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Departments of Exercise and Sport Sciences, Pharmaceutics, and Physiology, University of Florida, Gainesville, Florida 32611; Boston University Medical Center, Myocardial Biology Unit, Boston, Massachusetts 02118; and Department of Medicine, Division of Cardiology, University of Arkansas Health Science Center, Little Rock, Arkansas 72205
Submitted 18 December 2003 ; accepted in final form 2 March 2004
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ABSTRACT |
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antioxidants; heart; ischemia; oxidative stress; reperfusion
Compelling evidence indicates that endurance exercise is protective against I/R-induced myocardial stunning and infarction (4, 12, 13, 18, 25, 27, 31, 36, 40, 41). Interestingly, both short-term (1–5 days) and long-term (8–12 wk) exercise training have been shown to improve myocardial I/R tolerance equally (19, 36). Exercise-induced cardioprotection is evidenced by a decrease in cardiac arrhythmias, myocardial oxidative injury, myocyte death, and cardiac contractile dysfunction during reperfusion following ischemia (4, 12, 13, 18, 25, 27, 31, 36, 40, 41).
Although it is clear that exercise improves myocardial tolerance against an I/R injury, the biochemical mechanism responsible for this exercise-induced cardioprotection has remained elusive. Growing evidence suggests that exercise-related increases in myocardial antioxidant capacity could play a key role (23–25, 27, 36, 40, 41). In particular, exercise has been shown to increase manganese superoxide dismutase (MnSOD) activity in the myocardium (19, 23, 35, 36, 41). This is significant because MnSOD is a primary antioxidant enzyme located in the mitochondria that dismutates superoxide radicals.
Recent experiments using transgenic animals suggest that overexpression of myocardial MnSOD attenuates I/R-induced infarction (15, 28). Furthermore, evidence has also linked exercise-induced increases in ventricular MnSOD activity with protection against myocardial infarction (41). Nonetheless, because the relative contribution of pathways responsible for cardiac injury in myocardial stunning and infarction may differ, it is unclear if an exercise-induced increase in MnSOD activity is essential to provide cardioprotection during an I/R insult resulting in myocardial stunning. Therefore, we determined if an exercise-induced increase in myocardial MnSOD activity is required to achieve protection against myocardial stunning. Based on theory and published work, we hypothesized that preventing the exercise-induced increase in MnSOD activity would significantly decrease the exercise-induced protection against I/R-induced myocardial stunning.
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METHODS |
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We selected the male Sprague-Dawley rat for these I/R experiments because it is a well-accepted model for studying exercise-induced myocardial adaptations and myocardial responses to I/R and it is the best-characterized model for isolated, perfused hearts in small mammals (12, 13, 30, 34, 35). Rats (4 mo old) were randomly assigned to one of four experimental groups (n = 20/group): 1) sedentary control (S-C); 2) exercise trained, no treatment (E-C); 3) exercise trained, antisense oligonucleotide (E-AS); and 4) exercised trained, mismatch oligonucleotide (E-MM). Each of these groups was further divided into two experimental groups: sham or in vitro myocardial I/R. During the experimental period, all groups were maintained on a 12:12-h light-dark cycle and provided food (AIN93 diet) and water ad libitum. All experimental procedures performed were in accordance with the rules and regulations of the University of Florida Animal Care and Use Committee.
Exercise Training Protocol
The animals that participated in the exercise training protocol were habituated to treadmill exercise for 5 days (5–45 min/day). After 2 days of rest, animals then performed 3 consecutive days of treadmill exercise for 60 min/day, at 30 m/min, 0% grade [estimated work rate 
70% maximum oxygen consumption (
O2 max)] (29). All hearts were studied 24 h following the final exercise bout.
In Vitro Working Heart Protocol
To investigate myocardial function before and after ischemia, we selected the in vitro working heart model. This model is a highly reproducible preparation and can investigate cardiac performance without the confounding effects of other organ systems, the systemic circulation, and a host of peripheral complications (38). Importantly, the working heart model permits cardiac pump function to be measured while controlling cardiac filling pressures and afterload.
