INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GENERATION OF REACTIVE OXYGEN SPECIES (O·, H₂O₂, and ·OH) and nitrogen species (·NO and peroxynitrite) have been established as an important etiological mechanism for myocardial ischemia-reperfusion (I/R) injury (15, 26, 42, 48). Depending on the condition of ischemia and reperfusion, these oxidants can be produced by several cellular sources, including xanthine oxidase (50), the mitochondrial electron transport chain (15), and nitric oxide (NO) synthetase (48), and overwhelm antioxidant defense system. There is strong evidence that oxidative damage to cellular components of the myocardium is the direct cause of impaired functional performance in the postischemic heart (1, 8, 34).

Regular physical exercise has been shown to be an effective way to improve myocardial resistance to both functional and biochemical impairment induced by I/R, although there is still some controversy (25, 44, 45). Training adaptation against I/R injury may be related to a variety of cellular mechanisms, such as a more developed myocardial vasculature (38), a greater high-energy phosphate reserve (30), a reduced coronary resistance (45), improved Ca²⁺ sensitivity and handling (32, 33), and an enhanced antioxidant defense capacity (14, 19, 35). However, most of the data have been derived from studies using an isolated perfused heart model, wherein potential circulatory and neuroendocrine training adaptations are not taken into consideration. Few studies have compared myocardial responses to I/R in vivo between trained and untrained hearts, and data regarding heart performance are especially lacking (12, 17, 36).

Reduced glutathione (GSH) is a major nonenzymatic antioxidant in the heart and has been reported to play a vital role in myocardial protection against I/R (9, 17, 24). Depletion of endogenous GSH by inhibition of -glutamylcysteine synthetase (GCS) with buthionine sulfoximine (BSO) has been shown to intensify the oxidative damage observed in I/R hearts (3, 49). However, supplementation of exogenous GSH by intraperitoneal or intravenous injection has demonstrated a limited and controversial effect on increasing tissue GSH levels and improving heart functional performance under ischemic insult (46). A major obstacle is that a high plasma GSH concentration resulting from an exogenous source can pose a strong feedback inhibition on GCS and impair operation of the -glutamyl cycle (13). As an alternative, oral GSH ingestion has been advocated as a more effective method to increase tissue GSH concentration and redox status (7, 16). Previous studies (23, 31) have shown that I/R and endurance training could independently increase -glutamyl transpeptidase activity in rat hearts. This important adaptation may facilitate GSH breakdown and transmembrane transport, resulting in an increased GSH level in cardiomyocytes. According to our knowledge, the efficacy of GSH supplementation in conjunction with training in myocardial resistance to I/R has never been examined in the postischemic heart.

In the current study, we tested the hypothesis that dietary GSH supplementation, endurance training, and the combination of the two treatments would provide a greater protection against myocardial I/R injury using an open-chest intact rat heart model. Multifaceted measurements were used to evaluate heart performance, myocardial GSH status, antioxidant and -glutamyl cycle enzyme activities, and cellular oxidative damage. The results indicate that GSH supplementation in conjunction with training greatly improved myocardial resistance to I/R due to an improved antioxidant reserve and whole body GSH homeostasis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Female Sprague-Dawley rats (n = 72, age 6 wk, final age 4 mo) were purchased from Harlan Sprague Dawley (Indianapolis, IN). After arrival, the rats were individually caged at the Department of Animal Sciences, University of Wisconsin at Madison, in a temperature-controlled room (22°C) with a reverse 12:12-h light-dark cycle and maintained on a Purina chow diet and tap water before the experiment began. The animal treatment protocols were approved by the University of Wisconsin Research Animal Resource Center.

