Godelieve Vandeplassche

Singlet oxygen and myocardial injury: Ultrastructural, cytochemical and electrocardiographic consequences of photoactivation of rose bengal

Photoactivation of rose bengal leads to the generation of reactive oxygen intermediates (predominantly singlet oxygen with some superoxide anion) which are potentially injurious to biological systems. Isolated rat hearts were perfused aerobically at 37 degrees C with bicarbonate buffer for 10 min without rose bengal and for 10 min with rose bengal (500 nM). During the last 5 min of perfusion with rose bengal, hearts were globally illuminated (5500 lux) with light (530 to 590 nm) and electrocardiographic changes were detected within 2.7 +/- 0.3 s (approximately 15 beats) of the onset of illumination. All hearts developed ventricular premature beats, ventricular tachycardia and complete atrioventricular block after 20.2 +/- 6.6, 68.0 +/- 29.7 and 184.3 +/- 20.9 s, respectively. Photoactivation by rose bengal also resulted in severe ultrastructural damage including intracellular clarifications, swelling of mitochondria with disruption and clumping of cristae and the development of contraction band necrosis. Extensive degranulation of mast cells was also observed. These changes were most evident in myocytes adjacent to large epicardial blood vessels. Cytochemical studies demonstrated that there was a loss of the calcium which is normally localized at the inner sarcolemmal surface, and the appearance of intramitochondrial calcium precipitates. In control hearts (no illumination and/or no rose bengal), arrhythmias did not develop and tissue morphology and calcium distribution remained normal. In additional studies, rose bengal-perfused hearts were illuminated regionally for 10 min over an area (approximately 6 mm2) of the left ventricle. Extensive tissue injury and calcium overload developed in the area of maximum illumination.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method to study activated oxygen species induced damage in cardiomyocytes and protection by Ca2+-antagonists

It has been proposed that oxygen derived free radicals contribute to reperfusion injury in ischemic tissue: radical induced lipid peroxidation is believed to cause membrane destruction, eventually evolving to cell death. A method is introduced which investigates the effect of exogenously generated reactive O2 species on isolated Ca2+-tolerant rat cardiomyocytes. Singlet oxygen (O2(1)), generated by photo-excitation of the photosensitive dye rose bengal, induced the transformation of elongated rod-shaped cells into hypercontracted rounded cardiomyocytes. These shape changes were prevented by removal of extracellular Ca2+ or by addition of radical scavengers. Pre-treatment with various classes of Ca2+-antagonists dose-dependently reduced the number of hypercontracted cardiomyocytes after exposure to O2(1). Compounds not active on the slow Ca2+-channel (e.g. flunarizine-like) provided a better degree of protection than the genuine slow Ca2+-channel blockers (e.g. dihydropyridines). Ultrastructurally, cardiomyocytes exposed to O2(1) showed a loss of cytochemically demonstrable sarcolemma-associated Ca2+ and the presence of clustered Ca2+-deposits in the mitochondria. Drug pre-treated cells displayed a Ca2+-distribution pattern comparable to unchallenged control cells.

Mitochondrial hydrogen peroxide generation by NADH-oxidase activity following regional myocardial ischemia in the dog.

Recently, an exogenous NADH-oxidase has been shown to be a source of oxygen derived toxic species in heart mitochondria. This enzyme uses NADH and oxygen to form superoxide radicals and hydrogen peroxide. Growing evidence suggests that oxygen radicals and hydrogen peroxide may contribute to cardiac damage during ischemia or hypoxia. The activity of the enzyme NADH-oxidase could play an important role in the damage caused by oxygen derived toxic species, especially since cellular defense mechanisms against free radicals are depleted under ischemic conditions. In this study, a cytochemical method was used to visualize hydrogen peroxide, the reaction product of NADH-oxidase activity, in normal and ischemic dog myocardium. The NADH-oxidase reaction product was present in weak amounts in mitochondria from normoxic myocardium. In viable ischemic areas a high degree of activity was observed in the mitochondria. In infarcted tissue mitochondria contained few or no reaction product at all. The results support the hypothesis that hydrogen peroxide and oxygen radicals produced in the mitochondria by a high NADH-oxidase activity may contribute to the mitochondrial damage observed during ischemia when NADH is no longer oxidized by the respiratory chain and cellular defense mechanisms are impaired.

Pathophysiology of cardiomyocytes.

