Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 21, 2005). doi:10.1152/ajpregu.00636.2004
Voluntary exercise attenuates DOX cardiotoxicity
VOLUNTARY EXERCISE PROTECTS AGAINST ACUTE DOXORUBICIN CARDIOTOXICITY IN THE ISOLATED PERFUSED RAT HEART
Adam J. Chicco, Carole M. Schneider and Reid Hayward School of Sport and Exercise Science, and the Rocky Mountain Cancer Rehabilitation Institute University of Northern Colorado, Greeley, CO 80639
RUNNING HEAD: Voluntary exercise attenuates DOX cardiotoxicity
Correspondence should be addressed to: Reid Hayward, PhD School of Sport and Exercise Science University of Northern Colorado Greeley, CO 80639 Phone : (970) 351-1821 Fax: (970) 351-1762 Email:
[email protected]
1 Copyright © 2005 by the American Physiological Society.
Voluntary exercise attenuates DOX cardiotoxicity
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Voluntary exercise attenuates DOX cardiotoxicity ABSTRACT The clinical use of doxorubicin (DOX) is limited by a dose-dependent cardiotoxicity. The purpose of this study was to determine whether voluntary exercise training would confer protection against DOX cardiotoxicity in the isolated perfused rat heart. Female Sprague-Dawley rats were randomly assigned to standard holding cages (SED) or cages with running wheels (VEX) for 8 weeks. Twenty-four hours following the sedentary or voluntary running period, rats were anesthetized with sodium pentobarbital and hearts were isolated and perfused with oxygenated Krebs-Henseleit (KH) buffer at a constant flow of 15 ml⋅min-1. Following a 20-min stabilization period, hearts were paced at 300 bpm and perfused with KH buffer containing 10 µM DOX for 60 min. A set of control hearts from SED and VEX rats were perfused under identical conditions without DOX for the same period. DOX perfusion led to significant decreases in left ventricular (LV) developed pressure, +dP/dt, and -dP/dt, and significant increases in LV lipid peroxidation in sedentary rats compared to non-DOX controls (P < 0.05). Prior voluntary exercise training attenuated these DOX-induced effects, and was associated with a significant increase (78%, P < 0.05) in heat shock protein (HSP72), but not MnSOD or CuZnSOD protein expression in the hearts of wheel run animals. These data indicate that chronic physical activity may provide resistance against the cardiac dysfunction and oxidative damage associated with DOX exposure, and provide novel evidence of HSP72 induction in the heart following voluntary exercise.
KEYWORDS: Adriamycin, cardioprotection, heart, physical activity, cardiac function
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Voluntary exercise attenuates DOX cardiotoxicity
Doxorubicin (DOX) is a highly effective antineoplastic antibiotic commonly used to treat a variety of solid tumors and leukemias. Unfortunately, the clinical use of DOX is limited by a dose-dependent cardiotoxicity that may progress to chronic cardiomyopathy and congestive heart failure (CHF) (50). Characterization and prevention of DOX cardiotoxicity (DCT) has been the subject of intense research for decades, but no single intervention has proven to be completely successful in ameliorating the damaging effects of DOX on the heart while maintaining its antineoplastic efficacy. DOX cardiomyopathy and CHF typically develop following multiple intravenous DOX treatments over a period of several months in humans. Incidence of chronic cardiomyopathy appears to be highly dependent on the cumulative DOX dose administered (50), and has been recently reported to be as high as 26% in patients receiving 550 mg/m2 (54). Evidence from human (37) and animal (30) studies suggest that acute myocardial dysfunction and/or injury occurring early during the course of DOX treatment is highly predictive of later cardiomyopathy. Furthermore, it appears that the chronic manifestations of DCT may result from myocardial injuries associated with acute DOX exposure (40, 58). This has led to the use of a wide variety of experimental models aimed at exploring the mechanisms and circumvention of acute DCT in vivo and in vitro. Several investigators have utilized the isolated perfused rat heart model for this purpose, which has proven to provide a reliable and reproducible means of assessing the biochemical and functional aspects of acute DCT (19, 40, 42). Free radical formation has been consistently reported to play a pivotal role in several of the acute and chronic manifestations of DCT (23, 27, 30, 34). DOX is believed to interact with intracellular redox enzymes such as nicotinamide adenine dinucleotide
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Voluntary exercise attenuates DOX cardiotoxicity dehydrogenase to produce superoxide radicals and increase the levels of cytotoxic reactive oxygen species (ROS) in cardiomyocytes (10). The resulting oxidative stress is known to initiate a myriad of destructive events in cardiomyocytes, and has been specifically linked to membrane lipid peroxidation (27, 34, 39), impaired Ca2+ handling (15), mitochondrial dysfunction (13), apoptosis (23), and DNA injury (30). Superoxide dismutase (SOD) is an endogenous antioxidant enzyme that protects the heart from superoxide by catalyzing its dismutation into hydrogen peroxide (H2O2), which is then converted to oxygen and water by catalase (CAT) and the glutathione peroxidase (GPx) system. Acute DCT has been shown to be enhanced when SOD activity is compromised (49) and is attenuated in transgenic mice overexpressing the mitochondrial isoform of SOD (MnSOD) (62). These studies further support the role of superoxide in DCT, and suggest that any treatment that increases myocardial SOD activity may protect the heart from DOX-induced injury. Heat shock proteins of the 70 kDa family (particularly the inducible isoform, HSP72) are believed to protect cardiomyocytes against injury during states of cellular stress (5, 21). HSP72 induction has been specifically associated with the prevention of apoptosis, necrosis, and oxidative injury in the myocardium following exposure to ischemia-reperfusion (I/R) (8, 45, 47), hydrogen peroxide (55), and DOX (17). HSP72 most likely protects cardiomyocytes by preserving protein structure and function during states of stress (21), and may selectively protect mitochondria from the damaging effects of ROS (41). Exercise training has been reported to increase SOD (2, 44) and HSP72 content (28, 45) in the myocardium. These adaptations have been associated with an exercise-
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Voluntary exercise attenuates DOX cardiotoxicity induced preservation of cardiac function and attenuation of myocardial lipid peroxidation observed following I/R (2, 45). Therefore, it is plausible that exercise training may also confer protection against the cardiac dysfunction and myocardial lipid peroxidation induced by DOX exposure. The authors are aware of only one study that has examined the effect of prior exercise training on DCT (20). These investigators reported that mice swim trained before, during and after DOX therapy in vivo had fewer myocardial lesions than untrained DOX-treated mice, but no assessment of cardiac function was conducted. The vast majority of studies that have examined the cardioprotective effects of exercise training have utilized a forced treadmill running or swimming model. While these protocols permit strict control of the duration and intensity of exercise training, animals appear to experience significant systemic stress which may have far-reaching physiological implications (32, 48). Therefore, we have employed a voluntary running model in an effort to isolate the effects of habitual physical activity from the additional stress associated with forced exercise protocols. The purpose of this study was to determine whether chronic voluntary exercise prior to DOX exposure protects the heart from the acute manifestations of DCT. We have utilized an established model of acute DCT (19, 42, 43) to examine the direct effects of DOX exposure on intrinsic cardiac function and lipid peroxidation in isolated perfused hearts from sedentary and physically active rats. Given prior evidence of their role in exercise-induced cardioprotection against oxidative stress, MnSOD, CuZnSOD and HSP72 content were also examined in hearts of sedentary and physically active animals.
