3-nitrotyrosine and Myocardial-Ischemia

Administration of low-dose endotoxin (lipopolysaccharide, LPS) 24 h before a lethal ischemia induces pharmacological late preconditioning. The exact mechanism of this phenomenon is not clear. Here we aimed to investigate whether low-dose LPS exerts late effects on peroxynitrite formation and activation of Akt, Erk, and STAT3 in the heart. Male Wistar rats were injected with LPS (S. typhimurium; 0.5 mg/kg i.p.) or saline. Twenty-four hours later, hearts were isolated, perfused for 10 min, and then used for biochemical analyses. LPS pretreatment enhanced cardiac formation of the peroxynitrite marker 3-nitrotyrosine. LPS pretreatment also increased cardiac levels of the peroxynitrite precursor nitric oxide (NO) and superoxide. The activities of Ca2+-independent NO synthase and xanthine oxidoreductase increased in LPS-pretreated hearts. LPS pretreatment resulted in significantly enhanced phosphorylation of STAT3 and non-significantly increased phosphorylation of Akt without affecting the activation of Erk. In separate experiments, isolated working hearts were subjected to 30 min global ischemia and 20 min reperfusion. LPS pretreatment significantly improved ischemia-reperfusion-induced deterioration of cardiac function. We conclude that LPS pretreatment enhances cardiac peroxynitrite formation and activates STAT3 24 h later, which may contribute to LPS-induced late preconditioning.

Topics: Animals; Endotoxins; Ischemic Preconditioning, Myocardial; Lactate Dehydrogenases; Lipopolysaccharides; Male; Myocardial Ischemia; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Peroxynitrous Acid; Rats; STAT3 Transcription Factor; Tyrosine

Myocardial infarction is the leading cause of death, occurs after prolonged ischemia of the coronary arteries. Restore blood flow is the first intervention help against heart attack. However, reperfusion of the arteries leads to ischemia/reperfusion injury (I/R). The fatty acid amide palmitoylethanolamide (PEA) is an endogenous compound widely present in living organisms, with analgesic and anti-inflammatory properties. The present study evaluated the effect of ultramicronized palmitoylethanolamide (PEA-um) treatment on the inflammatory process associated with myocardial I/R. Myocardial ischemia reperfusion injury was induced by occlusion of the left anterior descending coronary artery for 30 min followed by 2 h of reperfusion. PEA-um, was administered (10 mg/kg) 15 min after ischemia and 1 h after reperfusion. In this study, we demonstrated that PEA-um treatment reduces myocardial tissue injury, neutrophil infiltration, adhesion molecules (ICAM-1, P-selectin) expression, proinflammatory cytokines (TNF-α, IL-1β) production, nitrotyrosine and PAR formation, nuclear factor kB expression, and apoptosis (Fas-L, Bcl-2) activation. In addition to study whether the protective effect of PEA-um on myocardial ischemia reperfusion injury is also related to the activation of PPAR-α, in a separate set of experiments it has been performed myocardial I/R in PPARα mice. Genetic ablation of peroxisome proliferator activated receptor (PPAR)-α in PPAR-αKO mice exacerbated Myocardial ischemia reperfusion injury when compared with PPAR-αWT mice. PEA-um induced cardioprotection in PPAR-α wild-type mice, but the same effect cannot be observed in PPAR-αKO mice. Our results have clearly shown a modulation of the inflammatory process, associated with myocardial ischemia reperfusion injury, following administration of PEA-um.

Topics: Amides; Animals; Ethanolamines; Immunohistochemistry; Intercellular Adhesion Molecule-1; Interleukin-1beta; Male; Mice; Mice, Knockout; Microtomy; Myocardial Ischemia; Myocardial Reperfusion Injury; P-Selectin; Palmitic Acids; PPAR alpha; Rats, Wistar; Tumor Necrosis Factor-alpha; Tyrosine

