Myocardial Glutathione Research Articles

Beneficial effect of captopril against ischaemia-reperfusion injury it isolated guinea pig hearts

The study was designed to clarify whether captopril, an angiotensin-coverting enzyme inhibitor, will reduce the injury of global ischaemia and reperfusion after cardioplegic arrest in isolated guinea pig hearts, in a modified Langendorff model. The hearts were randomly allocated into four groups (n = 10 in each) and subjected to 90 min of normothermic global ischemia, followed by 30 min of reperfusion; in all groups, cardioplegic arrest was achieved by administering St. Thomas's Hospital cardioplegic solution (STHCS). The first group was utilized as the control group. In the second group, captopril (200 mumol/L) was added to STHCS. In the third group, oral pretreatment was carried out (0.3 mg/kg captopril was given twice a day for 10 days). In the fourth group, oral pretreatment was achieved and captopril-enriched solution was applied in the first 5 min of reperfusion. Although the study groups showed better recovery of contractility than the control group, in the fourth group the hearts had the best left ventricular contractile function, where contractile force (g contractility/g heart weight) was 55.4% +/- 3.8% of the preischameic values. Groups I, II, and III achieved 31.0% +/- 3.2%, 41.6% +/- 3.8%, and 48.3% +/- 3.9% of their preischaemic contractile force values. Creatine kinase leakage was significantly lower and postischaemic coronary flows, too, were significantly higher in the fourth group. Coronary flow after reperfusion increased from 48.5 +/- 6.7 to 65.2 +/- 7.1 ml/min g heart in group 4 (p < 0.05). Myocardial lipid peroxides and glutathione contents showed that there was a correlation between the depletion of myocardial glutathione content and increased lipid peroxidation. These preliminary results showed that: the addition of captopril to reperfusion solution and oral preconditioning improved post-ischameic myocardial function and decreased myocardial injury.

Vitamin E and Oxidative Stress in the Heart of the Cardiomyopathic Syrian Hamster

Myocardial deterioration is relentlessly progressive in almost all patients who develop overt symptoms. Many dilated cardiomyopathies are associated with a marked increase in cardiac sympathetic tone which may be toxic to myocytes. Microvascular spasm, leading to diffuse, focal reperfusion injury, also appears to be an important mechanism of cardiomyocyte loss in many models of dilated cardiomyopathy. Free radicals may mediate both catecholamine-induced damage and reperfusion injury. We hypothesized that myocardial antioxidant reserve may be significantly reduced in dilated cardiomyopathy and that α-tocopheryl acetate may be of benefit. The enzymes superoxide dismutase, catalase and glutathione peroxidase were measured in the myocardial tissue of control and cardiomyopathic hamsters in early (25–50 days) and late (275–320 days) stages of the cardiomyopathy. In another study, myocardial glutathione peroxidase activity and protein oxidation was measured in control and late stage cardiomyopathic hamsters receiving α-tocopheryl (70 mg/kg/day) or vehicle for 1 month. There were no significant differences in glutathione peroxidase activity between control and cardiomyopathic hamsters in the early stage of the cardiomyopathy. Superoxide dismutase and catalase activities did not change with aging; however, glutathione peroxidase decreased over 30% , α-tocopherol was reduced by approximately 50% and protein oxidation increased more than 2-fold in the hearts of late stage cardiomyopathic hamsters. α-Tocopheryl acetate administration restored α-tocopherol levels, glutathione peroxidase activity and protein oxidation to normal. We conclude that the decompensating heart has significantly limited antioxidant reserve and that this reserve is sensitive to the intake of antioxidant supplements.

Changes in myocardial thyroid hormone metabolism and alpha-glycerophosphate dehydrogenase activity in rats deficient in iodine and selenium.

Weanling Wistar rats were fed on diets prepared from grain from areas deficient in I and Se where Keshan disease in endemic. Rats were divided into four groups, each of twelve rats, and received a diet supplemented with: I, Se, I + Se or nothing. At 8 weeks after weaning, myocardial alpha-glycerophosphate dehydrogenase (EC 1.1.1.8; alpha-GPD) activity and indices of Se and thyroid hormone status were determined. The group supplemented with iodine had increased plasma thyroxine levels. There was no difference in plasma triiodothyronine concentration between the groups but triiodothyronine levels in heart were reduced in the Se-supplemented group. Se supplementation increased myocardial glutathione peroxidase activity (EC 1.11.1.9) and the type I 5'-deiodinase (EC 3.8.1.4) activity in rat liver, but no type I 5'-deiodinase activity was detected in heart. alpha-GDP activity in heart was increased in group supplemented with Se, I or both. There was a significant relationship (P < 0.05) between myocardial alpha-GDP activity and plasma thyroxine levels but not between alpha-GDP and myocardial glutathione peroxidase activity. The results indicate that iodine may be more important than Se in energy metabolism in the myocardium, which may give a new insight for the study of the aetiology of Keshan disease in areas where foodstuffs are deficient in both Se and I.

