allopurinol and Myocardial-Ischemia

Allopurinol as an effective inhibitor of the enzyme xanthine oxidase (XO) has been used for several decades for the treatment of patients with gout and hyperuricemia. Because the inhibition of XO limits the formation of radical oxygen species as well as uric acid (UA) production, allopurinol has been used experimentally for the treatment of conditions associated with ischemia and reperfusion (I/R) injury.Although there have been many ischemic organs treated in the laboratory with allopurinol, the heart has been of particular interest. Therefore, we emphasize our attention to the administration of XO inhibitors such as allopurinol on cardiac I/R as well as cardiac failure. Experimental data also support allopurinol as a possible consideration for biochemical support after acute myocardial infarction. Anker and associates (Circulation. 2003;107:1991-1997) have observed a direct correlation between uric acid levels and mortality in treated heart failure patients. Anker and associates showed a 100% mortality rate in patients with UA levels 800 micromol/L or less over a period of 3 years. Comparing this to a 27% mortality rate in patients with UA levels 400 micromol/L or less over a period of 10 years, it seems that the suppression of XO activity ameliorates myocardial inefficiency, and poor vascular flow may present innovative contributions to the future treatment of I/R heart failure patients. Our review focuses on the role of allopurinol on ischemic hearts as well as those with added chronic heart failure.

Topics: Allopurinol; Animals; Humans; Myocardial Ischemia; Xanthine Oxidase

A growing body of evidence suggests that oxygen radicals can mediate myocardial tissue injury during ischaemia and, in particular, during reperfusion. This review focuses on the role of neutrophil as a mediator of myocardial damage. Upon reperfusion, neutrophils accumulate and produce an inflammatory response in the myocardium that is responsible, in part, for the extension of tissue injury associated with reperfusion. It has shown that the inhibition of neutrophil accumulation and adhesion is associated with decreased infarct size. This strongly suggests that myocardial cells at risk region undergo irreversible changes upon reperfusion and accumulation of neutrophils. Several pharmacological agents (ibuprofen, allopurinol, prostacyclin, and prostaglandin E analogues) protect the myocardium from reperfusion injury. In addition, the mechanisms by which these agents act and directions of research that may lead to therapeutically useful approaches are also discussed in this review.

Topics: Allopurinol; Anti-Inflammatory Agents, Non-Steroidal; Epoprostenol; Free Radical Scavengers; Humans; Ibuprofen; Iloprost; Myocardial Ischemia; Myocardial Reperfusion Injury; Neutrophils; Prostaglandins E, Synthetic; Reactive Oxygen Species

Topics: Allopurinol; Antihypertensive Agents; Benzbromarone; Cerebrovascular Disorders; Humans; Hypertension; Hyperuricemia; Insulin Resistance; Myocardial Ischemia; Uricosuric Agents

Topics: Animals; Antioxidants; Arachidonic Acid; Electron Transport; Free Radicals; Humans; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Neutrophils; Oxygen; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Considerable research effort has been directed at elucidating the mechanisms underlying the pathophysiologic alterations associated with reperfusion (reoxygenation) of ischemic (hypoxic) tissues. As a consequence of this intensive effort, a large body of evidence has accumulated, implicating a role for reactive oxygen metabolites and activated granulocytes in the genesis of postischemic cellular dysfunction. Figure 1 summarizes a hypothesis that has been proposed to explain the interaction of xanthine oxidase-derived oxidants, granulocyte infiltration, and the microvascular and parenchymal cell dysfunction that occurs in postischemic tissues. According to this scheme, xanthine oxidase-derived oxidants, produced at reperfusion, initiate the formation and release of proinflammatory agents, which subsequently attract and activate granulocytes. The activated neutrophils adhere to the microvascular endothelium, extravasate, and release cytotoxic oxidants and proteases, which contribute to tissue dysfunction. The aim of this review is to summarize the evidence that we and others have accumulated in support of this hypothesis.

Topics: Acute-Phase Reaction; Animals; Granulocytes; Humans; Myocardial Ischemia; Myocardial Reperfusion Injury; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Reactive Oxygen Species; Xanthine Oxidase

Allopurinol is a urate-lowering therapy used to treat patients with gout. Previous studies have shown that allopurinol has positive effects on several cardiovascular parameters. The ALL-HEART study aimed to determine whether allopurinol therapy improves major cardiovascular outcomes in patients with ischaemic heart disease.. ALL-HEART was a multicentre, prospective, randomised, open-label, blinded-endpoint trial done in 18 regional centres in England and Scotland, with patients recruited from 424 primary care practices. Eligible patients were aged 60 years or older, with ischaemic heart disease but no history of gout. Participants were randomly assigned (1:1), using a central web-based randomisation system accessed via a web-based application or an interactive voice response system, to receive oral allopurinol up-titrated to a dose of 600 mg daily (300 mg daily in participants with moderate renal impairment at baseline) or to continue usual care. The primary outcome was the composite cardiovascular endpoint of non-fatal myocardial infarction, non-fatal stroke, or cardiovascular death. The hazard ratio (allopurinol vs usual care) in a Cox proportional hazards model was assessed for superiority in a modified intention-to-treat analysis (excluding randomly assigned patients later found to have met one of the exclusion criteria). The safety analysis population included all patients in the modified intention-to-treat usual care group and those who took at least one dose of randomised medication in the allopurinol group. This study is registered with the EU Clinical Trials Register, EudraCT 2013-003559-39, and ISRCTN, ISRCTN32017426.. Between Feb 7, 2014, and Oct 2, 2017, 5937 participants were enrolled and then randomly assigned to receive allopurinol or usual care. After exclusion of 216 patients after randomisation, 5721 participants (mean age 72·0 years [SD 6·8], 4321 [75·5%] males, and 5676 [99·2%] white) were included in the modified intention-to-treat population, with 2853 in the allopurinol group and 2868 in the usual care group. Mean follow-up time in the study was 4·8 years (1·5). There was no evidence of a difference between the randomised treatment groups in the rates of the primary endpoint. 314 (11·0%) participants in the allopurinol group (2·47 events per 100 patient-years) and 325 (11·3%) in the usual care group (2·37 events per 100 patient-years) had a primary endpoint (hazard ratio [HR] 1·04 [95% CI 0·89-1·21], p=0·65). 288 (10·1%) participants in the allopurinol group and 303 (10·6%) participants in the usual care group died from any cause (HR 1·02 [95% CI 0·87-1·20], p=0·77).. In this large, randomised clinical trial in patients aged 60 years or older with ischaemic heart disease but no history of gout, there was no difference in the primary outcome of non-fatal myocardial infarction, non-fatal stroke, or cardiovascular death between participants randomised to allopurinol therapy and those randomised to usual care.. UK National Institute for Health and Care Research.

Topics: Aged; Allopurinol; Coronary Artery Disease; Female; Gout; Humans; Male; Myocardial Infarction; Myocardial Ischemia; Prospective Studies; Stroke; Treatment Outcome; United Kingdom; Uric Acid

Ischaemic heart disease (IHD) is one of the most common causes of death in the UK and treatment of patients with IHD costs the National Health System (NHS) billions of pounds each year. Allopurinol is a xanthine oxidase inhibitor used to prevent gout that also has several positive effects on the cardiovascular system. The ALL-HEART study aims to determine whether allopurinol improves cardiovascular outcomes in patients with IHD.. The ALL-HEART study is a multicentre, controlled, prospective, randomised, open-label blinded end point (PROBE) trial of allopurinol (up to 600 mg daily) versus no treatment in a 1:1 ratio, added to usual care, in 5215 patients aged 60 years and over with IHD. Patients are followed up by electronic record linkage and annual questionnaires for an average of 4 years. The primary outcome is the composite of non-fatal myocardial infarction, non-fatal stroke or cardiovascular death. Secondary outcomes include all-cause mortality, quality of life and cost-effectiveness of allopurinol. The study will end when 631 adjudicated primary outcomes have occurred. The study is powered at 80% to detect a 20% reduction in the primary end point for the intervention. Patient recruitment to the ALL-HEART study started in February 2014.. The study received ethical approval from the East of Scotland Research Ethics Service (EoSRES) REC 2 (13/ES/0104). The study is event-driven and results are expected after 2019. Results will be reported in peer-reviewed journals and at scientific meetings. Results will also be disseminated to guideline committees, NHS organisations and patient groups.. 32017426, pre-results.

Topics: Allopurinol; Female; Free Radical Scavengers; Humans; Male; Middle Aged; Myocardial Ischemia; Prospective Studies; Research Design; Treatment Outcome; United Kingdom

This study sought to ascertain if high-dose allopurinol regresses left ventricular mass (LVM) in patients with ischemic heart disease (IHD).. LV hypertrophy (LVH) is common in patients with IHD including normotensive patients. Allopurinol, a xanthine oxidase inhibitor, has been shown to reduce LV afterload in IHD and may therefore also regress LVH.. A randomized, double-blind, placebo-controlled, parallel group study was conducted in 66 patients with IHD and LVH, comparing 600 mg/day allopurinol versus placebo therapy for 9 months. The primary outcome measure was change in LVM, assessed by cardiac magnetic resonance imaging (CMR). Secondary outcome measures were changes in LV volumes by CMR, changes in endothelial function by flow-mediated dilation (FMD), and arterial stiffness by applanation tonometry.. Compared to placebo, allopurinol significantly reduced LVM (allopurinol -5.2 ± 5.8 g vs. placebo -1.3 ± 4.48 g; p = 0.007) and LVM index (LVMI) (allopurinol -2.2 ± 2.78 g/m(2) vs. placebo -0.53 ± 2.5 g/m(2); p = 0.023). The absolute mean difference between groups for change in LVM and LVMI was -3.89 g (95% confidence interval: -1.1 to -6.7) and -1.67 g/m(2) (95% confidence interval: -0.23 to -3.1), respectively. Allopurinol also reduced LV end-systolic volume (allopurinol -2.81 ± 7.8 mls vs. placebo +1.3 ± 7.22 mls; p = 0.047), improved FMD (allopurinol +0.82 ± 1.8% vs. placebo -0.69 ± 2.8%; p = 0.017) and augmentation index (allopurinol -2.8 ± 5.1% vs. placebo +0.9 ± 7%; p = 0.02).. High-dose allopurinol regresses LVH, reduces LV end-systolic volume, and improves endothelial function in patients with IHD and LVH. This raises the possibility that allopurinol might reduce future cardiovascular events and mortality in these patients. (Does a Drug Allopurinol Reduce Heart Muscle Mass and Improve Blood Vessel Function in Patients With Normal Blood Pressure and Stable Angina?; ISRCTN73579730).

Topics: Aged; Allopurinol; Double-Blind Method; Female; Humans; Hypertrophy, Left Ventricular; Magnetic Resonance Imaging; Male; Manometry; Myocardial Ischemia

Increased production of free radicals under oxidative stress conditions plays a vital role in the impairment of endothelial function and also in the pathogenesis of ischemic heart diseases. Ischemia, followed by reperfusion, leads to the exacerbated formation of oxy- free radicals. These reactive oxygen species through a chain of reactions damage the cardiomyocytes and cause more injury to the myocardium. L-Arginine is reported to act as free radical scavenger, inhibits the activity of pro-oxidant enzymes and thus acts as an antioxidant and these roles of L-arginine are mediated by nitric oxide (NO). In the present study, the effect of oral administration of L-arginine (3 g/day for 7 days) on some antioxidant enzymes, total thiols, lipid peroxidation measured as malondialdehyde (MDA), and plasma ascorbate levels in myocardial ischemic patients was investigated. We observed an increase in the activity of superoxide dismutase (SOD), total thiols (T-SH) and plasma ascorbate levels and a decrease in the activity of xanthine oxidase (XO), MDA levels, carbonyl content and serum cholesterol in the patients on oral administration of L-arginine. The present study demonstrates that L-arginine administration may be beneficial to patients with myocardial ischemic disorders, such as acute myocardial infarction and acute angina.

Topics: Adult; Aged; Arginine; Ascorbic Acid; Case-Control Studies; Cholesterol; Free Radical Scavengers; Humans; Malondialdehyde; Middle Aged; Myocardial Ischemia; Oxidants; Sulfhydryl Compounds; Superoxide Dismutase; Xanthine Oxidase

Xanthine oxidase has been demonstrated to be an important factor in postischemic reperfusion injury. Allopurinol is the inhibitor of xanthine oxidase. In this study, we evaluated usefulness of allopurinol for prevention of myocardial reperfusion injury in open heart surgery. Twenty adult patients were divided into two groups; Ap-group (n = 10) were administered allopurinol for seven days just before operation and control-group (n = 10) were administered nothing. The dose of allopurinol was ranged 100-300 mg/day. The dose was determined according to renal function of patients. Coronary sinus blood hypoxanthine, xanthine, uric acid, lactate, pyruvate, CK, and CK-MB levels were measured at before CPB, 5 and 15 min. after aortic declamping, 5 min. after CPB. Hypoxanthine and xanthine levels in Ap-group were significantly higher than those in control-group at 5 and 15 min. after aortic declamping, 5 min. after CPB. Uric acid levels in Ap-group were significantly lower than those in control-group at 5 and 15 min. after aortic declamping, 5 min. after CPB. Lactate, pyruvate, CK, and CK-MB levels were not different between Ap-group and control group. We considered that allopurinol suppressed the reaction rate of xanthine oxidase. Therefore, the levels of intermediates, hypoxanthine and xanthine, were high and, the level of final product, uric acid was low in Ap-group. However, allopurinol had no efficacy for the level of lactate, pyruvate, CK, and CK-MB, in this study.

