Free radicals and myocardial ischemia and reperfusion injury

There is a growing body of evidence for the role of free radicals in mediating myocardial tissue injury during myocardial ischemia and in particular during the phase of myocardial reoxygenation. Associated with myocardial ischemia and reperfusion is the generation of oxygen-derived free radicals from a variety of sources that include the mitochondrial electron transport chain; the biosynthesis of prostaglandins; the enzyme xanthine oxidase; and circulating elements in the blood, with the polymorphonuclear neutrophil assuming a primary focus of attention. Experimental studies have shown that free radical scavengers (e.g., N-[2-mercaptopropionyl]glycine) and enzymes that scavenge or degrade reactive species of oxygen (superoxide dismutase or catalase) can reduce the mass of myocardial tissue that undergoes irreversible injury. Additionally allopurinol, which inhibits the enzyme xanthine oxidase, reduces ultimate infarct size, putatively by reducing the xanthine oxidase generation of superoxide anion. Neutrophils that enter the ischemically injured myocardium under the influence of chemotactic attraction and activation of the complement system generate and release highly reactive and cytotoxic oxygen derivatives that are destructive to the vascular endothelium and to the cardiac myocytes. Studies have documented that neutrophil depletion or suppression of neutrophil function (ibuprofen, nafazatrom, BW 755C, or more recently with prostacyclin or iloprost) results in a significant salvage of myocardial tissue that is subjected to a period of regional ischemia followed by reperfusion. Our current understanding of the events associated with myocardial ischemia suggests that within the ischemic myocardial region or area at risk, there is a population of cells that are reversibly injured and that reperfusion within a specified period (less than 3 hours) of time is capable of restoring the majority of the jeopardized cells to a normal status, but that the act of reperfusion itself will lead to the sudden demise of a fraction of the cells because of the cytotoxic effects of reactive species of oxygen derived from one or more of the sources indicated above. The efforts to minimize the amount of tissue that undergoes cell death as a result of myocardial ischemia demand that early reperfusion be established. However, the reintroduction of molecular oxygen and the circulating elements of the blood will be associated with an "explosive" and self-limited destruction of some of the myocardial cells in the area at risk.(ABSTRACT TRUNCATED AT 400 WORDS)     

Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone

Recent studies suggest that oxygen free radicals may mediate postischemic myocardial dysfunction ("stunning"), but all the evidence for this hypothesis is indirect. Thus, we used electron paramagnetic resonance (EPR) spectroscopy and the spin trap, alpha-phenyl N-tert-butyl nitrone (PBN), to directly investigate whether free radicals are produced after a 15-min coronary artery occlusion and subsequent reperfusion in 30 open-chest dogs. After intracoronary infusion of PBN, EPR signals characteristic of oxygen- and carbon-centered radical adducts were detected in the venous blood draining from the ischemic/reperfused vascular bed. The myocardial release of PBN adducts began during coronary occlusion but increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of radicals abated but did not cease, persisting up to 3 h after reflow. The EPR spectra (aH beta = 2.67-2.79 G, aN = 14.75-15.00 G) were consistent with the trapping by PBN of secondary oxygen- and carbon-centered radicals, such as alkoxy and alkyl radicals, which could be formed by reactions of primary oxygen radicals with membrane lipids. There was a linear, direct relationship between the magnitude of PBN adduct production and the degree of ischemic flow reduction. Recovery of contractile function (measured as systolic wall thickening) after reperfusion was greater (P less than 0.05) in dogs given PBN than in controls. This study demonstrates that reversible regional myocardial ischemia in the intact animal is associated with prolonged free radical generation, and that the intensity of such generation is related to the severity of ischemia. The results provide direct evidence to support the hypothesis that reactive oxygen metabolites contribute to the persistent contractile dysfunction (myocardial stunning) observed after brief ischemia in vivo.     

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.     

Effects of exogenous free radicals on electromechanical function and metabolism in isolated rabbit and guinea pig ventricle. Implications for ischemia and reperfusion injury.

