3-nitrotyrosine and Myocardial-Infarction

In the present study, we hypothesized that postcon (postconditioning) confers cardioprotection in vivo by reducing the production of ONOO- (peroxynitrite) and nitro-oxidative stress subsequent to the inhibition of the iNOS (inducible NO synthase). Patients with AMI (acute myocardial infarct) were randomly assigned to undergo percutaneous coronary intervention without (control) or with ischaemic postcon by three episodes of 30-s inflation and 30-s deflation of the angioplasty balloon. Animal models of MI/R (myocardial ischaemia/reperfusion) injury were induced in rats by occluding the left coronary artery for 40 min followed by 4-h reperfusion. Rats were randomized to receive vehicle, postcon (three cycles of 10-s reperfusion and 10-s coronary re-occlusion preceding full reperfusion), the selective iNOS inhibitor 1400W or postcon plus 3-morpholinosydnonimine (an ONOO- donor). Postcon in patients reduced iNOS activity in white blood cells, decreased plasma nitrotyrosine, a fingerprint of ONOO- and an index of nitro-oxidative stress, and improved cardiac function (P<0.01 compared with control). In rats, postcon reduced post-ischaemic myocardial iNOS activity and nitrotyrosine formation, reduced myocardial infarct size (all P<0.05 compared with control) and improved cardiac function. Administration of 1400W resembled, whereas 3-morpholinosydnonimine abolished, the effects of postcon. In conclusion, reduction in ONOO--induced nitro-oxidative stress subsequent to the inhibition of iNOS represents a major mechanism whereby postcon confers cardioprotection in vivo.

Topics: Aged; Angioplasty, Balloon, Coronary; Animals; Apoptosis; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Humans; Ischemic Postconditioning; Leukocyte Count; Male; Malondialdehyde; Middle Aged; Molsidomine; Myocardial Infarction; Myocardial Reperfusion Injury; Nitric Oxide Synthase Type II; Oxidative Stress; Rats; Rats, Sprague-Dawley; Treatment Outcome; Tyrosine; Ventricular Function, Left

Adjunctive interventions protect from reperfusion injury during primary percutaneous coronary intervention (PCI), but it is not known whether they are also protective during elective PCI. We sought to assess the efficacy of staccato reperfusion (SR) during PCI.. Thirty seven patients with recent acute coronary syndrome and target lesions of 85-100% were randomized to SR (n=18), consisting of 6 periods of 10-s balloon inflation/deflation (total time, 120 sec) or abrupt reperfusion (AR, n=19), consisting of a single continuous 120-s balloon inflation; subsequently, all underwent stent implantation. Left ventricular wall motion score was echocardiography determined at baseline, 10 days and 1 year later. The oxidative markers malondialdehyde (MDA) and nitrotyrosine were assessed at baseline, 3 and 18 min after PCI. Patients were also followed for 1 year for major events (death, non-fatal myocardial infarction or revascularization).. Wall motion score index (SR: 1.34+/-0.29 (baseline), 1.17+/-0.17 (10-day), 1.08+/-0.12 (1-year); AR: 1.33+/-0.22, 1.27+/-0.20, 1.24+/-0.22, respectively) improved significantly as a result of SR (F=8.951, p=0.002). Similarly, the biomarkers of oxidative injury, MDA (1.74+/-0.49 micromol/L in SR vs. 2.45+/-1.26 micromol/L in AR, p=0.002) and nitrotyrosine (5.23+/-5.58 nmol/L in SR vs. 9.79+/-7.83 nmol/L in AR, p=0.003) measured 18 min after PCI were significantly lower in SR. No major events occurred.. SR can improve long-term wall motion score during PCI, at least partly through the attenuation of a reperfusion-type oxidative injury that also occurs in these patients.

Topics: Acute Coronary Syndrome; Aged; Aged, 80 and over; Angioplasty, Balloon, Coronary; Biomarkers; Coronary Stenosis; Female; Follow-Up Studies; Humans; Male; Malondialdehyde; Middle Aged; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Oxidative Stress; Pilot Projects; Recovery of Function; Secondary Prevention; Stents; Time Factors; Treatment Outcome; Tyrosine; Ultrasonography; Ventricular Function, Left