Antioxidative effects of rubidate on ischemic myocardium in mice

Rubidate is a phenolic compound. The present investigation was designed to establish whether rubidate protects the heart muscle against the deleterious effects of the oxygen free radicals produced by isoproterenol (ISO)-induced myocardial ischemia in mice. ISO treatment (30 mg kg-1 subcutaneous injection once daily for 2 days) caused widespread and severe myocardial cell damage, decreased myocardial glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities and increased myocardial malondialdehyde (MDA) content. Rubidate pretreatment 500 mg-kg-1, 2000 mg.kg-1×7d protected GSH-Px, SOD activity in the hearts of ISO-treated mice. Rubidate 2000 mg-kg-1 diminished MDA production. However, rubidate 500 mg.kg-1 did not show such an effect. This study demonstrated that rubidate inhibited lipid peroxidation (LPO, expressed by malondialdehyde formation), scavenged-Oz - and H2O2. It may be concluded that rubidate exhibits an antioxidative activity via scavenging free radicals.

Postischemic injury in isolated rat hearts is not aggravated by prior depletion of myocardial glutathione.

The aim of this study was to test the hypothesis that a decreased myocardial concentration of reduced glutathione (GSH) during ischemia renders the myocardium more susceptible to injury by reactive oxygen species generated during early reperfusion. To this end, rats were pretreated with L-buthionine-S,R-sulfoximine (2 mmol/kg), which depleted myocardial GSH by 55%. Isolated buffer-perfused hearts were subjected to 30 min of either hypothermic or normothermic no-flow ischemia followed by reperfusion. Prior depletion of myocardial GSH did not lead to oxidative stress during reperfusion, as myocardial concentration of glutathione disulfide (GSSG) was not increased after 5 and 30 min of reperfusion. In addition, prior depletion of GSH did not exacerbate myocardial enzyme release, nor did it impair the recoveries of tissue ATP, coronary flow rate and left ventricular developed pressure during reperfusion after either hypothermic or normothermic ischemia. Even administration of the prooxidant cumene hydroperoxide (20 microM) to postischemic GSH-depleted hearts during the first 10 min of reperfusion did not aggravate postischemic injury, although this prooxidant load induced oxidative stress, as indicated by an increased myocardial concentration of GSSG. These results do not support the hypothesis that a reduced myocardial concentration of GSH during ischemia increases the susceptibility to injury mediated by reactive oxygen species generated during reperfusion. Apparently, myocardial tissue possesses a large excess of GSH compared to the quantity of reactive oxygen species generated upon reperfusion.

Deleterious Effects of Buthionine Sulfoximine on Cardiac Function During Continuous Endotoxemia

Sepsis has been associated with reversible cardiac injury. To determine whether this injury is mediated by generation of reactive oxidants, tissue glutathione (GSH)--the major intracellular antioxidant--was depleted before endotoxemia. Basal values of cardiac contractile function, perfusion, and cardiac output were measured 5-7 days postsurgery. Salmonella enteritidis endotoxin was continuously infused at 3 micrograms/kg/hr iv via an osmotic pump (Alzet Corp). Endotoxemia significantly reduced myocardial glutathione content (394 +/- 46) to 206 +/- 9 micrograms/g), indicating oxidant stress during endotoxemia. Buthionine sulfoximine (BSO) pretreatment significantly reduced cardiac glutathione in sham pigs from 394 +/- 46 to 199 +/- 26 micrograms/g; and in endotoxemic pigs, BSO pretreatment significantly reduced cardiac glutathione to 106 +/- 18 micrograms/g. Vehicle- and BSO-treated endotoxemic groups demonstrated similar cardiovascular responses to endotoxin challenge. Heart rate increases (122 +/- 15 to 140 +/- 17 bpm) and cardiac outputs decreases (1.50 +/- 0.24 to 1.11 +/- 0.35 l/min) were similar, indicating similar cardiovascular insults induced by endotoxemia. Percent short axis shortening and end-systolic pressure-diameter relation (ESPDR) were significantly reduced in BSO pretreated compared with vehicle-treated endotoxemic pigs. Results support a conclusion that endotoxemia-induced cardiac injury is mediated, in part, by free radical injury. This conclusion is based upon the finding that endogenous myocardial glutathione was depleted by continuous endotoxin infusion and that prior depletion of myocardial glutathione by buthionine sulfoximine exacerbated cardiac injury.