Ischemic preconditioning, which is mediated by cell signaling molecules, protects the heart from ischemia-reperfusion injury by limiting the infarct size. Oleuropein, the main polyphenolic constituent of olives, reduced the infarct size in normal and cholesterol-fed rabbits when it was administered at a nutritional dose. The aim of the present study was to compare the effects of oleuropein and preconditioning in terms of the cell signaling and metabolism pathways underlying myocardial protection. Rabbits were randomly divided into six groups: the control group received 5 % dextrose for six weeks, the preconditioning group was subjected to two cycles of preconditioning with 5 min ischemia/10 min reperfusion, the O6 group was treated with oleuropein for six weeks, the Chol group was fed a cholesterol-enriched diet and 5 % dextrose for six weeks, and the CholO6 and CholO3 groups were treated with cholesterol and oleuropein for six and three weeks, respectively; oleuropein was dissolved in 5 % dextrose solution and was administered orally at a dose of 20 mg × kg(-1) × day(-1). All animals were subsequently subjected to 30 min myocardial ischemia followed by 10 min of reperfusion. At that time, myocardial biopsies were taken from the ischemic areas for the assessment of oxidative and nitrosative stress biomarkers (malondialdehyde and nitrotyrosine), and determination of phosphorylation of signaling molecules involved in the mechanism of preconditioning (PI3K, Akt, eNOS, AMPK, STAT3). The tissue extracts NMR metabolic profile was recorded and further analyzed by multivariate statistics. Oxidative biomarkers were significantly reduced in the O6, CholO6, and CholO3 groups compared to the control, preconditioning, and Chol groups. Considering the underlying signaling cascade, the phosphorylation of PI3K, Akt, eNOS, AMPK, and STAT-3 was significantly higher in the preconditioning and all oleuropein-treated groups compared to the control and Chol groups. The NMR-based metabonomic study, performed through the analysis of spectroscopic data, depicted differences in the metabolome of the various groups with significant alterations in purine metabolism. In conclusion, the addition of oleuropein to a normal or hypercholesterolemic diet results in a preconditioning-like intracellular effect, eliminating the deleterious consequences of ischemia and hypercholesterolemia, followed by a decrease of oxidative stress biomarkers. This effect is exerted through inducing precondit

Topics: Animals; Cholesterol; Disease Models, Animal; Hypercholesterolemia; Iridoid Glucosides; Iridoids; Male; Malondialdehyde; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Olea; Oxidative Stress; Phosphatidylinositol 3-Kinases; Protective Agents; Rabbits; Signal Transduction; Tyrosine

To provide evidence for the protective role of inorganic nitrite against acute ischaemia and reperfusion-induced ventricular arrhythmias in a large animal model.. Dogs, anaesthetized with chloralose and urethane, were administered intravenously with sodium nitrite (0.2 µmol kg(-1) min(-1)) in two protocols. In protocol 1 nitrite was infused 10 min prior to and during a 25 min occlusion of the left anterior descending (LAD) coronary artery (NaNO2-PO; n = 14), whereas in protocol 2 the infusion was started 10 min prior to reperfusion of the occluded vessel (NaNO2-PR; n = 12). Control dogs (n = 15) were infused with saline and subjected to the same period of ischaemia and reperfusion. Severities of ischaemia and ventricular arrhythmias, as well as changes in plasma nitrate/nitrite (NOx) levels in the coronary sinus blood, were assessed throughout the experiment. Myocardial superoxide and nitrotyrosine (NT) levels were determined during reperfusion. Changes in protein S-nitrosylation (SNO) and S-glutathionylation were also examined.. Compared with controls, sodium nitrite administered either pre-occlusion or pre-reperfusion markedly suppressed the number and severity of ventricular arrhythmias during occlusion and increased survival (0% vs. 50 and 92%) upon reperfusion. There were also significant decreases in superoxide and NT levels in the nitrite treated dogs. Compared with controls, increased SNO was found only in NaNO2-PR dogs, whereas S-glutathionylation occurred primarily in NaNO2-PO dogs.. Intravenous infusion of nitrite profoundly reduced the severity of ventricular arrhythmias resulting from acute ischaemia and reperfusion in anaesthetized dogs. This effect, among several others, may result from an NO-mediated reduction in oxidative stress, perhaps through protein SNO and/or S-glutathionylation.