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Voluntary exercise attenuates DOX cardiotoxicity METHODS Animals and experimental design All experimental procedures were approved by the University of Northern Colorado Institutional Animal Care and Use Committee and were in compliance with guidelines established by the American Physiological Society. Female Sprague-Dawley rats (200-225 g) were randomly assigned to a sedentary or voluntary exercise group. Female rats were selected due to their greater propensity for voluntary running compared to males (60). All animals were housed in a temperature-controlled facility (19-21 °C) maintained on a 12:12-h light:dark cycle with food and water provided ad libitum. Rats in the voluntary exercise group were housed individually in cages with stainless-steel running wheels (1.06 m circumference, Mini-Mitter, Bend, OR) and were allowed free access to the wheel 24 h per day for 8 weeks. Sedentary rats were housed in standard holding cages without running wheels for the same period. The voluntary wheel running model was selected in an effort to isolate the effects of habitual physical activity from the systemic stress response observed in rats following forced (e.g., treadmill) exercise training (32, 33). Following the 8-week sedentary or voluntary running period, rats were randomly subdivided into four experimental groups: sedentary control (SED; n = 6), SED + DOX (n = 7), voluntary exercise (VEX; n = 8), or VEX + DOX (n = 7). Hearts from SED and VEX rats were perfused with Krebs-Henseleit (KH) buffer for the entire perfusion period. SED + DOX and VEX + DOX hearts were perfused under identical conditions with KH buffer containing 10 µM DOX (Bedford Labs, Bedford, PA).
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Voluntary exercise attenuates DOX cardiotoxicity Isolated heart perfusion protocol All rats were anesthetized with heparinized (1000 U⋅ kg-1) sodium pentobarbital (40 mg⋅kg-1 i.p.) and hearts were rapidly excised and placed in ice-cold KH buffer consisting of (in mM): NaCl 120, KCl 5.9, CaCl2 2.5, MgCl 1.2, NaHCO3 25, glucose 17, and EDTA, 0.5 (Sigma, St. Louis, MO). The aorta was then immediately cannulated for retrograde cardiac perfusion at 37 °C with oxygenated (95% O2-5% CO2) KH buffer at a constant perfusion rate of 15 ml⋅min-1. Following a 15-min stabilization period, hearts were electrically paced at 300 bpm (5 Hz) using a stimulus isolator (ADInstruments, Colorado Springs, CO) and allowed to stabilize for an additional 5 min before being perfused with KH buffer (SED and VEX) or KH buffer containing 10 µM DOX (SED + DOX and VEX + DOX) for an additional 60 min. This DOX perfusion protocol has been previously shown to provide a reproducible model of acute DOX cardiotoxicity in the isolated perfused rat heart (19, 42, 43). Measurement of LV Function To monitor left ventricular function during the perfusion period, a microtip catheter pressure transducer (Millar Instruments Inc., Houston, TX) was inserted into the left ventricular (LV) cavity through the mitral valve for continuous measurement of LV developed pressure (LVDP) and its maximal and minimal first derivatives (+dP/dt and dP/dt, respectively). Following the 20-min stabilization period, LV pressure data were recorded under paced conditions (300 bpm) at 10-min intervals using a PowerLab/8e data acquisition system (ADInstruments, Colorado Springs, CO). Immediately following the perfusion period, hearts were trimmed free of surrounding connective tissue and fat, blotted
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Voluntary exercise attenuates DOX cardiotoxicity dry and weighed. The left ventricle was then isolated, frozen in liquid nitrogen and stored at –80 °C for future biochemical analyses. Lipid peroxidation assay. To examine the effect of DOX perfusion on myocardial lipid peroxidation in sedentary and wheel run animals, malondialdehyde and 4-hydroxy-alkenals (MDA + 4-HAE) were analyzed in LV of all hearts following the perfusion experiments described above using a spectrophotometric assay kit (Calbiochem, San Diego, CA) according to the manufacturer’s instructions. Frozen LV tissue was homogenized in ice-cold 20 mM TrisHCl, pH 7.4, containing 5 mM butylated hydroxytoluene (to prevent lipid peroxidation during homogenization) at a dilution of 1:5 (w/v) and centrifuged at 3000 g for 10 min at 4 o
C. The supernatant was collected and used for future spectrophotometic analyses. MDA
+ 4-HAE is expressed relative to the protein content of sample supernatant determined by the method of Bradford (1) using bovine serum albumin as the standard. Left ventricular SOD and HSP72 content. To examine the effect of voluntary exercise on potential candidates of exerciseinduced cardioprotection, HSP72, CuZnSOD and MnSOD contents were determined in LV samples obtained from SED and VEX animals following the perfusion protocol described above using western immunoblotting methods previously described (2, 28). Briefly, LV samples were homogenized in ice-cold homogenizing buffer containing (in mM) 100 KCl, 50 MOPS, 5 MgCl2, 1 ATP, 1 EGTA (pH 7.4) and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Homgenates then diluted 1:3 (v/v) in lysis buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS (pH 7.4), allowed to incubate at room temperature for 10 min, then centrifuged at
