Free radical scavengers in myocardial ischemia

Reperfusion of ischemic myocardium is recognized as potentially beneficial because mortality is directly related to infarct size, and the latter is related to the severity and duration of ischemia. However, reperfusion is associated with extension of the injury that is additive to that produced by ischemia alone. The phenomenon of reperfusion injury is caused in large part by oxygen-derived free radicals from both extracellular and intracellular sources. The loci of oxygen-free radical formation include: myocardial sources (mitochondria), vascular endothelial sources (xanthine oxidase and other oxidases), or the inflammatory cellular infiltrate (neutrophils). Experimental studies have shown that free radical scavengers and agents that prevent free radical production can reduce myocardial infarct size in dogs subjected to temporary regional ischemia followed by reperfusion. Superoxide dismutase and catalase, which catalyze the breakdown of superoxide anion and hydrogen peroxide, respectively, limit experimental myocardial infarct size. The free radical scavenging agent N-(2-mercaptopropionyl)glycine (MPG) is reported to be effective in limiting infarct size. The ischemic-reperfused myocardium derives significant protection when experimental animals are pretreated with the xanthine oxidase inhibitor allopurinol. Neutrophils also serve as a significant source of oxygen-derived free radicals at the site of tissue injury. A number of agents have been shown to directly inhibit neutrophil-derived oxygen free radical formation and neutrophil accumulation within the reperfused myocardium. These agents include ibuprofen, nafazatrom, BW755C, prostacyclin, and iloprost. Thus, free radical scavengers and agents that prevent free radical formation can provide significant protection to the ischemic-reperfused myocardium.     

Application of high-dose propofol during ischemia improves postischemic function of rat hearts: effects on tissue antioxidant capacity

Previous studies have shown that reactive oxygen species mediated lipid peroxidation in patients undergoing cardiac surgery occurs primarily during cardiopulmonary bypass. We examined whether application of a high concentration of propofol during ischemia could effectively enhance postischemic myocardial functional recovery in the setting of global ischemia and reperfusion in an isolated heart preparation. Hearts were subjected to 40 min of global ischemia followed by 90 min of reperfusion. During ischemia, propofol (12 microg/mL in saline) was perfused through the aorta at 60 microL/min. We found that application of high-concentration propofol during ischemia combined with low-concentration propofol (1.2 microg/mL) administered before ischemia and during reperfusion significantly improved postischemic myocardial functional recovery without depressing cardiac mechanics before ischemia, as is seen when high-concentration propofol was applied prior to ischemia and during reperfusion. The functional enhancement is associated with increased heart tissue antioxidant capacity and reduced lipid peroxidation. We conclude that high-concentration propofol application during ischemia could be a potential therapeutic and anesthetic strategy for patients with preexisting myocardial dysfunction.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

Oxygen-derived free radicals and 'stunned myocardium'

A brief, transient period of coronary artery occlusion (less than 20 minutes in duration) followed by reperfusion does not result in irreversible myocyte injury or death, yet the regional contractile function and high energy phosphate content of the previously ischemic tissue remains depressed or 'stunned' for hours to days following reperfusion. It has been suggested that this prolonged postischemic dysfunction of viable, previously ischemic myocardium may be a consequence of oxygen-derived free radicals generated during occlusion or at the time of reperfusion. Recent evidence demonstrates that free radical scavenging agents such as superoxide dismutase (SOD) + catalase, N-2-mercaptopropionylglycine, and allopurinol, administered prior to coronary artery occlusion, significantly enhance recovery of regional contractile function of the stunned, previously ischemic tissue. This improved contractile function was not, however, accompanied by improvements in high energy phosphate metabolism: infusion of SOD + catalase did not preserve ATP stores in the previously ischemic tissue. These data support the hypothesis that oxygen-derived free radicals contribute, at least in part, to the phenomenon of the stunned myocardium. The source or mechanisms of free radical production in the setting of brief, transient ischemia, however, remains to be elucidated.     

Free fatty acid metabolism during myocardial ischemia and reperfusion

Long chain free fatty acids (FFA) are the preferred metabolic substrates of myocardium under aerobic conditions. However, under ischemic conditions long chain FFA have been shown to be harmful both clinically and experimentally. Serum levels of free fatty acids frequently are elevated in patients with myocardial ischemia. The proposed mechanisms of the detrimental effects of free fatty acids include: (1) accumulation of toxic intermediates of fatty acid metabolism, such as long chain acyl-CoA thioesters and long chain acylcarnitines, (2) inhibition of glucose utilization, particularly glycolysis, during ischemia and/or reperfusion, and (3) uncoupling of oxidative metabolism from electron transfer. The relative importance of these mechanisms remains controversial. The primary site of FFA-induced injury appears to be the sarcolemmal and intracellular membranes and their associated enzymes. Inhibitors of free fatty acid metabolism have been shown experimentally to decrease the size of myocardial infarction and lessen postischemic cardiac dysfunction in animal models of regional and global ischemia. The mechanism by which FFA inhibitors improve cardiac function in the postischemic heart is controversial. Whether the effects are dependent on decreased levels of long chain intermediates and/or enhancement of glucose utilization is under investigation. Manipulation of myocardial fatty acid metabolism may prove beneficial in the treatment of myocardial ischemia, particularly during situations of controlled ischemia and reperfusion, such as percutaneous transluminal coronary angioplasty and coronary artery bypass grafting. (Mol Cell Biochem 166: 85-94, 1997)     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.     