Topics: Administration, Intravenous; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Disease Models, Animal; Dogs; Hemodynamics; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Nitrates; Nitric Oxide; Nitrites; Protein Processing, Post-Translational; Sodium Nitrite; Superoxides; Tyrosine

Endothelial dysfunction of the coronary arteries caused by oxidative stress plays an important role in the pathogenesis of coronary vasospasm. However, it is not clear whether circulating biomarkers for oxidative stress are altered after coronary vasospasm. We investigated temporal changes in the levels of oxidative stress biomarkers after coronary vasospasm induced by intracoronary acetylcholine provocation testing, resulting in transient myocardial ischemia.. Thirty consecutive patients with suspected vasospastic angina pectoris (VSAP) were enrolled in the study. Patients were categorized into the VSAP-positive group (n=14) and the VSAP-negative group (n=16) on the basis of test results. Serum samples were examined for the levels of the oxidative stress markers 4-hydroxynonenal (HNE) and nitrotyrosine (NT) before, and 15min, 3h, and 12h after the provocation test. The serum HNE levels did not change in either group after the test. The serum NT levels in the VSAP-positive group significantly increased at 3h and 12h after the test (11.3±3.3μg/ml at 3h, p=0.015, and 12.1±5.7μg/ml at 12h, p=0.03), as compared with baseline (8.1±3.2μg/ml). In the VSAP-negative group, the serum NT levels significantly decreased from baseline at each of the 3 time points.. Serum NT significantly increased after coronary vasospasm induced by acetylcholine provocation, suggesting that serum NT could be a biomarker of transient myocardial ischemia and could contribute to the development of VSAP.

Topics: Acetylcholine; Aged; Aldehydes; Biomarkers; Coronary Vasospasm; Female; Humans; Male; Middle Aged; Myocardial Ischemia; Oxidative Stress; Tyrosine

Cardioprotective effects of anesthetic preconditioning and cyclosporine A (CsA) are lost with aging. To extend our previous work and address a possible mechanism underlying age-related differences, we investigated the role of oxidative stress in the aging heart by treating senescent animals with the oxygen free radical scavenger Tempol. Old male Fischer 344 rats (22-24 mo) were randomly assigned to control or Tempol treatment groups for 2 or 4 wk (T×2wk and T×4wk, respectively). Rats received isoflurane 30 min before ischemia-reperfusion injury or CsA just before reperfusion. Myocardial infarction sizes were significantly reduced by isoflurane or CsA in the aged rats treated with Tempol (T×4wk) compared with old control rats. In other experiments, young (4-6 mo) and old rats underwent either chronic Tempol or vehicle treatment, and the levels of myocardial protein oxidative damage, antioxidant enzymes, mitochondrial Ca(2+) uptake, cyclophilin D protein, and mitochondrial permeability transition pore opening times were measured. T×4wk significantly increased MnSOD enzyme activity, GSH-to-GSSH ratios, MnSOD protein level, mitochondrial Ca(2+) uptake capacity, reduced protein nitrotyrosine levels, and normalized cyclophilin D protein expression in the aged rat heart. T×4wk also significantly prolonged mitochondrial permeability transition pore opening times induced by reactive oxygen species in old cardiomyocytes. Our studies demonstrate that 4 wk of Tempol pretreatment restores anesthetic preconditioning and cardioprotection by CsA in the old rat and that this is associated with decreased oxidative stress and improved mitochondrial function. Our results point to a new protective strategy for the ischemic myocardium in the high-risk older population.

Topics: Aging; Animals; Antioxidants; Calcium; Cardiotonic Agents; Cyclic N-Oxides; Heart; Hemodynamics; Ischemic Preconditioning, Myocardial; Male; Mitochondria; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Oxidative Stress; Rats; Rats, Inbred F344; Spin Labels; Superoxide Dismutase; Tyrosine