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Voluntary exercise attenuates DOX cardiotoxicity 3000 g for 5 min. The supernatant was then assayed for total protein (1), and a volume of supernatant containing 50 µg (HSP72) or 3 µg (SOD isoforms) of LV protein was electrophoresed on polyacrylamide gels for separation of proteins by molecular weight. Proteins were then transferred to membranes (Amersham, Piscataway, NJ) and incubated with an alkaline phosophatase-conjugated polyclonal antibody specific for rat HSP72, or polyclonal antibodies specific for CuZnSOD or MnSOD (Stressgen, Victoria, BC, Canada). HSP72 blots were then reacted with 5-bromo-4-chloro-3-indolyl phosphatenitro blue tetrazolium substrate (Sigma Chemical, St. Louis, MO) (HSP72) and scanned for band density analysis. SOD isoform blots were incubated with a secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a chemiluminescent substrate (Western Lighting, PerkinElmer Life Sciences, Boston, MA) and developed on film. Quantification of relative HSP72 and SOD isoforms band densities were performed on computerized scans of immunoblots using ImageJ densitometry software (NIH, Bethesda, MD). VEX band density is expressed as a percentage of the mean SED band density from the same membrane.
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Voluntary exercise attenuates DOX cardiotoxicity Soleus citrate synthase activity. To examine any training effect resulting from the voluntary running protocol on skeletal muscle, the right soleus muscle was excised from sedentary and wheel run rats and analyzed for citrate synthase activity at 30 °C by the method of Srere (52). Statistical analyses and protein assay. All data are expressed as group means ± SE. Cardiac function parameters were analyzed using a two-way (VEX x DOX) analysis of variance (ANOVA) with repeated measures, followed by independent sample t-tests to examine individual group differences. In addition, within-group differences between baseline and time point measurements during perfusion were examined using paired t-tests with the Bonferroni correction for multiple comparisons. The MDA+4-HAE data were analyzed by a twoway ANOVA with independent sample t-tests post hoc. HSP72, SOD isoforms, and morphological data from SED and VEX animals were compared by independent sample t-tests. Significance was established at P < 0.05 for all statistical analyses.
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Voluntary exercise attenuates DOX cardiotoxicity RESULTS Voluntary exercise and animal characteristics. Animals in the running wheel cages voluntarily ran an average of 6880 ± 348 meters per day. Figure 1 illustrates the average distance run per week over the 8-week period. The effects of voluntary exercise on animal characteristics are presented in Table 1. No significant differences in body weight were observed between the sedentary and wheel run rats at the end of the 8-week study. However, wheel running led to significant cardiac hypertrophy, indicated by greater heart weight and heart-to-body weight ratio in the VEX compared to SED animals (P < 0.001). In addition, a skeletal muscle training effect was observed in the VEX animals, indicated by significantly higher soleus weight and citrate synthase activity compared to SED (P < 0.05). Effect of voluntary exercise and DOX perfusion on LV function At the end of the stabilization period, there were no differences in any of the selected parameters of LV function observed between the sedentary or wheel run animals, indicating that voluntary exercise did not significantly alter basal cardiac function (Table 2). Figure 2 illustrates the effect of VEX and DOX perfusion on LV function relative to baseline levels throughout the 60-min perfusion period. Perfusion with KH buffer alone had no significant effect on LV function in the SED or VEX hearts relative to baseline values, and there were no significant functional differences observed between these two groups. DOX perfusion led to a significant decline in all parameters of LV function in the hearts from sedentary animals throughout the perfusion period. LVDP decreased to 84% of baseline values after 30 min of DOX perfusion (P < 0.05), and steadily declined thereafter to 38% of baseline by 60 min. Similar decrements were
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Voluntary exercise attenuates DOX cardiotoxicity observed in LV -dP/dt and +dP/dt, which decreased to 46% and 50% of baseline by 60 min, respectively (P < 0.05). These DOX-induced decrements in LV function were attenuated in the hearts of wheel run rats. LVDP remained at baseline levels for 40 min in the hearts of VEX+DOX rats, then steadily declined to 60% of baseline after 60 min (P = NS). While -dP/dt and +dP/dt also declined in hearts of wheel-run animals after 60 min of DOX perfusion (decreased to 91% and 78% of baseline, respectively), these decreases were not statistically significant. Furthermore, relative values of LVDP, -dP/dt and +dP/dt were significantly higher in the VEX+DOX than SED+DOX hearts at several time points throughout the perfusion period (see Figure 2). These data suggest that VEX prior to DOX exposure results in an attenuation of DOX-induced LV dysfunction. Lipid peroxidation Two-way ANOVA analysis revealed a significant effect of DOX perfusion on myocardial lipid peroxidation indices. While there was no effect of VEX alone on MDA+4-HAE levels, there was a significant interaction effect of VEX and DOX (P < 0.05). When compared to SED and VEX hearts, MDA+4-HAE levels were significantly higher in the SED+DOX hearts (P < 0.05), but not the VEX+DOX hearts (Figure 3). These data suggest that voluntary exercise attenuates myocardial lipid peroxidation induced by DOX exposure. Left ventricular SOD and HSP72 content. No significant differences in LV MnSOD or CuZnSOD content were observed between SED and VEX animals (Figure 4). HSP72 content, however, was 78% higher in the hearts of VEX compared to SED (Figure 5), indicating that voluntary exercise induced HSP72 protein expression in the heart.