Cardiac [formula omitted] ATPase activity and its relation to myocardial glutathione status: Studies in the rat

Cardiac Na + K + ATPase (which has previously been suggested to be regulated by a thiol-dependent process) is inactivated during ischemia. Moreover, myocardial glutathione (which controls the redox state of protein-thiol groups) is depleted during ischemia and thus may contribute to the changes in Na + K + ATPase activity. The objectives of the present study were to use the rat to: (1) pharmacologically manipulate myocardial glutathione to mimic ischemia-reperfusion-induced glutathione depletion and (2) determine (in the absence of ischemia) the relationship between myocardial glutathione content and Na + K + ATPase activity. Tissue glutathione was depleted by injecting rats with diethylmaleate (0, 55, 110, 215, 430 or 860 mg/kg; i.p.) 30 min before study. Total glutathione content fell from 1.72±0.03 to 1.66±0.04, 1.50±0.05, 0.93±0.03, 0.23±0.04 and 0.21±0.02 μmol/g wet weight, respectively. There was a linear correlation ( r=0.96) between Na + K + ATPase activity and glutathione content in diethylmaleate-treated animals. A separate group of animals were treated with phorone (0, 25, 50, 100, 150, 200 or 250 mg/kg; i.p. 120 min before study) which also depletes glutathione. Myocardial glutathione fell from its control value of 1.74±0.03 to 1.52±0.04, 1.35±0.06, 1.14±0.05, 0.84±0.04, 0.64±0.04, 0.54±0.03 μmoles/g wet weight, respectively. In these animals Na + K + ATPase activity was also linearly-related ( r=0.98) to glutathione content. We also characterized the temporal relationship between diethylmaleate-induced glutathione depletion and Na + K + ATPase activity. Diethylmaleate (216 mg/kg; i.p.) was given to rats and at various times (0–200 min), after administration the hearts were removed and assayed for glutathione. During the first 30 min after administration glutathione fell from 1.74±0.03 to its lowest value of 0.90±0.04 μmol/g wet weight. It then progressively recovered to within control levels by 150 min after administration. Na + K + ATPase activity paralleled the recovery of glutathione status. In additional studies, the cell-permeant glutathione analogue YM 737 (glutathione isopropyl ester: 1 mmol/kg, i.p.) accelerated the recovery of tissue glutathione and evoked a commensurate and parallel increase in the rate of recovery of Na + K + ATPase activity. Our studies suggest there is a close coupling between tissue glutathione content and Na + K + ATPase activity and that this may be an important factor in ischemia and reperfusion-induced changes in Na + K + ATPase activity.

Probucol protects against adriamycin cardiomyopathy without interfering with its antitumor effect.

The usefulness of adriamycin (ADR), a potent antitumor antibiotic, is limited by the development of life-threatening cardiomyopathy and congestive heart failure. Subcellular changes leading to heart failure are suggested to be mediated by a drug-induced increase in free radicals and lipid peroxidation. In an earlier study, concurrent treatment with probucol (PROB), a lipid-lowering drug with strong antioxidant properties, was shown to offer only partial protection against ADR cardiomyopathy. The present study had two aims: to determine whether this protective effect can be improved further by extended treatment with PROB, and to determine whether PROB affects the antitumor properties of ADR. ADR (cumulative dose, 15 mg/kg body wt) was administered in rats in six equal injections (IP) over a period of 2 weeks. Three weeks after the end treatment, cardiomyopathy and congestive heart failure were characterized by ascites, congested liver, depressed cardiac function, elevated left ventricular end-diastolic pressure, and myocardial cell damage. Myocardial glutathione peroxidase (GSHPx) activity was decreased and lipid peroxidation was increased. Administration of PROB (cumulative dose, 120 mg/kg body wt) in 12 equal injections (IP), before and concurrent with ADR, completely prevented these cardiomyopathic changes, normalized left ventricular function, lowered mortality, and eliminated ascites. Treatment with PROB was also accompanied by an increase in myocardial GSHPx and superoxide dismutase activities with a concomitant decrease in lipid peroxidation. Tumor regression in syngeneic DBA/2 mice inoculated with L5178Y-F9 lymphoma cells in the ADR+PROB group was significant and comparable to the ADR group. These data show for the first time that PROB can provide complete protection against ADR cardiomyopathy without interfering with antitumor properties of the drug. This protective effect of PROB may be related to the maintenance of the antioxidant status of the heart.