Oxygen-derived free radicals have been implicated in the pathogenesis of cardiac dysfunction during ischemia, postischemic myocardial "stunning," and reperfusion injury. We investigated the effects of oxygen-derived free radicals on cardiac function in intact isolated rabbit hearts and single guinea pig ventricular myocytes. In the intact rabbit ventricle, exposure to free radical-generating systems caused increased cellular K+ efflux, shortening of the action potential duration, changes in tension, and depletion of high energy phosphates similar to ischemia and metabolic inhibition. In patch-clamped single ventricular myocytes, free radical-generating systems activated ATP-sensitive K+ channels, decreased the calcium current, and caused cell shortening by irreversibly inhibiting glycolytic and oxidative metabolism. The results suggest that free radicals generated during ischemia and reperfusion may contribute to electrophysiologic abnormalities and contractile dysfunction by inhibiting glycolysis and oxidative phosphorylation. Inhibition of metabolism by free radicals may be an important factor limiting functional recovery from an ischemic insult after reestablishment of effective blood flow.     

Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion

Cardiac mitochondria sustain damage during ischemia and reperfusion, contributing to cell death. The reversible blockade of electron transport during ischemia with amobarbital, an inhibitor at the rotenone site of complex I, protects mitochondria against ischemic damage. Amobarbital treatment immediately before ischemia was used to test the hypothesis that damage to mitochondrial respiration occurs mainly during ischemia and that protection of mitochondria during ischemia leads to decreased cardiac injury with reperfusion. Langendorff-perfused Fischer-344 rat hearts were treated with amobarbital (2.5 mM) or vehicle for 1 min immediately before 25 min of global ischemia. Both groups were reperfused for 30 min without additional treatment. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after reperfusion. Ischemia and reperfusion decreased state 3 and increased state 4 respiration rate in both SSM and IFM. Amobarbital treatment protected oxidative phosphorylation measured following reperfusion and improved the coupling of respiration. Cytochrome c content measured in SSM and IFM following reperfusion decreased in untreated, but not in amobarbital-treated, hearts. H(2)O(2) release from SSM and IFM isolated from amobarbital-treated hearts during reperfusion was markedly decreased. Amobarbital treatment before ischemia improved recovery of contractile function (percentage of preischemic developed pressure: untreated 51 +/- 4%, n = 12; amobarbital 70 +/- 4%, n = 11, p < 0.01) and substantially reduced infarct size (untreated 32 +/- 2%, n = 7; amobarbital 13 +/- 2%, n = 7, p < 0.01). Thus, mitochondrial damage occurs mainly during ischemia rather than during reperfusion. Reperfusion in the setting of preserved mitochondrial respiratory function attenuates the mitochondrial release of reactive oxygen species, enhances contractile recovery, and decreases myocardial infarct size.     

Global ischemic duration and reperfusion function in the isolated perfused rat heart

Post-ischemic myocardial dysfunction has been observed in a variety of clinical situations including cardiac arrest. Potentially survivable cardiac arrest following short-term global myocardial ischemia may be of insufficient duration to cause irreversible myocyte injury, but still results in contractile and bioenergetic dysfunction. The purpose of this study was to characterize the ischemic transition from reversible to irreversible injury in the isolated perfused rat heart. Isolated, buffer perfused, male Sprague-Dawley rat hearts underwent normothermic ischemia of 15, 20, 25 or 30 min with or without 30 min of reperfusion and were freeze clamped in liquid nitrogen for bioenergetic analysis of LV tissue. Post-ischemic LV function and measurements of bioenergetic recovery were made between groups and with non-ischemic controls. Baseline LV function was similar in all groups. Post-ischemic contractile function was markedly depressed in the 25 and 30 min ischemia groups with persistent depression of high-energy phosphates, total adenine nucleotide pool, myocardial oxygen consumption, elevated CK release and evidence of significant mitochondrial edema in the 30 min group. In contrast with longer ischemic periods, the reduction in LV contractile function after 15 and 20 min of ischemia was mild, with more complete bioenergetic recovery, minimal CK release, and normal appearing mitochondrial. This data suggests a period of transition from reversible to irreversible injury occurring at approximately 20 min of normothermic global ischemia in the isolated perfused rat heart.     