Oxidative stress and increased inflammation have been reported to be increased in subjects with diabetes and to be involved in the pathogenesis of cardiovascular complications after myocardial infarction (MI). It is well recognized that red wine has antioxidant and anti-inflammatory activities. We examined the effects of moderate red wine intake on echocardiographic parameters of functional cardiac outcome in addition to inflammatory cytokines and nitrotyrosine (oxidative stress marker), in subjects with diabetes after a first uncomplicated MI.. One hundred and fifteen subjects with diabetes who had sustained a first non-fatal MI were randomized to receive a moderate daily amount of red wine (intervention group) or not (control group). Echocardiographic parameters of ventricular dys-synchrony, circulating levels of nitrotyrosine, tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-18 (IL-18) and C-reactive protein (CRP) were investigated at baseline and 12 months after randomization.. After 1 year of diet intervention, concentrations of nitrotyrosine (P < 0.01), CRP (P < 0.01), TNF-alpha (P < 0.01), IL-6 (P < 0.01) and IL-18 (P < 0.01) were increased in the control group compared with the intervention group. In addition, myocardial performance index (P < 0.02) was higher, and transmitral Doppler flow (P < 0.05), pulmonary venous flow analysis (P < 0.02) and ejection fraction (P < 0.05) were lower in the control group, indicating ventricular dys-synchrony. The concentrations of nitrotyrosine, CRP, TNF-alpha and IL-6 were related to echocardiographic parameters of ventricular dys-synchrony.. In subjects with diabetes, red wine consumption, taken with meals, significantly reduces oxidative stress and pro-inflammatory cytokines as well as improving cardiac function after MI. Moderate red wine intake with meals may have a beneficial effect in the prevention of cardiovascular complications after MI in subjects with diabetes.

Topics: Adult; Aged; C-Reactive Protein; Cardiovascular Diseases; Cytokines; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Diet, Mediterranean; Follow-Up Studies; Heart; Humans; Middle Aged; Myocardial Infarction; Oxidative Stress; Prognosis; Treatment Outcome; Tyrosine; Wine

Myocardial extracellular matrix (ECM) is essential for proper cardiac function and structural integrity; thus, the disruption of ECM homeostasis is associated with several pathological processes. Female Wistar rats underwent surgical ovariectomy (OVX) or sham operation (SO) and were then divided into eight subgroups based on the type of diet (standard chow or high-triglyceride diet/HT) and exercise (with or without running). After 12 weeks, cardiac MMP-2 activity, tissue inhibitor of metalloproteinase-2, content of collagen type I, the level of nitrotyrosine (3-NT) and glutathione (GSH), and the ratio of infarct size were determined. Our results show that OVX and HT diet caused an excessive accumulation of collagen; however, this increase was not observed in the trained animals. Twelve weeks of exercise promoted elevation in the levels of 3-NT and GSH and similarly an increase in MMP-2 activity of both SO and OVX animals. The high infarct-size ratio caused by OVX and HT diet was mitigated by physical exercise. Our findings demonstrate that ovarian estrogen loss and HT diet caused collagen accumulation and increased ratio of the infarct size. However, exercise-induced cardiac remodeling serves as a compensatory mechanism by enhancing MMP-2 activity and reducing fibrosis, thus minimizing the ischemia/reperfusion injury.

Topics: Animal Nutritional Physiological Phenomena; Animals; Collagen Type I; Estrogens; Female; Glutathione; Matrix Metalloproteinase 2; Myocardial Infarction; Myocardium; Ovariectomy; Physical Conditioning, Animal; Rats, Wistar; Tissue Inhibitor of Metalloproteinase-2; Tyrosine; Ventricular Remodeling

The long-term use of proton pump inhibitors (PPIs) has been shown to increase the risk of cardiovascular mortality, however the molecular mechanisms are unknown. Superoxide has been implicated in the regulation of nerve growth factor (NGF), a mediator of sympathetic innervation. The purpose of this study was to determine whether PPIs increase ventricular arrhythmias through magnesium-mediated superoxide production in infarcted rats. Male Wistar rats were randomly assigned to receive vehicle, omeprazole, omeprazole + magnesium sulfate, or famotidine treatment for 4 weeks starting 24 hours after the induction of myocardial infarction by ligating the coronary artery. Increased myocardial superoxide and nitrotyrosine levels were noted post-infarction, in addition to a significant upregulation of NGF expression on mRNA and protein levels. Sympathetic hyperinnervation after infarction was confirmed by measuring myocardial norepinephrine and immunofluorescent analysis. Compared with the vehicle, omeprazole-treated infarcted rats had significantly reduced myocardial magnesium content, increased oxidant production, and increased sympathetic innervation, which in turn increased ventricular arrhythmias. These effects were prevented by the coadministration of magnesium sulfate. In an in vivo study, an omeprazole-induced increase in NGF was associated with a superoxide pathway, which was further confirmed by an ex vivo study showing the attenuation of NGF levels after coadministration of the superoxide scavenger Tiron. Magnesium sulfate did not further attenuate NGF levels compared with omeprazole + Tiron. Our results indicate that the long-term administration of PPIs was associated with reduced tissue magnesium content and increased myocardial superoxide production, which exacerbated ventricular arrhythmias after infarction. Magnesium may be a potential target for PPI-related arrhythmias after infarction.

