Normothermic Ischaemic Cardiac Arrest Research Articles

The role of alpha 1-adrenergic stimulation in inositol phosphate metabolism during post-ischaemic reperfusion

The aim of this study was to elucidate the mechanism of enhanced inositol phosphate metabolism during reperfusion. Inositol phosphate stores were prelabelled by perfusing isolated rat hearts for 1 h with [ 3H] inositol (1.5 uCi/ml). LiCl (10 mM) and prazosin (0.3 uM) were subsequently added 15 min before (i) 20 min control perfusion; (ii) 20 min normothermic ischaemic cardiac arrest (NICA); (iii) 20 min NICA followed by 1 min reperfusion. The ventricles were freeze-clamped before determination of isotopical incorporation of [ 3H] inositol into the inositol phosphates (Dowex anion exchange chromatography) and InsP 3 levels (Amersham InsP 3 assay system). In addition, noradrenaline release into the perfusate was also assessed (HPLC and electrochemical detection). The results showed: (i) increased noradrenaline release into the perfusate immediately after the onset of reperfusion; (ii) significant depression of [ 3H] inositol incorporation into inositol phosphates and InsP 3 levels after 20 min NICA; (iii) reperfusion caused an immediate significant increase in isotopical incorporation of [ 3H] inositol into inositol phosphates as well as InsP 3 levels; (iv) the alpha 1-adrenergic blocker, prazosin (0.3 uM), completely inhibited the reperfusion-induced increase in inositol phosphate metabolism. These observations suggested that increased alpha 1-adrenergic receptor stimulation by noradrenaline might be responsible for the stimulation of ventricular inositol phosphate metabolism during postischaemic reperfusion.

The protective effect of desferal on rat myocardial mitochondria is not prolonged after withdrawal of desferal.

Reperfusion of ischaemic myocardium is necessary to sustain tissue viability (without it the tissue becomes necrotic), but reperfusion, on the other hand, can damage cells which have survived ischaemia. There is now considerable evidence that oxygen radicals, especially hydroxyl radicals produced via the Haber-Weiss and Fenton reactions, are responsible for reperfusion damage. Various investigators have reported that desferal, an iron chelator, has a beneficial effect on the myocardium during ischaemia and reperfusion. The aim of this study was two-fold: i) whether superoxide anions in the absence of LMWI can impair mitochondrial function, and ii) whether the protective effect of desferal on the mitochondrial function persists after withdrawal of desferal. Experiments were done on isolated rat hearts subjected to normothermic ischaemic cardiac arrest (NICA), with or without desferal, followed by 15-min reperfusion with desferal, followed by 15-min perfusion without desferal, or a hypoxanthine/xanthine oxidase medium that generates superoxide anions (with or without desferrioxamine (desferal) in the perfusate). Mitochondrial function (QO2 (state 3), ADP/O and OPR) as well as LMWI were measured. Our results indicated that i) superoxide anions and/or hydrogen peroxide can, independently of LMWI, damage the mitochondria, and ii) withdrawal of desferal after the respiratory burst resulted in the same or more severe mitochondrial damage than without any desferal.

Increased myocardial inositol trisphosphate levels during α1-adrenergic stimulation and reperfusion of ischaemic rat heart

