J. de Leiris

The isolated perfused rat heart: A model for studying myocardial hypoxia or ischaemia

SummaryThe aim of this paper is to consider the advantages of using isolated heart preparations in studies designed to investigate the effect of hypoxia or ischaemia on myocardial cells. After a brief description of the two most frequently used experimental models of perfused hearts, namely the Langendorff preparation and the working heart preparation, some of the various methods used to induce hypoxia or ischaemia are described, as well as some of the possible techniques allowing to assess metabolic alterations occurring in these pathological situations. After discussing the limitations and advantages specific to the Langendorff and working heart preparations, the suitability of isolated heart models in studies on myocardial protection is then considered. To illustrate this point, the effect of intravenous administration of the slow calcium channel blocker bepridil (5 mg · kg−1) on post-ischaemic recovery of cardiac function and metabolism after global normothermic ischaemia of the isolated heart is reported. It is concluded that isolated heart preparations allow a fine control of experimental conditions with the advantage that functional and metabolic measurements can be easily made.

Trace elements and cardioprotection: increasing endogenous glutathione peroxidase activity by oral selenium supplementation in rats limits reperfusion-induced arrhythmias.

Oxyradicals have been implicated as a possible cause of reperfusion-arrhythmias (RA). However, the use of diverse exogenous oxyradical scavengers designed to reduce RA has given contradictory results. The aim of the present study was to determine whether enhancing the activity of the main endogenous enzyme involved in peroxide elimination in cardiac cells, namely glutathione peroxidase, may limit RA in isolated heart preparations by increasing their antioxidant status. For this purpose, a group of 15 male Wistar rats received a selenium enriched diet for ten weeks (1.5 mg Se/kg diet). Control animals (n = 15) received a standard diet containing 0.05 mg Se/kg diet. The incidence of early ventricular arrhythmias was investigated during the reperfusion period following 10 min regional ischemia induced ex-vivo by left coronary artery ligation. Our results show that selenium-supplementation significantly increased the global selenium status of the animals. In the isolated heart preparations, the selenium supplementation induced a significant reduction of the severity of RA as assessed by the arrhythmia score and the limitation of the incidence of both ventricular tachycardia (control: 91% vs selenium: 36%, p < 0.05) and irreversible ventricular fibrillation (control: 45% vs selenium: 0%, p < 0.05). These effects were associated with a significant increase in cardiac mitochondrial and cytosolic glutathione peroxidase activities in both the left and the right ventricles. These results illustrate the potential protective effect of selenium against ischemia-reperfusion injury and suggest that peroxides might play a key role in the genesis of some aspects of the reperfusion syndrome.

Cardiac Toxicity of Singlet Oxygen: Implication in Reperfusion Injury

Oxygen-derived free radicals (O2.-, H2O2, and .OH) that are produced during postischemic reperfusion are currently suspected to be involved in the pathogenesis of tissue injury. Another reactive oxygen species, the electronically excited molecular oxygen (1O2), is of increasing interest in the area of experimental research in cardiology. In this review are discussed the main potential sources of singlet oxygen in the organism, particularly in the myocardium, the various cardiovascular cytotoxic effects induced by this reactive oxygen intermediate, and the growing evidence of its involvement in ischemia/reperfusion injury.

EUK-8 a Synthetic Catalytic Scavenger of Reactive Oxygen Species Protects Isolated Iron-Overloaded Rat Heart from Functional and Structural Damage Induced by Ischemia/Reperfusion