Topics: Animals; Dose-Response Relationship, Drug; Heart; Magnesium; Male; Myocardial Infarction; Myocardium; Nerve Growth Factor; Norepinephrine; Proton Pump Inhibitors; Rats; Rats, Wistar; Signal Transduction; Superoxides; Sympathetic Nervous System; Tyrosine; Ventricular Remodeling

Myocardial ischemia/reperfusion injury (myocardial I/R injury) has a high disability rate and mortality. Novel treatments for myocardial I/R injury are necessary.. In order to explore the protective effect of hydromorphine on myocardial I/R injury, we illuminate the underlying mechanism of the protective effect.. Hydromorphine significantly reduced myocardial infarct size (IFN/AAR), CKMB (Creatine Kinase MB) and TN-T (Troponin T) release, and improved cardiac function compared with I/R group. However, these advantageous effects were partly suppressed in the presence of hydromorphine. Myocardial I/R injury significantly decreased the phosphorylation of Akt and eNOS, and down-regulated total nitric oxide and nitrotyrosine content, while these inhibitory effects were partly abolished by hydromorphine. Conversely, the activated effects of hydromorphine on the phosphorylation of Akt and eNOS, and NO release were totally reversed by LY294002, which, used individually, show the same influence on reperfusion injury.. These findings suggest that hydromorphine postconditioning may protect isolated rat heart against reperfusion injury via activating P13K/Akt/eNOS signaling.

Topics: Animals; Creatine Kinase, MB Form; Disease Models, Animal; Isolated Heart Preparation; Male; Morphine Derivatives; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Signal Transduction; Troponin T; Tyrosine

Alginate oligosaccharide (AOS) has recently demonstrated the ability to protect against acute doxorubicin cardiotoxicity and neurodegenerative disorders by inhibiting oxidative stress and endoplasmic reticulum (ER) stress-mediated apoptosis, which are both involved in myocardial ischemia/reperfusion (I/R) injury. In the present study, we investigated whether pretreatment with AOS protects against myocardial I/R injury in mice and explored potential cardioprotective mechanisms. AOS pretreatment significantly decreased the infarct size, reduced the cardiac troponin-I concentration, and ameliorated the cardiac dysfunction. Accompanied with the reduced cardiac injury, AOS pretreatment clearly decreased I/R-induced myocardial apoptosis. With regard to mechanism, AOS pretreatment markedly attenuated nitrative/oxidative stress, as evidenced by decreases in 3-nitrotyrosine content and superoxide generation, and downregulated inducible nitric oxide synthase, NADPH oxidase2, and 4-hydroxynonenal. Moreover, AOS pretreatment decreased myocardial apoptosis by inhibiting the ER stress-mediated apoptosis pathway, which is reflected by the downregulation of C/EBP homologous protein, glucose-regulated protein 78, caspase-12, and Bcl-2-associated X protein, and by the upregulation of the anti-apoptotic protein B-cell lymphoma-2. Collectively, these findings demonstrate that AOS renders the heart resistant to I/R injury, at least in part, by inhibiting nitrative/oxidative stress and ER stress-mediated apoptosis.

Topics: Alginates; Animals; Apoptosis; Cardiotonic Agents; Disease Models, Animal; Endoplasmic Reticulum Stress; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Nitrosative Stress; Oligosaccharides; Oxidative Stress; Superoxides; Tyrosine

Despite increased secondary cardiovascular events in patients with ischemic cardiomyopathy (ICM), the expression of innate cardiac protective molecules in the hearts of patients with ICM is incompletely characterized. Therefore, we used a nonbiased RNAseq approach to determine whether differences in cardiac protective molecules occur with ICM.. RNAseq analysis of human control and ICM left ventricular samples demonstrated a significant decrease in

Topics: Adult; Animals; Calcium Channels, L-Type; Calcium Signaling; Calcium-Binding Proteins; Cardiomyopathies; Case-Control Studies; Disease Models, Animal; Female; Genetic Predisposition to Disease; Humans; Male; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Oxidative Stress; Phenotype; Potassium Channels, Inwardly Rectifying; Reactive Nitrogen Species; Reactive Oxygen Species; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tyrosine; Ventricular Dysfunction, Left; Ventricular Function, Left; Ventricular Pressure

Recent studies have demonstrated that inflammatory cells are a component that plays a role in thrombus formation in ST-elevation myocardial infarction (STEMI). 3-nitrotyrosine (3-NO2-Tyr), a specific marker for protein modification by nitric oxide-derived oxidants, is increased in human atherosclerotic lesions. The purpose of this study was to determine the possible association of inflammatory markers of coronary thrombi with nitroxidative stress. Intracoronary thrombus (n=51) and blood from the systemic circulation were obtained by thromboaspiration in 138 consecutive STEMI patients presenting for primary percutaneous coronary intervention (PCI). Each blood and intracoronary thrombus were measured simultaneously the following biomarkers: C-reactive protein (CRP), 3-NO2-Tyr, soluble CD 40 ligand (sCD40L), vascular cellular adhesion molecule-1 (VCAM-1) and haemoglobin content (only in coronary thrombus). Time delay in minutes from symptom onset to PCI was 244 ± 324. Serum CRP was positively correlated to CRP content in the thrombus (r= 0.395; p = 0.02) and serum sCD40L was negatively correlated to sCD40L in the thrombus (r= -0.394; p = 0.02). Patients were divided into tertiles according to thrombi 3-NO2-Tyr concentration: 1(st)tertile (<0.146ng/mg), 2(nd)tertile (0.146-0.485ng/mg) and 3(rd)tertile (>0.485ng/mg). Thus, thrombus in the highest tertile had significantly higher levels of CRP (p=0.002), VCAM-1 (p=0.003) and haemoglobin (p=0.002). In conclusion, the present study demonstrated that coronary thrombi with higher levels of 3-NO2-Tyr content often contain more inflammatory markers which could have a direct impact on the efficacy of drugs or devices used for coronary reperfusion.

