Sylvie Pucheu

Effects of selenium deficiency on the response of cardiac tissue to ischemia and reperfusion

Over a 10-week period, female Wistar rats received a diet containing a low level of selenium, cofactor of the antioxidant enzyme glutathione peroxidase (GPx) in order to examine the influence of deficiency of this trace element (i) on tissue antioxidant enzyme defence systems, and (ii) on the susceptibility of the myocardium to ischemia-reperfusion injury. At the end of the dietary treatment, hearts were perfused at constant flow (11 ml/min) before being subjected to 15 min of global normothermic ischemia, followed by 30 min of reperfusion. The effects of selenium deficiency were estimated by studying functional recovery of various cardiac parameters (left ventricular developed pressure LVDevP, heart rate HR, and the product HR x LVDevP), as well as ultrastructural tissue characteristics. Furthermore, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were measured at the end of the reperfusion period. Results suggest that: (a) the activity of GPx is decreased by selenium deficiency while SOD activity remains unchanged, (b) the recovery of cardiac function and myocardial ventricular ultrastructure during reperfusion are altered in the selenium-deficient group compared to controls. These results illustrate the crucial role that selenium, the co-factor of one of the major antioxidant enzymes of the myocardium, plays in determining the vulnerability of the heart to ischemia and reperfusion.

Effect of Antioxidant Trace Elements on the Response of Cardiac Tissue to Oxidative Stressa

Abstract: It is now well established that several trace elements, because of their involvement in the catalytic activity and spatial conformation of antioxidant enzymes, may contribute to the prevention of oxidative stress such as occurs upon reperfusion of ischemic tissue. The aim of this paper is (1) to review the role of these trace elements (Cu, Mn, Se, and Zn) in antioxidant cellular defenses in the course of post‐ischemic reperfusion of cardiac tissue, (2) to provide experimental data suggesting that variations in trace element dietary intake may modulate the vulnerability of cardiac tissue to ischemia‐reperfusion, and (3) to discuss in more detail the effect of Mn ions, which seem to play a special protective role against reperfusion injury. Some results obtained from experiments in animal models of myocardial reperfusion have shown that the dietary intake of such trace elements can modulate cardiac activity of antioxidant enzymes and, consequently, the degree of reperfusion damage. In addition, experimental data on the protective effects of an acute treatment with Mn are presented. Finally, experimental evidence on the protective role of salen‐Mn complexes, which exhibit catalytic SOD‐ and CAT‐like activities against reperfusion injury, are described. These complexes should be of considerable interest in clinical conditions.

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.

ASSESSMENT OF RADICAL ACTIVITY DURING THE ACUTE PHASE OF MYOCARDIAL INFARCTION FOLLOWING FIBRINOLYSIS : UTILITY OF ASSAYING PLASMA MALONDIALDEHYDE

Numerous experimental and clinical studies have reported a role of radical forms of oxygen in the etiology of the manifestations of reperfusion of the ischemic myocardium. However, clinical results remain controversial. The aim of this study was to ascertain the existence of reperfusion-related radical stress after thrombolysis with a marker that is easy to use and reliable. Thirty patients hospitalized for acute myocardial infarction were involved in the study. Of these, 18 had been subjected to intravenous thrombolysis (Group I) and 12 had not (Group II). They were compared to two control groups who had no history of myocardial infarction. Of these, 16 were patients with coronary heart disease hospitalized for stable angina (Group III) and 17 were patients free of any known cardiovascular disease (Group IV). Radical activity was assessed in plasma samples taken from a peripheral vein over a 10-day period of hospitalization by measuring (1) malondialdehydes (MDA) concentrations using fluorometry techniques or HPLC, (2) the antioxidant activity of glutathione peroxidase (GPx) and (3) the concentration of various antiradical compounds (beta-carotene, vitamins A and E, uric acid). All patients in Group I had a patent artery on coronary angiography and showed a significant increase in plasma MDA when compared to those who had not been subjected to thrombolysis (3.15 +/- 0.62 and 2.70 +/- 0.40 mole/l of plasma, respectively). Furthermore, GPx plasma activity was also significantly increased following thrombolysis. By contrast, there was no significant alteration in the antiradical compounds measured. These data suggest that MDA measurements (an early measurement 1-2 days and a late measurement 5-7 days after reperfusion) by fluorometry is a good marker of radical stress during reperfusion in man. The assessment of this marker in patients might represent a simple and reliable test of reperfusion efficacy following thrombolysis, and it might enable one to test the effect of various antioxidant therapies associated with thrombolytic treatment.

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.