Exercise training. At the end of the 2-wk acclimation, the rats were randomly divided into an exercise training group (T; n = 36) and an untrained control group (U; n = 36). Rats began exercising 4 h after the beginning of the dark cycle. During week 1, the animals were acclimated to treadmill running on a Quinton rodent treadmill at 15 m/min, 0% grade, for 10 min/day, 5 days/wk. At the end of week 1, the rats were able to run at 16.5 m/min, 0% grade, for 30 min. Running speed, grade, and duration were progressively increased to 25 m/min, 15% grade, for 75 min/day by the end of week 4. This intensity was maintained during the rest of the 10-wk training period. Untrained rats were exposed to treadmill walking three times a week for <10 min to alleviate handling effect.

Dietary GSH supplementation. All rats were fed a Purina chow diet during the initial 6.5 wk. Thereafter, rats were switched to a purified control diet (AIN-93G, Teklad; Madison, WI) until week 8.5. Food consumption and body weight were monitored throughout this time period. At this point, one-half of the T and U groups of rats (n = 18) received a GSH-supplemented diet (AIN-93G with 5 g GSH/kg) for 17 days before heart surgery (GSH-S group). The other one-half group (n = 18) was pair fed a control AIN-93G diet until heart surgery (C group). Food intake and GSH consumption showed no difference between the two groups (see RESULTS).

Heart ischemia and reperfusion. Forty-eight hours after the last bout of training, each group of rats were randomly divided into two surgical categories: I/R or sham operation. Aseptic surgical procedures set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals were followed throughout all surgeries. Rat myocardial I/R was produced by the occlusion and release of the main descending branch of the left coronary artery (LCA) as previously described (17, 18, 22). Rats were anesthetized by an intraperitoneal injection of Nembutal (45 mg/kg body wt) and intubated with a 14-gauge polystyrene tube by tracheotomy. Rats were ventilated using a Harvard respirator (Harvard Apparatus) at a tidal volume of ~1.1 ml/100 g body wt and at a frequency of 75-85 strokes/min. The effectiveness of ventilation was confirmed with an ABL500 blood gas analyzer (Radiometer; Copenhagen, Denmark). The right jugular vein was cannulated with an intravenous catheter [polyethylene (PE)-50, 0.6-mm inner diameter (ID), Intramedic] for removal of blood samples. Rectal temperature was monitored throughout the experiment, and normothermia (37°C) was maintained by a heating pad controlled by a thermostatic water circulator (Gaymar). A lateral thoracotomy was performed to expose the heart. The main descending vessel of the LCA was looped by a single suture (Ethicon 6-0) ~1 mm from its origin. A 1-cm segment of PE tube (0.9 mm ID) was slid down both ends of the 6-0 suture to compress the LCA, causing a reversible occlusion of the blood flow to the left ventricle (LV). This procedure produces a clearly demarcated (cyanotic and bulged) area of acute ischemia corresponding to the distribution of the LCA distal to the occlusion. Ischemia was maintained for 45 min. Reperfusion was allowed by withdrawing the compression of the tube for 30 min. In the sham group, the LCA was looped with a suture but was not occluded.

Cardiovascular responses. LV pressure curves were monitored using a fluid-filled catheter (PE-50, Intramedic) inserted in the right carotid artery and advanced to the LV. The catheter was connected to a P10EZ miniature pressure transducer (Viggo-Spectramed) and a Gould WindoGraf transducer signal conditioner (Gould Instruments). The following cardiovascular parameters were monitored in all animals throughout the entire surgery: LV peak systolic (LVSP) and end-diastolic pressure (LVDP), heart rate (HR), HR-pressure double product (RPDP), LV contractility (±dP/dt), and electrocardiograph (ECG). ECG signals were recorded with three subcutaneously inserted brass electrodes connected to a Gould Windograf ECG signal transducer.

Blood and tissue collection. At three time points during the I/R or sham surgery (0, 45, and 75 min), 350 µl of whole blood were removed via the catheter inserted in the right jugular vein. A portion of this blood (<50 µl) was used to determine hematocrit values using a microcapillary centrifuge (International Equipment). The remainder of the blood (300 ml) was placed in an Eppendorf tube containing 150 ml of saline with 0.6% (wt/vol) heparin. Plasma was obtained by centrifugation of the blood sample in a microcentrifuge (Hermle) at 500 g for 2 min.