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Voluntary exercise attenuates DOX cardiotoxicity DISCUSSION The results of this study indicate that prior voluntary exercise training attenuates the cardiac dysfunction and lipid peroxidation induced by acute DOX exposure. To our knowledge, these data are the first to provide direct evidence of exercise-induced protection against the acute functional manifestations of DCT. In addition, we provide novel evidence of HSP72 induction in the rat heart by chronic voluntary exercise. Effect of DOX perfusion on LV function and lipid peroxidation To determine the effects of DOX exposure on intrinsic cardiac function in previously sedentary or physically active rats, we have monitored LV pressure characteristics of isolated hearts perfused with 10 µM DOX over a 60 min period. This model provides a rapid and reproducible means of assessing the cumulative effects of continuous DOX exposure at a concentration similar to peak levels observed in plasma following single i.v. treatments in humans (7, 12). In accordance with previous studies (19, 40, 42), we observed that perfusion of hearts with 10 µM DOX results in a progressive decline in LVDP, with parallel decrements in dP/dt over a 60 min perfusion period. The mechanisms by which DOX exposure induces cardiac dysfunction have not been fully elucidated. Prevailing hypotheses suggest that myocardial oxidative stress is a primary event in the etiology of DCT and is believed to initiate several of the deleterious cellular events reported following DOX treatment (6, 63). Oxidative destruction of fatty acids in cell and organelle membranes indicated by increased levels of lipid peroxidation products has been frequently reported following DOX exposure (26, 39, 53) and has long been considered to be a critical event in DCT (34). In accordance with these studies, we
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Voluntary exercise attenuates DOX cardiotoxicity observed a significant increase in MDA+4-HAE in the LV from sedentary animals exposed to DOX, which supports the association between lipid peroxidation and DOXinduced cardiac dysfunction. Cardioprotective effects of voluntary wheel running Voluntary exercise elicited significant cardiac hypertrophy and skeletal muscle adaptations characteristic of wheel-run female rats in prior studies (22, 61). Despite reports of favorable adaptations in cardiac muscle following voluntary exercise training (35, 59, 61), no differences in absolute parameters of LV function were observed between the SED and VEX hearts in the current study when perfused in the absence of DOX (see Table 2). However, this finding is in agreement with previous reports that exercise training does not alter baseline LV function in the isolated perfused rat heart under standard perfusion conditions despite eliciting significant cardiac hypertrophy and cardioprotection against I/R stress and hydrogen peroxide exposure (3, 45, 55). Similarly, no alteration in myocardial lipid peroxidation was detected in the VEX vs. SED hearts following KH buffer perfusion in the current study. This finding is also in agreement with previous investigations that reported no effect of 8-10 weeks of treadmill training on myocardial lipid peroxidation in unchallenged hearts, despite observing a significant training-induced attenuation of lipid peroxidation following I/R or ethanol treatment (16, 45). Collectively, these data suggest that the physiological benefits of these training-induced adaptations may be evident only in the context of myocardial stresses that exceed those encountered under normal resting conditions . Despite the numerous reports of exercise-induced cardioprotection following treadmill exercise training, few studies have explored the cardioprotective effects of
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Voluntary exercise attenuates DOX cardiotoxicity voluntary exercise training. Woodiwiss et al. reported a preservation of diastolic function after 16 weeks of voluntary wheel running in rats with streptozocin-induced diabetes mellitus (59). Friberg et al. provided evidence that chronic wheel running enhances cardiac contractile function in spontaneously hypertensive rats (11). However, the current study is the first to examine the effects of voluntary running on the deleterious effects of DOX exposure. Our results indicate that hearts isolated from wheel run animals resist the DOX-induced LV dysfunction and lipid peroxidation observed in the hearts of sedentary animals. While this is the first report of an exercise-induced cardioprotection against DOX exposure, several previous investigations have demonstrated similar cardioprotection against other forms of oxidative stress following chronic exercise (2, 45, 55). However, the vast majority of these studies utilized a forced treadmill exercise training model rather than voluntary running. We have employed a voluntary running model in an effort to isolate the effects of physical activity from the additional stress associated with forced exercise protocols (32, 48). Therefore, our results provide evidence of exercise-induced cardioprotection in the absence of the coercive stress that may accompany forced exercise training. Mechanisms of exercise-induced cardioprotection Myocardial antioxidant enzymes defend the heart against the damaging effects of ROS, and have been hypothesized to play an important role in exercise-induced resistance to oxidative stress (46) and in the attenuation of DOX cardiotoxicity (51). In particular, several studies have indicated that the presence of myocardial SOD is critical for the prevention of DOX cardiotoxicity (18, 49, 62). This is reasonable since SOD removes superoxide from cells, thereby providing the first line of defense against DOX-
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Voluntary exercise attenuates DOX cardiotoxicity induced oxidative stress. The activity and protein expression of myocardial SOD isoforms has been reported to be greater in the male and female rat heart following chronic (≥ 8 weeks) forced treadmill running in some (2, 16, 44, 45), but not all studies (8, 24). To our knowledge, the current study is the first to examine myocardial SOD isoforms in the healthy rat heart following chronic voluntary wheel running. We detected no effect of voluntary exercise on the protein expression of MnSOD or CuZnSOD in the LV of rats in the current study, suggesting that other mechanisms must be involved in the observed cardioprotection against DOX exposure. A putative explanation for the lack of SOD increase observed in our study and the equivocal findings of prior studies that used treadmill training may be differences in the duration and/or intensity of the selected exercise training protocols. Moran et al. (31) recently reported that while 24 weeks of treadmill exercise training elicited significant increases in myocardial SOD activity, no changes were detected after 12 weeks. Powers et al. (44, 45) and Brown et al. (2) reported increases in myocardial SOD activity and protein expression following high intensity treadmill running protocols (≥ 30 m⋅min-1, ≥10% grade) lasting 10 to >20 weeks, while other studies using less intense treadmill running protocols have reported no effect (8, 24). Though female rats have been reported to voluntarily achieve brief periods of high intensity running in the wheels (> 45 m⋅min-1), the running is intermittent in nature (38). Therefore, is it plausible that the voluntary wheel running protocol utilized in the current study did not provide the sustained high-intensity running that may be required to elicit increases in myocardial SOD protein. Although no changes in SOD isoforms were observed in this study, prior investigations have indicated that an increase in myocardial SOD is not required for