Reduction of ischemia--reperfusion induced myocardial infarct size in rats by caffeic acid phenethyl ester (CAPE)

Myocardial ischemia--reperfusion (MI/R) represents a clinically relevant problem associated with thrombolysis, angioplasty, and coronary bypass surgery. MI/R injury is known to occur on restoration of coronary flow after a period of myocardial ischemia. Injury of myocardium caused by I/R includes cardiac contractile dysfunction, arrhythmias, as well as irreversible myocyte damage. Prevention of myocardial death in acute coronary syndromes is the immediate goal of therapy. The main factor concerned with the experimental generation of reperfusion damage is oxygen-derived free radicals. This MI/R injury has been shown to be salvaged by supplementing antioxidants to diseased hearts. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, has antioxidant and anti-inflammatory properties, and may function in cardiac protection against I/R-induced damage. To test this hypothesis, we randomly assigned 14 male Wistar rats for necrosis experiments. To produce myocardial necrosis, the left main coronary artery was occluded for 30 min, followed by 120 min of reperfusion in anesthetized rats. CAPE (50 microM kg-1) was given intravenously 10 min before occlusion and continued during ischemia by infusion pump. The volume of infarct and the risk zone was determined by planimentry of each tracing and multiplying by the slice thickness. Infarct was normalized by expressing it as a percentage of the area at risk. Compared to control group, CAPE administration statistically reduced the myocardial infarct size/area of risk zone (50 +/- 4% and 32 +/- 6%, respectively) and the myocardial infarct size (23 +/- 3% and 9 +/- 4%, respectively) in rat model of ischemia-reperfusion. In conclusion, this result shows that CAPE is important in reducing I/R-induced myocardial damage.     

Effect of Oxygen Free Radicals on Cardiac Contractile Activity and Sarcolemmal Na(+)-Ca(2+) Exchange

BACKGROUND: Although oxygen free radicals have been shown to induce myocardial cell damage and cardiac dysfunction, the exact mechanism by which these radicals affect the heart function is not clear. Since the occurrence of intracellular Ca(2+) overload is critical in the genesis of cellular damage and cardiac dysfunction, and since the sarcolemmal Na(+)-Ca(2+) exchange is intimately involved in Ca(2+) movements in myocardium, this study was undertaken to examine the effects of oxygen free radicals on the relationship between changes in cardiac contractile force development and sarcolemmal Na(+)-Ca(2+) exchange activity. METHODS AND RESULTS: Isolated rat hearts were perfused with a medium containing xanthine plus xanthine oxidase for different times, and changes in contractile force as well as sarcolemmal Na(+)-(2+) exchange activity were monitored. Perfusion of the heart with xanthine plus xanthine oxidase resulted in a transient increase followed by a marked decrease in contractile activity; the resting tension was markedly increased. The xanthine plus xanthine oxidase-induced depression in developed tension, rate of contraction, and rate of relaxation, except the transient increase in contractile activity, was prevented by the addition of catalase, but not by superoxide dismutase, in the perfusion medium. A time-dependent depression in sarcolemmal Na(+)-Ca(2+) was also evident upon perfusing the heart with xanthine plus xanthine oxidase. This depression in Na(+)-dependent Ca(2+) uptake was associated with a decrease in the maximal velocity of reaction without any changes in the affinity of Na(+)-Ca(2+) exchanger for Ca(2+). The presence of catalase, unlike superoxide dismutase, prevented the decrease in sarcolemmal Na(+)-Ca(2+) exchange activity in hearts perfused with xanthine plus xanthine oxidase. CONCLUSIONS: The results support the view that a depression in the sarcolemmal Na(+)-Ca(2+) exchange activity may contribute to the occurrence of intracellular Ca(2+) overload and subsequent decrease in contractile activity. Furthermore, these actions of xanthine plus xanthine oxidase in the whole heart appear to be a consequence of H(2)O(2) production rather than the generation of superoxide radicals.     

The beneficial effect of 2′-deoxycoformycin in renal ischemia-reperfusion is mediated both by preservation of tissue ATP and inhibition of lipid peroxidation

Renal ischemia injures the renal tubular cell by disrupting the vital cellular metabolic machinery. Further cell damage is caused when the blood flow is restored by oxygen free radicals that are generated from xanthine oxidase. Oxygen radicals cause lipid peroxidation of cell and organelle membranes, disrupting the structural integrity and capacity for cell transport and energy metabolism. In the present study, the possible therapeutic usefulness of the adenosine deaminase inhibitor, 2'-deoxycoformycin (DCF), during renal ischemia and reperfusion injury was investigated. The effects of DCF on renal malondialdehyde (MDA) and ATP levels were studied after 45 min ischemia and 15 min subsequent reperfusion in rat kidneys. MDA levels remained unchanged during ischemia, but increased after the subsequent reperfusion. DCF pretreatment (2.0 mg/kg i.m.) decreased MDA and increased ATP levels during the ischemia-reperfusion period. DCF exerts a dual protective action by facilitating purine salvage for ATP synthesis and inhibiting oxygen radical-induced lipid peroxidation. These results suggest that DCF therapy could be beneficial in the treatment of ischemia-reperfusion renal injuries.     