Topics: Aged; Biomarkers; C-Reactive Protein; CD40 Ligand; Coronary Thrombosis; Enzyme-Linked Immunosorbent Assay; Female; Hemoglobins; Humans; Inflammation; Male; Middle Aged; Myocardial Infarction; Oxidative Stress; Percutaneous Coronary Intervention; Tyrosine; Vascular Cell Adhesion Molecule-1

Chronic heart failure is characterized by decreased exercise capacity with early exacerbation of fatigue and dyspnea. Intrinsic skeletal muscle abnormalities can play a role in exercise intolerance. Causal or contributing factors responsible for muscle alterations have not been completely defined. This study evaluated skeletal muscle oxidative stress and NADPH oxidase activity in rats with myocardial infarction (MI) induced heart failure.. Four months after MI, rats were assigned to Sham, MI-C (without treatment), and MI-NAC (treated with N-acetylcysteine) groups. Two months later, echocardiogram showed left ventricular dysfunction in MI-C; NAC attenuated diastolic dysfunction. In soleus muscle, glutathione peroxidase and superoxide dismutase activity was decreased in MI-C and unchanged by NAC. 3-nitrotyrosine was similar in MI-C and Sham, and lower in MI-NAC than MI-C. Total reactive oxygen species (ROS) production was assessed by HPLC analysis of dihydroethidium (DHE) oxidation fluorescent products. The 2-hydroxyethidium (EOH)/DHE ratio did not differ between Sham and MI-C and was higher in MI-NAC. The ethidium/DHE ratio was higher in MI-C than Sham and unchanged by NAC. NADPH oxidase activity was similar in Sham and MI-C and lower in MI-NAC. Gene expression of p47(phox) was lower in MI-C than Sham. NAC decreased NOX4 and p22(phox) expression.. We corroborate the case that oxidative stress is increased in skeletal muscle of heart failure rats and show for the first time that oxidative stress is not related to increased NADPH oxidase activity.

Topics: Acetylcysteine; Animals; Ethidium; Free Radical Scavengers; Glutathione Peroxidase; Heart Failure; Heart Ventricles; Male; Malondialdehyde; Muscle, Skeletal; Myocardial Infarction; NADPH Oxidase 4; NADPH Oxidases; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Superoxide Dismutase; Tyrosine

Remote ischemic perconditioning (RIPerC) has a promising therapeutic insight to improve the prognosis of acute myocardial infarction. Chronic comorbidities such as diabetes are known to interfere with conditioning interventions by modulating cardioprotective signaling pathways, such as e.g., mTOR pathway and autophagy. However, the effect of acute hyperglycemia on RIPerC has not been studied so far. Therefore, here we investigated the effect of acute hyperglycemia on cardioprotection by RIPerC.. Wistar rats were divided into normoglycemic (NG) and acute hyperglycemic (AHG) groups. Acute hyperglycemia was induced by glucose infusion to maintain a serum glucose concentration of 15-20 mM throughout the experimental protocol. NG rats received mannitol infusion of an equal osmolarity. Both groups were subdivided into an ischemic (Isch) and a RIPerC group. Each group underwent reversible occlusion of the left anterior descending coronary artery (LAD) for 40 min in the presence or absence of acute hyperglycemia. After the 10-min LAD occlusion, RIPerC was induced by 3 cycles of 5-min unilateral femoral artery and vein occlusion and 5-min reperfusion. After 120 min of reperfusion, infarct size was measured by triphenyltetrazolium chloride staining. To study underlying signaling mechanisms, hearts were harvested for immunoblotting after 35 min in both the NG and AHG groups.. Infarct size was significantly reduced by RIPerC in NG, but not in the AHG group (NG + Isch: 46.27 ± 5.31 % vs. NG + RIPerC: 24.65 ± 7.45 %, p < 0.05; AHG + Isch: 54.19 ± 4.07 % vs. 52.76 ± 3.80 %). Acute hyperglycemia per se did not influence infarct size, but significantly increased the incidence and duration of arrhythmias. Acute hyperglycemia activated mechanistic target of rapamycine (mTOR) pathway, as it significantly increased the phosphorylation of mTOR and S6 proteins and the phosphorylation of AKT. In spite of a decreased LC3II/LC3I ratio, other markers of autophagy, such as ATG7, ULK1 phopsphorylation, Beclin 1 and SQSTM1/p62, were not modulated by acute hyperglycemia. Furthermore, acute hyperglycemia significantly elevated nitrative stress in the heart (0.87 ± 0.01 vs. 0.50 ± 0.04 µg 3-nitrotyrosine/mg protein, p < 0.05).. This is the first demonstration that acute hypreglycemia deteriorates cardioprotection by RIPerC. The mechanism of this phenomenon may involve an acute hyperglycemia-induced increase in nitrative stress and activation of the mTOR pathway.