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Voluntary exercise attenuates DOX cardiotoxicity exercise-induced cardioprotection (8, 14, 25). While it is possible that increases in other antioxidant enyzmes (e.g., CAT and GPx) may have contributed to the cardioprotection observed in wheel run rats, exercise has been reported to attenuate lipid peroxidation and LV dysfunction following I/R in the absence of any increase in CAT or GPx (8, 14, 25). Therefore, it appears that while antioxidant enzymes clearly play a role in defending the heart against ROS, an increase in their expression may not required for the enhanced cardioprotection conferred by exercise training. Increasing evidence suggests that myocardial HSP72 induction plays a pivotal role in the exercise-induced cardioprotection against oxidative stress (14, 46, 47, 56). HSP72 induction has been shown to attenuate apoptosis, necrosis, and oxidative injury in the myocardium following I/R (14, 47) and DOX exposure (17). Interestingly, HSP72 induction by exercise training or heat stress has attenuated myocardial lipid peroxidation and preserved cardiac function following I/R without a concomitant increase in cardiac SOD, CAT, or GPx activity (8, 14, 57). While previous studies have reported 400-500% increases in cardiac HSP72 content following forced treadmill training (36, 47), a similar effect has not been observed following voluntary wheel running. To our knowledge, only two studies have previously examined cardiac HSP72 content following voluntary wheel running, and both reported no effect (4, 36). In contrast, we have observed a 78% higher HSP72 level in the hearts of wheel run rats compared to the hearts of sedentary rats (P < 0.001). These discrepant findings may be explained by the fact that the two aforementioned studies that reported no effect of wheel running on cardiac HSP72 used male rats. The present study is first to examine the effect of voluntary wheel running on myocardial HSP72 protein in female rats, which were selected due to their greater
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Voluntary exercise attenuates DOX cardiotoxicity propensity for voluntary exercise compared to males (60). Indeed, the females in the present study averaged 9- to 15-fold longer running distances per week than the males in the previous studies (4, 36). Therefore, a substantially greater volume of physical activity may account for the observed differences in HSP72 protein expression. However, it has been suggested that intensity, rather than duration of exercise is the critical factor for cardiac HSP72 induction (29, 36). Milne and Noble (29) reported that cardiac HSP72 induction following a 60 min treadmill running bout occurred only when rats ran at speeds of 24 m·min-1 or greater. The intensity (i.e., speed) and duration of wheel running bouts were not measured in our study, however, female rats have been reported to voluntarily attain running speeds of 48-68 m·min-1 for up to 5 min with heart rates exceeding 500 beats⋅min-1 (38). It is not known whether brief episodes of high intensity running occurring intermittently over a period of several weeks provides sufficient stimulus for HSP72 induction in the heart. Further investigation is required to elucidate the mechanisms by which exercise intensity, duration, or their interaction may influence HSP72 induction the rat heart. While our data suggest a potential role of cardiac HSP72 induction in the cardioprotection observed in the wheel run rats of the present study, evidence from prior studies suggest that other mechanisms may be involved. Joyeux et al. (19) examined the effect of heat stress (42 °C for 15 min) on acute DOX cardiotoxicity in the isolated rat heart similar to the model used in the current study. They reported that while prior heat stress protected the myocardium against electrophysiological injury, it did not attenuate DOX-induced decreases in LVDP to the extent observed in our study. Although cardiac HSP72 was not determined in this study, Demirel et al. (9) reported a nearly 7-fold
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Voluntary exercise attenuates DOX cardiotoxicity increase in cardiac HSP72 content following an identical heat stress protocol. Furthermore, recent evidence from Lennon et al. (25) suggests that sustained increases in HSP72 are not essential for exercise-induced protection during myocardial stunning. Therefore, it is plausible that mechanisms other than HSP72 may have contributed to the observed exercise-induced cardioprotection against DOX exposure. Summary and Conclusions In summary, our data provide the first direct evidence of an exercise-induced protection against the cardiac dysfunction induced by acute DOX exposure. This functional cardioprotection was associated with an attenuation of DOX-induced lipid peroxidation and higher levels of HSP72 protein in the hearts of wheel run rats. While these data suggest a possible role of HSP72 induction in the cardioprotection conferred by voluntary exercise, the precise mechanisms of protection remain to be elucidated. In conclusion, our results indicate that chronic physical activity provides resistance against DOX cardiotoxicity, thereby potentially improving the therapeutic value of DOX in individuals who have been previously active. Limitations and Future Study We have employed an established model of acute DCT in the isolated rat heart to examine the direct effects of DOX exposure on intrinsic cardiac function in previously sedentary or physically active rats. While our data clearly indicate that prior physical activity is associated with a reduction in lipid peroxidation and preservation of cardiac function during acute DOX exposure, the protective effect of exercise training against chronic DOX treatment in vivo cannot be determined by our experiments. As previously mentioned, clinical presentation of DOX cardiomyopathy typically occurs following
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Voluntary exercise attenuates DOX cardiotoxicity completion of several i.v. DOX injections over a period of several months. Therefore, further investigation is needed to determine the effect of exercise training before, during or after chronic DOX treatment in vivo to fully explore the potential benefits of physical activity on the acute and chronic cardiotoxicity of DOX treatment.