Topics: Aged; Allopurinol; Creatine Kinase; Female; Heart Defects, Congenital; Heart Diseases; Humans; Isoenzymes; Male; Middle Aged; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Xanthine Oxidase

Topics: Allopurinol; Cardiovascular Diseases; Cardiovascular System; Coronary Artery Disease; Heart; Humans; Myocardial Ischemia

Allopurinol is commonly prescribed for gout, and its clinical use may expand with ongoing trials assessing its potential cardiorenal benefits. Because heart disease has been suggested to be a risk factor for allopurinol-associated severe cutaneous adverse reactions, we sought to confirm this association in a Canadian general population cohort.. We used population data from British Columbia, Canada, to identify all incident allopurinol users between 1997 and 2015. We examined the association between heart disease (ischemic heart disease and heart failure) and the risk of hospital admission for severe cutaneous adverse reactions, adjusting for known and purported risk factors. We also evaluated the joint effects of combined clinical and demographic risk factors.. Among 130 325 allopurinol initiators, 109 hospital admissions occurred for allopurinol-associated severe cutaneous adverse reactions. The multivariable relative risk among those with heart disease was 1.55 (95% confidence interval 1.01-2.37). Patients with heart disease and chronic kidney disease who were started on an allopurinol dosage of greater than 100 mg/d had an 11-fold higher risk. Allopurinol initiation at a lower dosage among patients with heart disease and chronic kidney disease resulted in a fivefold reduction in risk. Older women with heart disease from regions with large Asian populations had a 23-fold higher risk of allopurinol-associated severe cutaneous adverse reactions than younger men without heart disease from other regions.. Heart disease is independently associated with risk of allopurinol-associated severe cutaneous adverse reactions, similar to chronic kidney disease, and low-dosage allopurinol initiation may substantially mitigate this risk. Risk factors for these rare but serious reactions should be considered when initiating allopurinol.

Topics: Age Factors; Aged; Allopurinol; Asian People; British Columbia; Dose-Response Relationship, Drug; Drug Eruptions; Ethnicity; Female; Gout; Gout Suppressants; Heart Failure; Hospitalization; Humans; Incidence; Male; Middle Aged; Myocardial Ischemia; Renal Insufficiency, Chronic; Risk Factors; Severity of Illness Index; Sex Factors

Left ventricular hypertrophy (LVH), as assessed by measurement of left ventricular mass (LVM), is one of the most important cardiovascular risk factors. It is commonly present in patients with ischemic heart disease (IHD), irrespective of the level of blood pressure; recently, oxidative stress has been shown to be an important factor in its development. The question then arises: can this risk factor be modified by antioxidant treatment (e.g., with allopurinol, a xanthine oxidase inhibitor)?. This is an observational study with a cross-sectional design which explored the association between long-term (>12 months) allopurinol therapy and LV mass index (LVMI) as well as geometry in patients generally receiving standard treatments for IHD. The primary endpoint was LVMI measurement (by 2D-echocardiography) and secondary endpoints included the association of allopurinol use with LV function (ejection fraction), blood pressure, glycemic control, and lipid profile.. Ninety-six patients on standard anti-ischemic drug treatment (control group) and 96 patients who were additionally taking allopurinol (minimum dose 100 mg/day) were enrolled. Both groups were matched for age, sex, height, and co-morbidities, but poorer kidney function in the allopurinol group required further sub-group analysis based on renal function. Allopurinol treatment was associated with the lowest LVMI in the patients with normal serum creatinine (median LVMI; 70.5 g/m. In our population, treatment with allopurinol (presumably because of its anti-oxidant properties) has shown a tendency to be associated with smaller LVM in IHD patients with normal serum creatinine, along with better glycemic control.

Topics: Aged; Aged, 80 and over; Allopurinol; Antioxidants; Biomarkers; Blood Glucose; Case-Control Studies; Creatinine; Cross-Sectional Studies; Female; Glycated Hemoglobin; Humans; Hypertrophy, Left Ventricular; Male; Middle Aged; Myocardial Ischemia; Time Factors; Treatment Outcome; Ventricular Function, Left; Ventricular Remodeling

Pathogenesis of cardiac microvascular ischemia-reperfusion (IR) injury is associated with excessive mitochondrial fission. However, the upstream mediator of mitochondrial fission remains obscure. Bax inhibitor 1 (BI1) is linked to multiple mitochondrial functions, and there have been no studies investigating the contribution of BI1 on mitochondrial fission in the setting of cardiac microvascular IR injury. This study was undertaken to establish the action of BI1 on the cardiac microvascular reperfusion injury and figure out whether BI1 sustained endothelial viability via inhibiting mitochondrial fission. Our observation indicated that BI1 was downregulated in reperfused hearts and overexpression of BI1 attenuated microvascular IR injury. Mechanistically, reperfusion injury elevated the levels of xanthine oxidase (XO), an effect that was followed by increased reactive oxygen species (ROS) production. Subsequently, oxidative stress mediated F-actin depolymerization and the latter promoted mitochondrial fission. Aberrant fission caused mitochondrial dysfunction and ultimately activated mitochondrial apoptosis in cardiac microvascular endothelial cells. By comparison, BI1 overexpression repressed XO expression and thus neutralized ROS, interrupting F-actin-mediated mitochondrial fission. The inhibitory effect of BI1 on mitochondrial fission sustained endothelial viability, reversed endothelial barrier integrity, attenuated the microvascular inflammation response, and maintained microcirculation patency. Altogether, we conclude that BI1 is essential in maintaining mitochondrial homeostasis and alleviating cardiac microvascular IR injury. Deregulated BI1 via the XO/ROS/F-actin pathways plays a causative role in the development of cardiac microvascular reperfusion injury.

Topics: Actins; Animals; Cells, Cultured; Heart; Male; Membrane Proteins; Mice; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Dynamics; Myocardial Ischemia; Myocardial Reperfusion Injury; Oxidative Stress; Reactive Oxygen Species; Xanthine Oxidase

Ventricular arrhythmias induced by ischemic heart disease are the main cause of sudden cardiac death. Ischemia can cause life-threatening arrhythmias by modulating connexin 43 (Cx43), a principal cardiac gap junction channel protein. The present study investigates whether nitrite can attenuate ischemia-induced ventricular arrhythmias and dephosphorylation of Cx43 in a rat model.. Rats were medicated with normal saline (control, n = 10), nitrite (0.015, 0.15, and 1.5 mg/kg, n = 9 or 10 each), and 0.15 mg/kg nitrite with either the nitric oxide scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (cPTIO; n = 9) or allopurinol (xanthine oxidoreductase inhibitor, n = 9). We determined the severity of ventricular arrhythmias based on arrhythmia scores and levels of phosphorylated Cx43.. The median arrhythmia score may have been lower in the group given 0.15 mg/kg nitrite (4 [interquartile range {IQR}, 4-5]) than that in the control group (7.5 [IQR, 5.25-8]; P = 0.013). There was no difference among the control, the given 0.015 mg/kg nitrite (7 [IQR, 5-8]), and 1.5 mg/kg nitrite (7 [IQR, 5.5-7.75]; P = 0.95). The arrhythmia scores in the cPTIO (6 [IQR, 5-8]; P = 0.030) and allopurinol (7 [IQR, 5-8]; P = 0.005) groups may have been higher than that in 0.15 mg/kg nitrite group. Immunoblotting revealed that the level of phosphorylated Cx43 in the group given 0.15 mg/kg nitrite, but not in the other treated groups, was significantly higher compared with the control group (P = 0.007).. Nitrite may have attenuated acute ischemia-induced ventricular arrhythmias and Cx43 dephosphorylation in rats. Nitric oxide, which might be generated by xanthine oxidoreductase via nitrite reduction, appears to play a crucial role in this antiarrhythmic effect.

Topics: Allopurinol; Animals; Arrhythmias, Cardiac; Blood Gas Analysis; Connexin 43; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Hemodynamics; Imidazoles; Male; Myocardial Ischemia; Nitric Oxide; Phosphorylation; Rats; Rats, Wistar; Sodium Nitrite; Ventricular Dysfunction

Nuclear myosin regulates gene transcription and this novel function might be modulated through phosphorylation of the myosin regulatory light chain (p-MLC20). Nonmuscle MLC20 (nmMLC20) is also present in the nuclei of cardiomyocytes and a potential nmMLC20 binding sequence has been identified in the promoter of the xanthine oxidase (XO) gene. Thus, we investigated its function in the regulation of XO transcription after myocardial ischemia/reperfusion (IR). In a rat model of myocardial IR and a cardiomyocyte model of hypoxia/reoxygenation (HR) injury, the cardiac or cell injury, myosin light chain kinase (MLCK) content, XO expression and activity, XO-derived products, and level of nuclear p-nmMLC20 were detected. Coimmunoprecipitation (co-IP), chromatin immunoprecipitation, DNA pull-down, and luciferase reporter gene assays were used to decipher the molecular mechanisms through which nmMLC20 promotes XO expression. IR or HR treatment dramatically elevated nuclear p-nmMLC20 level, accompanied by increased XO expression, activity, and products (H2O2 and uric acid), as well as the IR or HR injury; these effects were ameliorated by inhibition of MLCK or knockdown of nmMLC20. Our findings from these experiments demonstrated that nuclear p-nmMLC20 binds to the consensus sequence GTCGCC in the XO gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and hence activates XO gene transcription. These results suggest that nuclear p-nmMLC20 plays an important role in IR/HR injury by transcriptionally upregulating XO gene expression to increase oxidative stress in myocardium. Our findings demonstrate nuclear nmMLC20 as a potential new therapeutic target to combat cardiac IR injury.

Topics: Animals; Apoptosis; Blotting, Western; Cell Nucleus; Cell Proliferation; Cells, Cultured; Chromatin Immunoprecipitation; Gene Expression Regulation, Enzymologic; Hydrogen Peroxide; Immunoprecipitation; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myosin Light Chains; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Xanthine Oxidase

We have previously shown that hyperuricemia plays an important role in the vascular complications of insulin resistance (IR). Here we investigated the effect of xanthine oxidase (XO) inhibition on the cardiac complications of IR.. IR was induced in rats by a high fructose high fat diet for 12 weeks. Allopurinol, a standard XO inhibitor, was administered in the last 4 weeks before cardiac hemodynamics and electrocardiography, serum glucose, insulin, tumor necrosis factor alpha (TNFα), 8-isoprostane, uric acid, lactate dehydrogenase (LDH) and XO activity were measured. Expression of cardiac angiotensin II (AngII) and angiotensin receptor 1 (AT1) were assessed by immunofluorescence.. IR animals had significant hyperuricemia which was inhibited by allopurinol administration. IR was associated with impaired ventricular relaxation (reflected by a decreased diastolic pressure increment and prolonged diastolic duration) and XO inhibition greatly attenuated impaired relaxation. IR was accompanied by cardiac ischemia (reflected by increased QTc and T peak trend intervals) while XO inhibition alleviated the ECG abnormalities. When subjected to isoproterenol-induced ischemia, IR hearts were less resistant (reflected by larger ST height depression and higher LDH level) while XO inhibition alleviated the accompanying ischemia. In addition, XO inhibition prevented the elevation of serum 8-isoprostane and TNFα, and blocked elevated AngII and AT1 receptor expression in the heart tissue of IR animals. However, XO inhibition did not affect the developed hyperinsulinemia or dyslipidemia.. XO inhibition alleviates cardiac ischemia and impaired relaxation in IR through the inhibition of low grade inflammation and the angiotensin system.

Topics: Administration, Oral; Allopurinol; Angiotensin II; Animals; Diet, High-Fat; Electrocardiography; Enzyme Inhibitors; Hemodynamics; Inflammation; Insulin Resistance; Isoproterenol; Male; Myocardial Ischemia; Myocardium; Oxidative Stress; Rats, Wistar; Ultrasonography; Uric Acid; Xanthine Oxidase

Ventricular tachycardia or fibrillation (VT/VF) of focal origin due to triggered activity (TA) from delayed afterdepolarizations (DADs) is reproducibly inducible after anterior coronary artery occlusion. Both VT/VF and TA can be blocked by reducing reactive oxygen species (ROS). We tested the hypothesis that inhibition of NADPH oxidase and xanthine oxidase would block VT/VF.. 69 dogs received apocynin (APO), 4 mg/kg intraveneously (IV), oxypurinol (OXY), 4 mg/kg IV, or both APO and OXY (BOTH) agents, or saline 3 h after coronary occlusion. Endocardium from ischemic sites (3-D mapping) was sampled for Rac1 (GTP-binding protein in membrane NADPH oxidase) activation or standard microelectrode techniques. Results (mean±SE, * p<0.05): VT/VF originating from ischemic zones was blocked by APO in 6/10 *, OXY in 4/9 *, BOTH in 5/8 * or saline in 1/27; 11/16 VT/VFs blocked were focal. In isolated myocardium, TA was blocked by APO (10(-6) M) or OXY (10(-8) M). Rac1 levels in ischemic endocardium were decreased by APO or OXY.. APO and OXY suppressed focal VT/VF due to DADs, but the combination of the drugs was not more effective than either alone. Both drugs inhibited ischemic Rac1 with inhibition by OXY suggesting ROS-induced ROS. The inability to totally prevent VT/VF suggests that other mechanisms also contribute to ischemic VT.

Topics: Acetophenones; Action Potentials; Animals; Blotting, Western; Disease Models, Animal; Dogs; Female; Male; Myocardial Ischemia; NADPH Oxidases; Oxypurinol; rac1 GTP-Binding Protein; Tachycardia, Ventricular; Ultrasonography; Ventricular Fibrillation; Xanthine Oxidase

Topics: Allopurinol; Female; Humans; Hypertrophy, Left Ventricular; Male; Myocardial Ischemia

Increased production of oxygen free radicals and decreased oxidant capacity occur in coronary artery diseases (CAD) This pro-oxidant shift in intracellular redox state may induce cell death by either direct cell membrane damage by lipic peroxidation or apoptosis through activation of transcription factors. These changes occur not only in cardiomyocytes, bu also in cardiac sympathetic nerves, which are very sensitive to oxidative damage. Patients with heart failure encountel reduced peripheralblood flow at rest, during exercise and in response to endothelium-dependentvasodilators. Current treatments of cardiomyopathy, a degenerative condition of the myocardium frequently associated with heart failure have done little to enhance patient survival. Decreased myocardial contractility and altered regulation of peripheral circulation along with oxidative conditions are important contributors to the symptoms and prognosis of the disease process. Nitric oxide formed from L-arginine (2-amino-5 guanidinovaleric acid) metabolism in endothelial cells contributes to regulation of blood flow under these conditions. L-Arginine is the precursor of nitric oxide, an endogenous messenger molecule involved in a variety of endothelium-mediated physiological effects in the vascular system. In the present study, we investigated the effect of oral administration of L-arginine (3 g/day) on the intracellular redox status of the patients of ischemic cardiomyopathy aged 45-60 yrs. The enzymatic and non-enzymatic antioxidant parameters like superoxide dismutase, catalase, total thiols (TSH) and ascorbic acid along with pro-oxidant parameters, such as xanthine oxidase, as well as index of oxidative stress as protein carbonyl content and malondialdehyde (a marker of lipid peroxidation) were investigated in the plasma and RBC lysate. L-Arginine (3 g/day) administration was found to improve the levels of these parameters in the patients and regulate the blood flow, as evident by the improved blood pressure of the patients. Thus, it is inferred that L-arginine attenuates the oxidative stress conditions along with maintaining the blood pressure rate of patients suffering from cardiomyopathy.