SummaryThe effects of EUK-8, a synthetic, catalytic scavenger of reactive oxygen species, on isolated iron-overloaded rat hearts submitted to ischemia-reperfusion were studied. In the absence of EUK-8, functional parameters (systolic and diastolic pressures, oxygen consumption as estimated by the product heart rate times left ventricular diastolic pressure) were severely impaired 1 minute and 15 minutes after reperfusion following a 15 minute ischemic episode. Dimethylthiourea (10 mM), a hydroxyl radical scavenger, had a minimally protective effect. In contrast, EUK-8 at a concentration of 50 μM in the perfusion medium maintained these parameters at close to their preischemia values. Electron microscopic analysis of heart tissues after 15 minutes ischemia followed by 15 minutes reperfusion showed extensive damage to mitochondria and sarcomeres in untreated hearts, while the extent of damage was significantly lower in EUK-8-treated hearts. The functional and structural protection afforded by EUK-8 were significantly better than those induced by dimethylthiourea. These data suggest that EUK-8 may be therapeutically useful in preventing heart damage induced by ischemia-reperfusion, for example, during thrombolytic treatment of myocardial infarction.

Effects of adriamycin on chronic cardiotoxicity in selenium-deficient rats

SummaryAdriamycin (doxorubicin) is an antineoplastic drug used to treat various cancers; however, its chronic use is unfortunately accompanied by cardiotoxicity. This toxicity can be reduced by antioxidant agents such as selenium, and it is particularly interesting that cancer patients are usually deficient in this trace element, which suggests that its supplementation could contribute to beneficial treatment.We have examined the effect of adriamycin on chronic cardiotoxicity in 6-week selenium deficiency in rats. Selenium-deficient rats showed a considerable reduction of selenium levels and of selenium-containing glutathione peroxidase. Cardiac lipid peroxides increased slightly in the deficient rats, whereas plasma and heart lipid peroxides increased markedly in adriamycin-treated rats. This increase was greatly accentuated in selenium deficiency. These results suggest that free radical mechanism may be contributing to adriamycin toxicity, and above all show the importance of balancing the selenium levels in adriamycin-treated subjects to limit its harmful myocardial action. A decrease in adriamycin cardiotoxicity with no concomitant decrease in its antineoplastic activity would be of considerable value by improving the therapeutic benefit of the drug.

Effect of iron overload in the isolated ischemic and reperfused rat heart

SummaryIt has been suggested that iron might play a pivotal role in the development of reperfusion-induced cellular injury through the activation of oxygen free radical producing reactions. The present study examined the effects of myocardial iron overload on cardiac vulnerability to ischemia and reperfusion. Moreover, the effect of the iron chelator deferoxamine in reversing ischemia-reperfusion injury was studied. Animals were treated with iron dextran solution (i.m. injection, 25 mg every third day during a 5 week period). The control group received the same treatment without iron. Isolated rat hearts were perfused at constant flow (11 ml/min) and subjected to a 15 minute period of global normothermic ischemia followed by reperfusion for 15 minutes. The effects of iron overload were investigated using functional and biochemical parameters, as well as ultrastructural characteristics of the ischemic-reperfused myocardium compared with placebo values. The results suggest that (a) a significant iron overload was obtained in plasma and hepatic and cardiac tissues (×2.5, ×16, and ×8, respectively) after chronic intramuscular administration of iron dextran (25 mg); (b) during normoxia, iron overload was associated with a slight reduction in cardiac function and an increase in lactate dehydrogenase (LDH) release (×1.5); (c) upon reperfusion, functional recovery was similar whether the heart had been subjected to iron overload or not. However, in the control group left ventricular end-diastolic pressure remained higher than in preischemic conditions, an effect that was not observed in the iron-overloaded group. Moreover, LDH release was markedly increased in the iron-loaded group (×4.2); (d) iron overload was associated with a significant worsening of the structural alterations observed during reperfusion, particularly at the mitochondrial and sarcomere level; (e) after 15 minutes of reperfusion, the activity of the anti-free-radical enzyme, glutathione peroxidase (GPX), was significantly reduced in ironoverloaded hearts, whereas catalase activity was increased; (e) the overall modifications observed in the presence of iron overload were prevented by deferoxamine. In conclusion, this study underlines the possible role of cardiac iron in the development of injury associated with ischemia and reperfusion, and the possible importance of the use of an iron-chelating agent in antiischemic therapy.

