli E, Adamson PB, Verrier RL, Foreman RD, andSchwartz PJ. Exercise training confers anticipatory protection fromsudden death during acute myocardial ischemia. Circulation 89: 548–552,1994.

19. Husain K and Hazelrigg SR. Oxidative injury due to chronic nitric oxidesynthase inhibition in rat: effect of regular exercise on the heart. BiochimBiophys Acta 1587: 75–82, 2002.

20. Husain K and Somani SM. Response of cardiac antioxidant system toalcohol and exercise training in the rat. Alcohol 14: 301–307, 1997.

21. Hutter JJ, Mestril R, Tam EK, Sievers RE, Dillmann WH, and WolfeCL. Overexpression of heat shock protein 72 in transgenic mice decreasesinfarct size in vivo. Circulation 94: 1408–1411, 1996.

22. Hutter MM, Sievers RE, Barbosa V, and Wolfe CL. Heat-shock proteininduction in rat hearts. A direct correlation between the amount ofheat-shock protein induced and the degree of myocardial protection.Circulation 89: 355–360, 1994.

1304 LOSS OF EXERCISE-INDUCED CARDIOPROTECTION

J Appl Physiol • VOL 96 • APRIL 2004 • www.jap.org

on June 17, 2012jap.physiology.org

Dow

nloaded from

23. Kanter MM, Hamlin RL, Unverferth DV, Davis HW, and Merola AJ.Effect of exercise training on antioxidant enzymes and cardiotoxicity ofdoxorubicin. J Appl Physiol 59: 1298–1303, 1985.

24. Kihlstrom M. Protection effect of endurance training against reoxygen-ation-induced injuries in rat heart. J Appl Physiol 68: 1672–1678, 1990.

25. Kim JD, Yu BP, McCarter RJ, Lee SY, and Herlihy JT. Exercise anddiet modulate cardiac lipid peroxidation and antioxidant defenses. FreeRadic Biol Med 20: 83–88, 1996.

26. Lawler JM, Powers SK, Hammeren J, and Martin AD. Oxygen cost oftreadmill running in 24-month-old Fischer-344 rats. Med Sci Sports Exerc25: 1259–1264, 1993.

27. Liu J, Yeo HC, Overvik-Douki E, Hagen T, Doniger SJ, Chyu DW,Brooks GA, Ames BN, and Chu DW. Chronically and acutely exercisedrats: biomarkers of oxidative stress and endogenous antioxidants. J ApplPhysiol 89: 21–28, 2000.

28. Locke M, Tanguay RM, Klabunde RE, and Ianuzzo CD. Enhancedpostischemic myocardial recovery following exercise induction of HSP72. Am J Physiol Heart Circ Physiol 269: H320–H325, 1995.

29. Marber MS, Latchman DS, Walker JM, and Yellon DM. Cardiac stressprotein elevation 24 hours after brief ischemia or heat stress is associatedwith resistance to myocardial infarction. Circulation 88: 1264–1272,1993.

30. Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, and DillmannWH. Overexpression of the rat inducible 70-kD heat stress protein in atransgenic mouse increases the resistance of the heart to ischemic injury.J Clin Invest 95: 1446–1456, 1995.

31. McCord JM and Fridovich I. The reduction of cytochrome c by milkxanthine oxidase. J Biol Chem 243: 5753–5760, 1968.

32. Mestril R and Dillmann WH. Heat shock proteins and protection againstmyocardial ischemia. J Mol Cell Cardiol 27: 45–52, 1995.

33. Mokelke EA, Palmer BM, Cheung JY, and Moore RL. Endurancetraining does not affect intrinsic calcium current characteristics in ratmyocardium. Am J Physiol Heart Circ Physiol 273: H1193–H1197, 1997.

34. Ohkuwa T, Sato Y, and Naoi M. Glutathione status and reactive oxygengeneration in tissues of young and old exercised rats. Acta Physiol Scand159: 237–244, 1997.

35. Oishi Y, Taniguchi K, Matsumoto H, Ishihara A, Ohira Y, and RoyRR. Muscle type-specific response of HSP60, HSP72, and HSC73 duringrecovery after elevation of muscle temperature. J Appl Physiol 92: 1097–1103, 2002.

36. Paroo Z, Haist JV, Karmazyn M, and Noble EG. Exercise improvespostischemic cardiac function in males but not females: consequences ofa novel sex-specific heat shock protein 70 response. Circ Res 90: 911–917,2002.

37. Powers SK, Criswell D, Lawler J, Martin D, Lieu FK, Ji LL, and HerbRA. Rigorous exercise training increases superoxide dismutase activity inventricular myocardium. Am J Physiol Heart Circ Physiol 265: H2094–H2098, 1993.

38. Powers SK, Demirel HA, Vincent HK, Coombes JS, Naito H, Hamil-ton KL, Shanely RA, and Jessup J. Exercise training improves myocar-dial tolerance to in vivo ischemia-reperfusion in the rat. Am J PhysiolRegul Integr Comp Physiol 275: R1468–R1477, 1998.

39. Powers SK, Locke and Demirel HA. Exercise, heat shock proteins, andmyocardial protection from I-R injury. Med Sci Sports Exerc 33: 386–392,2001.

40. Radford NB, Fina M, Benjamin IJ, Moreadith RW, Graves KH, ZhaoP, Gavva S, Wiethoff A, Sherry AD, Malloy CR, and Williams RS.Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.Proc Natl Acad Sci USA 93: 2339–2342, 1996.

41. Somani SM, Frank S, and Rybak LP. Responses of antioxidant systemto acute and trained exercise in rat heart subcellular fractions. PharmacolBiochem Behav 51: 627–634, 1995.

42. Somani SM and Rybak LP. Comparative effects of exercise training ontranscription of antioxidant enzyme and the activity in old rat heart. IndianJ Physiol Pharmacol 40: 205–212, 1996.

43. Starnes JW and Bowles DK. Role of exercise in the cause and preventionof cardiac dysfunction. Exerc Sport Sci Rev 23: 349–373, 1995.

44. Sutherland FJ and Hearse DJ. The isolated blood and perfusion fluidperfused heart. Pharmacol Res 41: 613–627, 2000.

45. Tate CA, Helgason T, Hyek MF, McBride RP, Chen M, RichardsonMA, and Taffet GE. SERCA2a and mitochondrial cytochrome oxidaseexpression are increased in hearts of exercise-trained old rats. Am JPhysiol Heart Circ Physiol 271: H68–H72, 1996.

46. Taylor RP, Harris MB, and Starnes JW. Acute exercise can improvecardioprotection without increasing heat shock protein content. Am JPhysiol Heart Circ Physiol 276: H1098–H1102, 1999.

47. Terblanche SE. The effects of exhaustive exercise on the activity levelsof catalase in various tissues of male and female rats. Cell Biol Int 23:749–753, 2000.

48. Yamashita N, Baxter GF, and Yellon DM. Exercise directly enhancesmyocardial tolerance to ischaemia-reperfusion injury in the rat through aprotein kinase C mediated mechanism. Heart 85: 331–336, 2001.

49. Yamashita N, Hoshida S, Otsu K, Asahi M, Kuzuya T, and Hori M.Exercise provides direct biphasic cardioprotection via manganese super-oxide dismutase activation. J Exp Med 189: 1699–1706, 1999.

50. Ytrehus K. The ischemic heart—experimental models. Pharmacol Res42: 193–203, 2000.

1305LOSS OF EXERCISE-INDUCED CARDIOPROTECTION

J Appl Physiol • VOL 96 • APRIL 2004 • www.jap.org

on June 17, 2012jap.physiology.org

Dow

nloaded from