Topics: Antioxidants; Arginine; Ascorbic Acid; Cardiomyopathies; Catalase; Coronary Artery Disease; Female; Free Radicals; Humans; Male; Middle Aged; Models, Biological; Myocardial Ischemia; Oxidants; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Thyrotropin; Xanthine Oxidase

Previously, we showed that oral allopurinol increased survival in mice with post-ischemic cardiomyopathy and attributed this outcome to an improvement of excitation-contraction coupling that boosted contractility. In this study, we tested the sustainability of this enhanced contraction associated with decreased oxidative damage over an extended time. Mice were divided into three groups: sham-operated control, myocardial infarction-heart failure (MI-HF), and oxypurinol-treated heart failure (Oxy-HF). After 9-11 months, echocardiography showed that mice treated with oxypurinol (1mM in drinking water) had significantly higher left ventricle fractional contraction and fractional wall thickening during systole than did mice in the MI-HF group (left ventricle fractional contraction: 28.4+/-2.2% vs. 19.9+/-2.3%, P<0.05; left ventricle fractional wall thickening: 45.0+/-4.0% vs. 23.5+/-2.0%, P<0.05). Left ventricular diastolic dimension, however, remained enlarged (0.50+/-0.04 vs. 0.54+/-0.05 cm, not significant). Twitch force was significantly higher at any given external Ca(2+) concentration in the Oxy-HF group than in the MI-HF group (P<0.01); amplitudes of intracellular Ca(2+) transients were also higher in the Oxy-HF group but were not statistically different from those of the MI-HF group. Force-frequency relation was improved in the Oxy-HF group. Muscle in the Oxy-HF group exhibited increases in myofilament Ca(2+) responsiveness, as evidenced by significantly higher maximal Ca(2+)-activated force (77.8+/-12.7 vs. 36.4+/-4.4 mN/mm(2), P<0.01). Finally, lipid peroxidation and myofilament oxidation were suppressed in the Oxy-HF group. These results indicate that the beneficial effects of antioxidation can be sustained by long-term treatment with oxypurinol after ischemic heart failure, with significantly improved cardiac contractility.

Topics: Animals; Calcium; Enzyme Inhibitors; Excitation Contraction Coupling; Heart; Heart Failure; Male; Mice; Myocardial Contraction; Myocardial Ischemia; Myocardium; Oxidation-Reduction; Oxidative Stress; Oxypurinol; Time Factors; Xanthine Oxidase

Nitric oxide (NO) may limit myocardial ischemia-reperfusion injury by slowing the mitochondrial metabolism. We examined whether rat heart contains catalysts potentially capable of reducing nitrite to NO during an episode of regional myocardial ischemia produced by temporary coronary artery occlusion. In intact Sprague-Dawley rats, a 15-min coronary occlusion lowered the nitrite concentration of the myocardial regions exhibiting ischemic glucose metabolism to approximately 50% that of nonischemic regions (185 +/- 223 vs. 420 +/- 203 nmol/l). Nitrite was rapidly repleted during subsequent reperfusion. The heart tissue tested in vitro acquired a substantial ability to consume nitrite when made hypoxic at neutral pH, and this ability was slightly enhanced by simultaneously lowering the pH to 5.5. More than 70% of this activity could be abolished by flushing the coronary circulation with crystalloid to remove trapped erythrocytes. Correspondingly, erythrocytes demonstrated the ability to reduce exogenous nitrite to NO under hypoxic conditions in vitro. In erythrocyte-free heart tissue, the nitrite consumption increased fivefold when the pH was lowered to 5.5. Approximately 40% of this pH-sensitive increase in nitrite consumption could be blocked by the xanthine oxidoreductase inhibitor allopurinol, whereas lowering the Po(2) sufficiently to desaturate myoglobin accelerated it further. We conclude that rat heart contains several factors capable of catalyzing ischemic nitrite reduction; the most potent is contained within erythrocytes and activated by hypoxia, whereas the remainder includes xanthine oxidoreductase and other pH-sensitive factors endogenous to heart tissue, including deoxymyoglobin.

Topics: Allopurinol; Animals; Catalysis; Disease Models, Animal; Enzyme Inhibitors; Erythrocytes; Glucose; Glycogen; Hydrogen-Ion Concentration; Hypoxia; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Myoglobin; Nitric Oxide; Nitrites; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Time Factors; Xanthine Dehydrogenase

The hypothesis was tested that endothelin-1 (ET-1)-induced superoxide (O(2)(-)) generation mediates post-ischemic coronary endothelial injury, that ischemic preconditioning (IPC) affords endothelial protection by preventing post-ischemic ET-1, and thus O(2)(-), generation, and that opening of the mitochondrial ATP-dependent potassium channel (mK(ATP)) triggers the mechanism of IPC. Furthermore, the study was aimed at identifying the source of O(2)(-) mediating the endothelial injury. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min washout of mK(ATP) opener diazoxide (0.5 mM). Coronary flow responses to acetylcholine (ACh) served as a measure of endothelium-dependent vascular function. Myocardial outflow of ET-1 and O(2)(-) and functional recoveries were followed during reperfusion. NADPH oxidase and xanthine oxidase (XO) activities were measured in cardiac homogenates. IR augmented ET-1 and O(2)(-) outflow and impaired ACh response. All these effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK(ATP) blocker) abolished the effects of IPC and diazoxide. Superoxide dismutase and tezosentan (a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. IR augmented also the activity of NADPH oxidase and XO. Apocynin treatment, that resulted in NADPH oxidase inhibition, prevented XO activation and O(2)(-) generation in IR hearts. The inhibition of XO, either by allopurinol or feeding the animals with tungsten-enriched chow, prevented post-ischemic O(2)(-) generation, although these interventions had no effect on the NADPH activity. In addition, the post-ischemic activation of NADPH oxidase and XO, and O(2)(-) generation were prevented by IPC, tezosentan, thenoyltrifluoroacetone (mitochondrial complex II inhibitor), and tempol (cell-membrane permeable O(2)(-) scavenger). In guinea-pig heart: (i) ET-1-induced O(2)(-) generation mediates post-ischemic endothelial dysfunction; (ii) IPC and diazoxide afford endothelial protection by attenuating the ET-1, and thus O(2)(-) generation, and the mK(ATP) opening triggers the protection; (iii) the NADPH oxidase maintains the activity of XO, and the XO-derived O(2)(-) mediates the endothelial injury, and (iv) ET-1 and O(2)(-) (probably of mitochondrial o

Topics: Animals; Endothelin-1; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Guinea Pigs; Ischemic Preconditioning, Myocardial; Mitochondria, Heart; Myocardial Ischemia; Myocardium; NADPH Oxidases; Superoxides; Xanthine Oxidase

A major limitation of stem cell transfer is early donor-cell death. Here, we seek to enhance myocardial repair following injury through transplantation of cardiomyocyte-enriched human embryonic stem cells (hESC) and recipient treatment with cytoprotective (allopurinol+uricase) and anti-inflammatory (ibuprofen) agents.. We injected 10(6) (15% hESC-derived cardiomyocytes) green fluorescent protein (GFP+) hESC in the infarcted area following left anterior descending artery (LAD)-ligation in SCID-beige mice. In Group I, 1.6 mg allopurinol and 0.2 mg of uricase were injected i.p. for 3 days prior to cell transplantation. In Group II, 0.35 mg/ml of ibuprofen were added to the drinking water before and after cell implantation. In Group III, the LAD was ligated and allopurinol/uricase was administered without cell treatment. In Group IV, ibuprofen was added to the drinking water and the LAD was ligated without additional cell treatment. In Group V, only cells were transplanted. Group VI involved infarcted controls and Group VII involved sham-operated mice (all groups: n=5). We evaluated heart function (ejection fraction (EF)) by MRI (4.7 T) 3 weeks later. The hearts were harvested for histology.. Differentiated hESC formed clusters and expressed alpha-sarcomeric actin and Connexin 43. Cell treatment improved heart function, which was best in the ibuprofen- and allopurinol-treated groups (+cell transfer), compared to the infarcted controls [EF: Group I: 76.6+/-8.6%, Group II: 78.6+/-7.3%, Group III: 58.1+/-5.7%, Group IV: 53.9+/-5.2%, Group V: 57.7+/-7.5%, Group VI: 43.5+/-4.3%, and Group VII: 66.3+/-7.8%]. We did not observe tumors in any group.. Allopurinol/uricase and ibuprofen enhance differentiated hESC-engraftment and myocardial restoration following transplantation into the injured heart.

Topics: Acute Disease; Allopurinol; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Combined Modality Therapy; Disease Models, Animal; Female; Fluorescent Antibody Technique; Heart; Humans; Ibuprofen; Mice; Mice, SCID; Microscopy, Confocal; Myocardial Ischemia; Myocytes, Cardiac; Stem Cell Transplantation; Stroke Volume; Urate Oxidase

We previously showed that caffeic acid phenethyl ester (CAPE) attenuated NO production, reduced apoptosis, diminished serum CK and AST activities, and is cardio-protective in rat heart subjected to ischemia/reperfusion (I/R). We now studied the short-term cardio-protective effect of CAPE in an I/R rat heart model.. Wistar rats were divided into four groups: (i) sham-operated, (ii) I/R, (iii) I/R+CAPE, and (iv) I/R+glutathione. CAPE (10 micromol/kg) was infused i.v. 10 min before occlusion of the left anterior descending coronary artery (8 min) followed by reperfusion (8 min).. SOD (p < 0.010) and CAT (p < 0.014) activities were increased and GSH-Px (p < 0.019) activity decreased in the I/R group in cardiac tissues compared with the sham-operated group. There were no effects of CAPE on antioxidant enzyme activities after I/R. Glutathione administration led to decreased SOD activity (p < 0.024) and increased GSH-Px (p < 0.014) activity after I/R. The most prominent effects of CAPE were seen in XO and ADA activities, which were increased after I/R compared with the sham-operated group (p < 0.029 and p < 0.001, respectively). When CAPE was administered, XO and ADA activities were decreased compared with the I/R group (p < 0.045 and p < 0.001). ADA activity was also decreased in the I/R+glutathione group compared with the I/R group. No differences in basal heart rate or systolic or diastolic pressure were noted among the experimental groups.. This study demonstrates that CAPE exerts cardio-protective effects in short-term I/R of rat heart. This protective effect may be mediated by a combination of decreased XO activity and direct antioxidant effects.

Topics: Acute Disease; Adenosine Deaminase; Animals; Antioxidants; Caffeic Acids; Cardiotonic Agents; Catalase; Glutathione Peroxidase; Male; Malondialdehyde; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitric Oxide; Phenylethyl Alcohol; Rats; Rats, Wistar; Superoxide Dismutase; Xanthine Oxidase

Reactive oxygen species, in particular superoxide, have been closely linked to the underlying pathophysiology of ischemic cardiomyopathy: superoxide not only mediates mechanoenergetic uncoupling of the myocyte but also adversely impacts on myocardial perfusion by depleting endothelial-derived nitric oxide bioavailability. Xanthine oxidase generates superoxide upon oxidation of hypoxanthine and xanthine and has been detected in cardiac myocytes and coronary endothelial cells of patients with ischemic heart disease. Here we investigated the effects of oxypurinol, a xanthine oxidase inhibitor, on myocardial contractility in patients with ischemic cardiomyopathy. Twenty patients (19 males, 66+/-8 years) with stable coronary disease, severely suppressed systolic function (left ventricular ejection fraction 22+/-2%), and nonelevated uric acid plasma levels received a single intravenous dose of oxypurinol (400 mg). Cardiac MRI studies, performed before and 5.2+/-0.9 h after oxypurinol administration, revealed a reduction in end-systolic volumes (-9.7+/-4.2%; p=0.03) and an increase in left ventricular ejection fraction (+17.5+/-5.2%; p=0.003), whereas 6 patients (6 males, 63+/-3.8 years, ejection fraction 26+/-5%) who received vehicle only did not show significant changes in any of the parameters studied. Oxypurinol improves left ventricular function in patients with ischemic cardiomyopathy. These results underscore the significance of reactive oxygen species as important pathophysiological mediators in ischemic heart failure and point toward xanthine oxidase as an important source of reactive species that serve to modulate the myocardial redox state in this disease.

Topics: Aged; Cardiomyopathies; Cardiotonic Agents; Enzyme Inhibitors; Female; Humans; Hypoxanthine; Magnetic Resonance Imaging, Cine; Male; Middle Aged; Myocardial Contraction; Myocardial Ischemia; Oxypurinol; Reactive Oxygen Species; Uric Acid; Xanthine; Xanthine Oxidase

Organic nitrates have been used clinically in the treatment of ischemic heart disease for more than a century. Recently, xanthine oxidase (XO) has been reported to catalyze organic nitrate reduction under anaerobic conditions, but questions remain regarding the initial precursor of nitric oxide (NO) and the link of organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of XO-mediated biotransformation of organic nitrate, studies using electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, NO electrode, and immunoassay were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered the reduction of organic nitrate to nitrite anion (NO2-). Studies of the pH dependence of nitrite formation indicated that XO-mediated organic nitrate reduction occurred via an acid-catalyzed mechanism. In the absence of thiols or ascorbate, no NO generation was detected from XO-mediated organic nitrate reduction; however, addition of L-cysteine or ascorbate triggered prominent NO generation. Studies suggested that organic nitrite (R-O-NO) is produced from XO-mediated organic nitrate reduction. Further reaction of organic nitrite with thiols or ascorbate leads to the generation of NO or nitrosothiols and thus stimulates the activation of sGC. Only flavin site XO inhibitors such as diphenyleneiodonium inhibited XO-mediated organic nitrate reduction and sGC activation, indicating that organic nitrate reduction occurs at the flavin site. Thus, organic nitrite is the initial product in the process of XO-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.