Topics: Animals; Apoptosis Regulatory Proteins; Arrhythmias, Cardiac; Autophagy; Autophagy-Related Protein 7; Autophagy-Related Protein-1 Homolog; Beclin-1; Heat-Shock Proteins; Hyperglycemia; Intracellular Signaling Peptides and Proteins; Ischemic Preconditioning, Myocardial; Myocardial Infarction; Myocardial Reperfusion Injury; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Sequestosome-1 Protein; Severity of Illness Index; Signal Transduction; Stress, Physiological; TOR Serine-Threonine Kinases; Tyrosine; Ubiquitin-Activating Enzymes

The core region of a myocardial infarction is notoriously unsupportive of cardiomyocyte survival. However, there has been less investigation of the potentially beneficial spontaneous recruitment of endogenous bone marrow progenitor cells (BMPCs) within infarcted areas. In the current study we examined the role of tissue oxygenation and derived toxic species in the control of BMPC engraftment during postinfarction heart remodeling.. For assessment of cellular origin, local oxygenation, redox status, and fate of cells in the infarcted region, myocardial infarction in mice with or without LacZ(+) bone marrow transplantation was induced by coronary ligation. Sham-operated mice served as controls. After 1 week, LacZ(+) BMPC-derived cells were found inhomogeneously distributed into the infarct zone, with a lower density at its core. Electron paramagnetic resonance (EPR) oximetry showed that pO2 in the infarct recovered starting on day 2 post-myocardial infarction, concomitant with wall thinning and erythrocytes percolating through muscle microruptures. Paralleling this reoxygenation, increased generation of reactive oxygen/nitrogen species was detected at the infarct core. This process delineated a zone of diminished BMPC engraftment, and at 1 week infiltrating cells displayed immunoreactive 3-nitrotyrosine and apoptosis. In vivo treatment with a superoxide dismutase mimetic significantly reduced reactive oxygen species formation and amplified BMPC accumulation. This treatment also salvaged wall thickness by 43% and left ventricular ejection fraction by 27%, with significantly increased animal survival.. BMPC engraftment in the infarct inversely mirrored the distribution of reactive oxygen/nitrogen species. Antioxidant treatment resulted in increased numbers of engrafted BMPCs, provided functional protection to the heart, and decreased the incidence of myocardial rupture and death.

Topics: Animals; Antioxidants; Apoptosis; Bone Marrow Cells; Bone Marrow Transplantation; Cell Differentiation; Cell Movement; Disease Models, Animal; Lac Operon; Male; Mice; Mice, Transgenic; Myocardial Infarction; Myocytes, Cardiac; Reactive Nitrogen Species; Reactive Oxygen Species; Stem Cell Transplantation; Stem Cells; Time Factors; Tyrosine; Ventricular Remodeling

The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress-induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion.. Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and inhibition of PKG (KT5823) or NOS (l-NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2(-)) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS.. These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2(-) production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling.

Topics: Animals; Biopterins; Cell Death; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Ischemic Postconditioning; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitrates; Nitric Oxide Synthase Type III; Nitrites; Oxidative Stress; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Superoxides; Time Factors; Tyrosine

Myocardial ischemia induces 5-lipoxygenase (LOX) translocation and leukotriene production in the heart. Leukotrienes increase inflammatory responses aggravating, thereby, ischemia-reperfusion (I/R) injury. This study aimed to investigate whether the selective 5-LOX inhibitor 11-keto-β-boswellic acid (11-keto BA), in three different dose levels, exert a protective effect on myocardial I/R injury in an in vivo rat heart model. Sixty male Wister rats were used in this study and divided into five equal groups (n=12): GP1, sham-operated receiving normal saline; Gp 2, rats were subjected to 45 min left anterior descending coronary artery ligation followed by 4 h reperfusion to serve as I/R group. Gps 3-5 received 11-keto BA in doses 250, 500, 1,000 mg/kg, respectively, via an oral gavage for 7 days then were exposed to I/R. I/R injury induced a significant elevation in myeloperoxidase activity and gene expression of intracellular adhesion molecules, cyclooxygenase-2, 5-lipooxygenasae, nuclear factor kappa-beta, tumor necrosis factor alpha, nuclear factor (erythroid-derived 2)-like 2, and hemeoxygenease-1 consequently with reduction in glutathione peroxidase in heart tissues. Furthermore, immunohistochemical examination of the heart tissues showed positive immuostaining for both 3-nitrotyrosine and caspase-3 with DNA-ladder formation in all diseased rats. 11-keto BA in three dose levels exerted dose dependent cardioprotective effect manifested by dose-dependent reduction in serum lactate dehydrogenase and infract size through mechanisms related to enhancement of antioxidant capacity and prevention of inflammatory cascades.