Biochemical analysis. GSH and oxidized glutathione (GSSG) concentrations were determined using HPLC according to Reed et al. (37) with slight modifications (21). Citrate synthase (EC 4.1.3.7) activity was measured according to Shepherd and Garland (41). Activities of the myocardial antioxidant enzyme superoxide dismutase (SOD; EC 1.15.1.1), glutathione peroxidase (GPX; EC 1.11.1.9), glutathione reductase (EC 1.6.4.2), and catalase (EC 1.11.1.6) were determined spectrophotometrically in the LV as previously described (18). -Glutamyl transpeptidase (GGT; EC 2.3.2.2) activity was measured spectrophotometrically at 37°C according to Meister et al. (29). GCS (E.C. 6.3.2.2 ) activity was measured spectrophotometrically according to Seelig and Meister (39). Plasma lactate dehydrogenase (LDH; EC 1.1.1.27) activity was measured as previously cited (18). Lipid peroxidation was determined in the LV by measuring malondialdehyde (MDA) as previously described (20). Caution was exercised to minimize artificial oxidation during assay. Briefly, 10 mM butylated hydroxytoluene and 200 mM ferrous sulfate were added to the tissue homogenates. Sealed tubes were incubated for 15 min at 99°C. MDA content was subsequently calculated against a standard curve using 1,1,3,3-tetraethoxypropane. Protein concentration in the heart and liver was determined by the Bradford method using bovine serum albumin as the standard (6).

Data analysis. LVSP, HR, HR-pressure double product, ±dP/dt, and plasma LDH data were analyzed using a four-way ANOVA with repeated measures (Systat; Evanston, IL). The four independent variables were surgery, diet, training, and time. Three-way ANOVA was used to examine effects of surgery, diet, and training on myocardial and liver GSH status and antioxidant enzyme activities. When a significant main effect or interaction was found, a Fisher's least-significant difference test was employed to determine the significance of group differences. The significance level was P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

General. The body weights of the rats were not affected by GSH supplementation or training (Table 1). Training increased heart weight by 9.6% (P < 0.05) in C rats and by 7.8% (P < 0.05) in GSH-S rats. The heart-to-body weight ratio was increased with training by 7.7% and 7.6% (P < 0.05) in C and GSH-S rats, respectively. Daily food consumption during the entire experimental period (data not shown) and the last 17 days of training (Table 1) was not different among various groups of rats. GSH consumption, calculated from food intake, also showed no difference between T and U rats.

Cardiovascular response. HR was significantly decreased by ~18% (P < 0.01) with the occlusion of the LCA in all groups of rats and remained suppressed during the entire ischemic period (Fig. 1). No group differences were observed. After reperfusion, HR returned to preischemic levels in all rats.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Heart rate response to ischemia-reperfusion (I/R) and sham surgery (S) in trained (T) and untrained (U) rats fed either a control (C) or reduced glutathione (GSH)-supplemented diet (G). #P < 0.01, I/R vs. sham rats at a given time point.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Response of peak left ventricular systolic pressure to I/R and sham surgery in trained and untrained rats fed either a control or GSH-supplemented diet. #P < 0.01, I/R vs. sham rats at a given time point. *P < 0.05, T/G vs. U/G rats. +P < 0.05, T/G vs. T/C or U/C rats.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Response of left ventricle contractility (+dP/dt) to I/R and sham surgery in trained and untrained rats fed either a control or GSH-supplemented diet. #P < 0.01, I/R vs. sham rats at a given time point. *P < 0.05, T/G vs. U/G or U/C rats. + P < 0.05, T/G vs. T/C rats.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Response of heart rate-pressure double product to I/R and sham surgery in trained and untrained rats fed either a control or GSH-supplemented diet. #P < 0.01, I/R vs. sham rats at a given time point. +P < 0.05, T/G or U/G vs. T/C or U/C rats.