Topics: Anions; Benzaldehydes; Binding Sites; Biotransformation; Catechols; Electrochemistry; Electrodes; Electron Spin Resonance Spectroscopy; Enzyme Activation; Flavins; Glutathione Transferase; Guanylate Cyclase; Hydrogen-Ion Concentration; Immunoassay; Kinetics; Models, Chemical; Myocardial Ischemia; NAD; Nitrates; Nitric Oxide; Nitrites; Protein Binding; S-Nitrosothiols; Time Factors; Xanthine; Xanthine Oxidase

The guanidine compound ME10092 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine), which possesses a strong cardioprotective effect to ischemia-reperfusion, was assessed for different pharmacological actions that may underlie its cardioprotective effect. In the living rat ME10092 decreased the blood pressure and heart rate in a dose-dependent manner. We found ME10092 to bind to alpha 1- and alpha 2-adrenoreceptors with moderate affinity (Ki values 1-4 microM), and to block adrenaline-elicited contractile responses in isolated guinea pig aortas. Our results indicate that ME10092 possesses a certain anti-oxidant profile. Thus, in a competitive manner and with low affinity it inhibited the bovine milk xanthine oxidase enzyme, as well as NAD(P)H oxidase driven oxyradical formation in membrane fractions isolated from the rat brain. By using electron paramagnetic resonance we here show that, after its systemic administration, ME10092 modulates the nitric oxide (NO) content in several tissues of the rat in a time-dependent manner. However, in vitro ME10092 inhibited the activities of nitric oxide synthases nNOS and eNOS, but not that of iNOS. Our data give evidence that the cardioprotective effect of ME10092 could be mediated through pharmacological mechanisms that include some modulation of NO production, as well as possible inhibition of radical formation during ischemia-reperfusion.

Topics: Animals; Aorta; Blood Pressure; Brain; Cardiotonic Agents; Chlorocebus aethiops; COS Cells; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Guanidines; Guinea Pigs; Heart Rate; Humans; Ileum; Injections, Intravenous; Liver; Male; Muscle Contraction; Muscle, Smooth; Myocardial Ischemia; NAD; NADPH Oxidases; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Reperfusion Injury; Xanthine Oxidase

Pinacidil solutions have been shown to have significant cardioprotective effects. Pinacidil activates both sarcolemmal and mitochondrial potassium-adenosine triphosphate (K(ATP)) channels. This study was undertaken to compare pinacidil solution with University of Wisconsin (UW) solution and to determine if the protective effect of pinacidil involved mitochondrial or sarcolemmal K(ATP) channels.. Thirty-two rabbit hearts received one of four preservation solutions in a Langendorff apparatus: (1) UW; (2) a solution containing 0.5 mmol/L pinacidil; (3) pinacidil with Hoechst-Marion-Roussel 1098 (HMR-1098), a sarcolemmal channel blocker; and (4) pinacidil with 5-hydroxydecanote, a mitochondrial channel blocker. Left ventricular pressure-volume curves were generated by an intraventricular balloon. All hearts were placed in cold storage for 8 hours, followed by 60 minutes of reperfusion.. Postischemic developed pressure was better preserved by pinacidil than by UW. This cardioprotective effect was eliminated by 5-hydroxydecanote and diminished by HMR-1098. Diastolic compliance was better preserved by pinacidil when compared with UW. This protection was abolished by the addition of 5-hydroxydecanote and moderately decreased by HMR-1098.. Our results support the superiority of pinacidil over UW after 8 hours of storage. The cardioprotective role of pinacidil is mediated primarily by the mitochondrial K(ATP) channel.

Topics: Adenosine; Allopurinol; Animals; Benzamides; Cardiotonic Agents; Coronary Circulation; Decanoic Acids; Drug Evaluation, Preclinical; Female; Glutathione; Heart; Heart Ventricles; Hydroxy Acids; Insulin; Ion Transport; Male; Membrane Proteins; Mitochondria, Heart; Myocardial Contraction; Myocardial Ischemia; Organ Preservation; Organ Preservation Solutions; Pinacidil; Potassium Channels; Pressure; Rabbits; Raffinose; Random Allocation; Sarcolemma; Tissue and Organ Harvesting; Ventricular Function, Left

Nitric oxide (NO.) is thought to protect against the damaging effects of myocardial ischemia-reperfusion injury, whereas xanthine oxidoreductase (XOR) normally causes damage through the generation of reactive oxygen species. In the heart, inorganic nitrite (NO(2)(-)) has the potential to act as an endogenous store of NO., liberated specifically during ischemia. Using a detection method that we developed, we report that under ischemic conditions both rat and human homogenized myocardium and the isolated perfused rat heart (Langendorff preparation) generate NO. from NO(2)(-) in a reaction that depends on XOR activity. Functional studies of rat hearts in the Langendorff apparatus showed that nitrite (10 and 100 microM) reduced infarct size from 47.3 +/- 2.8% (mean percent of control +/- SEM) to 17.9 +/- 4.2% and 17.4 +/- 1.0%, respectively (P < 0.001), and was associated with comparable improvements in recovery of left ventricular function. This protective effect was completely blocked by the NO. scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO). In summary, the generation of NO. from NO(2)(-), by XOR, protects the myocardium from ischemia-reperfusion injury. Hence, if XOR is presented with NO(2)(-) as an alternative substrate, the resultant effects of its activity may be protective, by means of its production of NO. , rather than damaging.

Topics: Animals; Cardiotonic Agents; Humans; Hydrogen-Ion Concentration; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitrites; Oxidation-Reduction; Rats; Rats, Wistar; Xanthine; Xanthine Oxidase

Long duration ischemia in hypothermic conditions followed by reperfusion alters membrane transport function and in particular Na,K-ATPase. We compared the protective effect of two well-described cardioplegic solutions on cardiac Na,K-ATPase activity during reperfusion after hypothermic ischemia. Isolated perfused rat hearts (n = 10) were arrested with CRMBM or UW cardioplegic solutions and submitted to 12 hr of ischemia at 4 degrees C in the same solution followed by 60 min of reperfusion. Functional recovery and Na,K-ATPase activity were measured at the end of reperfusion and compared with control hearts and hearts submitted to severe ischemia (30 min at 37 degrees C) followed by reflow. Na,K-ATPase activity was not altered after 12 hr of ischemia and 1 hr reflow when the CRMBM solution was used for preservation (55 +/- 2 micromolPi/mg prot/hr) compared to control (53 +/- 2 micromol Pi/mg prot/hr) while it was significantly altered with UW solution (44 +/- 2 micromol Pi/mg prot/hr, p < 0.05 vs control and CRMBM). Better preservation of Na,K-ATPase activity with the CRMBM solution was associated with higher functional recovery compared to UW as represented by the recovery of RPP, 52 +/- 12% vs 8 +/- 5%, p < 0.05 and coronary flow (70 +/- 2% vs 50 +/- 8%, p < 0.05). The enhanced protection provided by CRMBM compared to UW may be related to its lower K+ content.

Topics: Adenosine; Allopurinol; Animals; Cardioplegic Solutions; Glutathione; Insulin; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Organ Preservation Solutions; Potassium; Protective Agents; Raffinose; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Temperature; Time Factors

It has been reported that the xanthine oxidase inhibitor, allopurinol, has a protective effect on ischemia - reperfusion injury, but the precise mechanism of its action is still unclear. Therefore, in the present study the mechanisms of the myocardial protection of allopurinol were evaluated in isolated perfused rat hearts. Allopurinol significantly inhibited myocardial xanthine oxidase activity, and improved left ventricular dysfunction after ischemia - reperfusion. In addition, the lactate dehydrogenase content in the coronary effluent obtained after reperfusion was significantly decreased. ATP, ADP, AMP and IMP significantly decreased, whereas inosine, hypoxanthine and xanthine significantly increased after ischemia in both the control and allopurinol groups. The concentration of xanthine was significantly decreased after ischemia - reperfusion in the allopurinol group; however, allopurinol did not affect the other purine metabolites. To evaluate the accumulation of oxidative stress, thiobarbituric acid reactive substances (TBARS) production in myocardial tissue was measured and allopurinol significantly decreased TBARS formation after ischemia - reperfusion. Finally, myocardial hydroxyl radicals were directly measured by electron spin resonance spectroscopy with the nitroxide radical 4-hydroxy-2, 2,6,6-tetramethyl-piperidine-N-oxyl. Hydroxyl radicals significantly increased immediately after reperfusion, but were significantly decreased in the allopurinol group. In conclusion, allopurinol reduced myocardial injury after ischemia-reperfusion by suppressing oxidative stress, but not by salvage of ATP. These findings may lead to the development of new therapeutic strategies for myocardial ischemia - reperfusion injury.

Topics: Adenine Nucleotides; Allopurinol; Animals; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Heart; Hydroxyl Radical; In Vitro Techniques; L-Lactate Dehydrogenase; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Oxidative Stress; Perfusion; Pressure; Purines; Rats; Spin Labels; Thiobarbituric Acid Reactive Substances; Ventricular Function, Left; Xanthine Oxidase

Activation of cardiac sympathetic afferents during myocardial ischaemia causes angina and induces important cardiovascular reflex responses. Reactive oxygen species (ROS) are important chemical stimuli of cardiac afferents during and after ischaemia. Iron-catalysed Fenton chemistry constitutes one mechanism of production of hydroxyl radicals. Another potential source of these species is xanthine oxidase-catalysed oxidation of purines. Polymorphonuclear leukocytes (PMNs) also contribute to the production of ROS in some conditions. The present study tested the hypothesis that both xanthine oxidase-catalysed oxidation of purines and neutrophils provide a source of ROS sufficient to activate cardiac afferents during ischaemia. We recorded single-unit activity of cardiac afferents innervating the ventricles recorded from the left thoracic sympathetic chain (T1-5) of anaesthetized cats to identify the afferents' responses to ischaemia. The role of xanthine oxidase in activation of these afferents was determined by infusion of oxypurinol (10 mg kg(-1), I.V.), an inhibitor of xanthine oxidase. The importance of neutrophils as a potential source of ROS in the activation of cardiac afferents during ischaemia was assessed by the infusion of a polyclonal antibody (3 mg ml(-1) kg(-1), I.V.) raised in rabbits immunized with cat PMNs. This antibody decreased the number of circulating PMNs and, to a smaller extent, platelets. Since previous data suggest that platelets release serotonin (5-HT), which activates cardiac afferents through a serotonin receptor (subtype 3,5-HT3 receptor) mechanism, before treatment with the antibody in another group, we blocked 5-HT3 receptors on sensory nerve endings with tropisetron (300 microg kg(-1), I.V.). We observed that oxypurinol significantly decreased the activity of cardiac afferents during myocardial ischaemia from 1.5 +/- 0.4 to 0.8 +/- 0.4 impulses s(-1). Similarly, the polyclonal antibody significantly reduced the discharge frequency of ischaemically sensitive cardiac afferents from 2.5 +/- 0.7 to 1.1 +/- 0.4 impulses s(-1). However, pre-blockade of 5-HT3 receptors eliminated the influence of the antibody on discharge activity of the afferents during ischaemia. This study demonstrates that ROS generated from the oxidation of purines contribute to the stimulation of ischaemically sensitive cardiac sympathetic afferents, whereas PMNs do not play a major role in this process.

Topics: Action Potentials; Adrenergic Fibers; Animals; Antibodies; Cats; Enzyme Inhibitors; Female; Indoles; Leukocyte Count; Male; Myocardial Ischemia; Myocardium; Neurons, Afferent; Neutrophils; Oxypurinol; Peroxidase; Serotonin Antagonists; Tropisetron; Xanthine Oxidase

The cardioprotective effects of carvedilol (CV) may be explained in part by interactions with heart mitochondria. The objective of this work was to study the protection afforded by CV against oxidative stress induced in isolated heart mitochondria by hypoxanthine and xanthine oxidase (HX/XO), a well-known source of reactive oxygen species (ROS) in the cardiovascular system.. Mitochondria were isolated from Wistar rat hearts (n = 8) and incubated with HX/XO in the presence and in the absence of calcium. Several methods were used to assess the protection afforded by CV: evaluation of mitochondrial volume changes (by measuring changes in the optical density of the mitochondrial suspension), calcium uptake and release (with a fluorescent probe, Calcium Green 5-N) and mitochondrial respiration (with a Clark-type oxygen electrode).. CV decreased mitochondrial damage associated with ROS production by HX and XO, as verified by the reduction of mitochondrial swelling and increase in mitochondrial calcium uptake. In the presence of HX and XO, CV also ameliorated mitochondrial respiration in the active phosphorylation state and prevented decrease in the respiratory control ratio (p < 0.05) and in mitochondrial phosphorylative efficiency (p < 0.001).. The data indicate that CV partly protected heart mitochondria from oxidative damage induced by HX and XO, which may be useful during myocardial ischemia and reperfusion. It is also suggested that mitochondria may be a priority target for the protective action of some compounds.

Topics: Animals; Antioxidants; Carbazoles; Carvedilol; Hypoxanthine; Male; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion; Propanolamines; Rats; Rats, Wistar; Xanthine Oxidase

University of Wisconsin solution (UW) provides effective heart preservation under hypothermic conditions, but it can be deleterious at warmer temperatures. Re-warming during the implantation of the graft may be a problem. This study examined the damaging effect of peri-operative warm ischemia in a transplant setting and recovery from such damage. The amelioration of damage by rinsing the graft before re-warming and transplantation was also examined.. Rat donor hearts were preserved for 2 hr (0 degrees C) as follows: Series A was preserved with colloid-free UW (MUW), St. Thomas' solution (ST), or calcium-supplemented MUW (MUW+Ca) followed by either transplantation or warming (22 degrees C) for 10 min before transplantation. Series B was preserved with MUW, rinsed with fresh MUW, ST, MUW+Ca, or low-potassium MUW before warming and transplantation. All heart isografts were transplanted heterotopically with an indwelling left intraventricular balloon-tipped catheter. Graft function was measured 1 and 7 days after transplantation.. Grafts re-warmed rapidly during implantation. Function (left ventricular developed pressure, contractility, and relaxation) was significantly and persistently diminished in MUW-preserved grafts subjected to additional warming before transplantation. Preservation with ST was less effective than MUW despite being unaffected by warming. Preservation with MUW+Ca and rinsing with fresh MUW or ST before re-warming allowed recovery of function within 7 days despite significantly diminished function on day 1.. This study demonstrated that an increase in the peri-transplant warm ischemic period was detrimental when hearts were preserved with MUW. Preservation with calcium-supplemented MUW or rinsing the heart with fresh MUW or ST before transplantation ameliorated this damage.