Evidence of cytosolic iron release during post‐ischaemic reperfusion of isolated rat hearts Influence on spin‐trapping experiments with DMPO

Previous studies of oxygen‐derived free radical generation based on spin‐trapping methods have shown a signal formed of six bands (sextet) using electron spin resonance spectrometry (ESR) or coronary effluents collected during post‐ischaemic reperfusion of isolated hearts perfused with 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO). The origin of this signal has recently become controversial. In the present study we show that, in the rat, this sextet and cytosolic iron release occur simultaneously, and that this signal can be inhibited by the iron chelator desferrioxamine. It also appears that the iron release is not protein bound, and could therefore have a marked catalytic activity. This may be responsible for the production or an artefactural signal observed as the sextet.

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.

Relationship between severity of ischemia and oxidant scavenger enzyme activities in the isolated rat heart

It is currently believed that reperfusion injury of the ischemic or hypoxic myocardium can be attributed, at least in part, to an overproduction of reactive oxygen species (ROS). The aim of the present study was to determine whether ischemia (of different severity or duration) followed by reperfusion can affect the activity of endogenous scavenger enzymes in isolated perfused rat hearts. Isolated Langendorff perfused rat hearts were subjected to either total (10, 20 or 30 min; zero-flow) or partial (30, 60 or 90 min; low-flow of 0.10 or 0.35 ml/min) ischemia, followed by 10 min of reperfusion. Enzymatic activities of total superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were determined in cardiac tissues at the end of the perfusion protocol. Basal scavenger enzyme activities measured in control hearts (perfused under normoxic conditions) were 33.90 +/- 4.88, 31.20 +/- 5.32 and 1.61 +/- 0.29 IU/mg protein (mean +/- SD, n = 6 per group) for SOD, catalase and GPx respectively. Our results indicate that neither total SOD, GPx, nor catalase myocardial activities were changed whatever the perfusion protocol followed. The present study shows that the endogenous pool of catalytic ROS scavengers is not dramatically altered during ischemia or upon reperfusion. This suggests that ROS scavengers are not directly involved in the development of ischemia/reperfusion injuries. These results also support the premise that excessive radical generation does not occur in this model, where the isolated heart is subjected to ischemia.

Effect of ischemia/reperfusion sequence on cytosolic iron status and its release in the coronary effluent in isolated rat hearts.

The hypothesis that oxygen-derived free radicals play an important role in myocardial ischemic and reperfusion injury has received a lot of support. In the presence of catalytic amounts of transition metals such as iron, superoxide anions, and hydrogen peroxide can be transformed into a highly reactive hydroxyl radical °OH (Haber-Weiss reaction). In view of this, we have undertaken this study to investigate whether iron is involved in the reperfusion syndrome and therefore could aggravate free radicals injury. Coronary effluent iron concentrations and cardiac cytosolic iron levels were evaluated in rat hearts subjected to an ischemia/reperfusion sequences. In the case of total ischemia, iron concentration in coronary effluents peaked immediately in the first sample collected upon reperfusion. However, in the case of partial ischemia, iron concentration in coronary effluents peaked rather exclusively during ischemia period. Cardiac cytosolic iron level augmented significantly after 30 min of total ischemia and non significantly in the other ischemia protocols compared to perfused control hearts. It also appears that the iron released is not protein-bound, and could therefore have a marked catalytic activity. The results of the present study suggest that in the oxygen paradox, iron plays an important role in inducing alterations during reoxygenation.

Time-course of changes in plasma levels of trace elements after thrombolysis during the acute phase of myocardial infarction in humans

AbstractIt has been suggested that the injury induced by reperfusion of the ischemic myocardium could result, in part, from the cytotoxic effects of oxygen free radicals. Since various trace elements are involved in several of the reactions leading to free radical production, we have measured plasma levels of copper, zinc, selenium, and iron:1.In 18 patients (mean age 60 yr old) subjected to thrombolytic therapy within 6 h after the onset of a myocardial infarction (G1);2.In 16 patients with coronary artery disease, but without a history of a previous myocardial infarction (MI) (mean age 50 yr old, G2); and3.In 50 healthy volunteers divided into two subgroups according to age (mean age 33 yr old, G3 and 55 yr old, G4). Plasma myosin levels were used to estimate quantitatively the extent of the infarcted mass. Plasma trace element levels were measured in blood samples following centrifugation and storage at −80°C.The main results were as followed: In G1 patients who have been subjected to thrombolysis, an important release of myosin was measured in plasma, with a peak at D6 (1678 vs 95 μU/L at H0). In those G1 patients after MI:1.A significant increase in plasma copper levels was observed from day 4 to day 10 postinfarction (×1.15 in reference to the baseline data at H0);2.A decrease in plasma zinc levels was observed and was maximum 12 h after the onset of the thrombolytic treatment;3.A decrease in selenium concentration was observed in G1, as well as in G2 patients, compared to the control groups (80% of G3 and G4 values); and4.A significant decrease in plasma iron levels was observed in G1 (67.8% of G3 and G4 control values) and was significant from H0 to day 7 (p<0.01). In conclusion, this study underlined the time-course evolution of plasma trace element levels in the followup of patients who have been subjected to thrombolysis following a MI and the potential prognostic implication of such variations.