Plasma LDH activity. The sham procedure elicited a similar (~80%, P < 0.05) increase in plasma LDH activity in all groups of rats (Fig. 5). After ischemia, LDH release was ~80% (P < 0.01) higher in I/R rats compared with sham rats. Greater increases in LDH activity were observed after reperfusion in all groups (P < 0.01). A 2.7-fold higher LDH activity was observed in U/C, T/C, and U/GSH-S rats compared with their sham counterparts (P < 0.01). The I/R-induced LDH release was 20% lower (P < 0.05) in T/GSH-S rats than that in the other three groups.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Plasma lactate dehydrogenase activity responses to I/R and sham surgery in trained and untrained rats fed either a control or GSH-supplemented diet. #P < 0.01, I/R vs. sham rats at 45 min and I/R vs. sham rats at 75 min. *P < 0.05, 75 vs. 0 min. +P < 0.05, T/G vs. U/G and T/C or U/C in sham rats.

Myocardial lipid peroxidation. I/R resulted in a 33%, 38%, and 33% (P < 0.05) increase in myocardial MDA content in U/C, U/GSH-S, and T/C hearts, respectively (Fig. 6). In contrast, GSH-S/T hearts demonstrated no significant increase in the MDA level after I/R.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Myocardial malondialdehyde content in response to I/R and sham surgery in trained and untrained rats fed either a control or GSH-supplemented diet. #P < 0.05, I/R vs. sham rats.

LV, liver, and plasma GSH status. The GSH content and GSH-to-GSSG ratio in LV were greater in GSH-S versus C hearts regardless of training or surgical treatment (P < 0.05; Table 2). Training increased GSH content as well as the GSH-to-GSSG ratio in GSH-S hearts subjected to either sham or I/R (P < 0.05). I/R decreased GSH content, increased GSSG content, and decreased the GSH-to-GSSG ratio in all treatment groups (P < 0.05); however, T/GSH-S hearts demonstrated the highest GSH-to-GSSG ratio after I/R compared with other treatment groups (P < 0.05).

Enzyme activity. The activities of total SOD, GPX, and glutathione reductase in the LV were increased in the T versus U rats (P < 0.05; Table 4). The magnitude of induction was not affected by GSH supplementation. These enzyme activities were unchanged with I/R. Catalase activity was not altered by any of the treatments. GGT activity in the LV was elevated with training in the I/R but not sham hearts (P < 0.05, interaction). Hepatic GCS activity was increased in T/GSH-S versus U/GSH-S rats (P < 0.05), whereas GGT activity was lowered by training (P < 0.05, main training effect).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Regular exercise has long been advocated as an effective way to improve myocardial function under physiological stress and in certain pathogenesis (38). Endurance training has been shown to attenuate heart I/R injury in isolated perfused hearts. Several researchers have shown that training enhances the ability of the heart to preserve LV developed pressure (4), high-energy phosphate content (5), and Ca²⁺ homeostasis (32, 33) in response to I/R. These training effects, however, were not demonstrated uniformly, and some studies (25, 45) showed no improvement in contractile function in endurance-trained rat hearts. One of the major limitations of the isolated perfused heart model is the removal of circulatory and neural input, which could also be critically influenced by training (38). The current open-chest anesthetized rat heart model was developed in an attempt to provide insight into cardiovascular responses to in vivo I/R after training using LVSP, dP/dt, and RPDP as parameters. However, these measurements are subjected to certain limitations. For example, LVSP is influenced by the size of the area at risk and by the presence or absence of infarction, which could occur in some hearts during a 45-min ischemic insult. dP/dt, indicative of pressure attained over time, depends largely on peak pressure and is sensitive to changes in preload and afterload, which could be altered by training adaptations of hormonal and vascular systems. With the use of a similar model, Powers et al. (36) showed that peak systolic pressure was better maintained during I/R in rats subjected to 10 wk of endurance training compared with controls. In the present study, training alone did not demonstrate clear-cut improvement of LVSP, dP/dt, or RPDP in I/R hearts. However, the duration of ischemia (45 min) and reperfusion (30 min) in our study were much longer than those reported by Powers et al. (36) (20 and 10 min). Prolonged I/R might have resulted in greater free radical generation and oxidative damage that could overwhelm training adaptations demonstrated under less stressful conditions. Another limitation with the current model is the use of heterogeneous LV tissues. Although measures were taken to harvest tissues from the areas at risk, some nonischemic samples were likely included for biochemical determinations. This might have diluted true I/R effects.