Topics: Adenosine; Allopurinol; Animals; Blood Pressure; Body Weight; Cardioplegic Solutions; Glutathione; Graft Survival; Heart Transplantation; Insulin; Male; Myocardial Contraction; Myocardial Ischemia; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Time Factors; Transplantation, Isogeneic

Topics: Allopurinol; Animals; Cardioplegic Solutions; Coronary Circulation; Disaccharides; Electrolytes; Energy Metabolism; Glutamates; Glutathione; Heart; Histidine; Mannitol; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Organ Preservation; Raffinose; Rats; Time Factors

To establish whether coronary perfusion with cardioplegic solutions results in better intraischaemic structural preservation of endothelial cells than of cardiomyocytes, we determined intraischaemic swelling of these two cell types in hearts differently arrested during global ischaemia at 5 degrees C. Cardiac arrest was induced in situ by aortic cross clamping or by additional coronary perfusion with various cardioplegic solutions. Parameters for cellular swelling were determined, i.e. barrier thickness of capillary endothelial cells and sum of the volume fractions (V(V)) of free sarcoplasm and mitochondria (V(VSp) + V(VMi)) in cardiomyocytes. In order to test the intraischaemic relative increase of cellular volume in both cell types, regression analyses were performed. The results show that the relative intraischaemic volume increase was similar in both cell types after perfusion with histidine-tryptophan-ketoglutarate solution, and significantly less pronounced in capillary endothelial cells after perfusion with University of Wisconsin solution. In hearts arrested with St. Thomas' Hospital solution, a significantly higher volume increase was determined in capillary endothelial cells. Thus, capillary endothelium does not generally show a higher structural preservation than cardiomyocytes during ischaemia. Instead, volume regulation in both types of cells depends on the type of cardioplegic solution used. These results should be taken into consideration in human transplantation medicine.

Topics: Adenosine; Allopurinol; Animals; Bicarbonates; Calcium Chloride; Capillaries; Cardioplegic Solutions; Cell Size; Dogs; Endothelium, Vascular; Female; Glucose; Glutathione; Heart Arrest, Induced; Humans; Insulin; Magnesium; Male; Mannitol; Microscopy, Electron; Mitochondria, Heart; Mitochondrial Swelling; Myocardial Ischemia; Myocardium; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Sodium Chloride

Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O(2)(-).). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O(2)(-)., implying the NADH concentration-to-NAD(+) concentration ratio ([NADH]/[NAD(+)]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD(+)]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 +/- 2%, 58 +/- 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O(2)(-). formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.

Topics: Animals; Antioxidants; Coumaric Acids; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guinea Pigs; Hydralazine; In Vitro Techniques; Lactic Acid; Mitochondria, Heart; Multienzyme Complexes; Myocardial Ischemia; Myocardium; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Pyruvic Acid; Rabbits; Reactive Oxygen Species; Xanthine Oxidase

The myocardial protective role of heat shock protein (HSP) has been demonstrated. Recently, we reported that ischemia/reperfusion induced a significant activation of heat shock factor (HSF) 1 and an accumulation of mRNA for HSP70 and HSP90. We examined the role of reactive oxygen species (ROSs) in the induction of stress response in the ischemic-reperfused heart.. Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element. Induction of mRNA for HSP70 and HSP90 was examined by Northern blot analysis. Repetitive ischemia/reperfusion, which causes recurrent bursts of free radical generation, resulted in burst activation of HSF1, and this burst activation was significantly reduced with either allopurinol 1 mmol/L (an inhibitor of xanthine oxidase) or catalase 2x10(5) U/L (a scavenger of H2O2). Significant activation of HSF1 was observed on perfusion with buffer containing H2O2 150 micromol/L or xanthine 1 mmol/L plus xanthine oxidase 5 U/L. The accumulation of mRNA for HSP70 or HSP90 after repetitive ischemia/reperfusion was reduced with either allopurinol or catalase.. Our findings demonstrate that ROSs play an important role in the activation of HSF1 and the accumulation of mRNA for HSP70 and HSP90 in the ischemic-reperfused heart.

Topics: Allopurinol; Animals; Antimetabolites; Catalase; DNA; DNA-Binding Proteins; Heat Shock Transcription Factors; In Vitro Techniques; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Transcription Factors

1. The potential for the N-hydroxyguanidine compound PR5 (N-(3, 4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models. 2. Administration of 1-10 mg kg-1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg-1 of PR5, respectively). 3. Administration of 3 mg kg-1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion. 4. Coronary occlusion/reperfusion (10 - 20 min) increased malondialdehyde (MDA) of rat hearts (0.88+/-0.13 for sham vs 1.45+/-0.12 nmol mg-1 protein for ischaemic; P<0.05). In rats where 3 mg kg-1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04+/-0.12; P<0.05 vs ischaemic). 5. PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326+/-32 mg for controls vs 137+/-21 mg for animals treated with 3x3 mg kg-1 of PR5 (P<0.01). 6. PR5 reduced the elevation of the ST-segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion. 7 We conclude that the N-hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life-threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N-hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.

Topics: Animals; Antihypertensive Agents; Arrhythmias, Cardiac; Cardiovascular Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanabenz; Guanidines; Hydroxylamines; Male; Malondialdehyde; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Oxidation-Reduction; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O(2) of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 +/- 0.1 to 2.3 +/- 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 +/- 0.1, as did the site I inhibitors rotenone (1.0 +/- 0.1), amytal (1.1 +/- 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 +/- 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-L-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 +/- 0.1) and decreased with exogenous SOD (1.1 +/- 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain.

Topics: Acetophenones; Allopurinol; Animals; Cells, Cultured; Chick Embryo; Cytosol; Enzyme Inhibitors; Heart; Kinetics; Methacrylates; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; NG-Nitroarginine Methyl Ester; Oxidation-Reduction; Reactive Oxygen Species; Rotenone; Superoxide Dismutase; Superoxides; Thiazoles

Optimal preservation of donor hearts remains a significant concern during transplantation. Organ shortage led to an increase in the use of damaged hearts.. To study the effect of preservation solutions on recovery of myocardial metabolism and function after warm ischemia, 10 dogs underwent 30 minutes of warm global ischemia under cardiopulmonary bypass. The animals were then administered 1 liter of Celsior (5 dogs), an extracellular crystalloid solution or 1 liter of University of Wisconsin solution (5 dogs), cooled at 4 degrees C, followed by 60 minutes of cold preservation and 30 minutes of warm blood reperfusion. Interstitial myocardial pH and pO2 changes were measured. The left ventricle dP/dt was measured before and after the ischemic episode, as where creatine kinase, troponine T and lactate serum levels.. Tissue pH averaged 6.9+/-0.1, 6.2+/-0.1, 6.7+/-0.1 and 6.8+/-0.1 before and after warm ischemia, following the 60 minutes of cold preservation and the reperfusion period in animals treated with the Celsior solution, compared to 6.8+/-0.1, 6.4+/-0.1, 7+/-0.1 and 6.8+/-0.2 respectively in dogs treated with the University of Wisconsin solution (p<0.05). Oxygen tension in the myocardium averaged 36+/-8 mmHg before warm ischemia and 59+/-31 mmHg after in animals that received Celsior compared to 30+/-10 mmHg and 49+/-7 mmHg in dogs treated with University of Wisconsin (p>0.05). Global myocardial function decreased significantly following reperfusion compared to baseline in both groups of animals. The serum levels of creatine kinase, troponine T and lactate increased significantly during the experiment although there was no significant difference between the 2 groups.. Both preservation solutions (Celsior and University of Wisconsin) resulted in suboptimal recovery of myocardial function and metabolism when administered after a period of warm ischemia. Strategies to improve recovery of damaged donor hearts remain to be appropriately defined.

Topics: Acid-Base Equilibrium; Adenosine; Allopurinol; Animals; Creatine Kinase; Dogs; Energy Metabolism; Glutathione; Insulin; Lactic Acid; Myocardial Ischemia; Organ Preservation; Organ Preservation Solutions; Potassium Compounds; Raffinose; Temperature; Troponin T; Ventricular Function, Left

Continuous hypothermic perfusion of donor hearts may provide extra protection for long ischemic times and suboptimal donors. The aim of three separate studies was to assess the effect of continuous hypothermic perfusion during simulated donor heart storage and implantation.. In study 1 twelve isolated rat hearts underwent 10 minutes of normothermic ischemia to simulate the effect of brain death on the heart and 5 hours of cardioplegic arrest, using University of Wisconsin solution. Six hearts were statically stored in University of Wisconsin solution at 2 degrees C, and six were perfused with University of Wisconsin solution. To assess the effect of simulated implantation, in study 2 an additional 12 hearts were statically stored for 5.5 hours in University of Wisconsin solution, six of which were rewarmed to a mean of 16 degrees C over the last 30 minutes of arrest. To assess the effect of simulated perfusion, in study 3 during implantation 12 hearts were rewarmed to a mean of 16 degrees C over the last 30 minutes of arrest, during which time six were perfused with 2 degrees C solution.. Hearts perfused during storage demonstrated greater recovery of prearrest power, 85.8% +/- 1.8%, than hearts preserved by static storage, 72.7% +/- 3.0% (p < 0.01). The simulated warm implantation period reduced recovery of power from 68.3% +/- 5.1% to 40.2% +/- 2.0% (p < 0.001). Perfusion during warm implantation improved recovery to 61.8% +/- 3.9% (p < 0.01). In all experiments improved function was accompanied by improved metabolic energy status.. During the implantation period of heart transplantation the donor heart sustains injury that could amount to 50% of total ischemic injury. Continuous perfusion during the cold storage phase and during simulated implantation improves recovery of the donor heart.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Blood Pressure; Body Water; Brain Death; Cardiac Output; Cardioplegic Solutions; Cryopreservation; Energy Metabolism; Glutathione; Heart; Heart Arrest, Induced; Heart Transplantation; Insulin; Male; Myocardial Contraction; Myocardial Ischemia; Myocardium; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Perfusion; Raffinose; Rats; Rats, Wistar; Rewarming

The study aims to determine a possible relationship between intracellular water, energy metabolism, functional recovery and membrane permeability, during and after hypothermic cardiac preservation. Isolated rat hearts were stored for 12 h at 4 degrees C with University of Wisconsin (UW), St Thomas Hospital (ST) and Krebs-Henseleit (KH) solutions, and were reperfused for 1 h. Cellular volumes were measured by 1H NMR of water and 59Co NMR of the extracellular marker cobalticyanide, and energetic profiles by 31P NMR spectroscopy. Storage in ST solution reduced ischemic swelling from 2.50 +/- 0.06 to 2.73 +/- 0.09 (P < 0.001 v 3.56 +/- 0.10 ml/g dry weight in KH), while UW solution caused cellular shrinkage to 2.12 +/- 0.08 ml/g dry weight. Intracellular ATP concentrations and pH values were higher in UW as compared to ST solution. At reperfusion, hearts stored in ST shrank while those stored in UW expanded, resulting in similar intracellular volumes. Storage with UW was superior to ST in post-ischemic function 65 +/- 5% (P < 0.01 v 49 +/- 4% with ST) and in recovery of ATP 46 +/- 3% (P < 0.001 v 25 +/- 4% with ST). Storage with both ST and UW solutions did not prevent interstitial edema. Sarcolemmal membrane integrity, as assessed by cellular swelling in response to a hypo-osmotic shock (210 mmol/l), was significantly improved by ST and UW solutions as compared to KH (P < 0.05). Creatine kinase efflux was reduced by ST and UW as compared to KH (P < 0.05), and by UW as compared to ST (P < 0.05). Coronary flow was higher following storage with UW than ST solutions. 66 +/- 6 and 45 +/- 4%, respectively (P < 0.01). According to these data, the beneficial effects of UW and ST solutions on hypothermic ischemic storage of rat hearts included prevention of cellular edema and preservation of sarcolemmal membrane integrity. It is concluded: (a) UW and ST solutions reduce ischemic and reperfusion cellular volumes: (b) both solutions, and UW in particular were efficient in preservation of membrane integrity: (c) prevention of cellular edema is not the single or main mechanism responsible for the improved preservation with UW and ST solutions.