Although stimulated [ 3H] inositol phosphate turnover has been demonstrated in isolated, perfused [ 3H] inositol prelabelled rat hearts, there is still no information regarding Ins(1,4,5)P 3 levels in intact cardiac muscle. Using a D-myo-Ins(1,4,5)P 3 assay system, Ins(1,4,5)P 3 levels were determined in isolated perfused rats hearts during ischaemia, reperfusion and α 1-adrenergic stimulation via noradrenaline (3 × 10 −5 m). Control hearts contained ± 674 pmols Ins(1,4,5)P 3/g dry heart weight. Myocardial Ins(1,4,5)P 3 levels were significantly decreased (± 389 pmols/g dry heart weight) after exposure to 20 mins of normothermic ischaemic cardiac arrest (NICA). Reperfusion produced a marked increase in Ins(1,4,5,)P 3 levels (± 1 115 pmols/g dry heart weight) after only 30 s. Noradrenaline caused a 3–4 fold increase in tissue Ins(1,4,5)P 3 levels within 30 s. After 20 mins stimulation with noradrenaline, the Ins(1,4,5)P 3 levels were still significantly elevated. The rise in tissue Ins(1,4,5)P 3 levels during reperfusion as well as during noradrenaline administration was counteracted by neomycin (0.5 × 10 −3 m), an inhibitor of phosphoinositidase specific phospholipase C. In both events neomycin restored the Ins(1,4,5)P 3 levels to control values. For correlation of tissue Ins(1,4,5)P 3 levels with mechanical events, noradrenaline (3 × 10 −5 m), in the presence of 10 m m LiCl, 10 −7 m propanolol and 10 −7 m atropine, was administered to isolated perfused rat hearts and the mechanical performance recorded over a period of 20 mins. Noradrenaline caused a significant increase in peak systolic pressure and work performance which was maintained for at least 10 mins, suggesting that the positive inotropic effects of noradrenaline may be provoked by Ins(1,4,5)P 3. Furthermore, the finding that 20 min NICA followed by 30 s reperfusion causes an immediate significant increase in Ins(1,4,5)P 3 content suggests a role for the phosphatidylinositol pathway in the intracellular Ca 2+ overloading, characteristic of ischaemia-reperfusion.

Normothermic ischemic cardiac arrest in the isolated working rabbit heart: Effects of dl-nebivolol and atenolol

The effect of pretreatment with selective beta 1-adrenoceptor blockers (dl-nebivolol or atenolol) on myocardial mechanical activity, mitochondrial function, morphology, and calcium cytochemistry was studied during normothermic ischemic arrest and reperfusion of isolated working rabbit hearts. The hearts subjected to 25 min of ischemia followed by 30 min of post-ischemic reperfusion showed typical signs of severe myocardial ischemic damage. The ultrastructural changes showed a good relation with the changes in mechanical activity and mitochondrial function. To determine whether these changes could be prevented or reduced by beta 1-adrenoceptor blockade, dl-nebivolol or atenolol (0.62 mg/liter) was added to the perfusate 30 min before the induction of ischemia. The results showed that dl-nebivolol exerted a protective effect on recovery of mechanical activity, on mitochondrial function during reperfusion as well as on the ultrastructure as examined at the end of the reperfusion period. On the other hand, atenolol failed to protect the myocardium against ischemia-reperfusion damage in the isolated working rabbit heart.

The effect of desferal on rat heart mitochondrial function, iron content, and xanthine dehydrogenase/oxidase conversion during ischemia-reperfusion

Cardiac mitochondrial function as measured by oxidative phosphorylation is impaired by ischemia; and, this deteriorates even further on reperfusion of the heart. Free oxygen radicals, especially the formation of hydroxyl radicals via the iron-catalyzed Haber-Weiss and Fenton reactions have been implicated in the reperfusion injury. In this study, the effect of desferrioxamine (desferal) in the perfusate on mitochondrial function of isolated rat hearts during different periods of normothermic ischemic cardiac arrest (NICA), and subsequent reperfusion was investigated. Mitochondrial functions measured were the QO2 (state 3); ADP/O ratio and oxidative phosphorylation; the mitochondrial, loosely bound (chelateable) iron (LB-iron); the xanthine dehydrogenase and xanthine oxidase activities. Inclusion of desferal in the perfusion solution significantly improved mitochondrial function during the different NICA periods, and prevented the deterioration of mitochondrial function resulting from reperfusion. Desferal did not significantly affect the LB-iron content of the mitochondria or the ratio of xanthine dehydrogenase/xanthine oxidase activities in the mitochondria during NICA or reperfusion. Our experiments suggest that iron, which is free to be chelated by desferal, plays a role in this injury to the rat myocardium.

Effect of normothermic ischemic cardiac arrest and of reperfusion on the free oxygen radical scavenger enzymes and xanthine oxidase (a generator of superoxide anions).