Probucol promotes endogenous antioxidants and provides protection against adriamycin-induced cardiomyopathy in rats.

The potential usefulness of adriamycin (ADR) is restricted because of its cardiotoxic side effects. Since free radicals and lipid peroxidation are suggested to be involved in ADR cardiomyopathy, we examined the beneficial effects of probucol, a lipid-lowering drug with strong antioxidant properties. ADR was administered to rats in six equal intraperitoneal injections over a period of 2 weeks (cumulative dose of 15 mg/kg). After a 3-week posttreatment period, cardiomyopathy and congestive heart failure were characterized by ascites, congested liver, depressed cardiac function, elevated left ventricular end-diastolic pressure, and myocardial cell damage. Myocardial glutathione peroxidase (GSHPx) activity was decreased, and lipid peroxidation was increased. Probucol (cumulative dose, 60 mg/kg IP) was administered in six equal injections over a 2-week period on days alternating with ADR treatment. Probucol significantly attenuated the myocardial effects of ADR, improved left ventricular function, and lowered mortality as well as the amount of ascites. Treatment with probucol was also accompanied by an increase in myocardial GSHPx and superoxide dismutase activities, with a concomitant decrease in lipid peroxidation. These data provide evidence that ADR cardiomyopathy is associated with an antioxidant deficit. Improved cardiac function resulting from treatment with probucol may be related to the maintenance of the antioxidant status of the heart. The study suggests potential usefulness of antioxidant (probucol) therapy in ADR cardiomyopathy.

Myocardial response to regional ischemia and reperfusion in vivo in rat heart.

Ischemia-reperfusion-induced myocardial oxidative changes were investigated in open-chest hearts of anesthetized rats. Surgical occlusion of the left anterior descending coronary artery for 30 min followed by 15 min reperfusion resulted in a significant decrease of reduced glutathione, an increase in glutathione disulfide, and an enhanced lipid peroxidation in rapidly frozen left ventricular tissues. Direct electron paramagnetic resonance spectroscopy revealed an increase in free radical concentration in ischemic cardiac tissues reperfused for 45 s, but the increase diminished at 15 min. these alterations were associated with decreased activities of myocardial glutathione peroxidase, glutathione reductase, and catalase. Ischemia resulted in a significant reduction of high-energy phosphate compounds and an accumulation of nucleotide degradation products, particularly adenosine, in the myocardium. Deterioration of cardiovascular function in reperfused animals was also evident. It is concluded that regional ischemia followed by reperfusion in situ can produce biochemical and physiological alterations consistent with free radical injury in rat hearts, and that an increased purine nucleotide degradation and a decreased antioxidant defense may be responsible for the observed changes.

Brief myocardial ischemia affects free radical generating and scavenging systems in dogs.

This study examined whether brief repeated myocardial ischemia altered free radical generating and scavenging activity in a dog model. In dogs preconditioned with four 5-min left anterior descending coronary artery (LAD) occlusions and reperfusions, we examined transcardiac changes in both the function of neutrophils, cells which are major free radical generators, and in myocardial antioxidant enzyme activity, as an indication of free radical scavenging. Neutrophil function was assessed by determining luminol-enhanced whole blood chemiluminescence (CL) induced by zymosan. Blood was taken simultaneously from the carotid artery and the cardiac vein running along the occluded LAD. Preconditioning with sublethal ischemia significantly reduced whole blood CL in the cardiac vein compared with the carotid artery after the first and fourth 5-min reperfusions, while there was no difference in neutrophil count between these sampling sites. Immediately after brief repeated ischemia and reperfusion, manganese-superoxide dismutase (SOD) activity was significantly enhanced, and glutathione reductase activity was markedly reduced in the ischemic, compared with the non-ischemic, myocardium. There were no differences in the myocardial activities of copper, zinc-SOD, glutathione peroxidase, and glutathione S-transferase between the ischemic and non-ischemic regions. Also, no difference was observed between the reduced myocardial glutathione levels in these regions, although the oxidized glutathione level was significantly higher in the ischemic regions of the subepicardial and subendocardial areas. We demonstrated that brief repeated ischemia affects free radical generating and scavenging systems in the ischemic myocardium.