Topics: Adenosine; Allopurinol; Animals; Bicarbonates; Calcium Chloride; Cell Membrane Permeability; Cryopreservation; Glutathione; Heart; Hot Temperature; Insulin; Intracellular Fluid; Magnesium; Myocardial Ischemia; Myocardial Reperfusion; Organ Preservation; Organ Preservation Solutions; Potassium Chloride; Raffinose; Rats; Rats, Sprague-Dawley; Sodium Chloride

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Cold Temperature; Creatine Kinase; Free Radical Scavengers; Glutathione; Heart; Heart Transplantation; Insulin; Ischemic Preconditioning, Myocardial; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Organ Preservation Solutions; Perfusion; Polyethylene Glycols; Pregnatrienes; Raffinose; Rats; Rats, Wistar; Superoxide Dismutase; Time Factors

We investigated whether xanthine oxidase-derived superoxide radical generation could be modified by interfering with adenosine transport and metabolism in reducing myocardial injury during post-ischemic reperfusion. Isolated rat hearts perfused at constant pressure were subjected to 20 min of pretreatment with test agents, followed by 40 min global ischemia and 30 min reperfusion with or without test agents. In hearts treated with adenosine deaminase inhibitor, erythro 9-(2-hydroxy-3-nonyl) adenine (EHNA), alone or together with a selective nucleoside transport blocker, p-nitrobenzylthioinosine (NBMPR), the accumulated amount of O-2. was significantly reduced [10.2+/-0.97, 11.6+/-2.4, 8.1+/-0.51, respectively, v 31.6+/-2.1 (s. e.) nmol/wet g/30 min in ischemic control, P<0.01]. A positive correlation between O-2. and inosine release was observed in the initial 5 min of reperfusion in hearts treated with either EHNA or NBMPR ( r=0.475, P<0.05). Furthermore, the accumulated amount of LDH release showed positive correlation with that of O-2. among the same groups (r=0.474, P<0.05). Both EHNA and NBMPR had the cardioprotective effect on the recovery of left ventricular end-diastolic pressure (LVEDP), ATP repletion, and build up of endogenous adenosine. This study suggests that : (1) adenosine metabolism can be manipulated towards the formation of O-2. during reperfusion, and it has an important bearing on the cardiac recovery of ischemic myocardium, (2) the generation of O-2. is related to only inosine release during initial reperfusion.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Animals; Enzyme Inhibitors; Heart; Heart Rate; In Vitro Techniques; Isoenzymes; L-Lactate Dehydrogenase; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Organophosphates; Purines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stroke Volume; Superoxides; Thioinosine; Xanthine Oxidase

Tissue factor is a transmembrane protein that activates the extrinsic coagulation pathway by binding factor VII. Endothelial cells, being in contact with circulating blood, do not normally express tissue factor. Here we provide evidence that oxygen free radicals induce tissue factor messenger RNA transcription and expression of tissue factor procoagulant activity in endothelial cells in culture. Isolated, perfused rabbit hearts exposed to exogenous oxygen free radicals also showed a marked increase in tissue factor activity within the coronary circulation. Furthermore, in ex vivo and in vivo hearts subjected to ischemia and reperfusion, a condition associated with a production of oxygen free radicals in large amounts, a marked increase in tissue factor activity occurred. This phenomenon could be abolished by oxygen radical scavengers. This increase in tissue factor activity during postischemic reperfusion was accompanied by a significant decrease in coronary flow, suggesting that increase in tissue factor activity with the consequent activation of the coagulation cascade might impair coronary flow during reperfusion and possibly contribute to the occurrence of reperfusion injury.

Topics: Animals; Blotting, Northern; Cells, Cultured; Coronary Circulation; Cycloheximide; Endothelium, Vascular; Free Radical Scavengers; Free Radicals; Gene Expression; Heart; In Vitro Techniques; Myocardial Ischemia; Myocardial Reperfusion; Oxygen; Rabbits; Regional Blood Flow; RNA, Messenger; Thromboplastin; Xanthine; Xanthine Oxidase; Xanthines

The scavenging effect of Chinonin on NO and oxygen free radicals and its protective effect on myocardium from the ischemia-reperfusion injury was studied with electron spin resonance (ESR) and chemiluminescence techniques. Chinonin can effectively inhibit the oxidative activity of ONOO-, (the IC50 = 7 x 10 (-5) mmol/L) and scavenge oxygen free radicals generated from the reaction of xanthine and xanthine oxidase (the IC50 = 2/5 x 10(-4) mmol/l). It is difficult to find another antioxidant which can scavenge so effectively both ONOO- and oxygen free radicals simultaneously. In the system of ischemia-reperfusion injury of myocardium, Chinonin can, in parallel, scavenge the NO and oxygen free radicals generated from the ischemia-reperfused myocardium, and decrease the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart and therefore protect the heart from ischemia-reperfusion injury. The protective effect of 0.1 mmol/l Chinonin is similar to that of 1500 U/ml SOD and catalase.

Topics: Animals; Arginine; Catalase; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Free Radical Scavengers; Free Radicals; Glycosides; Heart; Hemoglobins; In Vitro Techniques; Kinetics; Luminescent Measurements; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Xanthenes; Xanthine Oxidase; Xanthones

In the presence of its substrates hypoxanthine and xanthine, xanthine oxidase generates oxygen free radicals that cause postischemic injury. Recently, it has been demonstrated that the burst of xanthine oxidase-mediated free radical generation in the reperfused heart is triggered by a large increase in substrate formation, which occurs secondary to the degradation of adenine nucleotides during ischemia. It is not known, however, whether blocking this substrate formation is sufficient to prevent radical generation and functional injury. Therefore, studies were performed in isolated rat hearts in which xanthine oxidase substrate formation was blocked with the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and measurements of contractile function and free radical generation were performed. Chromatographic measurements of the intracellular adenine nucleotide pool showed that preischemic administration of EHNA blocked postischemic hypoxanthine, xanthine, and inosine formation. Electron paramagnetic resonance spin trapping measurements of free radical generation showed that inhibition of adenosine deaminase with EHNA blocked free radical generation and that it also increased the recovery of contractile function by more than 2-fold. Exogenous infusion of hypoxanthine and xanthine totally reversed the protective effects of EHNA. These results demonstrate that blockade of xanthine oxidase substrate formation by adenosine deaminase inhibition can prevent free radical generation and contractile dysfunction in the postischemic heart.

Topics: Adenine; Adenine Nucleotides; Adenosine Deaminase Inhibitors; Animals; Coronary Circulation; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Female; Free Radicals; Hydroxyl Radical; Myocardial Ischemia; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Animals; Cardiac Output; Cardioplegic Solutions; Glutathione; Heart; Heart Rate; Hemodynamics; In Vitro Techniques; Insulin; Lactates; Male; Myocardial Ischemia; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Systole; Time Factors

Topics: Adenosine Triphosphate; Allopurinol; Animals; Cardioplegic Solutions; Creatine Kinase; Glutathione; Heart; Heart Arrest; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Myocardial Ischemia; Myocardium; Organ Preservation; Phosphates; Phosphocreatine; Phosphorus; Purines; Raffinose; Rats; Time Factors

1. Myocardial ischaemia and reperfusion can evoke excitation of cardiac vagal afferent nerve endings and activation of a cardiogenic depressor reflex (Bezold-Jarisch effect). We postulate that oxygen free radicals, which are well known to be produced during ischaemia and reperfusion, contribute to this excitation. 2. Activity from vagal afferent fibres in rats, whose endings were located in the walls of all four chambers of the heart, was recorded in response to topical application of pro-oxidant chemicals to the surface of the heart. Activity was also recorded from vagal afferent fibres, whose endings were located in the left ventricle, in response to occlusion of the left anterior coronary artery (LAC) for 30 min and subsequent reperfusion. A majority of the recorded fibres were classified as chemosensitive C-fibre endings due to their irregular discharge under resting conditions, their activation in response to the topical application of capsaicin (1-10 micrograms) to the surface of the heart encompassing the receptive field and their conduction velocities. 3. Topical application of either H2O2 or xanthine/xanthine oxidase to the heart activated 50% of the chemosensitive endings and did not directly affect cardiac mechanoreceptors. This effect was reproducible, dose-dependent and was not due to [H+]. 4. Administration of the superoxide radical scavenging enzyme, superoxide dismutase (20000 U/kg, i.v.), decreased the response of fibres to xanthine/xanthine oxidase but had no effect on the activation caused by H2O2. The antioxidants deferoxamine (20 mg/kg, i.v.) or dimethylthiourea (10 mg/kg, i.v.), which scavenge the hydroxyl radical, abolished the responses to xanthine/xanthine oxidase and H2O2. Administration of indomethacin (5 mg/kg, i.v.) had no effect on the afferent response to H2O2. 5. In response to ligation of the left anterior coronary (LAC), the activity of chemosensitive endings within the ischaemic zone increased within the first 2 min of occlusion. Endings outside the ischaemic zone were not affected at the beginning of ischaemia. Reperfusion activated only chemosensitive endings responsive to topical H2O2. These reperfusion-sensitive endings were located both within and outside the ischaemic zone of the left ventricle. 6. Indomethacin (5 mg/kg, i.v.) prevented activation of chemosensitive endings at the beginning of LAC occlusion regardless of their sensitivity to H2O2 but had no effect on the response to reperfusion. Conversely, deferoxa

Topics: Animals; Capsaicin; Chemoreceptor Cells; Coronary Vessels; Heart; Hydrogen Peroxide; Myocardial Ischemia; Myocardial Reperfusion; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Vagus Nerve; Xanthine; Xanthine Oxidase; Xanthines

The hypothermic simple immersion technique has been widely used to preserve the donor heart for transplantation. However, there is still controversy as to which temperature provides the best protection against prolonged ischemia. The low molecular weight solutes within cells depress the freezing point to -0.6 degree C. In practice, however, most cells do not freeze internally unless they are cooled below -10 degrees C. We investigated the effects of subzero nonfreezing storage at -1 degree C on the preservation of myocardial metabolism and function.. Isolated Wistar rat hearts were subjected to 6 hours of preservation with the intracellular type University of Wisconsin solution; the hearts of the subzero group were preserved at subzero nonfreezing (-1 degree C) temperature, and the hearts of the control group were at 4 degrees C. Recovery of cardiac function, myocardial adenine nucleotides content, and myocardial water content were evaluated after preservation.. Subzero group resulted in significantly better aortic flow, cardiac output, and aortic systolic pressure than in the control group. Myocardial adenosine triphosphate, adenosine diphosphate, and total adenine nucleotides at end-storage were significantly better preserved in subzero group when compared with the control group. Myocardial water content at reperfusion significantly increased in the control group compared with the subzero group.. Storage in the intracellular type solution at subzero nonfreezing (-1 degree C) temperature as compared with 4 degrees C appears to prolong myocardial preservation with respect to the enhancement of postischemic functional recovery, preservation of myocardial adenine nucleotides during ischemia, and prevention of myocardial edema at reperfusion.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Body Water; Cryopreservation; Glutathione; Heart; Insulin; Male; Myocardial Ischemia; Myocardium; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Wistar; Temperature

Single or multiple brief periods of regional or global ischemia and reperfusion prior to a prolonged ischemic insult showed cardioprotective effects. Although this phenomenon (ischemic preconditioning [IPC]) has been described in ischemic reperfusion models, the effect of IPC on heart preservation has not been previously reported. We, thus, investigated the effect of IPC on heart preservation. Hearts isolated from male Wistar rats (250-350 g) were mounted on a Langendorff apparatus to estimate baseline function (aortic flow, coronary flow, cardiac output, heart rate, systolic pressure, and rate pressure product). All hearts were divided into 5 groups. In groups 1 and 4, the hearts were subjected to 8 and 12 hr of preservation, respectively. The hearts in group 2 were subjected to a single 2.5-minute cycle of normothermic global ischemic episode (IPC) before 8 hr of preservation. In groups 3 and 5, the hearts were subjected to two 2.5-min IPC cycles and stored for 8 or 12 hr. The hearts were arrested with University of Wisconsin solution and stored at 4 degrees C. Following storage, the hearts were reperfused and measured postpreservative function to assess cardiac functional recovery. Lactate and troponin-T leakage in the coronary perfusate was also measured. In group 3, the treatment of two 2.5-min IPC cycles significantly increased cardiac output, but the treatment of single 2.5-min IPC cycle did not affect the result. In the extended preservation group (group 5), the recovery (%) of both coronary flow and cardiac output were significantly increased compared with group 4. Furthermore, lactate leakage was significantly reduced in groups 2 and 3. These results suggest that IPC improves cardiac functional recovery following simple cold storage and has cardioprotective effects in rat heart preservation.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Hemodynamics; Insulin; Male; Myocardial Ischemia; Myocardial Reperfusion; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar

With microdialysis, we monitored cardiac interstitial fluid (ISF) levels of allopurinol, its metabolites, and the adenine nucleotide breakdown products (ANBP), inosine, hypoxanthine (HYP), xanthine (Xa), uric acid (UA) in dogs that received 1 and 10 mg/kg allopurinol intravenously (i.v.). Half-life (t1/2) of drug penetration into the heart was dose independent (1.8 min), whereas for the 10-mg/kg dose terminal elimination t1/2 (96 min) was much prolonged and ISF clearance (9.6 l/min kg) was reduced as compared with that induced by 1 mg/kg (28 min and 30.4 l/min kg) probably due to capacity limitation of allopurinol conversion to oxypurinol by Xa dehydrogenase/oxydase (Xa D/O). Inhibition of Xa D/O activity by allopurinol resulted in a dose-dependent increase in ISF HYP and Xa levels and a decrease in UA level. For a 10-mg/kg dose, maximal effect was attained approximately 40 min after drug injection. Allopurinol (1 mg/kg) given 30 min after the start of 40-min coronary artery occlusion during ischemia entered the ischemic zone ISF very slowly as compared with that of the control zone; the no-reflow phenomenon was evident because the levels became similar in both zones only 15 min after initiation of reperfusion. To examine cardioprotective efficiency, we administered allopurinol (10 mg/kg) 40 min before 40-min occlusion; it had little effect on total ANBP release during ischemia but facilitated washout of ANBP from the ischemic zone during reperfusion, thus manifesting protective efficacy against reperfusion injury and no-reflow. As shown by the lack of ischemia-induced increase in ISF Xa, myocardial Xa D/O activity was completely blocked by allopurinol.

Topics: Adenine Nucleotides; Allopurinol; Animals; Dogs; Female; Half-Life; Male; Microdialysis; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Oxypurinol; Purines; Xanthine Oxidase; Xanthines

Previous work has suggested tolerance to ischemic injury in newborn myocardium. Although various mechanisms for this protection have been proposed, a link between oxidant-antioxidant factors, stress protein expression, and protection from cardiac ischemia/reperfusion (I/R) injury has not been made in newborn myocardium. We hypothesized total newborn myocardial resistance to I/R is related to decreased oxygen radical producing potential, increased free radical scavenging capacity and augmented stress protein expression. The purposes of the study were to examine in newborn and adult rat hearts (1) functional recovery from I/R, (2) catalase and xanthine oxidase (XO) activities, and (3) heat shock protein 72 (HSP 72) expression.. Isolated rat hearts (7 to 10 days versus 60 days) were perfused on a nonworking Langendorff apparatus at 60 mm Hg (Krebs-Henseleit buffer, pH 7.4, 37 degrees C) and subjected to 20 minutes of global ischemia and 40 minutes of reperfusion. Left ventricular developed pressure was recorded by using a left ventricular catheter. Catalase and XO were measured by means of standard assays, and HSP 72 was assessed with in situ immunohistochemistry.. Newborn rat hearts had greater percentage functional recovery of left ventricular developed pressure after I/R (66.0% +/- 4.2% versus 44.3% +/- 3.5%; p < 0.05). The newborn myocardium also had increased catalase activity (1027.9 +/- 20.6 units/gm versus 707.3 +/- 38.7 units/gm; p < 0.05), whereas the activity of XO was decreased relative to the adult (0.23 +/- 0.01 mU/gm versus 7.6 +/- 1.4 mU/gm; p < 0.05). Furthermore, the expression of HSP 72 was greater in the newborn than the adult control.. Relative to adult hearts, newborn rat hearts are more tolerant to a global ischemic insult followed by reperfusion. This improved functional recovery is associated with decreased oxidant production potential (XO), increased scavenging capacity (catalase), and augmented stress protein expression (HSP 72).