Our working heart preparation followed methods described by Bowles et al. (12, 13). Briefly, hearts were perfused at 37°C with a modified Krebs-Henseleit buffer containing (in mM) 1.25 CaCl2, 130 NaCl, 5.4 KCl, 11 glucose, 0.5 MgCl2, 0.5 NaH2PO4, and 25 NaHCO3 and aerated with 95% O2-5% CO2. Buffer pH was 7.4–7.45. Animals were anesthetized with pentobarbital sodium at a dose of 100 mg/kg, and a 100-IU injection of heparin was made into the hepatic vein. After a surgical plane of anesthesia was reached, the heart was rapidly excised, placed in cold saline (4°C), and weighed. The heart was then placed in a temperature-controlled chamber and the aorta was secured on a stainless steel catheter and perfused in a retrograde or Langendorff mode at 80 cmH2O. Excess tissue was trimmed, weighed, and subtracted from the gross weight to obtain a final heart weight. All subsequent cardiac performance values were normalized for heart weight. The left atrium was cannulated through the pulmonary veins. After 15 min of retrograde reperfusion, the heart was switched to the working heart mode and preischemic function was evaluated at 14 cmH2O (atrial filling pressure) with an 80-cm aortic column.
Global, normothermic ischemia was induced by simultaneously clamping both the aortic and atrial lines for 25 min. During ischemia, the heart was enclosed in a sealed, water-jacketed chamber maintained at 37°C. After the ischemic period, hearts were initially retrograde perfused for 15 min and then switched to working heart mode for an additional 15 min at 80 cmH2O.
Our selection of 25-min global ischemia was designed to produce myocardial stunning with little or no nonreversible injury in the heart. Nonetheless, the working heart model of I/R injury is incapable of differentiating reversible from nonreversible injury produced by the ischemia event (1). Therefore, while it seems likely that our ischemia protocol is primarily a "stunning event," it is possible that our I/R insult resulted in a small level of nonreversible injury (i.e., infarction) in our experimental hearts.
Cardiac Contractile Measurements
Cardiac contractile measurements were made every 5 min during baseline (preischemia) and reperfusion. Measurements of coronary flow (CF) and aortic overflow (AF) were performed at 10-min intervals throughout the experiment. Cardiac output (CO) was defined as the sum of these two flows (CO = CF + AF). Peak systolic pressure, diastolic pressure, the rate of pressure development and decline (±dP/dt), and heart rate were measured via a pressure transducer (Harvard Instruments) connected to the aortic cannula. Data were recorded using a customized data-acquisition system.
Tissue Preparation
At the completion of either the sham or I/R protocol, the left ventricular free wall was immediately sectioned into five vertical strips cut from base to apex, frozen in liquid nitrogen, and stored at –80°C for subsequent biochemical analysis. Before freezing, the heart was rinsed with a cold antioxidant buffer (50 mM NaHPO4, 0.1 mM butylated hydroxytoluene, and 0.1 mM EDTA).
Inhibition of Myocardial MnSOD Protein Translation
Inhibition of MnSOD translation was achieved via a 22-mer phosphorothioate (5'-CACGCCGCCCGACACAACATTG-3') antisense oligodeoxynucleotide (AS-ODN). The AS-ODN was delivered via an intraperitoneal injection (10 mg/kg) immediately postexercise. Experiments in our laboratory (unpublished data) and the work of others (41) indicate that the timing of administration and the daily dose of this AS-ODN provide inhibition of the exercise-induced increase of MnSOD activity in myocardial tissue. A mismatch control oligonucleotide (5'-CACTCCTCCCAGCACAACAGTC-3') was used in our experiment to verify the specificity of the antisense effect. This control was administered in the same fashion and dose as the AS-ODN. All oligonucleotides were prepared by GenoMechanix (Gainesville, FL).
Biochemical Analysis of Antioxidant Enzyme Activity
To assess the effect of exercise training on myocardial antioxidant capacity, a section of the left ventricular free wall from the sham surgery groups was homogenized in 100 mM cold phosphate buffer with 0.05% bovine serum albumin (1:20 wt/vol; pH 7.4). Homogenates were centrifuged at 400 g for 10 min at 4°C. The supernatant was decanted and assayed to determine total protein content along with the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities. Protein content was determined using methods described by Bradford (14). Total SOD and MnSOD activity were measured by the method of McCord and Fridovich (32). Copper zinc SOD was determined by the subtraction of MnSOD from total SOD. GPx activity was measured by the method of Flohe and Gunzler (21) and CAT by the method of Aebi (1). All biochemical analyses were performed in quadruplicate at 25°C and samples from all experimental groups were assayed on the same day to avoid interassay variation. The coefficients of variation for SOD, GPx, and CAT assays ranged between 2 and 5%. Left ventricular reduced and oxidized glutathione concentration was assayed using a kit (Cayman Chemical, Ann Arbor, MI).