Another effective way to ameliorate myocardial postischemic injury is to boost endogenous antioxidant defense capacity (11). As a major antioxidant, GSH and its analogs such as N-acetyl cysteine and GSH ethyl ester have been experimentally supplemented in an attempt to enhance intracellular GSH concentration and to protect against myocardial oxidative damage from I/R (10, 40, 43). The rationale for exogenous GSH supplementation lies in the critical role of the -glutamyl cycle and the finding that myocardial GGT activity increases with I/R, which facilities GSH transport from plasma to cardiomyocytes (31). Singh et al. (43) studied the effect of GSH supplementation in an in vivo pig model and found that intravenous infusion of GSH 2 h before I/R significantly increased myocardial GSH content and improved resistance to I/R injury as assessed by functional recovery, infarct size, and tissue morphology. In isolated perfused rat hearts, GSH supplementation was found to significantly enhance postischemic recovery of contractile function without appreciable increase in myocardial thiol content (40). Recently, GSH supplementation was shown to reduce levels of peroxynitrite due to the formation of the NO donor S-nitroso-glutathione, thereby reducing myocardial I/R injury (10). Most of the previous GSH supplementation studies involve acute GSH injection or infusion. Although a rapid increase in plasma GSH levels may facilitate the removal of reactive oxygen species generated at the vascular endothelial cells, this also poses a strong inhibition on GCS, the rate-limiting enzyme of the -glutamyl cycle, and hence the intracellular resynthesis of GSH (27, 28). Thus acute GSH supplement appeared to have only limited success in increasing GSH content and reducing reactive oxygen species generation in the cell. In the present study, dietary GSH supplementation for only 17 days was found to significantly raise GSH content at rest and the GSH-to-GSSG ratio after I/R in the untrained hearts, without affecting other major antioxidant enzyme activities. However, these effects were relatively small and did not influence the extent of I/R-induced impairments of LVSP and dP/dt, myocardial lipid peroxidation, or LDH release. Thus dietary GSH supplementation alone, at least at the current dosage and duration, does not seem to be an effective way to attenuate heart I/R injury in vivo. It is possible, however, that longer period of GSH supplementation could reveal additional benefits, which warrants further study.

The main finding in the present study is that dietary GSH supplementation in conjunction with 10 wk of endurance training can increase myocardial antioxidant defense and attenuate oxidative damage induced by in vivo I/R. This can in turn improve heart functional recovery from the I/R insult in an intact rat heart. Important myocardial adaptations took place at both cellular and organ levels as a result of combined training and GSH supplementation, which according to our knowledge have never been reported before. Support for this conclusion came from several lines of experimental data. First, T/GSH-S hearts demonstrated lesser a degree of oxidative damage after I/R, as indicated by a higher LV GSH-to-GSSG ratio and a smaller increase in lipid peroxidation. T/GSH-S rats increased myocardial GSH content (without altering GSSG) and the GSH-to-GSSG ratio either with or without I/R treatment. The net increase in GSH content was small; however, the better-preserved myocardial redox status could provide subtle protection to organelles and proteins that are sensitive to redox changes and oxidative stress. For example, Bauer et al. (2) reported that the pCa 5.0 for force development was increased in skinned cardiomyocytes exposed to a higher level of GSH, whereas GSSG decreased the force by 54% at pCa 5.6. It appears that the sensitivity of contractile protein to Ca²⁺ was compromised as the intracellular environment becomes more oxidized. Turan et al. (47) showed in rat papillary muscles that a decrease in the thiol redox ratio could reduce Ca²⁺ current magnitude and hence force production. In addition, Ca²⁺ handling by intracellular organelles was hampered, as shown by a rise in the basal Ca²⁺ concentration and a decrease in the Ca²⁺ spike amplitude. The adverse effects were reversed by dithiothereitol treatment.