Topics: Aging; Animals; Animals, Newborn; Antioxidants; Blood Pressure; Catalase; Diastole; Heart; Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Male; Myocardial Ischemia; Myocardium; Oxidoreductases; Phenotype; Rats; Rats, Sprague-Dawley; Ventricular Function, Left; Xanthine Oxidase

While the free radical-generating enzyme xanthine oxidase is a central mechanism of injury in postischemic tissues, questions remain regarding how xanthine oxidase-mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation of that of its substrates xanthine and hypoxanthine. Therefore, studies were performed in isolated rat hearts correlating the magnitude and time course of radical generation with alteration in xanthine oxidase and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy and correlated with spectrophotometric assays of tissue xanthine oxidase activity and chromatographic measurements of tissue and effluent concentrations of xanthine oxidase substrates and products. Xanthine oxidase was present in preischemic hearts and slightly increased during 30-min global ischemia. Hypoxanthine and xanthine were not present prior to ischemia but accumulated greatly during ischemia due to ATP degradation. These substrate concentrations rapidly declined over the first 5 min of reperfusion matching the observed time course of radical generation, whereas xanthine oxidase activity was largely unchanged. Both substrates were also observed in the coronary effluent during the first 5 min of reflow along with the product uric acid. Thus, the burst of xanthine oxidase-mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation that occurs secondary to the degradation of ATP during ischemia.

Topics: Adenosine Triphosphate; Animals; Female; Free Radicals; Myocardial Ischemia; Myocardium; Rats; Rats, Sprague-Dawley; Xanthine Oxidase

This work evaluates the myocardial protective potential of potassium cardioplegia on ischaemically arrested and reperfused hearts by two cardioplegic solutions: the University of Wisconsin solution (UW) and the standard crystalloid solution of St. Thomas' Hospital (ST). Evaluation of myocardial preservation was based on creatine kinase and lactate releases and on high-energy phosphate preservation of isolated rabbit hearts after 4 hours' hypothermic ischaemia. A morphometric ultrastructural evaluation of mitochondria in cardiomyocytes was also performed. The hearts of 24 rabbits were normothermally perfused with oxygenated Krebs-Henseleit solution for 30 min (Langendroff preparation), and the baseline contractile performance and biochemical parameters were evaluated. The hearts were then arrested and stored in the cardioplegic solutions (12 UW and 12 ST) at 4 degrees C for 4 hours. The hearts were then rewarmed and reperfused with oxygenated Krebs-Henseleit solution for further 30 min. At the end of reperfusion, creatine phosphate and high energy phosphates were higher with UW (p < 0.05); creatine kinase release during reperfusion was significantly lower with UW both at 15 min (p < 0.01) and at 30 min (p < 0.05). Lactate release during the first 15 min of reperfusion was about doubled (p < 0.05) with respect to controls in both groups; at 30 min this increase had almost vanished (+8%) with UW but not with ST (+30%). Ultrastructural morphometry did not show any significant difference at the level of mitochondria between the two treatments. The results indicate, for UW, an improved myocardial preservation associated with relative retention of high-energy phosphates and higher recovery of mechanical function, accelerated metabolic recovery and reduced stress of cell membranes.

Topics: Adenosine; Allopurinol; Animals; Bicarbonates; Calcium Chloride; Cardioplegic Solutions; Creatine Kinase; Cryopreservation; Energy Metabolism; Glutathione; In Vitro Techniques; Insulin; Lactic Acid; Magnesium; Myocardial Ischemia; Myocardium; Organ Preservation Solutions; Phosphates; Potassium Chloride; Rabbits; Raffinose; Sodium Chloride; Time Factors

Although the formation of oxygen-derived free radicals (or reactive oxygen species; ROS) and the release of endogenous opioid peptides (EOP) have been independently reported to be the major arrhythmogenic factors in ischemic hearts, possible relations between these two factors have seldom been investigated. Thus, we studied whether the ROS and EOP were related in the progression of ischemia-induced arrhythmias. Isolated rat hearts perfused in the Langendorff mode were treated with dynorphin A1-13 (kappa EOP receptor agonist), and/or allopurinol (xanthine oxidase inhibitor), before the onset of ischemia induced by ligating the left coronary arteries. Ischemic period lasted for 30 min, during which cardiac rhythms were recorded. At the end of ischemia, hearts were analyzed for the glutathione and ascorbate levels. Allopurinol (100 nmoles/heart) was effective in reducing the severity of arrhythmia (arrhythmia score: Mean +/- SEM 3.00 +/- 0.80 for allopurinol, 5.75 +/- 0.41 for placebo, p < 0.01), while dynorphin (10 micrograms/heart) potentiated the arrhythmia (6.71 +/- 0.52, p < 0.05 vs. placebo). Coadministration of allopurinol and dynorphin was capable of reducing arrhythmia (5.57 +/- 0.65) when compared with the administration of dynorphin alone (6.71 +/- 0.52, p < 0.05). Tissue oxidative stress was evaluated by the concentrations of glutathione (GSH) and ascorbate. Allopurinol did not significantly elevate tissue GSH concentrations (1.46 +/- 0.05 mumoles/g wet wt) in ischemic hearts, while dynorphin alone significantly decreased the GSH concentrations (0.96 +/- 0.08, p < 0.05) when compared with the placebo (1.32 +/- 0.03). The dynorphin-induced GSH decrease cannot be reversed by coadministration with allopurinol (0.90 +/- 0.104). Allopurinol significantly elevated tissue ascorbate levels (0.16 +/- 0.01) when compared with placebo (0.10 +/- 0.01, p < 0.05). Interestingly, dynorphin alone also elevated the tissue ascorbate concentrations (0.16 +/- 0.02). Coadministration of allopurinol and dynorphin further spiked the ascorbate levels (0.28 +/- 0.05, p < 0.01). In conclusion, the results suggested that ischemia-induced arrhythmia mechanisms might involve the formation of superoxide and other ROS, which were probably generated from the release of EOP (or EOP/EOP receptor interactions). Superoxide, the formation of which can be inhibited by allopurinol that exerted antiarrhythmic effect, was probably scavenged by ascorbate in myocardial ischemia. The ROS r

Topics: Allopurinol; Animals; Arrhythmias, Cardiac; Ascorbic Acid; Dynorphins; Female; Free Radicals; Glutathione; In Vitro Techniques; Models, Cardiovascular; Myocardial Ischemia; Opioid Peptides; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Ischemia/reperfusion (I/R) and preconditioning of the heart by coronary artery occlusions increase expression of heat shock protein 70 (HSP 70). Because free radicals are generated during I/R, we hypothesized that the oxidant stress might contribute to an increased expression of HSP 70. Isolated rat hearts were perfused with free radical-generating systems such as xanthine/xanthine oxidase (X/XO), irradiated rose bengal (RB) generating singlet oxygen, and H2O2 for 15 min followed by 30 min of recovery period. Significant decrease in developed pressure and coronary flow occurred after perfusion with X/XO, H2O2, and RB. During I/R, the developed pressure and coronary flow were 60 +/- 8 and 80 +/- 5%, respectively, of control, which improved significantly with superoxide dismutase. The expression of HSP 70 mRNA increased over 13-fold in hearts perfused with X/XO, 6- to 7-fold with RB, and over 5-fold with H2O2. With I/R, an over 10-fold increase in HSP 70 mRNA was observed, which decreased significantly in the presence of superoxide dismutase. These results demonstrate that oxidant stress directly increases HSP 70 mRNA in the rat heart. It is concluded that one of the potential mechanisms of expression of HSP 70 by I/R may be oxygen radicals.

Topics: Animals; Coronary Circulation; Free Radicals; HSP70 Heat-Shock Proteins; Hydrogen Peroxide; In Vitro Techniques; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Oxidative Stress; Oxygen; Perfusion; Rats; Rats, Sprague-Dawley; RNA, Messenger; Rose Bengal; Singlet Oxygen; Xanthine; Xanthine Oxidase; Xanthines

Oxygen radicals accumulated during ischemia and reperfusion may affect coronary contractility by endothelium dependent and independent pathways one of which may involve Na(+)-pump. Here we report a contractility assay for Na(+)-pump in pig coronary artery and use it to examine the effects of hydrogen peroxide and superoxide. Coronary artery rings contracted in a K(+)-free Krebs solution and relaxed upon subsequent exposure to K+. The relaxation approximated a single exponential decay whose rate constant depended on [K+]2. This K(+)-induced relaxation was abolished by ouabain and was attributed to Na(+)-pump. In tissues pretreated with peroxide, the rate of relaxation of the K(+)-free contracted arteries decreased with an IC50 = 1.6 +/- 0.6 mmol/l for peroxide. Another set of tissues was pretreated with the superoxide generating system containing 0.3 mmol/l xanthine + varying concentrations of xanthine oxidase (XO) and precontracted in K(+)-free Krebs solution. The rate of the K(+)-induced relaxation decreased with IC50 = 24 +/- 8 mU/ml for XO. Thus, using the relaxation assay we conclude that exposing coronary arteries to oxygen radicals can damage Na(+)-pumps.

Topics: Animals; Coronary Vessels; Hydrogen Peroxide; In Vitro Techniques; Kinetics; Muscle Contraction; Muscle, Smooth, Vascular; Myocardial Ischemia; Myocardial Reperfusion; Ouabain; Potassium; Sodium-Potassium-Exchanging ATPase; Superoxides; Swine; Time Factors; Xanthine; Xanthine Oxidase; Xanthines

It is currently believed that reactive oxygen species are produced in the heart post-ischemia reperfusion, causing pathophysiological disorders. Studies reported in the literature dealing with this subject have generated contradictory findings. The aim of this study was to assess the catalytic activity of the superoxide anion-producing enzyme xanthine oxidase, and the level of lipid peroxides in isolated rat heart muscle undergoing ischemia of varying duration and severity followed by reperfusion. Three levels of ischemia were investigated: total, and partial at either 0.10 or 0.35 ml/min (residual flow rate). Three different periods of ischemia were examined in each case. After each period of ischemia, followed by 10 min of reperfusion, the heart was frozen in liquid nitrogen. Xanthine oxidase activity and lipid peroxide levels were assayed in the cardiac homogenate and in the centrifuged supernatant, respectively. In the different experimental protocols studied here, both cardiac xanthine oxidase and lipid peroxide levels remained statistically unchanged compared to the continuously perfused control hearts. Moreover, in a recent study (Boucher et al., FEBS Lett. 203, 261-264, 1992), we were unable to detect reactive oxygen species in perfusate upon reperfusion of ischemic rat hearts. These results suggest that changes in xanthine oxidase activity during myocardial ischemia-reperfusion, and lipid peroxidation, as assessed by measuring thiobarbituric acid reactants and lipid hydroperoxides, are not predominant phenomena in ischemia-reperfusion-induced injury, at least in the experimental model used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Electron Spin Resonance Spectroscopy; Free Radicals; In Vitro Techniques; L-Lactate Dehydrogenase; Lipid Peroxides; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Rats; Rats, Wistar; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Isolated canine hearts were preserved for 12 h at 5 degrees C followed by normothermic reperfusion for 2 h. Dogs were divided into two groups: group 1 (n = 7) received a nondepolarizing preservation solution in multidose, and group 2 (n = 6) received single-flushed University of Wisconsin (UW) solution, both administered in multidose fashion. At the end of reperfusion, the myocardial adenosine triphosphate concentration and left ventricular systolic and diastolic function were preserved better in groupl than in group 2. Myocardial mitochondrial ultrastructural integrity was identical in the two groups. These results suggested that in a 12-h heart preservation, nondepolarizing solution administered in multidose fashion protects the myocardium from the deleterious effects of hypothermia and cardioplegia better than UW solution.

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cardioplegic Solutions; Dogs; Glutathione; Heart; Heart Arrest, Induced; Insulin; Mitochondria, Heart; Models, Animal; Myocardial Ischemia; Myocardium; Organ Preservation; Organ Preservation Solutions; Phosphocreatine; Raffinose; Reperfusion; Ventricular Function, Left

Open chest dogs undergoing 30-min occlusion of the left anterior descending coronary artery (LAD), followed by 20-min reperfusion, received silibinin (2 mg/kg body weight), allopurinol (100 mg for two days as pretreatment, 20 mg/kg body weight during ischemia and reperfusion), superoxide dismutase (SOD, 5 and 0.5 mg/kg body weight, starting from the last minute of ischemia over 6 min). Control and treated dogs were comparable with respect to myocardial regional contractile force (strain gauge), malondialdehyde (MDA) and creatinine kinase (CK) levels of sinus coronarius blood samples, heart rate, and blood pressure. Allopurinol and large doses of SOD produced significant improvement in contractility and decreased MDA levels, which might suggest free radical mediated reactions during reperfusion.

Topics: Allopurinol; Animals; Antioxidants; Blood Pressure; Dogs; Female; Heart Rate; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Silymarin; Superoxide Dismutase; Ventricular Function, Left

Previous studies have shown that myocardial membranes, isolated from ischemic myocardial tissue, showed marked changes in microviscosity. To evaluate the contribution of free radical production and concomitant lipid peroxidation to these changes in microviscosity, the in vitro effects of two radical producing systems (H2O2/FeCl2 and xanthine oxidase/hypoxanthine/FeCl3) were investigated separately on the microviscosity of sarcolemmal, mitochondrial and sarcoplasmic reticulum membranes. In all three membranes both these free radical producing systems caused formation of malondialdehyde as quantitated by the thiobarbituric acid test. The sensitivity of the membranes to free radical damage differed: the sarcolemma was more sensitive to H2O2 damage, while mitochondrial malondialdehyde production was highest with xanthine oxidase. H2O2/FeCl2 caused a reduction in microviscosity (i.e. increased fluidity) of all three membranes, whereas the xanthine oxidase system increased mitochondrial and sarcolemmal microviscosity and reduced that of the sarcoplasmic reticulum. The similarity between ischemia-induced membrane microviscosity changes and those induced in vitro by xanthine oxidase, indicate a possible causal role for superoxide and hydroxyl free radicals produced during ischemia.