Measurement of Lactate Dehydrogenase Activity
To determine the amount of lactate dehydrogenase (LDH) released from the hearts exposed to I/R, coronary effluent was collected immediately before ischemia and during minutes 5–7 of reperfusion. LDH activity in the coronary effluent was determined by the method of Bergmeyer et al. (3). LDH activity in the coronary effluent was normalized to heart weight, and LDH release was expressed as the percent increase in LDH release from heart (i.e., preischemia to reperfusion).
Data Analysis
To test our hypotheses, we performed a one-way ANOVA to determine if group differences existed. Significant group differences were evaluated via a Tukey post hoc analysis (Tukey test). Significance was established at P < 0.05.
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RESULTS |
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The physical characteristics of the animals in both the sham and I/R groups are contained in Table 1. Notice that heart weights, used to normalize CO, are provided for the I/R animals within Table 1. Although animals were randomly assigned to experimental groups, the S-C sham group contained the heaviest animals (358 ± 4.7 g) and their weight was significantly greater than other experimental groups (E-C 338 ± 2.9 g; E-MM 341 ± 5.5 g; E-AS 341 ± 4.7 g). Nonetheless, the body weight-to-heart weight ratio in the S-C animals did not differ from other experimental groups. Note that young adult male Sprague-Dawley rats gain weight rapidly at this age and, therefore, these group variances in weight do not reflect differences in age between the animals.
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Myocardial antioxidant capacity. To determine the effectiveness of the antisense oligonucleotide, left ventricular MnSOD activity was measured in S-C and exercise-trained groups. Compared with S-C, MnSOD activity was elevated in the hearts of the E-C and E-MM groups, whereas MnSOD activity did not differ between E-AS and S-C animals (Fig. 1). These results indicate that the antisense oligonucleotide effectively prevented the exercise-induced increase in MnSOD activity, whereas the mismatch oligonucleotide (control) had no effect on myocardial MnSOD activity. Furthermore, CAT activity was significantly increased in all trained groups regardless of treatment compared with S-C. In contrast, acute exercise training did not alter cardiac GSH content in any experimental group (Table 2).
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140–490% across the experimental groups. Group comparisons revealed that LDH release was significantly lower (P < 0.05) in all exercise-trained groups compared with the S-C group (Fig. 2).
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The functional characteristics of the isolated, perfused hearts can be found in Table 3. Cardiac performance parameters measured during preischemia (baseline) showed no significant differences between groups. In postischemic hearts, I/R injury decreased the amount of heart work performed in each experimental group. When comparing absolute cardiac work across groups, hearts from E-C animals performed more work and had a greater systolic pressure and rate-pressure product than S-C animals following the ischemic challenge. Furthermore, although cardiac performance parameters in both the E-MM and E-AS groups tended to be greater than the S-C group, these differences did not reach significance. However, when external cardiac work [systolic pressure (SP) x CO] was expressed as percent recovery of baseline, all exercise-trained groups produced significantly more cardiac work at 30 min of reperfusion compared with S-C (Fig. 3). This marker of contractile performance is important, as it normalizes each group to its baseline (preischemia) values (Fig. 3). In addition, all exercise-trained groups performed more cardiac work than S-C at all time points measured throughout reperfusion (data not shown). Finally, percent recovery of CO was only significant (P < 0.05) for E-AS (93%) compared with S-C (78%), whereas strong trends (P = 0.07) existed for both E-C (87%) and E-MM (87%).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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This is the first study to determine if an increase in myocardial MnSOD activity is essential to achieve exercise-induced protection against myocardial stunning. Based on theory and published work, we hypothesized that an exercise-induced increase in heart MnSOD activity is a requirement for exercise-mediated cardioprotection against stunning. Our results do not support this hypothesis and reveal that an exercise-induced increase in cardiac MnSOD activity is not a prerequisite to attain exercise-mediated protection against I/R-induced myocardial stunning. This novel finding provides new insight into the mechanisms responsible for exercise-induced cardioprotection. Indeed, while the work of Yamashita et al. (41) demonstrates that elevated MnSOD activity in the heart is mandatory to attain exercise-induced protection against myocardial infarction, the current data indicate that other mechanisms of cardioprotection are responsible for the exercise-mediated protection against myocardial stunning.