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Voluntary exercise attenuates DOX cardiotoxicity ACKNOWLEDGEMENTS This study was supported by grants from the American Cancer Society and the University of Northern Colorado Sponsored Programs and Academic Research Center to RH. We wish to acknowledge Russell Moore, David Brown, Korinne Jew, and Micah Johnson at the University of Colorado, Boulder for their technical advice and assistance with western blotting experiments.
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Voluntary exercise attenuates DOX cardiotoxicity 15. Harris RN and Doroshow JH. Effect of doxorubicin-enhanced hydrogen peroxide and hydroxyl radical formation on calcium sequestration by cardiac sarcoplasmic reticulum. Biochem Biophys Res Commun 130: 739-745, 1985. 16. Husain K and Somani SM. Response of cardiac antioxidant system to alcohol and exercise training in the rat. Alcohol 14: 301-307, 1997. 17. Ito H, Shimojo T, Fujisaki H, Tamamori M, Ishiyama S, Adachi S, Abe S, Marumo F, and Hiroe M. Thermal preconditioning protects rat cardiac muscle cells from doxorubicin-induced apoptosis. Life Sci 64: 755-761, 1999. 18. Ito H, Torii M, and Suzuki T. Decreased superoxide dismutase activity and increased superoxide anion production in cardiac hypertrophy of spontaneously hypertensive rats. Clin Exp Hypertens 17: 803-816, 1995. 19. Joyeux M, Godin-Ribuot D, Faure P, Demenge P, and Ribuot C. Heat stress protects against electrophysiological damages induced by acute doxorubicin exposure in isolated rat hearts. Cardiovasc Drugs Ther 15: 219-224, 2001. 20. Kanter MM, Hamlin RL, Unverferth DV, Davis HW, and Merola AJ. Effect of exercise training on antioxidant enzymes and cardiotoxicity of doxorubicin. J Appl Physiol 59: 1298-1303, 1985. 21. Kiang JG and Tsokos GC. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther 80: 183-201, 1998. 22. Kriketos AD, Pan DA, Sutton JR, Hoh JF, Baur LA, Cooney GJ, Jenkins AB, and Storlien LH. Relationships between muscle membrane lipids, fiber type, and enzyme activities in sedentary and exercised rats. Am J Physiol 269: R1154-1162, 1995. 23. Kumar D, Kirshenbaum L, Li T, Danelisen I, and Singal P. Apoptosis in isolated adult cardiomyocytes exposed to adriamycin. Ann N Y Acad Sci 874: 156-168, 1999. 24. Leeuwenburgh C, Hollander J, Leichtweis S, Griffiths M, Gore M, and Ji LL. Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific. Am J Physiol 272: R363-369, 1997. 25. Lennon SL, Quindry J, Hamilton KL, French J, Staib J, Mehta JL, and Powers SK. Loss of exercise-induced cardioprotection after cessation of exercise. J Appl Physiol 96: 1299-1305, 2004. 26. Li T, Danelisen I, and Singal PK. Early changes in myocardial antioxidant enzymes in rats treated with adriamycin. Mol Cell Biochem 232: 19-26, 2002. 27. Li T and Singal PK. Adriamycin-induced early changes in myocardial antioxidant enzymes and their modulation by probucol. Circulation 102: 2105-2110, 2000. 28. Locke M, Tanguay RM, Klabunde RE, and Ianuzzo CD. Enhanced postischemic myocardial recovery following exercise induction of HSP 72. Am J Physiol 269: H320-325, 1995. 29. Milne KJ and Noble EG. Exercise-induced elevation of HSP70 is intensity dependent. J Appl Physiol 93: 561-568, 2002. 30. Monti E, Prosperi E, Supino R, and Bottiroli G. Free radical-dependent DNA lesions are involved in the delayed cardiotoxicity induced by adriamycin in the rat. Anticancer Res 15: 193-197, 1995. 31. Moran M, Delgado J, Gonzalez B, Manso R, and Megias A. Responses of rat myocardial antioxidant defences and heat shock protein HSP72 induced by 12 and 24-week treadmill training. Acta Physiol Scand 180: 157-166, 2004.