Postconditioning (PostC) modifies the early post-ischemic pH, redox environment, and activity of enzymes. We hypothesized that early acidosis in PostC may affect superoxide dismutase (SOD) and catalase (CAT) activities, may reduce 3-nitrotyrosine (3-NT) protein levels, and may increase S-nitrosylated (SNO) protein levels, thus deploying its protective effects. To verify this hypothesis, we studied the early (7(th) min) and late (120(th) min) phases of reperfusion (a) endogenous SOD and CAT activities and (b) 3-NT protein levels and SNO protein levels. Isolated rat hearts underwent 30-min ischemia/120-min reperfusion (I/R) or PostC (5 cycles of 10-s I/R at the beginning of 120-min reperfusion) either with or without exogenous CAT or SOD infused during the initial 3 min of reperfusion. The effects of early reperfusion with acid buffer (AB, pH 6.8) on endogenous antioxidant enzymes were also tested. Pressure, infarct size, and lactate dehydrogenase release were also measured. At the 7(th) min, PostC induced a significant decrease in SOD activity with no major change both in Mn and Cu/Zn SOD levels and in CAT activity and level. PostC also reduced 3-NT and increased SNO levels. Exogenous SOD, but not CAT, abolished PostC cardioprotection. In late reperfusion (120-min), I/R increased SOD activity but decreased CAT activity and Cu/Zn SOD levels; these effects were reversed by PostC; 3-NT was not affected, but SNO was increased by PostC. AB reproduced PostC effects on antioxidant enzymes. The conclusions are as follows: PostC downregulates endogenous SOD and preserves CAT activity, thus increasing SNO and reducing 3-NT levels. These effects are triggered by early post-ischemic acidosis. Yet acidosis-induced SOD downregulation may limit denitrosylation, thus contributing to PostC triggering. Hence, exogenous SOD, but not CAT, interferes with PostC triggering. Prolonged SOD downregulation and SNO increase may contribute to PostC and AB beneficial effects.

Topics: Acidosis; Animals; Catalase; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Rats; Rats, Wistar; Superoxide Dismutase; Tyrosine

The role of iron in the pathogenesis of cardio-vascular disorders is still controversial. We studied the effects of iron perturbations on myocardial injury upon temporary ischemia/reperfusion. C57BL/6J male mice were injected with iron dextran for 2 weeks while controls received saline. Mice were then subjected to 30 min of myocardial ischemia and subsequent reperfusion for 6-24 h. Tissue damage was quantified histologically and by troponin T determination. The expressions of tumor necrosis factor-alpha (TNF-alpha), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) were investigated in non-ischemic and ischemic regions of both groups. After myocardial ischemia and reperfusion, troponin T levels, as a marker of myocardial damage, were significantly reduced in iron-treated mice as compared to control mice (P < 0.05). Under the same conditions the infarction area and damage score were significantly lower in iron-treated animals. In parallel, TNF-alpha and SOD expressions were increased in infarcted regions of iron-treated mice as compared to controls, whereas myocardial iNOS expression was significantly lower in iron-treated mice. Although, iron challenge increased radical formation and TNF-alpha expression in vivo, this did not result in myocardial damage which may be linked to the parallel induction of SOD. Importantly, iron treatment inhibited iNOS expression. Since, an increased nitric oxide (NO) formation has been linked to cardiac damage after acute myocardial infarction, iron may exert short time cardio-protective effects after induction of ischemia/reperfusion via decreasing iNOS formation.

Topics: Animals; Iron; Male; Mice; Mice, Inbred C57BL; Myocardial Ischemia; Nitric Oxide Synthase Type II; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Tyrosine

A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.

Topics: Animals; Electron Transport Complex I; Male; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion Injury; Proteomics; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfhydryl Compounds; Tyrosine

The beneficial effects of reperfusion therapies have been limited by the amount of ischemic damage that occurs before reperfusion. To enable development of interventions to reduce cell injury, our research has focused on understanding mechanisms involved in cardiac cell death after ischemia/reperfusion (I/R) injury. In this context, our laboratory has been investigating the role of the receptor for advanced-glycation end products (RAGE) in myocardial I/R injury.. In this study we tested the hypothesis that RAGE is a key modulator of I/R injury in the myocardium. In ischemic rat hearts, expression of RAGE and its ligands was significantly enhanced. Pretreatment of rats with sRAGE, a decoy soluble part of RAGE receptor, reduced ischemic injury and improved functional recovery of myocardium. To specifically dissect the impact of RAGE, hearts from homozygous RAGE-null mice were isolated, perfused, and subjected to I/R. RAGE-null mice were strikingly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH, improved functional recovery, and increased adenosine triphosphate (ATP). In rats and mice, activation of the RAGE axis was associated with increases in inducible nitric oxide synthase expression and levels of nitric oxide, cyclic guanosine monophosphate (cGMP), and nitrotyrosine.. These findings demonstrate novel and key roles for RAGE in I/R injury in the heart. The findings also demonstrate that the interaction of RAGE with advanced-glycation end products affects myocardial energy metabolism and function during I/R.