Exercise-Induced Cardioprotection
This study reinforces the notion that short-term (i.e., days) exercise training provides cardioprotection against I/R-induced myocardial stunning. Several mechanisms have been proposed for this form of exercise-induced cardioprotection. Of the proposed mechanisms, an increase in myocardial antioxidant capacity has been argued to be an important protective mediator as short-term training does not anatomically alter coronary collateral circulation (41). Furthermore, exercise-mediated cardioprotection against myocardial stunning can occur in the absence of an increase in heat shock proteins (23, 40). Therefore, we hypothesized that an elevation in antioxidant capacity is required to provide protection against an I/R-induced myocardial stunning and in particular, that increased levels of MnSOD would be essential to this cardioprotection.
Several key observations led to this hypothesis. First, radicals and other reactive oxygen species are important contributors to I/R-induced injury in the heart (2, 5–11, 22). Second, the cardioprotection associated with both short- and long-term (weeks) exercise training is accompanied by an increase in myocardial antioxidant capacity (i.e., MnSOD activity) (19, 23, 24, 26, 27, 35–37). Furthermore, increasing antioxidant capacity (e.g., gene transfer or exogenous antioxidants) can decrease I/R-induced cardiac damage (15–17, 20, 24, 28, 37). Finally, prevention of the exercise-induced increase in cardiac MnSOD activity eliminates the exercise-induced protection against I/R-induced myocardial infarction (41). Hence, based on these findings, the hypothesis that an increase in myocardial MnSOD activity is essential for exercise-mediated protection against myocardial stunning appears logical.
Nonetheless, our data do not support this postulate. Indeed, in our experiments, treatment with antisense oligonucleotides against MnSOD mRNA prevented an exercise-induced increase in cardiac MnSOD activity but did not abate exercise-induced cardioprotection against myocardial stunning. In contrast to these findings, Yamashita et al. (41) demonstrated that antisense inhibition of exercise-induced MnSOD activity removed the normally observed exercise-induced cardioprotection against infarction. The explanation for this discrepancy is likely due to differences in the experimental model of I/R injury. For example, the current study investigated myocardial stunning using an in vitro model, whereas Yamashita et al. (41) studied infarction via an in vivo model. It is clear that myocardial stunning and infarction are very different cardiac insults and the mechanisms responsible for cellular injury from these insults may also differ (9). Infarction results in both necrotic and apoptotic cell death, whereas stunning causes temporary contractile dysfunction with little or no cell death (9). Therefore, it seems probable that MnSOD could play a more important protective role during long-duration ischemia and reperfusion resulting in infarction due to greater oxidative damage inflicted on the myocardium. Regardless of the explanation for this divergent finding between the current study and the work of Yamashita et al. (41), the present data reveal, for the first time, that an exercise-mediated increase in myocardial MnSOD activity is not essential to produce exercise-induced protection against myocardial stunning in vitro.
Exercise-Induced Protection Against Myocardial Stunning: Potential Mechanisms
If an exercise-induced increase in myocardial MnSOD activity is not required to achieve exercise-mediated protection against myocardial stunning, what is the mechanism(s) responsible for this form of cardioprotection? A logical place to search for an answer may reside within an understanding of those factors that contribute to myocardial stunning. The two primary postulates to explain the pathogenesis of myocardial stunning are the radical hypothesis and the calcium hypothesis (9). The radical hypothesis argues that myocardial stunning results from myocardial oxidative injury due to radicals and other reactive species produced during ischemia and reperfusion. This oxidative injury could impair myocardial contractility due to a reduced responsiveness of contractile filaments to calcium (9). The calcium hypothesis is based on knowledge that I/R results in a transient calcium overload in the myocardium that activates calcium-activated proteases (e.g., calpains) resulting in degradation of key cytoskeletal proteins. Note that the radical and calcium hypotheses are not mutually exclusive because calcium overload in cells can contribute to radical production and oxidation of calcium transport proteins can promote disturbances in calcium homeostasis (9). Therefore, it is feasible that the mechanism responsible for exercise-induced protection against myocardial stunning is linked to exercise-mediated expression of antioxidants and/or calcium-handling proteins.
In the current experiments, exercise significantly increased myocardial CAT activity in all exercise-trained groups. This antioxidant enzyme removes hydrogen peroxide from cells and, therefore, could play a role in the protection of the heart from I/R-mediated oxidative injury. Nonetheless, exercise-induced increases in myocardial CAT activity are not a consistent finding in the literature. For example, work from our group (24) and others (25) confirmed that exercise-induced cardioprotection against I/R injury can occur without an increase in myocardial CAT activity. Therefore, it seems likely that other antioxidants may be involved in cardioprotection. Moreover, in the current experiments, it is possible that prevention of exercise-induced increases in myocardial MnSOD activity may result in an upregulation of other antioxidant pathways that may, or may not, be normally upregulated in the heart in response to endurance exercise. In this regard, preliminary experiments in our laboratory suggest that exercise elevates cardiac mRNA levels of several antioxidant proteins in the heart other than CAT and MnSOD (e.g., thioredoxin reductase). Future experiments are required to determine if one or more of these antioxidants are essential to achieve exercise-induced protection against myocardial stunning.