Early and late effects of leukopenic reperfusion on the recovery of cardiac contractile function. Studies in the transplanted and isolated blood-perfused rat heart.

Since there is considerable evidence that leukocytes contribute to tissue injury during ischemia and reperfusion, the present study was designed to: (1) determine whether reperfusion in vivo with leukopenic blood affords protection in a model of reversible hypothermic ischemia, (2) determine the duration of any protection, (3) characterize the relation between protection and duration of leukopenic perfusion, and (4) assess the effect of leukopenic reperfusion on myocardial glutathione content. Rat hearts (n = 12 per group) were excised, immediately arrested with an infusion (2 minutes at 4 degrees C) of St Thomas' cardioplegic solution, and subjected to 4 hours of global ischemia (4 degrees C). The hearts were then transplanted (1 hour additional ischemic time) into the abdomen of saline-treated or leukopenic recipients. Leukopenia was induced by intraperitoneal administration of mustine hydrochloride (2 mg/kg) 3 days before study. Hearts were then reperfused in situ for 1, 4, or 24 hours, after which they were excised and either processed for histological examination (n = 4 per group) or perfused aerobically with bicarbonate buffer for 20 minutes, and contractile function was assessed (n = 8 per group); at the end of this period, some hearts (n = 5 per group) were taken for metabolite analysis. After 1 hour of reperfusion, contractile function in the saline-treated control group was significantly reduced compared with aerobic controls that had not been subjected to ischemia (left ventricular developed pressure [LVDP], 108 +/- 5 vs 126 +/- 3 mm Hg at an end-diastolic pressure of 12 mm Hg; P < .05). However, in the hearts with leukopenic reperfusion, LVDP (119 +/- 2 mm Hg) was similar to that of aerobic controls. This benefit, however, was lost after 4 and 24 hours of reperfusion. Cardiac compliance was not influenced by leukopenia. Coronary flow recovered significantly better in the leukopenic hearts during the first 4 hours of reperfusion (11.8 +/- 0.5 vs 9.3 +/- 0.4 mL/min at 1 hour and 10.0 +/- 0.5 vs 8.0 +/- 0.4 mL/min at 4 hours, P < .05), but again this benefit was lost after 24 hours of reperfusion. The myocardial contents of reduced and oxidized glutathione after 1, 4, and 24 hours of reperfusion were similar in saline-treated and leukocyte-depleted animals. In additional studies, the period of ischemia was extended to 8 hours, and similar results were obtained, with improved recovery of contractile function and coronary flow but not cardiac compliance in the leukopenic group after 1 hour of reperfusion. In further studies with the isolated blood-perfused rat heart, ischemia was induced for 8 hours; this was followed first by reperfusion for 0, 2, 10, 30, or 60 minutes with leukopenic blood and then by perfusion with blood from saline-treated animals for 60, 58, 50, 30, or 0 minutes, respectively. Reperfusion with leukopenic blood for 2 minutes did not improve the recovery of LVDP (106 +/- 7 vs 96 +/- 10 mm Hg in controls; NS) but when continued for 10, 30, or 60 minutes resulted in significant improvements (137 +/- 5, 138 +/- 3, and 150 +/- 10 mm Hg, respectively). Although coronary flow tended to be greater in all leukopenic groups, by the end of 60 minutes of reperfusion, only those hearts reperfused with leukopenic blood for the entire reperfusion period showed a significant improvement (3.4 +/- 0.3 vs 2.5 +/- 0.2 mL/min in controls; P < .05). Histological studies revealed no intravascular aggregation of leukocytes or features of myocyte necrosis. Reperfusion with leukopenic blood accelerated the rate of recovery of cardiac function after reversible myocardial injury but did not lead to a sustained increase in the eventual extent of recovery. Reperfusion with leukopenic blood for the first 10 minutes of reflow is sufficient to obtain this benefit.