Second, the elevated myocardial GSH status was accompanied by training adaptations of the antioxidant enzymes related to the cellular GSH/GSSG cycle and the -glutamyl cycle. An increased SOD activity in the trained heart facilitates the dismutation of O·, whereas a higher GPX activity could enhance the ability of the cell to remove hydroperoxides. The combination of the two adaptations ultimately reduce the chances of ·OH generation. Furthermore, higher glutathione reductase activity probably contributed to the greater GSH-to-GSSG ratio observed in the T/GSH-S heart. Of particular interest was the finding that GGT activity was increased in trained hearts subjected to I/R, consistent with previous reports (23, 31). Taken together, our data suggest that the improved myocardial GSH status in T/GSH-G hearts is attributed to increases in enzyme activities in both the GSH/GSSG cycle and the -glutamyl cycle during I/R.

Third, I/R caused a lesser extent of myocardial LDH release in the T/GSH-S hearts compared with those subjected to training or GSH supplementation alone. This observation suggests that fewer of the T/GSH-S hearts had suffered from myocardial infarction during I/R, and if infarction did occur, its size was probably smaller. In the current study, plasma LDH level was used as a marker of myocardial damage. There is a good consistency in the literature that plasma LDH (as well as creatine kinase) levels correlate well with infarction size (15, 42), which is not subjected to influences by peripheral hemodynamic changes. Our time course data showing a greater increase in LDH activity after reperfusion indicate that reactive oxygen species generation not only retard the myocardial ability to generate force but also affected the integrity of the cardiomyocyte membrane.

Finally, T/GSH-S hearts demonstrated a greater ability to recover LVSP, dP/dt, and RPDP during reperfusion after the initial 45-min ischemia compared with any of the other treatment groups. By the end of the 30-min reperfusion period, LVSP and +dP/dt in T/GSH-S hearts approached those of the sham hearts. This increased protection demonstrated by the GSH-S and T hearts may be the result of both myocardial adaptations, such as enhanced high-energy phosphate reserve, coronary collateral blood flow, Ca²⁺ homeostasis, antioxidant defense, and peripheral adaptations, such as vascular proliferation and hormonal changes (30, 38). The later changes can affect the preload and afterload of the myocardial contraction, thus improving LVSP and dP/dt recovery.

In addition to the above-mentioned adaptations, training and GSH supplementation elicited changes in liver and plasma GSH status that may also contribute to a greater myocardial protection against I/R. Hepatic GSH content was increased with training, which likely was caused by the observed increase in GCS activity, the rate-limiting enzyme for GSH synthesis, and to a lesser extent by a decrease of GGT activity (28). I/R resulted in a net efflux of liver GSH and an increase in plasma GSH concentration, presumably under the influence of vasopressin and catecholamine stimulation. It is interesting to note that plasma GSSG levels were dramatically elevated (3-fold) by I/R, a reflection of enhanced GSSG efflux from the myocardium and other organs under oxidative stress, as well as increased reactive oxygen species generated from myocardial endothelial cells (50). GSH-S rats, however, demonstrated a significantly lower plasma GSSG level and higher GSH-to-GSSG ratio. These changes suggest that endothelial reactive oxygen species production might be attenuated with GSH supplementation.

In summary, data from the present study indicate that training in conjunction with a short-term dietary GSH supplementation can enhance myocardial resistance to I/R induced damage in anesthetized rat. This important adaptation may result from a preservation of heart GSH homeostasis and increased antioxidant protection, which improved myocardial functional recovery from initial I/R insult.