Topics: Animals; Cyclic GMP; Energy Metabolism; Male; Mice; Mice, Knockout; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Synthase Type II; Rats; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Tyrosine; Up-Regulation

This study examined the potential deleterious effect of high-dose nitroglycerin (NTG) on cardiac function and cellular injury after ischemia (30 min) and reperfusion (120 min) in isolated perfused rat hearts. Low-dose (0.75 microg/h), medium-dose (3.75 microg/h), high-dose (15 microg/h) NTG or high-dose NTG plus glutathione (GSH, 1 mmol/L) was administrated at the time of reperfusion. Administration of high-dose NTG significantly exacerbated cardiac reperfusion injury as evidenced by increased creatine kinase and lactate dehydrogenase activity in coronary effluent, increased cardiomyocyte apoptosis and necrosis, and decreased cardiac function recovery after reperfusion. Compared with the vehicle group, formation of nitrotyrosine, a footprint for peroxynitrite (ONOO) production, was markedly increased in the hearts treated with medium-dose or high-dose NTG. Most interestingly, cotreatment with GSH blocked high-dose NTG-induced ONOO formation and attenuated myocardial ischemia/reperfusion injury. Taken together, our present results demonstrated that administration of high-dose NTG aggravated, rather than attenuated myocardial ischemia/reperfusion injury likely via increasing ONOO formation. Coadministration of GSH may reverse the advert action of high-dose NTG.

Topics: Animals; Apoptosis; Glutathione; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitroglycerin; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Tyrosine

Thioredoxin (Trx) is an oxidoreductase that prevents free radical-induced cell death in cultured cells. Here we assessed the mechanism(s) underlying the cardioprotective effects of Trx in vivo.. The effects of myocardial ischemia (30 min) and reperfusion were measured in mice, with assays of myocardial apoptosis, superoxide production, NOx and nitrotyrosine content, and myocardial infarct size. Recombinant human Trx (rhTrx, 0.7-20 mg kg(-1), i.p.) was given 10 min before reperfusion.. Treatment with 2 mg kg(-1) rhTrx significantly decreased myocardial apoptosis and reduced infarct size (P<0.01). Nitrotyrosine content of cardiomyocytes was markedly reduced in rhTrx-treated animals (P<0.01). To further identify the mechanisms by which rhTrx may exert its anti-nitrative effect, iNOS expression and production of NOx and superoxide were determined. Treatment with rhTrx had no significant effect on iNOS expression or NOx content in the ischemic/reperfused heart. However, it markedly upregulated mSOD and reduced tissue superoxide content. To further establish a causative link between the anti- peroxynitrite effect and the cardioprotective effect of rhTrx, cultured adult cardiomyocytes were incubated with SIN-1, a peroxynitrite donor, (50 microM for 3 h) resulting in a nitrotyrosine content comparable to that seen in the ischemic/reperfused heart and causing significant cardiomyocyte apoptosis (P<0.01). Treatment with rhTrx markedly decreased SIN-1 induced apoptosis (P<0.01).. These results demonstrate that Trx is a novel anti-apoptotic and cardioprotective molecule that exerts its cardioprotective effects by reducing ischemia/reperfusion-induced oxidative/nitrative stress.

Topics: Animals; Apoptosis; Male; Mice; Molsidomine; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Superoxides; Thioredoxins; Tyrosine

Nitric oxide (NO) plays a vital role in vascular homeostasis and in the pathophysiology of coronary heart disease. Its metabolites, nitrite and nitrate, have vasculoprotective properties, whereas peroxynitrite, an oxidant metabolite of NO, is cytotoxic and can aggravate myocardial damage during ischaemic reperfusion injury. Peroxynitrite nitrates free and protein bound tyrosine residues to produce nitrotyrosine. The measurement of nitrotyrosine provides an indirect estimation of plasma peroxynitrite concentrations.. To measure plasma nitrotyrosine concentrations to see whether peroxynitrite could contribute to myocardial dysfunction during myocardial ischaemia induced by an exercise tolerance test (ETT).. Plasma free nitrotyrosine concentrations were compared before and after exercise in 29 subjects with a positive ETT and 34 subjects with a negative ETT.. Plasma nitrotyrosine concentrations were similar in patients with exercise induced myocardial ischaemia and controls.. Peroxynitrite does not contribute to the myocardial dysfunction in reversible myocardial ischaemia.