Topics: Animals; Free Radicals; Hypoxanthine; Hypoxanthines; Intracellular Membranes; Lipid Peroxidation; Male; Malondialdehyde; Membrane Fluidity; Mitochondria, Heart; Myocardial Ischemia; Myocardium; Phosphatidylcholines; Phosphatidylethanolamines; Rats; Rats, Wistar; Reactive Oxygen Species; Viscosity; Xanthine Oxidase

Oxygen-derived free radicals are known to take part in cardiac injury during post-ischemic reperfusion (I/R). Xanthine oxidase (XO) is closely associated with the generation of superoxide radicals. We have determined the distribution of XO in rat myocardium after ischemia (I) and I/R by immunocytochemical method using murine monoclonal antibody against XO (bovine milk) and by enzyme histochemistry (EHC) in situ. Frozen sections of periodate-lysine-paraformaldehyde (PLP) fixed myocardium after 15, 60 and 90 min ischemia and 15 min ischemia and 30 min reperfusion were processed for immunocytochemistry and EHC. In other experiments, rats were treated with allopurinol, an inhibitor of XO, and hearts were processed for immunocytochemistry. By immunoperoxidase and immunofluorescence methods, a deep staining of interstitial cells, capillary and small blood vessels was observed, but the staining intensity of these cells was increased after reperfusion, in comparison to the normal and ischemic heart tissue. In the electron microscope, an immunoperoxidase reaction product was seen in the cytoplasm of interstitial, endothelial and smooth muscle cells. Similarly, EHC studies by nitroblue tetrazolium staining showed an increase in enzymatic activity in the tissue after reperfusion. The allopurinol-treated I/R tissue exhibited reduced staining. The data suggest that XO activity increases during ischemia but intensifies after reperfusion. The enzyme is localized in interstitial cells, coronary vessel endothelium and smooth muscle cells. XO is constantly present in the interstitial cells of the myocardium and it is a new finding not previously reported. It is further suggested that myocardial interstitium may be one of the major sites where oxygen derived radicals are generated during ischemia.

Topics: Allopurinol; Animals; Endothelium, Vascular; Immunohistochemistry; Male; Muscle, Smooth, Vascular; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Xanthine Oxidase

The effects of tetrahydroberberine (THB) on ischemic and reperfused myocardium were studied in comparison with verapamil (Ver). In anesthetized rats, THB and its analogues, l-THP and l-SPD, reduced the infarct size after 4 h of left anterior descending coronary artery (LAD) ligation. In Langendorff hearts, in common with Ver, THB 1 and 10 mumol.L-1 markedly decreased the incidences of ventricular tachycardia (VT) and ventricular fibrillation (VF) in the reperfusion period. The malondialdehyde content and xanthine oxidase activity were also decreased in global ischemic-reperfused hearts pretreated with THB (P < 0.01, or P < 0.05). It suggested that THB could protect the myocardium from ischemic and reperfusion injury.

Topics: Animals; Anti-Arrhythmia Agents; Berberine; Berberine Alkaloids; In Vitro Techniques; Male; Malondialdehyde; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Sprague-Dawley; Verapamil; Xanthine Oxidase

The effect of 5'-nucleotidase inhibitor (AMP-C) and xanthine oxidase inhibitor (Allopurinol: ALLO) on myocardial functional recovery and the restoration of myocardial high energy phosphates after 15 min of normothermic global ischemic insult, was studied in the isolated isovolemic Langendorff rat heart model. Fifty nine rats were divided into 4 groups: Group I; saline, Group II; AMP-C plus ALLO, Group III; AMP-C, Group IV; ALLO. Intermittent infusion of drugs was delivered in 3 ml of solution at 5 min intervals during ischemia. Percent recovery of left ventricular systolic function was as follows: Group I; 74.2 +/- 3.6%, Group II; 87.7 +/- 1.7%, Group III; 83.5 +/- 3.1%, Group IV; 86.4 +/- 2.6%. Improved recovery was statistically significant only in Group II (p < 0.05 vs Group I). Suppression of reactive hyperemia was seen with reperfusion in the groups which had been treated with AMP-C (i.e., Groups II and III). Myocardial adenine nucleotides and purines were measured in 6 hearts in each group using high performance liquid chromatography. Myocardial ATP levels was 0.89 +/- 0.16 nmol/mg left ventricular wet weight in Group I, 1.37 +/- 0.12 in Group II (p < 0.05 vs Group I), 1.42 +/- 0.17 in Group III (p < 0.05) and 1.17 +/- 0.15 in Group IV. This study demonstrates that intermittent infusion of AMP-C plus ALLO during global myocardial ischemia results in improved myocardial functional recovery and improved preservation of high energy phosphates.

Topics: 5'-Nucleotidase; Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Allopurinol; Animals; Coronary Circulation; In Vitro Techniques; Male; Myocardial Ischemia; Myocardium; Purines; Rats; Rats, Inbred Lew; Vascular Resistance; Ventricular Function, Left; Xanthine Oxidase

The effectiveness of the University of Wisconsin solution and the Collins' M solution for preservation of rat hearts was compared by examining histologic appearance, tissue water content, and mitochondrial respiratory functions after prolonged hypothermic storage and subsequent heterotopic transplantation. Survival of transplanted hearts after 5 days of reperfusion was markedly lowered by storage in Collins' M solution for 15 hours. Hearts stored in University of Wisconsin solution for 10 hours showed no increase in myocardial necrosis after 5 days of reperfusion, whereas hearts stored in University of Wisconsin solution for 15 hours and Collins' M solution for 10 and 15 hours showed a significant increase in tissue necrosis. University of Wisconsin solution reduced tissue swelling during hypothermic storage, whereas Collins' M solution did not cause such reduction. The yield of mitochondrial protein after reperfusion was significantly decreased by storage in either solution, especially after 15 hours in Collins' M solution. Mitochondrial oxidative phosphorylation was significantly inhibited by storage, especially by storage in Collins' M solution and subsequent reperfusion. These results indicate that myocardial injury, after prolonged ischemia and reperfusion, results in a decrease in functionally and structurally intact mitochondria that is dependent on preservation conditions. University of Wisconsin solution protects isolated hearts against ischemia and reperfusion injury possibly by preventing cellular and mitochondrial deterioration.

Topics: Adenosine; Allopurinol; Animals; Body Water; Cardioplegic Solutions; Cold Temperature; Glutathione; Graft Survival; Heart Transplantation; Hypertonic Solutions; Insulin; Male; Mitochondria, Heart; Myocardial Ischemia; Myocardium; Necrosis; Organ Preservation; Organ Preservation Solutions; Oxidative Phosphorylation; Raffinose; Rats; Rats, Inbred Lew

1. We have assessed the effect of allopurinol, amlodipine and propranolol pretreatment on both endothelium-dependent and endothelium-independent coronary vasodilatation in vivo, by comparing pre-ischaemic responses with those measured after 60 min of coronary artery occlusion and 30 min of reperfusion in anaesthetized dogs. 2. In 15 untreated dogs ischaemia and reperfusion attenuated the increases in coronary blood flow produced by either acetylcholine (0.01-0.05 micrograms kg-1, i.a.) or glyceryl trinitrate (0.05-0.2 micrograms kg-1, i.a.), to an average of 39 +/- 4% and 42 +/- 5% of the pre-ischaemic control response, respectively (both P < 0.05). 3. In 5 dogs treated with allopurinol (25 mg kg-1, orally, 24 h previously, plus 50 mg kg-1, i.v., 5 min before occlusion), the increases in coronary blood flow after ischaemia and reperfusion (acetylcholine: 78 +/- 12%, glyceryl trinitrate: 60 +/- 3% of pre-ischaemic response) were significantly larger than post-ischaemic responses in untreated dogs (both P < 0.05). 4. Similarly, amlodipine treatment (3 micrograms kg-1 min-1, i.v., starting 90 min before occlusion) in 5 dogs improved post-ischaemic increases in blood flow (acetylcholine: 58.5%, glyceryl trinitrate: 66 +/- 6% of pre-ischaemic response, significantly greater than post-ischaemic responses in untreated dogs, P < 0.05). 5. In contrast, in a further 6 dogs pretreated with propranolol (1 mg kg-1, i.v., 30 min before occlusion,plus 0.5 mg kg-1 h-1, i.v.), blood flow responses after ischaemia and reperfusion were not different from post-ischaemic responses in untreated dogs (acetylcholine: 46 +/- 6%, glyceryl trinitrate: 46 +/-6% of pre-ischaemic response).6. These results suggest that allopurinol and amlodipine protect against the post-ischaemic impairment of endothelium-dependent and endothelium-independent coronary vasodilatation in vivo by mechanisms additional to endothelial protection.

Topics: Allopurinol; Amlodipine; Anesthetics; Animals; Dogs; Endothelium, Vascular; Female; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Propranolol; Vasodilator Agents

Topics: Allopurinol; Animals; Blood Pressure; Chromatography, High Pressure Liquid; Coronary Circulation; Heart; In Vitro Techniques; Male; Myocardial Ischemia; Myocardium; Oxygen Consumption; Purines; Rats; Rats, Wistar

Contracture of the arrested myocardium during prolonged storage of the heart results in both systolic and diastolic dysfunction, and is a major limitation to extended preservation. We studied the effects of a reversible contractile inhibitor, 2,3-butanedione monoxime (BDM), on myocardial ischemic tolerance. Isolated rabbit hearts were flushed with University of Wisconsin (UW) solution with and without 30 mmol/L BDM and 1 mmol/L CaCl, stored at 4 degrees C for 24 hours, and subsequently reperfused for 60 minutes. Left ventricular pressure-volume relationships and adenine nucleotide content were determined before reperfusion. Left ventricular systolic pressure, diastolic volume, and adenine nucleotide content were measured after reperfusion. Hearts stored in UW solution underwent contracture and adenosine triphosphate (ATP) depletion during storage, and exhibited systolic dysfunction, impaired diastolic relaxation, and poor ATP regeneration upon reperfusion. The addition of calcium worsened contracture and ATP depletion (p < 0.005) and slightly improved function and ATP regeneration (p = not significant). Hearts stored in the presence of BDM experience no contracture during storage; ATP was preserved (10.7 versus 15.7 nmol/mg; p < 0.05), and left ventricular systolic pressure and ATP content recovered to 74% and 93% of control on reperfusion, respectively (p < 0.005). Left ventricular diastolic volume remained depressed, however, although less than with UW solution (0.87 versus 0.45 mL; p < 0.001). When both BDM and calcium were included in the UW solution, calcium-stimulated ATP hydrolysis and contracture were prevented, left ventricular systolic pressure returned to 87% of control, and left ventricular diastolic volume and ATP content returned to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Calcium Chloride; Compliance; Coronary Circulation; Diacetyl; Glutathione; Heart Rate; Heart Ventricles; In Vitro Techniques; Insulin; Myocardial Ischemia; Myocardium; Organ Preservation; Organ Preservation Solutions; Rabbits; Raffinose; Solutions

The effectiveness of the University of Wisconsin solution on extended myocardial preservation was examined in this study using phosphorus 31-nuclear magnetic resonance spectroscopy. Isolated perfused rat hearts were arrested and stored in four preservation solutions: group 1, modified Krebs-Henseleit solution; group 2, modified St. Thomas' Hospital solution; group 3, oxygenated modified St. Thomas' Hospital solution containing 11 mmol/L glucose; and group 4, University of Wisconsin solution. The changes in myocardial high energy phosphate profiles and the intracellular pH values were measured during 12 hours of cold (4 degrees C) global ischemia and 90 minutes of normothermic reperfusion. Following ischemia, the hearts were assessed for hemodynamic recovery and myocardial water content. During ischemia, adenosine triphosphate depletion was observed in all groups; however, after 5 hours of ischemia, the adenosine triphosphate levels were significantly higher in group 3 compared with the other groups (adenosine triphosphate levels at 6 hours in mumol/gm dry weight: group 3, 7.6; group 4, 3.2; group 2, < 1; p < 0.025). The tissue water content at the end of ischemia was lower with the University of Wisconsin solution compared with the modified St. Thomas' Hospital solution or the oxygenated modified St. Thomas' Hospital solution (in ml/gm dry weight: group 4, 3.0; group 2, 4.4; group 3, 3.9; p < 0.05). The adenosine triphosphate repletion during reperfusion was greater with the University of Wisconsin solution compared with the modified St. Thomas' Hospital solution or the oxygenated modified St. Thomas' Hospital solution (12 mumol/gm dry weight in group 4; 8.1 in group 2; 9.0 in group 3; p < 0.05). Similar findings were obtained for the recovery of left ventricular pressure (in percent of preischemic control: group 4, 70%; group 2, 42%; group 3, 52%; p < 0.01) and coronary flow (group 4, 61%; group 2, 49%; group 3, 49%; p < 0.05). These data suggest that preservation with the University of Wisconsin solution affords improved hemodynamic recovery, enhanced adenosine triphosphate repletion, and reduced tissue edema upon reperfusion; however, oxygenated St. Thomas' Hospital solution with glucose is associated with the preservation of higher myocardial adenosine triphosphate levels during prolonged cold global ischemia. In conclusion, these data indicate that the University of Wisconsin solution might improve graft tolerance of ischemia in clinical heart transp

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Bicarbonates; Body Water; Calcium Chloride; Cardioplegic Solutions; Glutathione; Heart; Hemodynamics; Hydrogen-Ion Concentration; Insulin; Magnesium; Magnetic Resonance Spectroscopy; Male; Myocardial Ischemia; Myocardium; Organ Preservation; Organ Preservation Solutions; Phosphocreatine; Phosphorus; Potassium Chloride; Raffinose; Rats; Rats, Sprague-Dawley; Sodium Chloride; Solutions; Treatment Outcome