Delayed protection of the ischemic heart — from pathophysiology to therapeutic applications

Summary— Preconditioning the heart with brief episodes of ischemia paradoxically increases its resistance to subsequent ischemic episodes, and markedly limits infarct size. Although preconditioning is now considered as the most powerful antiischemic intervention known, its beneficial effects are short-lived since they are lost if the reperfusion period after preconditioning is extended past 2–3 h. There is, however, some evidence of a delayed phase of protection, manifest 24 h after the initial preconditioning stimulus, associated with a decrease in infarct size, a prevention of postischemic contractile dysfunction (stunning) and a reduction in endothelial injury. The delayed beneficial effects of preconditioning resemble those induced by prior heat stress, and might be related to the expression of stress proteins (heat shock proteins or HSP). Evidence for a role of HSP derives from observations showing that brief ischemia is a potent stimulus for HSP expression. Moreover, transfection of isolated cells with HSP or overexpression of HSP in transgenic mice renders the myocytes more resistant to ischemia. Once produced, HSP are believed to facilitate protein synthesis, stabilize newly formed proteins and repair denatured ones. Alternatively, delayed preconditioning may be mediated by antioxidant enzymes such as superoxide dismutase or catalase, which are also upregulated by ischemia, and this could lead to a lesser production of oxygen-derived free radicals during reperfusion. Indeed, in isolated myocytes, prevention of hypoxia-induced expression of superoxide dismutase (using an antisense oligonucleotide) abolished the delayed protective effect of preconditioning. Importantly, recent in vivo evidence suggests that the delayed protection may be mediated by adenosine, through activation of A1-receptors, and by stimulation of protein kinase C. Finally, although the exact mechanisms by which preconditioning induces delayed protection are still mostly unknown, the fact that the expression of protective proteins such as HSP can be induced by many other means than ischemia suggests that it is possible to pharmacologically stimulate this expression and thus possibly mimic the endogenous protective pathway. This could lead to the development of new pharmacological interventions which induce delayed myocardial protection in clinical situations such as angioplasty, coronary bypass surgery or even in patients at high risk of infarction.     