Topics: Adult; Aged; Exercise Test; Exercise Tolerance; Female; Humans; Male; Middle Aged; Myocardial Ischemia; Peroxynitrous Acid; Tyrosine

Nitric oxide (NO) production is increased in postischemic myocardium, and NO can control mitochondrial oxygen consumption in vitro. Therefore, we investigated the role of endothelial NO synthase (eNOS)-derived NO on in vivo regulation of oxygen consumption in the postischemic heart.. Mice were subjected to 30 minutes of coronary ligation followed by 60 minutes of reperfusion. Myocardial oxygen tension (Po2) was monitored by electron paramagnetic resonance oximetry. In wild-type, N-nitro-L-arginine methyl ester (L-NAME)-treated (with 1 mg/mL in drinking water), and eNOS knockout (eNOS-/-) mice, no difference was observed among baseline myocardial Po2 values (8.6+/-0.7, 10.0+/-1.2, and 10.1+/-1.2 mm Hg, respectively) or those measured at 30 minutes of ischemia (1.4+/-0.6, 2.3+/-0.9, and 3.1+/-1.4 mm Hg, respectively). After reperfusion, myocardial Po2 increased markedly (P<0.001 versus baseline in each group) but was much lower in L-NAME-treated and eNOS-/- mice (17.4+/-1.6 and 20.4+/-1.9 mm Hg) than in wild-type mice (46.5+/-1.7 mm Hg; P<0.001). A transient peak of myocardial Po2 was observed at early reperfusion in wild-type mice. No reactive hyperemia was observed during early reperfusion. Endothelial NO decreased the rate-pressure product (P<0.05), upregulated cytochrome c oxidase (CcO) mRNA expression (P<0.01) with no change in CcO activity, and inhibited NADH dehydrogenase (NADH-DH) activity (P<0.01) without alteration of NADH-DH mRNA expression. Peroxynitrite-mediated tyrosine nitration was higher in hearts from wild-type mice than in eNOS-/- or L-NAME-treated hearts.. eNOS-derived NO markedly suppresses in vivo O2 consumption in the postischemic heart through modulation of mitochondrial respiration based on alterations in enzyme activity and mRNA expression of NADH-DH and CcO. The marked myocardial hyperoxygenation in reperfused myocardium may be a critical factor that triggers postischemic remodeling.

Topics: Animals; Cell Respiration; Disease Models, Animal; Electron Transport Complex IV; Male; Mice; Mice, Knockout; Myocardial Ischemia; NADH Dehydrogenase; Nitric Oxide; Nitric Oxide Synthase Type III; Oximetry; Oxygen; Oxygen Consumption; Tyrosine; Up-Regulation

This study examined the effects of nitrate tolerance (NT) on myocardial ischemia reperfusion (MI/R) injury and elucidated the potential mechanisms involved. Furthermore, the effects of GSH on postischemic myocardial apoptosis in NT rats were investigated.. Male Sprague-Dawley rats were randomized to receive nitroglycerin (60 microg/kg/h) or saline for 12 h followed by 40 min of MI and 4 h of reperfusion. Myocardial apoptosis, infarct size, nitrotyrosine formation, plasma CK and LDH activity, and cardiac function were determined. MI/R resulted in significant apoptotic cell death, which was further increased in animals with NT. In addition, NT further increased plasma CK and LDH activity, enlarged infarct size, and impaired cardiac functional recovery after ischemia. Myocardial nitrotyrosine, a footprint for cytotoxic reactive nitrogen species formation, was further enhanced in the NT heart after MI/R. Treatment of NT animals with exogenous GSH inhibited nitrotyrosine formation, reduced apoptosis, decreased infarct size, and improved cardiac functional recovery.. Our results demonstrate that nitrate tolerance markedly enhances MI/R injury and that increased peroxynitrite formation likely plays a role in this pathologic process. In addition, our results suggest that GSH could decrease peroxynitrite formation and reduce MI/R injury in nitrate tolerant hearts.