Topics: Animals; Arachidonate 5-Lipoxygenase; Cardiotonic Agents; Caspase 3; Cyclooxygenase 2; DNA Fragmentation; Gene Expression Regulation; Glutathione Peroxidase; Heme Oxygenase (Decyclizing); Intercellular Adhesion Molecule-1; Lipoxygenase Inhibitors; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; NF-E2-Related Factor 2; NF-kappa B; Oxidation-Reduction; Peroxidase; Rats; Rats, Wistar; Triterpenes; Tumor Necrosis Factor-alpha; Tyrosine

Inhaled nitric oxide (NO) has been reported to decrease the infarct size in cardiac ischemia-reperfusion (I/R) injury. However, reactive nitrogen species (RNS) produced by NO cause myocardial dysfunction and injury. Because H₂ is reported to eliminate peroxynitrite, it was expected to reduce the adverse effects of NO. In mice, left anterior descending coronary artery ligation for 60 min followed by reperfusion was performed with inhaled NO [80 parts per million (ppm)], H₂ (2%), or NO + H₂, starting 5 min before reperfusion for 35 min. After 24 h, left ventricular function, infarct size, and area at risk (AAR) were assessed. Oxidative stress associated with reactive oxygen species (ROS) was evaluated by staining for 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal, that associated with RNS by staining for nitrotyrosine, and neutrophil infiltration by staining for granulocyte receptor-1. The infarct size/AAR decreased with breathing NO or H₂ alone. NO inhalation plus H₂ reduced the infarct size/AAR, with significant interaction between the two, reducing ROS and neutrophil infiltration, and improved the cardiac function to normal levels. Although nitrotyrosine staining was prominent after NO inhalation alone, it was eliminated after breathing a mixture of H₂ with NO. Preconditioning with NO significantly reduced the infarct size/AAR, but not preconditioning with H₂. In conclusion, breathing NO + H₂ during I/R reduced the infarct size and maintained cardiac function, and reduced the generation of myocardial nitrotyrosine associated with NO inhalation. Administration of NO + H₂ gases for inhalation may be useful for planned coronary interventions or for the treatment of I/R injury.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Administration, Inhalation; Aldehydes; Animals; Antioxidants; Cardiotonic Agents; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Gases; Hydrogen; Immunohistochemistry; Inhalation; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neutrophil Infiltration; Nitric Oxide; Oxidative Stress; Receptors, Cell Surface; Time Factors; Tyrosine; Ventricular Function, Left

Myocardial ischemic postconditioning (PosC) describes an acquired resistance to lethal ischemia-reperfusion (I/R) injury afforded by brief episodes of I/R applied immediately after the ischemic insult. Cardioprotection is conveyed by parallel signaling pathways converging to prevent mitochondria permeability transition. Recent observations indicated that PostC is associated with free radicals generation, including nitric oxide (NO(.)) and superoxide (O2 (.-)), and that cardioprotection is abrogated by antioxidants. Since NO. And O2 (. -) react to form peroxynitrite, we hypothesized that postC might trigger the formation of peroxyntrite to promote cardioprotection in vivo. Rats were exposed to 45 min of myocardial ischemia followed by 3h reperfusion. PostC (3 cycles of 30 seconds ischemia/30 seconds reperfusion) was applied at the end of index ischemia. In a subgroup of rats, the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (FeTPPS) was given intravenously (10 mg/kg(-1)) 5 minutes before PostC. Myocardial nitrotyrosine was determined as an index of peroxynitrite formation. Infarct size (colorimetric technique and plasma creatine kinase-CK-levels) and left ventricle (LV) function (micro-tip pressure transducer), were determined. A significant generation of 3-nitrotyrosine was detected just after the PostC manoeuvre. PostC resulted in a marked reduction of infarct size, CK release and LV systolic dysfunction. Treatment with FeTPPS before PostC abrogated the beneficial effects of PostC on myocardial infarct size and LV function. Thus, peroxynitrite formed in the myocardium during PostC induces cardioprotective mechanisms improving both structural and functional integrity of the left ventricle exposed to ischemia and reperfusion in vivo.

Topics: Animals; Cardiotonic Agents; Cytoprotection; Heart; Ischemic Postconditioning; Male; Metalloporphyrins; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peroxynitrous Acid; Rats; Rats, Wistar; Tyrosine