Effect of glutathione on canine myocardial ischaemia without reperfusion.

The present study was to evaluate the effect of exogenous glutathione on myocardial damage resulting from permanent (no reperfusion) coronary ligation (3 or 6 h) in anaesthetized dogs. Haemodynamics, infarct size and myocardial glutathione content were determined. Erythrocyte superoxide dismutase (SOD) activity was also determined in coronary venous blood samples. Glutathione was administered by the intraperitoneal route, 100 mg kg-1 as initial dose given 5 min before coronary ligation, and successive doses of 25 mg kg-1 every 40 min throughout the study period. Saline-treated dogs showed myocardial infarction, a decrease in myocardial glutathione content, and a transient increase in SOD activity. Three hours occlusion in glutathione-treated dogs resulted in a small reduction of infarct size, and no changes in myocardial glutathione content and SOD activity. By contrast, administration of glutathione failed to reduce infarct size and failed to prevent myocardial glutathione decrease in dogs subjected to 6 h occlusion. These results indicate that exogenous glutathione is of minor beneficial effect for myocardial damage resulting from permanent coronary occlusion and suggest that endogenous glutathione has a limited role in protecting against myocardial ischaemia without reperfusion.

Myocardial glutathione depletion impairs recovery of isolated blood-perfused hearts after global ischaemia

This study was performed to determine whether depletion of myocardial glutathione would impair recovery of left ventricular function of blood-perfused, isolated hearts after reversible ischaemic injury. Cats were treated with either vehicle or buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the synthesis of glutathione. The feline isolated hearts were perfused with the blood of normal donor cats before and after 40 min of global myocardial ischaemia. The myocardial concentration of glutathione of the BSO group, 178±38 ng/mg tissue, was significantly less than that of the control group, 292±38 ng/mg tissue ( P<0.05). The peak left ventricular developed pressure (LVDP) 1 h after reperfusion, expressed as a fraction of the peak LVDP before ischaemia, was 0.87±0.10 for the control group and 0.64±0.08 for the BSO group ( P=0.05 vs. control). The peak left ventricular dP dt after reperfusion, expressed as a fraction of the peak dP dt before ischaemia, was 1.08±0.14 for the control group and 0.78±0.09 for the BSO group ( P=0.05 vs. control). The myocardial creatine kinase activity of the BSO group, 1046±46 U/g tissue, was not significantly different from that of the control group, 1038±17 U/g tissue ( P=0.87). Thus, depletion of myocardial glutathione resulted in impaired post-ischaemic contractile function that cannot be attributed to a greater extent of irreversible cell injury.

Effect of oxygen tension and cardiovascular operations on the myocardial antioxidant enzyme activities in patients with tetralogy of Fallot and aorta-coronary bypass

Since the chronically cyanotic myocardium appears to be more susceptible to reperfusion injury after cardiac operations than the noncyanotic myocardium, we studied the association between the preoperative arterial oxygen tension and the myocardial superoxide dismutase, catalase, and glutathione peroxidase activities. Fourteen patients with tetralogy of Fallot scheduled for elective operations had baseline arterial blood gas measurements done before operation. During the operation right ventricular biopsy specimens were taken for enzyme analysis immediately before cold blood cardioplegic arrest and 20 minutes after crossclamp removal. The tissue antioxidant enzyme activities of the patients with tetralogy of Fallot were compared with the myocardial results in 15 adults with stable angina pectoris having elective aorta-coronary artery bypass graft operations. Myocardial tissues removed from two patients with hypertrophic obstructive cardiomyopathy who had corrective operations were analyzed for antioxidant activities. There were no changes in myocardial antioxidant enzyme activities during the operation in the patients with tetralogy of Fallot and coronary artery bypass graft. The myocardial superoxide dismutase, catalase, and glutathione peroxidase activities correlated (0.82, 0.68, and 0.89, respectively) significantly (p values were less than 0.01, 0.05, and 0.01, respectively) with the preoperative arterial oxygen tensions in the patients with tetralogy of Fallot. The myocardial glutathione peroxidase activities were at least four times higher in the myocardium of patients with coronary artery bypass graft and hypertrophic obstructive cardiomyopathy than in that of those with tetralogy of Fallot. This study provides putative evidence that the myocardium of patients with tetralogy of Fallot is a risk of oxygen-derived free radical injury during and immediately after corrective cardiovascular operations.