Evidence of direct toxic effects of free radicals on the myocardium

The hypothesis that oxygen-derived free radicals do indeed play a role in myocardial ischemic and reperfusion injury has received a lot of support. Experimental results have shown that free radical scavengers can protect against certain aspects of myocardial ischemic injury and that on reperfusion the heart approaches a level that is more normal than those hearts not receiving additional scavenging agents. Superoxide dismutase, catalase, glutathione peroxidase, hydroxyl radical scavengers and iron chelators such as desferrioxamine have proven successful in providing an increased level of recovery. These results indicate, as would be expected, that superoxide, hydrogen peroxide and hydroxyl radicals may all, at some point, either contribute to the injury or be important in generating a subsequent radical which causes damage. In addition, solutions capable of generating free radicals have been shown to cause damage to myocardial cells and the vascular endothelium that is similar to the damage observed during myocardial ischemic and reperfusion injury. Alterations in function, structure, flow, and membrane biochemistry have been documented and compared to ischemic injury. The continued investigation of the role of free radicals in ischemic injury is warranted in the hope of further elucidating the mechanisms involved in free radical injury, the sources of their generation, and in defining a treatment that will provide significant protection against this particular aspect of ischemic damage.     

Free radicals, lipid peroxidation and muscular ischemia]

The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because of the relative resistance of this tissue to normothermic ischemia. Under normal conditions small quantities of oxygen free radicals are produced but they are quenched by intracellular free radical scavenging enzymes (superoxide dismutase, catalase and glutathione peroxidase) or alpha-tocopherol. The increase in malondialdehyde suggests increased lipid peroxidation initiated by free radical reactions. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. The results of recent studies indicate that: a) oxygen free-radicals mediates, at least in part, the increased microvascular permeability produced by reoxygenation, b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia. Additional evidence support the hypothesis of the interrelationship between ischemic tissue and inflammatory cells. So capillary plugging by granulocytes and oxygen free radical formation may contribute to the ischemic injury.     

Free radical centers in the dog myocardial tissue in regional ischemia

The effect of regional ischemia on canine myocardial in situ free radical species was studied by the EPR method. Rapid fixation of heart muscle samples by freezeclamping was performed at the following physiological states: native myocardial blood circulation, regional ischemia with the presence of collateral circulation, total ischemia, and postischemic reperfusion. EPR spectra of the samples at -40 degrees C exhibited two free radical signals from the semireduced forms of ubiquinone and flavine coenzymes. Upon transition from normal blood supply to regional ischemia, an increase in the contribution of the flavine signal was registered, but reperfusion resulted in the recovery of the characteristics of EPR signals. It was found that the increase in the intensity of collateral circulation in the ischemic area led to an increase in the portion of ubisemiquinone in the integral EPR signal, whereas in total ischemia this signal was not registered. It was shown that the changes in spectral characteristics of integral free radical signals are accompanied by changes in their relaxation parameters.     

Oxygen radical mechanisms of brain injury following ischemia and reperfusion.

This review addresses current understanding of oxygen radical mechanisms as they relate to the brain during ischemia and reperfusion. The mechanism for radical production remains speculative in large part because of the difficulty of measuring radical species in vivo. Breakdown of lipid membranes during ischemia leads to accumulation of free fatty acids. Decreased energy stores during ischemia result in the accumulation of adenine nucleotides. During reperfusion, metabolism of free fatty acids via the cyclooxygenase pathway and metabolism of adenine nucleotides via the xanthine oxidase pathway are the most likely sources of oxygen radicals. Although leukocytes have been found to accumulate in some models of ischemia and reperfusion, their mechanistic role remains in question. Therapeutic strategies aimed at decreasing brain injury have included administration of radical scavengers at the time of reperfusion. Efficacy of traditional oxygen radical scavengers such as superoxide dismutase and catalase may be limited by their inability to cross the blood-brain barrier. Lipid-soluble antioxidants appear more efficacious because of their ability to cross the blood-brain barrier and because of their presence in membrane structures where peroxidative reactions can be halted.     