Topics: Adaptation, Physiological; Animals; Apoptosis; Blood Pressure; Caspase 3; Creatine Kinase; Dietary Supplements; Enzyme Activation; Glutathione; Heart Rate; Heart Ventricles; L-Lactate Dehydrogenase; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Nitrates; Nitroglycerin; Rats; Recovery of Function; Tyrosine; Vasodilation

Aging and estrogen deficiency increase the risk for developing cardiovascular disease (CVD). Oxidative stress has also been implicated in the pathophysiology of CVD and in ischemia-reperfusion (I/R) injury. We tested the hypothesis that chronic in vivo estrogen treatment or superoxide inhibition with the SOD mimetic EUK-8 improves cardiac functional recovery after I/R in the aged female rat. Sprague-Dawley rats (12-14 mo) were used as follows: intact (n = 6), ovariectomized + placebo (OVX, n = 6), OVX + EUK-8 (EUK-8, 3 mg/kg, n = 6), and OVX + estrogen (1.5 mg/pellet, 60 days release, n = 6). Perfused isolated hearts were subjected to global ischemia (25 min) followed by reperfusion (40 min). Functional recovery after I/R and myocardial protein expression of NADPH oxidase (p22, p67, and gp91(phox)), inducible nitric oxide synthase (NOS), endothelial NOS, and SOD1, as well as nitrotyrosine levels (as a marker for peroxynitrite), were assessed. Compared with OVX, EUK-8 and estrogen markedly improved functional recovery after I/R, which was associated with a decrease in NADPH oxidase expression and nitrotyrosine staining. However, estrogen increased inducible NOS expression, whereas EUK-8 had little effect. There were no significant changes in endothelial NOS and SOD1 expression among the groups. These results indicate that EUK-8 and estrogen improved cardiac recovery after I/R. Given the controversy surrounding hormone replacement therapy, EUK-8 may be an alternative to estrogen in protecting those at risk for myocardial ischemia in the aging population.

Topics: Aging; Animals; Body Weight; Cardiotonic Agents; Estrogens; Ethylenediamines; Female; Heart; Heart Ventricles; Myocardial Ischemia; Myocardial Reperfusion Injury; NADPH Oxidases; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Organ Size; Organometallic Compounds; Ovariectomy; Rats; Rats, Sprague-Dawley; Recovery of Function; Superoxide Dismutase; Tyrosine; Uterus

Hypercholesterolemia (HC) and atherosclerosis can elicit oxidative stress, coronary endothelial dysfunction, and myocardial ischemia, which may induce growth-factor expression and lead to myocardial neovascularization. We tested the hypothesis that chronic antioxidant intervention in HC would attenuate neovascularization and preserve the expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF).. Three groups of pigs (n=6 each) were studied after 12 weeks of normal or 2% HC diet or HC+antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily). Myocardial samples were scanned ex vivo with a novel 3D micro-CT scanner, and the spatial density and tortuosity of myocardial microvessels were determined in situ. VEGF mRNA, protein levels of VEGF and VEGF receptor-1, HIF-1alpha, nitrotyrosine, and superoxide dismutase (SOD) were determined in myocardial tissue. The HC and HC+antioxidant groups had similar increases in serum cholesterol levels. HC animals showed an increase in subendocardial spatial density of microvessels compared with normal (160.5+/-11.8 versus 95.3+/-8.2 vessels/cm2, P<0.05), which was normalized in HC+antioxidant (92.5+/-20.5 vessels/cm2, P<0.05 versus HC), as was arteriolar tortuosity. In addition, HC induced upregulation of VEGF, HIF-1alpha, and nitrotyrosine expression and decreased SOD expression and activity, all of which were preserved by antioxidant intervention.. Changes in myocardial microvascular architecture invoked by HC are accompanied by increases in HIF-1alpha and VEGF expression and attenuated by antioxidant intervention. This underscores a role of increased oxidative stress in modulating myocardial microvascular architecture in early atherogenesis.

Topics: Animals; Antioxidants; Arteriosclerosis; Ascorbic Acid; Cardiotonic Agents; Coronary Circulation; Diet, Atherogenic; Dinoprost; Enzyme Induction; Female; Gene Expression Profiling; Gene Expression Regulation; Heart; Hypercholesterolemia; Hypoxia-Inducible Factor 1, alpha Subunit; Imaging, Three-Dimensional; Myocardial Ischemia; Neovascularization, Pathologic; Oxidative Stress; Superoxide Dismutase; Swine; Tomography, X-Ray Computed; Transcription Factors; Tyrosine; Vascular Endothelial Growth Factor A; Vitamin E