Effect of zinc deficiency on lipid peroxidation status and infarct size in rat hearts

The objective of this study was to investigate the effect of dietary zinc on endogenous production of lipid peroxides, and on myocardial infarct size in rats. Male rats were fed a zinc-deficient diet containing 4 ppm zinc, or a standard diet containing 60 ppm zinc. After 3 weeks of diet, half of the animals underwent occlusion of the left coronary artery. The remaining animals underwent sham operation without occlusion. Forty-eight hours later, the hearts were sampled and lipid peroxide levels and infarct size were evaluated. Coronary occlusion was associated with an increase in cardiac lipid peroxide levels which were more pronounced in the zinc deficient group. However, infarct size appeared to be independent from zinc deficiency, despite the free radical-mediated lipid peroxide augmentation reported here. The pharmacological limitation of infarct size in rats with permanent coronary occlusion is discussed.

Ischemia and reperfusion injury in isolated rat heart: Effect of reperfusion duration on xanthine oxidase, lipid peroxidation, and enzyme antioxidant systems in myocardium

SummaryThe aim of this work was to assess the catalytic activity of xanthine oxidase, the level of lipid peroxides and enzymic antioxidant systems in isolated rat heart muscle subjected to a globally partial ischemia followed by varying durations of reperfusion.After 40 min of globally partial ischemia (residual perfusion flow rate: 0.1 ml/min), four different durations of reperfusion were investigated (0, 20, 40, and 60 min). After each experimental ischemia/reperfusion sequence, the heart was frozen in liquid nitrogen. Lipid peroxides were assayed in the cardiac homogenate and the catalytic of xanthine oxidase and enzymic antioxidant systems (glutathione peroxidase, superoxide dismutase and catalase) were determined in the centrifuged supernatant. In the different experimental protocols studied in this work, there was no significant increase in the activity of cardiac xanthine oxidase or in the level of lipid peroxides when compared to the non reperfused or to the continuously perfused hearts. Indeed, enzymic antioxidant systems were also not significantly modified in the different periods of reperfusion when compared to control hearts (continuously perfused hearts).These results suggest that xanthine oxidase is apparently not a major source of free radicals in the course of an ischemia-reperfusion sequence in heart muscle, in particular, if we consider the early phases of reperfusion. The process of lipid peroxidation, assessed by assaying thiobarbituric acid reactants, is not a predominant phenomenon of reperfusion-induced injury, at least in the experimental model used here. However, enzymic antioxidant systems investigated in this study do not seem modified. This could mean that the small quantity of oxygen free radicals produced does not overwhelm the enzymic antioxidant systems of myocardium which is in agreement with peroxidatized lipid results.

Insulin resistance modifies plasma fatty acid distribution and decreases cardiac tolerance to in vivo ischaemia/reperfusion in rats

1. The early stage of insulin resistance, also termed the ‘prediabetic state’, is characterized by the development of hyperinsulinaemia, which maintains normoglycaemia under fasting conditions. The metabolic disorders induced in myocardial cells during this stage of the disease may constitute a basis for an alteration of the tolerance of the heart to ischaemia and reperfusion.

Ability of N-tert-butyl alpha phenylnitrone (PBN) to be used in isolated perfused heart spin trapping experiments: Preliminary studies

SummaryThe aim of this study was to investigate the possibility of oxygen-free radical spin trapping with PBN, in models of isolated perfused hearts. Preliminary studies reported here demonstrate that (i) PBN may be precisely measured with UV spectroscopy, (ii) commercially available PBN does not show any ESR signal, (iii) PBN does not trap significant amounts of free radicals in a perfusion medium oxygenated for at least 3 h, and (iv) when added at 15 or 56 mM in the perfusion medium, PBN is a highly toxic compound, whereas no toxic effect was observed with 3mM-containing perfusate.