Oxygen free radicals and myocardial damage: Protective role of thiol-containing agents

It has been suggested that the sudden presence of oxygen during reperfusion after a period of ischemia may be toxic for the myocardial cell. The oxygen molecule is capable of producing reactions in the cell, forming highly reactive free radicals, and inducing lipid peroxidation of membranes, altering their integrity and increasing their fluidity and permeability. The ischemic and reperfused cardiac cell is the prime candidate for this reaction sequence and may explain the molecular mechanism underlying the pathologic events related to membrane dysfunction and calcium homeostasis. However, the myocardium has a series of defense mechanisms including the enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase plus other endogenous antioxidants such as vitamin E, ascorbic acid, and cysteine to protect the cell against the cytoxic oxygen metabolites. The prerequisite for oxygen free radical involvement in ischemia and reperfusion damage is that ischemia alters the defense mechanisms against oxygen toxicity. It is known that ischemia may impair mitochondrial SOD and, with reperfusion, oxidative stress may occur as shown by tissue accumulation and release of oxidized glutathione. This tripeptide molecule is the cofactor of glutathione peroxidase, the enzyme that removes hydrogen and lipid peroxides. Its formation and subsequent release is a reliable index of oxidative damage. In our study, we investigated the effects of N-acetylcysteine on oxidative damage in the isolated rabbit heart. N-acetylcysteine increases, in a dose-dependent manner (from 10 −7 to 10 −5 M), the myocardial glutathione content and provides an important degree of protection against ischemia and reperfusion. Oxidative stress does not occur, mitochondrial function is maintained, enzyme release is reduced, and contractile recovery is increased. Similarly, we administered N-acetylcysteine in the pulmonary artery of coronary artery disease patients undergoing coronary bypass grafting (150 mg/kg in 1 hour followed by 150 mg/kg in 4 hours). The degree of oxidative stress on reperfusion was reduced and recovery of cardiac function improved. In this article, we review the cardioprotective role of thiol-containing agents.

Protective effect of N-acetylcysteine on ischaemia-induced myocardial damage in canine heart

The glutathione redox pathway is an important antioxidant system in the myocardium. N-Acetylcysteine is a low molecular weight glutathione precursor that has been used clinically to replenish glutathione stores. The present study was aimed at evaluating the protective effect of N-acetylcysteine on myocardial damage resulting from permanent coronary occlusion (without reperfusion) in anaesthetized dogs. N-Acetylcysteine (150 mg kg-1 i.v.) administered 2 min before occlusion reduced infarct size in dogs subjected to 24 h ischemia. The infarct size as a percentage of the area at risk was 86.8 +/- 3.6% (n = 11) in control (saline-treated) dogs and 68.2 +/- 2.4% (n = 7; P less than 0.05 vs control) in N-acetylcysteine-treated animals. Haemodynamic variables (heart rate, mean arterial pressure and rate-pressure product) were similar in the control and the treated group. Regional myocardial blood flow was determined with radioactive microspheres in ischaemic and non-ischaemic zones before occlusion and 3 h post-occlusion. N-Acetylcysteine did not influence the regional distribution of myocardial blood flow. The myocardial content of reduced glutathione was significantly (P less than 0.05) decreased 3 h post-occlusion (0.53 +/- 0.19 mumol/g-1; n = 5) compared to either pre-occlusion values (0.94 +/- 0.03 mumol/g-1; n = 8) or values 3 h post-occlusion in sham-operated animals (0.93 +/- 0.15 mumol/g-1; n = 5). Depletion of myocardial glutathione 3 h post-occlusion was not observed in dogs treated with N-acetylcysteine (0.87 +/- 0.11 mumol/g-1; n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Relation between myocardial glutathione content and extent of ischemia-reperfusion injury.