1. Myocardial injury caused by ischaemia and reperfusion comes from multiple pathogenic events, including endothelial damage, neutrophil extravasation into tissue, mast cell activation, and peroxidation of cell membrane lipids. These events are followed by myocardial cell alterations resulting eventually in cell necrosis. An enhanced formation of reactive oxygen species is widely accepted as a stimulus for tissue destruction and cardiac failure. 2. In this study, we have investigated the cardioprotective effects of M40403 in myocardial ischaemia-reperfusion injury. M40403 is a low molecular weight, synthetic manganese containing superoxide dismutase mimetic (SODm) that selectively removes superoxide anion. Ischaemia was induced in rat hearts in vivo by ligating the left anterior descending coronary artery. Thirty minutes after the induction of ischaemia, the ligature was removed and reperfusion allowed to occur for at least 60 min. M40403 (0.1-1 mg kg(-1)) was given intravenously 15 min before ischaemia. 3. The results obtained in this study showed that M40403 significantly reduced the extent of myocardial damage, mast cell degranulation and the incidence of ventricular arrhythmias. Furthermore, M40403 significantly attenuated, in a dose-dependent manner, neutrophil infiltration in the myocardium as well as the associated induction of lipid peroxidation. Calcium overload seen post-reperfusion of the ischaemic myocardium was also reduced by M40403. 4. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in cardiac tissue taken after reperfusion: this was attenuated by M40403. Moreover reperfused cardiac tissue sections showed positive staining for P-selectin and for anti-intercellular adhesion molecule (ICAM-1) in the vascular endothelial cells. M40403 treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in these tissues. No staining for nitrotyrosine, P-selectin or ICAM-1 was found in cardiac tissue taken at the end of the ischaemic period. 5. Overall, M40403 treatment reduced the morphological signs of myocardial cell injury and significantly improved survival. 6. Taken together, these results clearly indicate that M40403 treatment exerts a protective effect against ischaemia-reperfusion-induced myocardial injury, supporting a key role for superoxide anion in reperfusion injuries. This suggests that synthetic enzymes of SOD such as M40403, offer a novel therapeutic approach for the treatment of ischaemic hea

Topics: Animals; Cardiotonic Agents; Immunohistochemistry; Intercellular Adhesion Molecule-1; Male; Manganese; Molecular Mimicry; Myocardial Ischemia; Myocardial Reperfusion Injury; Organometallic Compounds; P-Selectin; Peroxidase; Rats; Rats, Wistar; Superoxide Dismutase; Tyrosine

Exposure of isolated rat hearts to 30 min global ischemia followed by 60 min reperfusion resulted in a significant 80% increase (p <.05) in actin content of carbonyl groups, which was associated with significant depression (p <.05) of postischemic contractile function. This result supports the hypothesis that one mechanism of postischemic contractile dysfunction may be oxidation of contractile proteins.

Topics: Actins; Animals; Blood Pressure; Heart; Heart Rate; Immunoblotting; In Vitro Techniques; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardial Stunning; Myocardium; Oxidation-Reduction; Perfusion; Rats; Rats, Sprague-Dawley; Troponin T; Tyrosine; Ventricular Function, Left

The role for peroxynitrite (ONOO(-)) in the mechanism of preconditioning is not known. Therefore, we studied effects of preconditioning and subsequent ischemia/reperfusion on myocardial ONOO(-) formation in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three intermittent periods of global ischemia/reperfusion of 5 min duration each) followed by a test ischemia/reperfusion (30 min global ischemia and 15 min reperfusion). When compared to nonpreconditioned controls, preceding preconditioning improved postischemic cardiac performance and significantly decreased test ischemia/reperfusion-induced formation of free nitrotyrosine measured in the perfusate as a marker for cardiac endogenous ONOO(-) formation. During preconditioning, however, the first period of ischemia/reperfusion increased nitrotyrosine formation, which was attenuated after the third period of ischemia/reperfusion. We conclude that classic preconditioning inhibits ischemia/reperfusion-induced cardiac formation of ONOO(-) and that subsequent periods of ischemia/reperfusion result in a gradual attenuation of ischemia/reperfusion-induced ONOO(-) generation. This mechanism might be involved in ischemic adaptation of the heart.

Topics: Animals; Aorta; Blood Flow Velocity; In Vitro Techniques; Ischemic Preconditioning, Myocardial; L-Lactate Dehydrogenase; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Nitrates; Rats; Rats, Wistar; Time Factors; Tyrosine; Ventricular Function, Left