脑缺血再灌注引起大鼠脑细胞水肿及脑组织超氧化物歧化酶活性的变化

背景脑缺血再灌注产生的自由基主要是黄嘌呤氧化酶,导致梗死灶容积细胞肿胀.目的观察脑缺血再灌注引起脑自由基清除酶超氧化物歧化酶活性的变化,探讨嘌呤氧化酶抑制剂别嘌呤醇对缺血再灌注脑组织细胞含水量的影响.设计完全随机对照实验.单位沈阳医学院附属中心医院神经内科,中国医科大学附属第一医院神经外科,辽宁省肢体伤残矫形医院.材料实验于2003-05/2004-04在沈阳医学院附属中心医院实验室完成.选择Wistar大鼠40只,随机分为4组,每组10只.缺血+别嘌呤醇组脑缺血6 h,灌胃给予100mg/kg别嘌呤醇;缺血+悬浊剂组脑缺血6 h,灌胃给予相同剂量的悬浊剂(恶喹酸溶液);缺血再灌注+别嘌呤醇组脑缺血6 h,再灌注2 h,灌胃给予100 mg/kg别嘌呤醇;缺血再灌注+悬浊剂组脑缺血6 h,再灌注2 h,灌胃给予相同剂量的悬浊剂.其中别嘌呤醇组于脑缺血前48 h,24h和1 h共3次分别以100 mg/kg剂量灌胃给予别嘌呤醇.悬浊剂组同样方法给予悬浊剂.方法缺血+别嘌呤醇组、缺血+悬浊剂组的大鼠于闭塞后6 h,缺血再灌注+别嘌呤醇组、缺血再灌注+悬浊剂组于灌注后2 h测量脑组织含水量.脑组织过氧化物歧化酶分布采用过氧化物歧化酶免疫染色观察.主要观察指标①脑组织过氧化物歧化酶分布.②各组大鼠脑组织含水量.结果40只大鼠全部进入结果分析.①脑组织过氧化物歧化酶分布缺血+悬浊剂组、缺血再灌注+悬浊剂组铜锌超氧化物歧化酶染色,缺血灶内可见全部明显的染色增强.缺血+悬浊剂组的锰过氧化物歧化酶染色、缺血灶内可见血管周围轮状染色增强.同时可见染色增强的血管壁和神经细胞.缺血再灌注+悬浊剂组缺血灶内染色呈现弥漫性、稍低下.缺血+别嘌呤醇组和缺血再灌注+别嘌呤醇组铜锌过氧化物歧化酶都没有变化、在缺血+别嘌呤醇组可见血管周围染色增强,缺血再灌注+别嘌呤醇组未见弥漫性改变.缺血+悬浊剂组、缺血再灌注+悬浊剂组小动脉内皮细胞核肿大、中层肌细胞膨大、血管膜扩大,脑组织血管周围显著呈海绵状.缺血+别嘌呤醇组、缺血再灌注+别嘌呤醇组这些变化减轻.②各组大鼠脑组织含水量缺血+别嘌呤醇组,缺血再灌注+别嘌呤醇组低于缺血+悬浊剂组和缺血再灌注+悬浊剂组[(78.56±0.30)%,(79.08±0.33)%;(78.85±0.49)%,(79.86±0.49)%,(P＜0.05)].结论别嘌呤醇可通过对过氧化物歧化酶的抑制有效减轻脑缺血再灌注后的组织损伤.     

Reperfusion after acute coronary occlusion in dogs impairs endothelium-dependent relaxation to acetylcholine and augments contractile reactivity in vitro.

Endothelial injury may contribute to the augmented coronary vascular tone seen in myocardial ischemia by impairing endothelial production or release of vasodilators. In vitro reactivity of arterial rings was studied after 60 min of coronary occlusion and 60 min of reperfusion in anesthetized dogs. Ischemia without reperfusion blunted contractile reactivity to potassium chloride (KCl), whereas ischemia plus reperfusion augmented contractile responses to both KCl and ergonovine. The response to acetylcholine, an endothelium-dependent vasodilator, was abolished in reperfused arteries, whereas the response to nitroprusside, an endothelium-independent vasodilator, was intact. Verapamil pretreatment restored KCl contractile responses to normal in reperfused coronary rings and partially restored endothelium-dependent relaxation. Electron microscopy revealed a nondenuding epicardial coronary endothelial injury in reperfused arteries. These data support the hypothesis that reperfusion of ischemic myocardium augments reactivity to vasoconstrictor agents by causing endothelial cell damage, excessive calcium influx, and loss of modulating vasodilator function.     

Normothermic liver ischemia in rats: xanthine oxidase is not the main source of oxygen free radicals

We studied the effect of allopurinol (ALL) on the activity of xanthine dehydrogenase (XDH), xanthine oxidase (XOX), superoxide dismutase (SOD), and catalase (CAT) in rat liver during ischemia followed by 60 min of reperfusion. We induced 60-min ischemia in the median and left lobes by clamping the hepatic artery and portal branches. The percentage XOX relative to total oxidase activity increased significantly in the control group, from 10% during the stabilization period to 18% after 60 min of reperfusion. The XDH activity decreased during reperfusion. Activity of both XDH and XOX was almost completely blocked by ALL. The activity of SOD and CAT did not differ significantly between the ALL group and controls after 60 min of reperfusion. ALL treatment did not affect liver injury parameters, as concentrations of lactate dehydrogenase (LDH) and alanine transferase (ALT) increased in plasma after ischemia, both in controls and in the ALL-treated group. We concluded that ischemia promotes conversion of XDH to XOX during reperfusion. XOX may not be the main source of free radical production, since intracellular scavengers (SOD and CAT) did not differ significantly between controls and the ALL-treated group, despite the fact that ALL blocked XOX activity completely.     