Isolated cardiomyocytes lend themselves very well to the quantification of pathological damage and to the determination of reversible versus irreversible changes. These single cells were used to study the cellular response to a variety of pathologic stimuli that impair structure and function. Degenerative alterations are accompanied by hyperactivation and irreversible rounding up of otherwise quiescent rod-shaped cells. Stereotypic degenerative changes and loss of sarcolemma-bound Ca2+ were seen during prolonged severe hypoxia, exposure to either depolarizing concentrations of potassium, veratrine, acylcarnitines, cationic amphiphiles, free-radical-generating systems, cardiac glycosides, or uncouplers of oxidative phosphorylation. Since the presence of extracellular Ca2+ is a prerequisite to obtain cell degeneration in most of these aggressive insults and since cellular Ca2+ overload parallels the damage, we studied the influence of representative compounds of the various subclasses of Ca2+ antagonists: verapamil, nifedipine, nicardipine, and diltiazem (Ca2+ blockers with high affinity for cardiac slow Ca2+ channels), cinnarizine, flunarizine, lidoflazine, and mioflazine (Ca2+ blockers with no affinity for cardiac slow Ca2+ channels). The non-slow-channel-blocking drugs were generally superior in protection against the imposed insults suggesting that prevention of Ca2+ overload is not correlated with slow channel blockade. For the latter group of drugs, other (hitherto not elucidated) mechanisms of membrane-drug interactions seem to be responsible for the preservation of Ca2+ homeostasis during the induction of pathological Ca2+ influx.

Effects of β-adrenoceptor antagonists on cardiac function in ischemic-reperfused myocardium of the isolated working rabbit heart

The effects of beta-adrenoceptor antagonists (dl-nebivolol, atenolol and propranolol) and of 1-nebivolol on cardiodynamics and mitochondrial oxidative phosphorylation were studied in the isolated working rabbit heart subjected to normothermic global ischemia, followed, in some cases, by reperfusion. The hearts were pretreated with the different drugs (0.32 mg/l) 30 min before the start of ischemia, dl-Nebivolol and propranolol provided protection for both cardiodynamic and mitochondrial functions, as did l-nebivolol, which lacks beta-adrenoceptor blocking properties, while atenolol failed to protect mechanical activity and cardiac mitochondria against the effects of ischemia and post-ischemic reperfusion. Catecholamine depletion with reserpine did not have a beneficial effect on the recovery of cardiodynamic and mitochondrial function during post-ischemic reperfusion. It is concluded that the beneficial effects of beta-blockers on the ischemic and reperfused myocardium can not be explained by a specific beta-blocking action alone.

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

SummaryThe effect of pretreatment with selective β1-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 β1-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.

Differential effects of thromboxane A2 synthase inhibition, singly or combined with thromboxane A2/prostaglandin endoperoxide receptor antagonism, on occlusive thrombosis elicited by endothelial cell injury or by deep vascular damage in canine coronary arteries.

In open-chest dogs, cyclic flow reductions (CFRs, 5.1-6.6/hr in controls; n = 24) caused by platelet deposition/dislodgment at sites of endothelial cell injury in critically stenosed left anterior descending coronary arteries (59% flow reduction) were attenuated to the same extent either by single thromboxane A2 (TXA2) synthase inhibition (0.31 mg/kg i.v. ridogrel; CFR, 0.16 +/- 0.16/hr; n = 6; p less than 0.05) or by a comparatively modest degree of TXA2/prostaglandin endoperoxide receptor antagonism on top of TXA2 synthase inhibition (5 mg/kg i.v. ridogrel; CFR, 0.22 +/- 0.1/hr; n = 10; p less than 0.05). By contrast, occlusive thrombosis on deep vascular damage elicited by intraluminal stimulation (150-microA anodal constant current) in nonpreconstricted canine coronary arteries (time to occlusion, 237.1 +/- 13.9 minutes; n = 7; incidence of occlusion within 300 minutes, six of seven experiments) was not affected by platelet cyclooxygenase inhibition (5 mg/kg i.v. acetylsalicylic acid; n = 7), single TXA2 synthase inhibition (1.25 mg/kg i.v. ridogrel; n = 7), or single TXA2/prostaglandin endoperoxide receptor antagonism (10 mg/kg + 10 mg/kg/hr i.v. sulotroban for 300 minutes; n = 5). However, such an occlusive thrombus formation was significantly reduced by combined TXA2 synthase/prostaglandin endoperoxide receptor inhibition (5 mg/kg i.v. ridogrel; time to occlusion greater than 300 minutes, n = 7; incidence of occlusion within 300 minutes, one of seven experiments; p less than 0.05). This study reveals 1) a differential efficacy of TXA2 synthase inhibition, singly or combined with TXA2/prostaglandin endoperoxide receptor antagonism, depending on the extent of the vessel wall lesion triggering thrombosis and the size of the thrombus required to obstruct the vascular lumen and 2) a significant synergism in preventing occlusive thrombosis of extensively damaged coronary arteries between strong TXA2 synthase inhibition and comparatively modest TXA2/prostaglandin endoperoxide receptor antagonism with ridogrel.