Several laboratories investigated exercise-induced changes in myocardial calcium handling. For example, although exercise does not alter the number of L-type calcium channels in the heart (33), exercise training does increase the rate of calcium transport by calcium ATPase in the sarcoplasmic reticulum of rat hearts (39). At present, other than sarcoplasmic reticulum calcium ATPases, limited information exists regarding exercise-mediated changes in other calcium-handling proteins in the heart. It seems possible that exercise training may alter calcium-handling proteins in such a way as to protect against I/R-induced calcium overload. This is a testable hypothesis and could provide an exciting area for future research.
Conclusions
This is the first investigation to evaluate the role of cardiac MnSOD activity in exercise-induced cardioprotection against I/R-induced myocardial stunning. Our results indicate that an increase in cardiac MnSOD activity is not essential to achieve exercise-induced cardioprotection against stunning. This new and important finding differs from a previous report indicating that exercise-induced improvements in myocardial MnSOD activity are required to achieve protection against myocardial infarction (41). We interpret these divergent results as an indication that the mechanism(s) responsible for exercise-induced cardioprotection against myocardial stunning and infarction differ. Nonetheless, although an increase in cardiac MnSOD activity is not essential for protection against stunning, it remains possible that exercise-induced cardioprotection against myocardial stunning is due, in part, to enhanced myocardial antioxidant capacity. Indeed, exercise-induced increases in one or more elements of the cardiac antioxidant system could play an important role in protection against myocardial stunning. Furthermore, it is possible that exercise-induced changes in one or more calcium-handling proteins could also contribute to exercise-mediated cardioprotection against myocardial stunning. Therefore, future experiments should explore the hypothesis that exercise-induced protection against myocardial stunning is a polygenic phenomenon that results in cardioprotection against both I/R-induced oxidative injury and calcium overload.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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-lipoic acid on skeletal muscle contractile properties. J Appl Physiol 90: 1424–1430, 2001.This article has been cited by other articles:
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  J. P. French, K. L. Hamilton, J. C. Quindry, Y. Lee, P. A. Upchurch, and S. K. Powers Exercise-induced protection against myocardial apoptosis and necrosis: MnSOD, calcium-handling proteins, and calpain FASEB J, August 1, 2008; 22(8): 2862 - 2871. [Abstract] [Full Text] [PDF]
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 A. N. Kavazis, J. M. McClung, D. A. Hood, and S. K. Powers Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H928 - H935. [Abstract] [Full Text] [PDF]
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 J. C. Quindry, K. L. Hamilton, J. P. French, Y. Lee, Z. Murlasits, N. Tumer, and S. K. Powers Exercise-induced HSP-72 elevation and cardioprotection against infarct and apoptosis J Appl Physiol, September 1, 2007; 103(3): 1056 - 1062. [Abstract] [Full Text] [PDF]
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 D. B. Thorp, J. V. Haist, J. Leppard, K. J. Milne, M. Karmazyn, and E. G. Noble Exercise training improves myocardial tolerance to ischemia in male but not in female rats Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R363 - R371. [Abstract] [Full Text] [PDF]
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J. L. Staib, J. C. Quindry, J. P. French, D. S. Criswell, and S. K. Powers Increased temperature, not cardiac load, activates heat shock transcription factor 1 and heat shock protein 72 expression in the heart Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R432 - R439. [Abstract] [Full Text] [PDF]
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R. P. Taylor, M. E. Olsen, and J. W. Starnes Improved postischemic function following acute exercise is not mediated by nitric oxide synthase in the rat heart Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H601 - H607. [Abstract] [Full Text] [PDF]
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H. Hwang, P. J. Reiser, and G. E. Billman Effects of exercise training on contractile function in myocardial trabeculae after ischemia-reperfusion J Appl Physiol, July 1, 2005; 99(1): 230 - 236. [Abstract] [Full Text] [PDF]
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Z.-Q. Jin, H.-Z. Zhou, G. Cecchini, M. O. Gray, and J. S. Karliner MnSOD in mouse heart: acute responses to ischemic preconditioning and ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2986 - H2994. [Abstract] [Full Text] [PDF]
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