Comparison of three different A1 adenosine receptor antagonists on infarct size and multiple cycle ischemic preconditioning in anesthetized dogs

A(1) adenosine receptor (AR) antagonists are effective diuretic agents that may be useful for treating fluid retention disorders including congestive heart failure. However, antagonism of A(1)ARs is potentially a concern when using these agents in patients with ischemic heart disease. To address this concern, the present study was designed to compare the actions of the A(1)AR antagonists CPX (1,3-dipropyl-8-cyclopentylxanthine), BG 9719 (1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine), and BG 9928 (1,3-dipropyl-8-[1-(4-propionate)-bicyclo-[2,2,2]octyl]xanthine) on acute myocardial ischemia/reperfusion injury and ischemic preconditioning (IPC) in an in vivo dog model of infarction. Barbital-anesthetized dogs were subjected to 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion, after which infarct size was assessed by staining with triphenyltetrazolium chloride. IPC was elicited by four 5-min occlusion/5-min reperfusion cycles produced 10 min before the 60-min occlusion. Multiple-cycle IPC produced a robust reduction ( approximately 65%) in infarct size; this effect of IPC on infarct size was not abrogated in dogs pretreated with any of the three AR antagonists. Surprisingly, in the absence of IPC, pretreatment with CPX or BG 9928 before occlusion or immediately before reperfusion resulted in significant reductions ( approximately 40-50%) in myocardial infarct size. However, treatment with an equivalent dose of BG 9719 had no similar effect. We conclude that the A(1)AR antagonists BG 9719, BG 9928, and CPX do not exacerbate cardiac injury and do not interfere with IPC induced by multiple ischemia/reperfusion cycles. We discuss the possibility that the cardioprotective actions of CPX and BG 9928 may be related to antagonism of A(2B)ARs.     

State of the science of cardioprotection: Challenges and opportunities--proceedings of the 2010 NHLBI Workshop on Cardioprotection

The National Heart, Lung, and Blood Institute convened a Workshop on September 20-21, 2010, "New Horizons in Cardioprotection," to identify future research directions for cardioprotection against ischemia and reperfusion injury. Since the early 1970s, there has been evidence that the size of a myocardial infarction could be altered by various interventions. Early coronary artery reperfusion has been an intervention that consistently reduces myocardial infarct size in animal models as well as humans. Most cardiologists agree that the best way to treat acute ST-segment elevation myocardial infarction is to reperfuse the infarct artery as soon as possible and to keep the infarct artery patent. In general, stenting is superior to angioplasty, which is superior to thrombolysis. There is no accepted adjunctive therapy to acutely limit myocardial infarct size along with reperfusion that is routinely used in clinical practice. In the Kloner experimental laboratory, some adjunctive therapies have reproducibly limited infarct size (regional hypothermia, preconditioning, cariporide, combinations of the above, remote preconditioning, certain adenosine agonists, and late sodium current blockade). In clinical trials, a host of pharmacologic adjunctive therapies have failed to either reduce infarct size or improve clinical outcome. Potential reasons for the failure of these trials are discussed. However, some adjunctive therapies have shown promise in data subanalyses or subpopulations of clinical trials (adenosine, therapeutic hypothermia, and hyperoxemic reperfusion) or in small clinical trials (atrial natriuretic peptide, ischemic postconditioning, and cyclosporine, the mitochondrial permeability transition pore inhibitor). A recent clinical trial with remote conditioning induced by repetitive inflation of a brachial artery cuff begun prior to hospitalization showed promise in improving myocardial salvage and there are several reports in the cardiothoracic literature, suggesting that remote preconditioning protects hearts during surgery. Thus, in 2011, there is hope that applying some of the body's own conditioning mechanisms may provide protection against ischemic damage.     