Recent evidence suggests that free oxygen radicals are produced by ischaemic tissues, accounting for at least part of the damage that results. These free oxygen radicals are produced by xanthine oxidase, amongst others, and removed by scavenger enzymes (catalase, superoxide dismutase and glutathione peroxidase) and anti-oxidants. As mitochondria are oxygen-utilising organelles, they are capable of producing free oxygen radicals. Our results indicate that the removal of free oxygen radicals are not diminished during ischaemia, but the activity of the free oxygen radical generator, xanthine oxidase, is increased. This could lead to an increased superoxide anion concentration.

Protective effects of amiodarone pretreatment on mitochondrial function and high energy phosphates in ischaemic rat heart

The effects of the antianginal and antiarrhythmic drug amiodarone on mitochondrial function and high-energy phosphate content were assessed during normothermic ischaemic cardiac arrest and reperfusion in Langendorff-perfused rat heart. Total ischaemia for 30 min at 37 degrees C produced highly significant changes in mitochondrial oxidative phosphorylation and high-energy phosphate content. Pretreatment of the rats with one single dose of amiodarone (20 mg/kg i.v., 30 min before killing) markedly attenuated the deleterious effect of ischaemia on mitochondrial function and slightly reduced ATP depletion. In normally perfused hearts, amiodarone pretreatment did not modify any parameter of mitochondrial respiratory function nor did it influence high-energy phosphate or glycogen content. After reperfusion for 15 min, amiodarone-treated hearts showed improved recovery of mitochondrial oxidative phosphorylation and tissue high-energy phosphate content as compared to control hearts. Pretreatment of hearts with amiodarone did not reduce ischaemia-induced leakage of total adenylic nucleotides but highly significantly reduced lactate dehydrogenase release during reperfusion. These results indicate that amiodarone could exert substantial protection on the infarcting myocardium.

Normothermic ischemic cardiac arrest and reperfusion of the isolated working rat heart: Effect of carbocromene pretreatment on functional and metabolic recovery

Normothermic Ischemic Cardiac Arrest and Reperfusion of the Isolated Working Rat Heart: Effect of Carbocromene Pretreatment on Functional and Metabolic Recovery. This paper describes the effect of carbocromene pretreatment on the functional recovery of the isolated rat heart submitted to ischemic cardiac arrest and reperfused. In a previous study we showed that hearts isolated from rats which had been pretreated for 8 days with a daily oral administration of carbocromene at 15 mg/kg body weight, exhibit higher mechanical performances (aortic output, coronary flow, cardiac work) than hearts isolated from untreated animals. This was associated with a greater tissue content of high energy phosphates and glycogen. In the present work, carbocromene-pretreated hearts are submitted to 30 minutes of normothermic no-flow ischemia after being arrested by a 2 min high potassium, substrate-free perfusion, containing 2 mg/l carbocromene (cardioplegic solution). After reperfusion, post-ischemic recovery of function is significantly better in treated hearts as compared to control untreated preparations submitted to a similar protocol. Although no significant difference can be demonstrated in the metabolic status of either groups of preparations after 30 min of reperfusion, lactate dehydrogenase release, taken as an index of myocardial cell damage, is significantly reduced in the carbocromene-treated group. In view of these results it is suggested that carbocromene pretreatment and/or the addition of carbocromene to cardioplegic solutions could be beneficial in improving functional recovery after temporary ischemic cardiac arrest.

Normothermic ischaemic cardiac arrest of the isolated perfused rat heart: effects of trifluoperazine and lysolecithin on mechanical and metabolic recovery.

To evaluate the hypothesis that maintenance of the integrity of myocardial membrane systems and prevention of Ca2+ influx into the cell are significant in the survival of ischaemic tissue, the effect of trifluoperazine and lysolecithin, were tested on the recovery of globally ischaemic rat hearts. Trifluoperazine increases membrane stabilization, inhibits calmodulin and binds to other Ca2+-dependent proteins. Lysolecithin, on the other hand, has a detergent action on myocardial cell membranes and facilitates Ca2+ ingress in ischaemic tissue. With trifluoperazine (2.45 microM), added before induction of ischaemia or during reperfusion only, hearts subjected to 40 min normothermic ischaemic cardiac arrest recovered mechanically. Untreated hearts failed after 20 min of ischaemia. The drug had no effect on tissue high energy phosphate levels or mitochondrial oxidative phosphorylation. Conversely, lysolecithin (2.5-10 microM) caused all hearts to fail after being subjected to 15 min ischaemia. Mechanical failure during reperfusion of such hearts was associated with a significant reduction in tissue ATP and CrP levels. Trifluoperazine counteracted the harmful effects of lysolecithin to a limited extent.