Cardioprotection requires flipping the 'posttranslational modification' switch

Minimizing damage during reperfusion of the heart following an ischemic event is an important part of the recovery process, as is preventing future recurrences; however, restoring blood perfusion to the heart following ischemia can lead to apoptosis, necrosis, and finally, diminished cardiac function. Exercise reduces risk of heart disease and has been shown to improve the recovery of the heart following ischemia and reperfusion. Brief intermittent ischemic events administered prior to or following a myocardial infarction have also been demonstrated to reduce the infarct size and improve cardiac function, thereby providing cardioprotection. Many signaling transduction pathways are known to regulate cardioprotection, including but not limited to calcium regulation, antioxidant scavenging, and kinase activation. Although posttranslational modifications (PTM) such as phosphorylation, O-GlcNAcylation, methylation, and acetylation are essential regulators of these pathways, their contributions are often overlooked in the literature. This review will examine how PTMS are important regulators of cardioprotection and demonstrate why they should be targeted when developing future therapies for the minimization of damage caused by cardiac ischemia and reperfusion.     

褪黑素与缺血再灌注损伤

褪黑素(melatonin,MT)具有强效抗氧化作用,在肠、肝脏、心脏、脑等器官的缺血再灌注损伤实验中具有清除自由基、保护线粒体、抗细胞凋亡等保护性作用。本文综合褪黑素应用于缺血再灌注损伤的动物实验的近几年相关文献,总结并分析褪黑素在缺血再灌注损伤动物实验中的保护作用及其相关机制。     

Effect of superoxide dismutase and catalase on myocardial energy metabolism during ischemia and reperfusion

Survival of cardiac patients undergoing heart surgery depends critically upon the recovery of myocardial energy metabolism during reperfusion of ischemic myocardium. The present study compares various parameters of myocardial energy metabolism using an isolated in situ pig heart. The left anterior descending (LAD) coronary artery was occluded for 60 min, followed by 60 min of global hypothermic cardioplegic arrest and 60 min of reperfusion. Free radical scavengers [superoxide dismutase SOD and catalase] were used to protect the ischemic heart from reperfusion injury. In both control and SOD plus catalase-treated groups, ATP, creatine phosphate (CP), ATP/ADP ratio, energy charge and phosphorylation potential dropped significantly during ischemic insult. After reperfusion, CP, ATP/ADP ratio and phosphorylation potential improved significantly, but they were restored to control level only in treated animals. In either case, free energy of ATP hydrolysis (delta G) lowered only by 5% during ischemia, but recovered promptly upon reperfusion. SOD and catalase also improved coronary blood flow and reduced creatine kinase release compared to those of untreated animals, suggesting improved myocardial recovery upon reperfusion. Our results suggest that SOD and catalase significantly improve the myocardial recovery during reperfusion by enhancing rephosphorylation steps, and the value of delta G is more critical compared to those of ATP and CP for myocardial recovery.     

Beneficial effects of different flavonoids, on functional recovery after ischemia and reperfusion in isolated rat heart

Three newly synthesised lipid peroxidation inhibitors (7, 11, 14) were evaluated for their effects on myocardial functional recovery during reperfusion after 30 min global ischemia in isolated rat hearts. The flavonoid compounds (7, 11, 14, rutin) reduce ischemia/reperfusion-induced cardiac dysfunction.     

When melatonin gets on your nerves: its beneficial actions in experimental models of stroke

This article summarizes the evidence that endogenously produced and exogenously administered melatonin reduces the degree of tissue damage and limits the biobehavioral deficits associated with experimental models of ischemia/reperfusion injury in the brain (i.e., stroke). Melatonin's efficacy in curtailing neural damage under conditions of transitory interruption of the blood supply to the brain has been documented in models of both focal and global ischemia. In these studies many indices have been shown to be improved as a consequence of melatonin treatment. For example, when given at the time of ischemia or reperfusion onset, melatonin reduces neurophysiological deficits, infarct volume, the degree of neural edema, lipid peroxidation, protein carbonyls, DNA damage, neuron and glial loss, and death of the animals. Melatonin's protective actions against these adverse changes are believed to stem from its direct free radical scavenging and indirect antioxidant activities, possibly from its ability to limit free radical generation at the mitochondrial level and because of yet-undefined functions. Considering its high efficacy in overcoming much of the damage associated with ischemia/reperfusion injury, not only in the brain but in other organs as well, its use in clinical trials for the purpose of improving stroke outcome should be seriously considered.     