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Voluntary exercise attenuates DOX cardiotoxicity 32. Moraska A, Deak T, Spencer RL, Roth D, and Fleshner M. Treadmill running produces both positive and negative physiological adaptations in Sprague-Dawley rats. Am J Physiol Regul Integr Comp Physiol 279: R1321-1329, 2000. 33. Moraska A and Fleshner M. Voluntary physical activity prevents stress-induced behavioral depression and anti-KLH antibody suppression. Am J Physiol Regul Integr Comp Physiol 281: R484-489, 2001. 34. Myers CE, McGuire WP, Liss RH, Ifrim I, Grotzinger K, and Young RC. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science 197: 165-167, 1977. 35. Natali AJ, Turner DL, Harrison SM, and White E. Regional effects of voluntary exercise on cell size and contraction-frequency responses in rat cardiac myocytes. J Exp Biol 204: 1191-1199, 2001. 36. Noble EG, Moraska A, Mazzeo RS, Roth DA, Olsson MC, Moore RL, and Fleshner M. Differential expression of stress proteins in rat myocardium after free wheel or treadmill run training. J Appl Physiol 86: 1696-1701, 1999. 37. Nousiainen T, Jantunen E, Vanninen E, and Hartikainen J. Early decline in left ventricular ejection fraction predicts doxorubicin cardiotoxicity in lymphoma patients. Br J Cancer 86: 1697-1700, 2002. 38. Overton JM, Tipton CM, Matthes RD, and Leininger JR. Voluntary exercise and its effects on young SHR and stroke-prone hypertensive rats. J Appl Physiol 61: 318324, 1986. 39. Pacher P, Liaudet L, Bai P, Mabley JG, Kaminski PM, Virag L, Deb A, Szabo E, Ungvari Z, Wolin MS, Groves JT, and Szabo C. Potent metalloporphyrin peroxynitrite decomposition catalyst protects against the development of doxorubicin-induced cardiac dysfunction. Circulation 107: 896-904, 2003. 40. Pelikan PC, Weisfeldt ML, Jacobus WE, Miceli MV, Bulkley BH, and Gerstenblith G. Acute doxorubicin cardiotoxicity: functional, metabolic, and morphologic alterations in the isolated, perfused rat heart. J Cardiovasc Pharmacol 8: 1058-1066, 1986. 41. Polla BS, Kantengwa S, Francois D, Salvioli S, Franceschi C, Marsac C, and Cossarizza A. Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc Natl Acad Sci U S A 93: 6458-6463, 1996. 42. Pouna P, Bonoron-Adele S, Gouverneur G, Tariosse L, Besse P, and Robert J. Development of the model of rat isolated perfused heart for the evaluation of anthracycline cardiotoxicity and its circumvention. Br J Pharmacol 117: 1593-1599, 1996. 43. Pouna P, Bonoron-Adele S, Gouverneur G, Tariosse L, Besse P, and Robert J. Evaluation of anthracycline cardiotoxicity with the model of isolated, perfused rat heart: comparison of new analogues versus doxorubicin. Cancer Chemother Pharmacol 35: 257-261, 1995. 44. Powers SK, Criswell D, Lawler J, Martin D, Lieu FK, Ji LL, and Herb RA. Rigorous exercise training increases superoxide dismutase activity in ventricular myocardium. Am J Physiol 265: H2094-2098, 1993. 45. Powers SK, Demirel HA, Vincent HK, Coombes JS, Naito H, Hamilton KL, Shanely RA, and Jessup J. Exercise training improves myocardial tolerance to in vivo ischemia-reperfusion in the rat. Am J Physiol 275: R1468-1477, 1998.
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Voluntary exercise attenuates DOX cardiotoxicity 46. Powers SK, Lennon SL, Quindry J, and Mehta JL. Exercise and cardioprotection. Curr Opin Cardiol 17: 495-502, 2002. 47. Powers SK, Locke, and Demirel HA. Exercise, heat shock proteins, and myocardial protection from I-R injury. Med Sci Sports Exerc 33: 386-392, 2001. 48. Rupp H. Differential effect of physical exercise routines on ventricular myosin and peripheral catecholamine stores in normotensive and spontaneously hypertensive rats. Circ Res 65: 370-377, 1989. 49. Sarvazyan NA, Askari A, and Huang WH. Effects of doxorubicin on cardiomyocytes with reduced level of superoxide dismutase. Life Sci 57: 1003-1010, 1995. 50. Singal PK and Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med 339: 900-905, 1998. 51. Singal PK, Iliskovic N, Li T, and Kumar D. Adriamycin cardiomyopathy: pathophysiology and prevention. Faseb J 11: 931-936, 1997. 52. Srere P. Citrate synthase. Methods Enzymol 13: 3-5, 1969. 53. Sun X and Kang YJ. Prior increase in metallothionein levels is required to prevent doxorubicin cardiotoxicity. Exp Biol Med (Maywood) 227: 652-657, 2002. 54. Swain SM, Whaley FS, and Ewer MS. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer 97: 2869-2879, 2003. 55. Taylor RP, Ciccolo JT, and Starnes JW. Effect of exercise training on the ability of the rat heart to tolerate hydrogen peroxide. Cardiovasc Res 58: 575-581, 2003. 56. Taylor RP and Starnes JW. Age, cell signalling and cardioprotection. Acta Physiol Scand 178: 107-116, 2003. 57. Tekin D, Xi L, Zhao T, Tejero-Taldo MI, Atluri S, and Kukreja RC. Mitogenactivated protein kinases mediate heat shock-induced delayed protection in mouse heart. Am J Physiol Heart Circ Physiol 281: H523-532, 2001. 58. Torti FM, Bristow MR, Howes AE, Aston D, Stockdale FE, Carter SK, Kohler M, Brown BW, Jr., and Billingham ME. Reduced cardiotoxicity of doxorubicin delivered on a weekly schedule. Assessment by endomyocardial biopsy. Ann Intern Med 99: 745-749, 1983. 59. Woodiwiss AJ, Kalk WJ, and Norton GR. Habitual exercise attenuates myocardial stiffness in diabetes mellitus in rats. Am J Physiol 271: H2126-2133, 1996. 60. Yamamoto T, Ohkuwa T, Itoh H, Sato Y, and Naoi M. Effect of gender differences and voluntary exercise on antioxidant capacity in rats. Comp Biochem Physiol C Toxicol Pharmacol 132: 437-444, 2002. 61. Yano H, Yano L, Kinoshita S, and Tsuji E. Effect of voluntary exercise on maximal oxygen uptake in young female Fischer 344 rats. Jpn J Physiol 47: 139141, 1997. 62. Yen HC, Oberley TD, Vichitbandha S, Ho YS, and St Clair DK. The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest 98: 1253-1260, 1996. 63. Zucchi R and Danesi R. Cardiac toxicity of antineoplastic anthracyclines. Curr Med Chem Anti-Canc Agents 3: 151-171, 2003.