The relation between the extent of myocardial injury sustained during reperfusion and total glutathione (GSH) content in the ischemic myocardium was examined in anesthetized open-chest pigs subjected to coronary occlusion for 45 minutes and reperfusion for 2 hours. In pigs infused with saline during reperfusion (n = 6) there was a decrease in myocardial GSH content from 380 +/- 48 micrograms/g in normally perfused myocardium to 182 +/- 36 micrograms/g in the ischemic reperfused myocardium (p less than 0.02). Myocardial infarct size (expressed as a percentage of the ischemic area) was 12.5 +/- 0.8%. There was a delay of recovery of contractile function before returning to 60% of preocclusion value. In pigs pretreated with buthionine sulfoximine (BSO) (n = 5), an inhibitor of cellular GSH synthesis, there was reduction in GSH content to 215 +/- 25 micrograms/gm in normally perfused myocardium and to 77 +/- 8 micrograms/gm in the ischemic reperfused myocardium. The extent of injury was greater as evidenced by an increase in infarct size to 30.4 +/- 4.0% (p less than 0.001), severe destructive changes in subepicardial ultrastructure, which were absent in saline-infused pigs, and persistence of dyskinesia throughout reperfusion. In pigs infused with glutathione intravenously (0.8 gm/kg) at a rate of 6.5 mg/kg/min (n = 6), 5 minutes before and continuously during reperfusion, there was an increase in GSH content to 582 +/- 67 micrograms/g in normally perfused myocardium and to 312 +/- 80 micrograms/g in ischemic reperfused myocardium. The increase in myocardial GSH was associated with a reduction in infarct size to 7.5 +/- 1.3% (p less than 0.05, compared with saline-infused pigs) and an early recovery of contractile function of the ischemic myocardium. GSH infusion into pigs pretreated with BSO (n = 4) failed to increase myocardial GSH content and failed to reduce the extent of myocardial injury. Thus, the extent of myocardial injury sustained during reperfusion is very dependent on the effectiveness of its antioxidant defenses. Markedly increased susceptibility to injury occurs when the GSH content in the ischemic myocardium becomes depleted.

Myocardial glutathione depletion impairs recovery after short periods of ischemia.

Isolated, isovolumic rat hearts, perfused by Krebs-Henseleit buffer at constant coronary flow rate, were used to explore the hypothesis that endogenous cardiac glutathione provides protection against myocardial dysfunction associated with short periods of ischemia. Experimental animals were depleted of cardiac glutathione to 35% of control levels by intraperitoneal injections of diethylmaleate (DEM). Left ventricular pressure, coronary perfusion pressure, and glutathione levels were measured in control and experimental hearts after 60 minutes of oxygenated perfusion and after 20 minutes of global, no-flow ischemia and 30 minutes of reperfusion. With each protocol, both control and glutathione-depleted hearts received either standard buffer or one supplemented with 2 mM glutathione. Recovery of systolic function after ischemia-reperfusion was impaired in DEM-treated hearts compared with controls. In addition, the rise in perfusion pressure and chamber stiffness was also greater in DEM-treated hearts compared with controls. Recovery in glutathione-depleted hearts was improved when the reperfusate was supplemented with glutathione. In addition, the supplemented reperfusate prevented the decrease in compliance and the increase in coronary perfusion pressure in the glutathione-depleted hearts. Ischemia-reperfusion alone were not associated with a significant alteration in myocardial glutathione levels. Prewashout myocardial levels of glutathione were elevated after reperfusion with glutathione-supplemented buffer but fell to baseline levels after a short washout period. These studies demonstrate that endogenous glutathione is important in protection of myocardium from injury after ischemia-reperfusion, presumably by modifying levels of active oxygen intermediates. The smaller changes in left ventricular pressure and coronary resistance after administration of GSH probably reflects an extracellular mechanism because benefit is seen soon after reperfusion.

Correlation between myocardial glutathione content and extent of ischemia-reperfusion injury

Considerable evidence suggests that myocardial injury may occur during reperfusion of the ischemic myocardium. Reactive oxygen species are generated during reperfusion which may play an important role in the genesis of myocardial reperfusion injury. Glutathione (GSH) is an important antioxidant in the heart. A decrease in myocardial GSH content has been observed during ischemia and reperfusion of the ischemic myocardiijm. We hypothesized that this depletion of GSH may be detrimental to the ability of the ischemic myocardium to protect itself against reactive oxygen species during reperfusion.In this study, anesthetized open chest pigs were subjected to coronary occlusion for 45 minutes and 2 hours reperfusion. Myocardial GSH was experimentally depleted by pretreatment with buthionine sulfoximine (BSO), a potent inhibitor of cellular GSH synthesis, and was augmented by intravenous administration of GSH. For ultrastructural study multiple subepicardial biopsies 3 mm deep were taken from myocardium supplied by the occluded artery. The biopsies were processed by routine procedures. Thin sections were stained with uranyl acetate and lead citrate and examined with a Philip EM 300 electron microscope.