Normothermic ischemic cardiac arrest of the isolated working rat heart. Effects of time and reperfusion on myocardial ultrastructure, mitochondrial oxidative function, and mechanical recovery.

The ischemic state of the myocardium of the isolated working rat heart after induction of normothermic ischemic cardiac arrest was assessed by the interrelationship among changes in myocardial ultrastructure, mitochondrial oxidative phosphorylation, and tissue high energy phosphate contents. At all time intervals (10-40 minutes) studied, the ultrastructural changes were more severe in the subendocardium than in the subepicardium. After 25-40 minutes of normothermic ischemic cardiac arrest, the mitochondrial oxygen uptake (state 3) became increasingly depressed, particularly in mitochondria isolated from the subendocardium. Mitochondrial oxidative function, as measured in vitro, did not correlate well with mitochondrial ultrastructural damage. In addition, the effects of coronary reperfusion on the ability of the ischemic heart to recover in terms of ultrastructure, mechanical, and metabolic function were evaluated. Hearts subjected to 10-40 minutes of normothermic ischemic cardiac arrest showed almost complete ultrastructural recovery of the subepicardium upon reperfusion; regression of ultrastructural changes occurred to a lesser extent in the subendocardium. Reperfusion for 30 minutes did not alleviate the depression in mitochondrial oxidative function, while tissue ATP levels did not return to control, preischemic levels. After 20 minutes of normothermic ischemic cardiac arrest, the mechanical performance of the working heart during reperfusion was significantly depressed, compared with pre-ischemic control values. Normal ultrastructure of the subendocardium always accompanied mechanical recovery, while improvement of mitochondrial oxidative function was not essential.

Normothermic ischaemic cardiac arrest and reperfusion of the isolated working heart: effect of chlorpromazine on functional, metabolic and morphological recovery.

The effects of chlorpromazine, an inhibitor of both Ca2+ flux and phospholipase activity, on myocardial ultrastructure, function and metabolism were assessed during normothermic ischaemic cardiac arrest and reperfusion of the isolated working rat heart. Normothermic ischaemic cardiac arrest produced significant changes in myocardial ultrastructure, high energy phosphate contents and mitochondrial oxidative phosphorylation within 20 min. Reperfusion of untreated hearts subjected to 20 and 25 min ischaemia failed to restore mitochondrial function, mechanical activity and ATP content to control, pre-ischaemic levels. Morphological signs of ischaemic injury regressed, especially in the subendocardial layer. Pretreatment of hearts with chlorpromazine did not prevent the ischaemia-induced changes in myocardial ultrastructure and mitochondrial function. However, during reperfusion the chlorpromazine-treated, totally ischaemic heats (20 to 25 min) exhibited improved coronary flow rates, and ultrastructural and mechanical recovery. The mitochondrial oxidative phosphorylation process and tissue high energy phosphate contents were not affected by the drug.

Normothermic ischaemic cardiac arrest of isolated working rat heart: effects of reserpine and propranolol on functional, metabolic and morphological recovery.

The ability to preserve myocardial structural and functional integrity during extended periods of total ischaemia has practical clinical significance. The role of endogenous catecholamines in the onset of irreversible damage in global ischaemia of the isolated rat heart was assessed by beta-blockade or catecholamine depletion. The effects of propranolol and reserpine pretreatment on myocardial ultrastructure, function and metabolism were studied during normothermic ischaemic arrest (NICA) and reperfusion of the isolated working rat heart. beta-Blockade as well as catecholamine depletion resulted in an increase in the percentage of totally ischaemic hearts which recovered mechanically upon reperfusion. In these studies mechanical recovery during reperfusion was associated with reversal of ultrastructural ischaemic alterations, but without an improvement in mitochondrial function. These findings support the concept that failure of mitochondria to recover functionally upon reperfusion is not the cause of either irreversible mechanical failure or ultrastructural damage of the ischaemic myocardium.