26
Voluntary exercise attenuates DOX cardiotoxicity
Running Distance (km)
70 60 50 40 30 20 10 0 1
2
3
4 5 6 Time (Weeks)
7
8
Figure 1
27
Voluntary exercise attenuates DOX cardiotoxicity
% of Baseline
120
A
100 80
*†
60
*†
40 20
*
*
0 0
% of Baseline
140
10
20
30
40
50
60
B
120 100 80
*
60
*
40
*
†
*
40
50
20
*†
0 0
% of Baseline
120
10
20
30
60
C
100 80
CON
60
VEX
40
*†
CON+DOX
20
*
†
*†
VEX+DOX
*†
0 0
10
20 30 40 50 Perfusion Time (min)
60
Figure 2
28
Voluntary exercise attenuates DOX cardiotoxicity
MDA + 4-HNE (pmol/mg protein)
600
*
500 400 300 200 100 0 SED
VEX
SED + DOX
VEX + DOX
Figure 3
29
Voluntary exercise attenuates DOX cardiotoxicity
A
VEX
SED
VEX
MnSOD Protein (arbitrary units)
25 kDa →
1.5
1
0.5
0
SED
B
VEX
VEX SED
VEX
CuZnSOD Protein (arbitrary units)
25 kDa →
1.5
1
0.5
0
SED
VEX
Figure 4 30
Voluntary exercise attenuates DOX cardiotoxicity
SED
VEX
72 kDa →
HSP72 Protein (arbitrary units)
2.5
*
2 1.5 1 0.5 0
SED
VEX
Figure 5
31
Voluntary exercise attenuates DOX cardiotoxicity Figure Legends Figure 1. Weekly running distances for rats in running wheel cages. Data are means ± SE. Figure 2. Response of left ventricular developed pressure (LVDP) (A), maximum rate of left ventricular pressure development (+dP/dt) (B), and maximum rate of left ventricular pressure decline (-dP/dt) (C) during the 60-min perfusion period. SED, sedentary hearts perfused with buffer only (n = 6); VEX, voluntary exercise hearts perfused with buffer only (n = 7); SED + DOX, sedentary hearts perfused with buffer + 10 µM DOX (n = 8); VEX, voluntary exercise hearts perfused with buffer + 10 µM DOX (n = 7). All data are expressed as mean % of baseline (0 min) values ± SE. * Significantly lower than baseline in the SED+DOX group (P < 0.05). † Significant difference between VEX+DOX and SED+DOX (P < 0.05). Figure 3. Left ventricular MDA + 4-HAE content following the 60 min perfusion period. Data are means ± SE. Two-way ANOVA revealed a significant VEX×DOX interaction. * Significantly greater than SED and VEX (P < 0.05). Figure 4. Left ventricular MnSOD (A) and CuZnSOD (B) content with representative blots from sedentary (SED; n = 6) and wheel run (VEX; n = 8) rats. No significant differences were detected between SED and VEX. Figure 5. Left ventricular HSP72 content with representative blots (A) and SOD activity (B) from sedentary (SED; n = 6) and wheel run (VEX; n = 8) rats. * Significantly greater than SED (P < 0.001).
32
Voluntary exercise attenuates DOX cardiotoxicity
Table 1. Effects of voluntary wheel running on animal characteristics. SED
VEX
P Value
266 ± 12
287 ± 4
0.12
1.09 ± 0.04
1.36 ± 0.04
< 0.001
4.12 ± 0.10
4.72 ± 0.11
< 0.001
Soleus wt, mg ⋅ g body wt
0.39 ± 0.01
0.44 ± 0.02
0.03
Soleus citrate synthase activity (µmol ⋅g wet wt-1 ⋅ min-1)
25.2 ± 2.3
30.2 ± 1.6
0.04
Final body wt, g Heart wt, g Heart/body wt ratio, mg ⋅ g
-1
-1
Values are means ± SE. SED, sedentary (n = 15); VEX, voluntary exercise (n = 13).
33
Voluntary exercise attenuates DOX cardiotoxicity Table 2. Absolute values of left ventricular function in isolated hearts. EDP (mmHg)
ESP (mmHg)
LVDP (mmHg)
+dP/dt (mmHg ⋅ sec-1)
–dP/dt (mmHg ⋅ sec-1)
SED
2.1 ± 0.1
86.6 ± 2.8
84.5 ± 3.2
2507 ± 96
-1610 ± 60
VEX
1.5 ± 0.1
88.4 ± 3.7
86.9 ± 6.7
2497 ± 73
-1698 ± 42
SED+DOX
2.3 ± 0.2
85.4 ± 2.9
83.1 ± 6.4
2332 ± 58
-1573 ± 40
VEX+DOX
2.0 ± 0.1
86.8 ± 3.1
84.8 ± 4.7
2445 ± 124
-1627 ± 61
SED
2.2 ± 0.1
91.8 ± 2.7
89.6 ± 3.1
2566 ± 100
-1826 ± 169
VEX
1.5 ± 0.1
89.8 ± 5.0
88.3 ± 5.2
2822 ± 351
-1817 ± 48
SED+DOX
14.6 ± 4.1*
84.4 ± 4.3
69.8 ± 2.9*
1909 ± 82*
-1272 ± 75*
VEX+DOX
7.9 ± 1.8
93.0 ± 5.0
85.1 ± 4.7
2288 ± 121
-1665 ± 93
SED
2.3 ± 0.1
95.3 ± 3.0
93.0 ± 3.6
2563 ± 109
-1753 ± 111
VEX
1.4 ± 0.1
95.3 ± 4.5
93.9 ± 2.0
3080 ± 518
-1954 ± 62
SED+DOX
59.6 ± 49.1*
90.1 ± 7.5
30.5 ± 1.4*
1168 ± 52*
-720 ± 44*
VEX+DOX
51.8 ± 70.2
102.6 ± 17.2
50.1 ± 7.7
1896 ± 179
-1481 ± 196
Baseline (0 min)
30 min perfusion
60 min perfusion
Values are means ± SE. EDP, end-diastolic pressure; ESP, end-systolic pressure; LVDP, left ventricular developed pressure (ESP-EDP); +dP/dt, maximum rate of pressure development; -dP/dt, maximum rate of pressure decline; SED, sedentary (n = 6); VEX, voluntary exercise (n = 8); SED+DOX, sedentary perfused with DOX (n = 7), VEX+DOX; VEX perfused with DOX (n = 7). * Significantly different than baseline level in the same group (P < 0.05).
34