Chronic administration of high dose opioids such as morphine is known to create intracellular oxidative stress via an opioid receptor dependent mechanism and this can interfere with cellular function. This study aimed at examining whether such changes can occur following short term exposure to high concentration of remifentanil, a potent short acting opioid. We conducted a experimental study using rat myocardium and systematically quantified tissue levels of superoxide anions, malondialdehyde (MDA) and nitrotyrosine following exposure to increasing duration (15 min, 1 or 2 h) or escalating doses of remifentanil (1 μg, 5 μg, 10 μg or 20 μg/kg/min). Concurrently the susceptibility of the heart to ischaemia reperfusion injury was assessed under the similar conditions. For any given duration of remifentanil infusion, there was increasing superoxide anions generated as the dose of remifentanil was increased. MDA concentrations were significantly increased when the animal was exposed to 10 μg/kg/min for 2h or 20 μg/kg/min for any duration. There was a trend towards an increased nitrotyrosine concentration with increasing dose of remifentanil, becoming significant when the dose was 20 μg/kg/min. The infarct limiting ability of remifentanil was compromised when the dihydroethidium fluorescence positive cell percentage exceeded 50%, MDA concentration greater than 2 nmol/mg of protein and nitrotyrosine content exceeding 1.5 μg/mg of protein. Short term high dose opioid exposure can induce oxidative changes seen previously only with chronic opioid use and this high oxidative stress environment corrupts the heart's sensitivity to be preconditioned by opioids.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Analgesics, Opioid; Animals; Deoxyguanosine; Dose-Response Relationship, Drug; Hemodynamics; Ischemic Preconditioning, Myocardial; Male; Malondialdehyde; Myocardial Infarction; Myocardium; Oxidative Stress; Piperidines; Rats; Rats, Sprague-Dawley; Remifentanil; Superoxide Dismutase; Superoxides; Tyrosine

Large body of evidences accumulated in clinical and epidemiological studies indicate that hearts of diabetic subjects are more sensitive to ischemia reperfusion injury (IRI), which results in a higher rate of mortality at post-operation than that of non-diabetes. However, experimental results are equivocal and point to either increased or decreased susceptibility of the diabetic hearts to IRI, especially at the early stage of the disease. The present study was designed to test the hypothesis that the duration/severity of the indexed ischemia is a major determinant of the vulnerability to myocardial IRI at early stage of diabetes.. Four weeks streptozotocin (STZ)-induced diabetic (D) and non-diabetic (C) Sprague-Dawley rats were randomly assigned to receive 30 or 45 min of left anterior descending artery ligation followed by 2 or 3 hours of reperfusion, respectively. Cardiac function was recorded by using Pressure-Volume (PV) conduction system. Myocardial infarct size was determined with triphenyltetrazolium chloride staining. Plasma Creatine kinase-MB (CK-MB), Lactate dehydrogenase (LDH) release, myocardial nitric oxide(NO) content and nitrotyrosine formation, 15-F(2t)-Isoprostane and plasma superoxide dismutase (SOD) were measured with colorimetric assays. Cardiomyocyte apoptosis was assessed by TUNEL staining. Myocardial TNFα, Caspase-3, STAT3, Akt, and GSK-3β were determined by Western blotting.. Prolongation of ischemia but not reperfusion from 30 min to 45 min significantly increased infarct size in D compared to C rats (P < 0.05), accompanied with significantly increased plasma CK-MB (P < 0.05). Prolongation of the duration of either ischemia or reperfusion significantly increased plasma LDH release and myocardial 15-F(2t)-Isoprostane and reduced plasma SOD activity, with concomitant reduction of myocardial NO and increase of nitrotyrosine formation in D relative to C (P < 0.05). Prolongation of ischemia and reperfusion significantly reduced left ventricular ejection fraction and increased the peak rate of pressure, accompanied with increased end systolic pressure in D relative to C rats (P < 0.05) but reduced phosphorylations of myocardial STAT3 at site Ser727 and Akt at site Ser473 as well as GSK-3β at Ser 9 (P < 0.05).. Diabetic hearts, even at early stage of the disease are more sensitive to IRI, and this increased severity of post-ischemic myocardial injury depends more on the duration of ischemia than that of reperfusion.

Topics: Animals; Biomarkers; Caspase 3; Creatine Kinase, BB Form; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Dinoprost; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; L-Lactate Dehydrogenase; Male; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Severity of Illness Index; STAT3 Transcription Factor; Stroke Volume; Superoxide Dismutase; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine; Ventricular Function, Left; Ventricular Pressure

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

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

Peroxynitrite (ONOO(-)) formation triggers oxidative/nitrative stress and contributes to exacerbated myocardial ischemia/reperfusion (MI/R) injury. Catalpol, an iridoid glycoside, abundantly found in the roots of Rehmannia glutinosa L. that is included in the family Phrymaceae in the order Lamiales, endemic to China, was found to have neuroprotective effects. However, the effect of catalpol on MI/R injury has not been identified.. This study investigated whether catalpol attenuates oxidative/nitrative stress in acute MI/R.. Adult male rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion and were treated with saline, catalpol (5 mg/kg, i.p., 5 min before reperfusion) or catalpol plus wortmannin (15 µg/kg intraperitoneally injected 15 min before reperfusion).. Pretreatment with catalpol significantly improved cardiac functions, reduced myocardial infarction, apoptosis and necrosis of cardiomyocytes after MI/R (all p < 0.05). Meanwhile, ONOO(-) formation was markedly reduced after catalpol treatment (3.01 ± 0.22 vs. 4.66 ± 0.53 pmol/mg protein in vehicle, p < 0.05). In addition, catalpol increased Akt and endothelial nitric oxide synthase phosphorylation, nitric oxide (NO) production, anti-oxidant capacity and reduced MI/R-induced inducible nitric oxide synthase expression and superoxide anion (·O(2)(-)) production in I/R hearts. PI3K inhibitor wortmannin not only blocked catalpol-induced Akt activation, but also attenuated all the beneficial effects of catalpol. Suppression of ONOO(-) formation by either catalpol or an ONOO(-) scavenger uric acid (5 mg/kg) reduced myocardial infarct size in MI/R rats.. In conclusion, catalpol affords cardioprotection against MI/R insult by attenuating ONOO(-) formation, which is attributable to increased physiological NO and decreased ·O(2)(-) production.