Poly(ADP-ribose) polymerase contributes to the development of myocardial infarction in diabetic rats and regulates the nuclear translocation of apoptosis-inducing factor

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 by oxidant-mediated DNA damage is an important pathway of cell dysfunction and tissue injury during myocardial infarction. Because diabetes mellitus can substantially alter cellular signal transduction pathways, we have now investigated whether the PARP pathway also contributes to myocardial ischemia/reperfusion (MI/R) injury in diabetes mellitus in rodents. Myocardial ischemia/reperfusion in control and streptozotocin-diabetic rats was induced by transient ligation of the left anterior descending coronary artery. PARP activation was inhibited by the isoindolinone derivative PARP inhibitor INO-1001. In diabetic rats, a more pronounced degree of myocardial contractile dysfunction developed, which also was associated with a larger infarct size, and significant mortality compared with nondiabetic rats. Inhibition of PARP provided a similar degree of myocardial protective effect in diabetic and nondiabetic animals and reduced infarct size and improved myocardial contractility. In diabetic rats, PARP inhibition reduced mortality during the reperfusion phase. There was marked activation of PARP in the ischemic/reperfused myocardium, which was blocked by INO-1001. In addition, there was a significant degree of mitochondrial-to-nuclear translocation of the cell death effector apoptosis-inducing factor (AIF) in myocardial infarction, which was blocked by pharmacological inhibition of PARP. The role of PARP in regulating AIF translocation in myocytes also was confirmed in an isolated perfused heart preparation. Overall, the current results demonstrate the importance of the PARP pathway in diabetic rats subjected to myocardial infarction and demonstrate the role of PARP in regulating AIF translocation in MI/R.     

Serum lipid peroxide levels in the course of coronary by-pass surgery.

Reperfusion of the ischemic myocardium is associated with the extension of already existing injury. Investigation of the reperfused myocardium under different conditions revealed ultrastructural changes as well as functional disturbances which were identified as free radical-mediated damage. Free radical scavengers and antioxidant agents have been shown to reduce the size of the infarct, in which a temporary regional ischemia was followed by reperfusion. Elaborately designed in vitro and animal models strongly suggest that reperfusion injury might be of clinical significance in operations involving extracorporeal circulation, as in coronary by-pass surgery. However, there is limited information on the relationship between reperfusion injury and subsequent clinical conditions. In our study, we determined lipid peroxide concentrations in timed serum samples of patients who had undergone coronary by-pass surgery. Preoperative lipid peroxide concentrations were 2.50 +/- 0.50 mumol/l. This value increased to 3.30 +/- 1.00 mumol/l after six hours and showed a second increase to 4.20 +/- 0.80 mumol/l 48 hours after the onset of reperfusion. These results suggest that postoperative increases in lipid peroxide concentrations can be of multifactorial origin and may be an indication of reperfusion injury to the myocardium. Further studies are under way to elucidate the relevance of this increase in different groups and clinical conditions.     

Oxygen free radicals in ischemic acute renal failure in the rat.

During renal ischemia, ATP is degraded to hypoxanthine. When xanthine oxidase converts hypoxanthine to xanthine in the presence of molecular oxygen, superoxide radical (O-2) is generated. We studied the role of O-2 and its reduction product OH X in mediating renal injury after ischemia. Male Sprague-Dawley rats underwent right nephrectomy followed by 60 min of occlusion of the left renal artery. The O-2 scavenger superoxide dismutase (SOD) was given 8 min before clamping and before release of the renal artery clamp. Control rats received 5% dextrose instead. Plasma creatinine was lower in SOD treated rats: 1.5, 1.0, and 0.8 mg/dl vs. 2.5, 2.5, and 2.1 mg/dl at 24, 48, and 72 h postischemia. 24 h after ischemia inulin clearance was higher in SOD treated rats than in controls (399 vs. 185 microliter/min). Renal blood flow, measured after ischemia plus 15 min of reflow, was also greater in SOD treated than in control rats. Furthermore, tubular injury, judged histologically in perfusion fixed specimens, was less in SOD treated rats. Rats given SOD inactivated by prior incubation with diethyldithiocarbamate had plasma creatinine values no different from those of control rats. The OH X scavenger dimethylthiourea (DMTU) was given before renal artery occlusion. DMTU treated rats had lower plasma creatinine than did controls: 1.7, 1.7, and 1.3 mg/dl vs. 3.2, 2.2, and 2.4 mg/dl at 24, 48, and 72 h postischemia. Neither SOD nor DMTU caused an increase in renal blood flow, urine flow rate, or solute excretion in normal rats. The xanthine oxidase inhibitor allopurinol was given before ischemia to prevent the generation of oxygen free radicals. Plasma creatinine was lower in allopurinol treated rats: 2.7, 2.2, and 1.4 mg/dl vs. 3.6, 3.5, and 2.3 mg/dl at 24, 48, and 72 h postischemia. Catalase treatment did not protect against renal ischemia, perhaps because its large size limits glomerular filtration and access to the tubular lumen. Superoxide-mediated lipid peroxidation was studied after renal ischemia. 60 min of ischemia did not increase the renal content of the lipid peroxide malondialdehyde, whereas ischemia plus 15 min reflow resulted in a large increase in kidney lipid peroxides. Treatment with SOD before renal ischemia prevented the reflow-induced increase in lipid peroxidation in renal cortical mitochondria but not in crude cortical homogenates. In summary, the oxygen free radical scavengers SOD and DMTU, and allopurinol, which inhibits free radical generation, protected renal function after ischemia. Reperfusion after ischemia resulted in lipid peroxidation; SOD decreased lipid peroxidation in cortical mitochondria after renal ischemia and reflow. We concluded that restoration of oxygen supply to ischemic kidney results in the production of oxygen free radicals, which causes renal injury by lipid peroxidation.     