Cytochemical evidence of NADH-oxidase activity in the isolated working rabbit heart subjected to normothermic global ischaemia

SummaryThe cytochemical localization of NADH-oxidase, a possible source of oxygen derived toxic species was studied in the isolated working rabbit heart subjected to normothermic global ischaemia. The activity of this oxidase could be important for the damage observed during ischaemia, when cellular defence mechanisms against free radicals are depleted. In non-ischaemic myocardium only small amounts of the NADH-oxidase reaction product were present in the mitochondria. Although the reaction product could already be observed after 45 min of incubation, prolonged incubation times up to 2 h were necessary to clearly define these reactive sites. The reaction product is substrate dependent and is not affected by cyanide. Exposure of the hearts to ischaemia resulted in an alteration of the enzyme activity depending on the degree of ischaemic damage. In ultrastructurally slightly altered areas a high degree of cytochemical study supports the hypothesis that hydrogen peroxide and oxygen radicals produced in the mitochondria by NADH-oxidase activity may contribute to the mitochondrial damage observed during ischaemia when NADH is no longer oxidized by the respiratory chain and cellular defence mechanisms are impaired.

Ultrastructural damage and Ca2(+)-shifts in the canine myocardium subjected to regional incomplete ischemia.

SummaryThe role of Ca2+ in the pathogenesis leading to ischemic myocardial cell death is still controversial. To gain insight into this phenomenon a cytochemical procedure, the phosphate pyroantimonate method, was used to localize different subcellular Ca2+-pools at the ultrastructural level. After 45 min of left anterior descending coronary artery (LAD) occlusion, the coronary arteries were perfused with triphenyltetrazoliumchloride staining (TTC) to identify viable ischemic and infarcted tissue. In non-ischemic tissue, Ca2+-deposits were confined to the sarcolemma, sarcolemma-derived vesicles, transverse tubules, and intercalated disks. In infarcted tissue (TTC-negative), the sarcolemma lost its Ca2+-binding capacity and mitochondria were either overloaded with Ca2+-precipitate or they contained amorphous densities. In viable ischemic areas (determined with the TTC-technique) the sarcolemma was virtually devoid of Ca2+-deposits. Mitochondria in this area frequently showed clumping of the cristae, associated with an accumulation of Ca2+-precipitate in between the clustered cristae. The results of this study indicate that Ca2+-shifts occur in ischemic myocardial cells before the occurrence of other ultrastructural signs of irreversible injury which, therefore, narrows the possibility that Ca2+-overload is only a consequence of ischemic cell death.

Functional and structural impairment in human, rat and guinea-pig atrial muscle in response to in vitro calcium overload: a cytochemical study on cellular calcium distribution

Critical accumulation of cellular calcium in ischaemic myocardium is involved in irreversible cell damage. In human right atrial trabeculae and in rat and guinea-pig left atria, we investigated whether direct calcium overload by increasing the extracellular calcium concentration, [Ca2+]o, leads to similar impairment of function and ultrastructure as observed after ischaemia. The force of contraction was measured during two consecutive cumulative increases in [Ca2+]o (1-25 mmol/l) separated by 30 min of incubation at low [Ca2+]o. Compared to the first Ca2+ challenge, the positive inotropic effect of increasing [Ca2+]o was depressed during the second one and the after-contractions, and the increase in resting tension developing with high [Ca2+]o tended to be larger. The ultrastructure of the tissue fixed immediately after excision was well preserved. When fixed after the second Ca2+ challenge, half of the cells were severely damaged with various signs of cellular Ca2+ overload similar to those observed after ischaemic damage: the sarcolemma lost its Ca(2+)-binding properties, sarcomeres showed contraction band necrosis, the mitochondria had disrupted cristae and contained either large clusters of Ca2+ precipitate or amorphous densities (Jennings granules). In many cells, calcium precipitates were present in the cytoplasm. The morphological and functional changes were similar in the three species studied. Our results suggest that the deterioration of atrial myocardium after challenge with high [Ca2+]o or after severe ischaemia may be traced back to a common mechanism, i.e. the sarcolemma loses its competence as a permeable barrier for Ca2+ and therefore facilitates excessive Ca2+ entry. However, for the direct demonstration of calcium precipitates as a sign of cytosolic Ca2+ overload, high [Ca2+]o are required with are not normally present in the myocardium.