Improved myocardial performance induced by clofibrate during reperfusion after acute myocardial infarction

The increase of cellular fatty acids appears to be one of the causes of the myocardial injury during ischemia and reperfusion. This study was designed to examine whether a hypolipidemic drug such as clofibrate can reduce the myocardial injury during ischemia and reperfusion. Clofibrate was fed to experimental pigs for 9 days. Isolated in situ hearts from both experimental and control pigs were subjected to 60 min of regional ischemia induced by occluding the left anterior descending coronary artery, followed by 60 min of global ischemia by hypothermic cardioplegic arrest and 60 min of reperfusion. The clofibrate feeding resulted in the better cardiac performance as judged by increased coronary blood flow, improved left ventricular function, and reduced myocardial injury as judged by creatine kinase release. Although the clofibrate-fed animals contained higher levels of thiobarbituric reactive materials, the free fatty acid levels of plasma and myocardium were much lower compared with control animals. The clofibrate feeding was also associated with increased peroxisomal catalase and beta-oxidation of fatty acids. These results suggest that decreased levels of free fatty acids in the plasma and the myocardium and increased catalase activity induced by antilipolytic therapy appear to provide beneficial effects to the myocardium during ischemia and reperfusion.     

Dinitrosyl iron complexes with glutathione in rat myocardial tissue during regional ischemia and postischemic reperfusion

Injection of dinitrosyl iron complexes with glutathione at the onset of 40-min regional myocardial ischemia in rat was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After introducing the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that in postischemic reperfusion, the rate of decrease in the content of these complexes in the ischemic area increases, which reflects effective scavenging of short-lived reactive oxygen species by the dinitrosyl iron complexes.     

Evaluation of the role of xanthine oxidase in myocardial reperfusion injury

The free radical-generating enzyme xanthine oxidase has been hypothesized to be a central mechanism of the injury which occurs in postischemic tissues; however, its importance remains controversial. Much attention has focused on the role of this enzyme in myocardial reperfusion injury. While xanthine oxidase has been observed in ischemic tissue homogenates, the presence and importance of radical generation by the enzyme in intact tissues are unknown. Therefore, we performed electron paramagnetic resonance, nuclear magnetic resonance and hemodynamic studies to measure the presence and significance of xanthine oxidase-mediated free radical generation in the isolated rat heart. When isolated perfused rat hearts were reperfused after 30 min of global ischemia, myocardial function and coronary flow were significantly improved in the presence of the definitive xanthine oxidase blocker oxypurinol. Free radical concentrations measured by spin-trapping with 5,5'-dimethyl-1-pyrroline-N-oxide were significantly decreased by oxypurinol and the energetic state of the heart was improved as reflected by an increased recovery of phosphocreatine and a higher phosphocreatine/Pi ratio. ATP recovery, however, was not altered, indicating that the improved functional and metabolic state of the heart was not due to ATP salvage. Spectrophotometric assays for the enzyme showed an increase in the amount of xanthine oxidase relative to dehydrogenase following ischemia, and a total available xanthine oxidase pool in the rat heart of approximately 150 milliunits/g of protein. Thus, xanthine oxidase is a significant source of the oxidative injury which occurs upon reperfusion of the ischemic rat heart.     

Tempol reduces infarct size in rodent models of regional myocardial ischemia and reperfusion.

Reactive oxygen species (ROS) contribute to ischemia-reperfusion injury of the heart. This study investigates the effects of tempol, a membrane-permeable radical scavenger on (i) the infarct size caused by regional myocardial ischemia and reperfusion of the heart in vivo (rat, rabbit) and in vitro (rat), and (ii) the cell injury caused by hydrogen peroxide (H2O2) in rat cardiac myoblasts (H9c2 cells). In the anesthetized rat, tempol reduced the infarct size caused by regional myocardial ischemia (25 min) and reperfusion (2 h) from 60 +/- 3% (control, n = 8) to 24 +/- 5% (n = 6, p < .05). In the anesthetized rabbit, tempol also attenuated the infarct size caused by myocardial ischemia (45 min) and reperfusion (2 h) from 59 +/- 3% (control, n = 6) to 39 +/- 5% (n = 5, p < .05). Regional ischemia (35 min) and reperfusion (2 h) of the isolated, buffer-perfused heart of the rat resulted in an infarct size of 54 +/- 4% (control n = 7). Reperfusion of hearts with buffer containing tempol (n = 6) caused a 37% reduction in infarct size (n = 6, p < .05). Pretreatment of rat cardiac myoblasts with tempol attenuated the impairment in mitochondrial respiration caused by H2O2 (1 mM for 4 h). Thus, the membrane-permeable radical scavenger tempol reduces myocardial infarct size in rodents.     

Dinitrosyl complexes of iron with glutathione in the rat myocardial tissue during regional ischemia and postischemic reperfusion

The injection of dinitrosyliron iron complexes with glutathione at the onset of 40-min rat regional myocardial ischemia was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After the introduction of the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that, as a result of postischemic reperfusion, the rate of the decrease in the content of these complexes in the ischemic area increases, which demonstrates the effective scavenging of short-lived reactive oxygen species by molecules of dinitrosyl iron complexes.