Allopurinol enhanced adenine nucleotide repletion after myocardial ischemia in the isolated rat heart.

Allopurinol, a competitive inhibitor of xanthine oxidase, has been shown to have a protective effect on ischemic myocardium, but its mechanism of action remains controversial. We used an isolated rat heart preparation to test the hypothesis that allopurinol could restore adenosine triphosphate (ATP) levels and improve the recovery of left ventricular function after global myocardial ischemia. Hearts were equilibrated for 30 min, subjected to 10 min of global, normothermic (37 degrees C) ischemia, and reperfused for 15, 30, and 60 min. Hearts treated with allopurinol (100 microM) exhibited greater ATP levels and improved function during reperfusion than did untreated control hearts. Hearts treated with hypoxanthine (100 microM), the substrate for xanthine oxidase, also showed increased ATP and functional recovery compared with controls. These results suggest that allopurinol may protect the globally ischemic myocardium by enhancing the salvage of hypoxanthine for reincorporation into adenine nucleotides.     

Recombinant superoxide dismutase reduces oxygen free radical concentrations in reperfused myocardium.

It has been proposed that oxygen free radicals mediate damage that occurs during postischemic reperfusion. Recombinant human superoxide dismutase (r-h-SOD) has been shown to be effective at reducing reperfusion injury, but it is not known if this infused enzyme actually reduces oxygen free radical concentrations in the myocardial tissue. Electron paramagnetic resonance spectroscopy was used to directly measure the effect of r-h-SOD on free radical concentrations in the postischemic heart. Hearts were freeze clamped at 77 degrees K after 10 min of normothermic global ischemia followed by 10 s of reflow with control perfusate (n = 7) or perfusate containing 60,000 U r-h-SOD (n = 7). The spectra of these hearts exhibited three different signals: signal A isotropic, g = 2.004, identical to the carbon-centered ubiquinone free radical; signal B anisotropic with axial symmetry, g parallel = 2.033, g perpendicular = 2.005, identical to the oxygen-centered alkyl peroxyl free radical; and the signal C an isotropic triplet, g parallel = 2.000, an = 24 G, similar to a nitrogen-centered free radical such as a peroxyl amine. With r-h-SOD administration the concentration of the oxygen free radical, signal B, was reduced 49% from 6.8 +/- 0.3 microM to 3.5 +/- 0.3 microM (P less than 0.01) and the concentration of the nitrogen free radical, signal C, was reduced 38% from 3.4 +/- 0.3 to 2.1 +/- 0.3 microM (P less than 0.01). The concentration of the carbon-centered free radical, signal A, however, was increased 51% from 3.3 +/- 0.2 to 5.0 +/- 0.2 microM (P less than 0.01). Identical reperfusion with peroxide-inactivated r-h-SOD did not alter the concentrations of free radicals indicating that the specific enzymatic activity of r-h-SOD is required to decrease the concentrations of reactive oxygen free radicals. Additional measurements performed varying the duration of reflow demonstrate a burst of oxygen free radical generation peaking at 10 s of reperfusion. r-h-SOD entirely abolished this burst. These studies demonstrate that superoxide-derived free radicals are generated during postischemic reperfusion and suggest that the beneficial effect of r-h-SOD is due to its specific enzymatic scavenging of superoxide free radicals.