Topics: Animals; Antioxidants; Apoptosis; Cardiotonic Agents; Disease Models, Animal; Down-Regulation; Enzyme Activation; Free Radical Scavengers; Injections, Intraperitoneal; Iridoid Glucosides; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxynitrous Acid; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine

Low concentrations of a hydrophilic nitric oxide donor (NOD) are reported to reduce myocardial reperfusion injury only when combined with a lipophilic antioxidant (AOX) to form a hybrid molecule (HYB). Here we tested whether liposoluble NOD requires to be combined with AOX to be protective. Isolated rat hearts underwent 30 minutes of ischemia and 120 minutes of reperfusion. To induce postconditioning, 1 μM solutions of the following liposoluble compounds were given during the first 20 minutes of reperfusion: NOD with weak (w-NOD) or strong NO-releasing potency (s-NOD); weak HYB built up with w-NOD and a per se ineffective AOX lead; strong HYB built up with s-NOD and the same AOX; mixtures of w-NOD plus AOX or s-NOD plus AOX. A significant reduction of infarct size with improved recovery of cardiac function was obtained only with weak HYB. We suggest that w-NOD requires the synergy with a per se ineffective AOX to protect. The synergy is possible only if the 2 moieties enter the cell simultaneously as a hybrid, but not as a mixture. It seems that strong HYB was ineffective because an excessive intracellular NO release produces a large amount of reactive species, as shown from the increased nitrotyrosine production.

Topics: Animals; Antioxidants; Cardiotonic Agents; Drug Synergism; Ischemic Postconditioning; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Donors; Rats; Rats, Wistar; Tyrosine

Metabolic diseases such as hyperlipidemia and diabetes attenuate the cardioprotective effect of ischemic preconditioning. In the present study, we examined whether another metabolic disease, prolonged uremia, affects ischemia/reperfusion injury and cardioprotection by ischemic preconditioning. Uremia was induced by partial nephrectomy in male Wistar rats. The development of uremia was verified 29 wk after surgery. Transthoracic echocardiography was performed to monitor cardiac function. At week 30, hearts of nephrectomized and sham-operated rats were isolated and subjected to a 30-min coronary occlusion followed by 120 min reperfusion with or without preceding preconditioning induced by three intermittent cycles of brief ischemia and reperfusion. In nephrectomized rats, plasma uric acid, carbamide, and creatinine as well as urine protein levels were increased as compared with sham-operated controls. Systolic anterior and septal wall thicknesses were increased in nephrectomized rats, suggesting the development of a minimal cardiac hypertrophy. Ejection fraction was decreased and isovolumic relaxation time was shortened in nephrectomized rats demonstrating a mild systolic and diastolic dysfunction. Infarct size was not affected significantly by nephrectomy itself. Ischemic preconditioning significantly decreased infarct size from 24.8 ± 5.2% to 6.6 ± 1.3% in the sham-operated group and also in the uremic group from 35.4 ± 9.5% to 11.9 ± 3.1% of the area at risk. Plasma ANG II and nitrotyrosine were significantly increased in the uremic rats. We conclude that although prolonged experimental uremia leads to severe metabolic changes and the development of a mild myocardial dysfunction, the cardioprotective effect of ischemic preconditioning is still preserved.

Topics: Angiotensin II; Animals; Biomarkers; Creatinine; Disease Models, Animal; Ischemic Preconditioning, Myocardial; Male; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nephrectomy; Proteinuria; Rats; Rats, Wistar; Stroke Volume; Time Factors; Tyrosine; Ultrasonography; Urea; Uremia; Uric Acid; Ventricular Function, Left

High-mobility group box 1 (HMGB1) is a nuclear protein actively secreted by immune cells and passively released by necrotic cells that initiates pro-inflammatory signalling through binding to the receptor for advance glycation end-products. HMGB1 has been established as a key inflammatory mediator during myocardial infarction, but the proximal mechanisms responsible for myocardial HMGB1 expression and release in this setting remain unclear. Here, we investigated the possible involvement of peroxynitrite, a potent cytotoxic oxidant formed during myocardial infarction, on these processes.. The ability of peroxynitrite to induce necrosis and HMGB1 release in vitro was evaluated in H9c2 cardiomyoblasts and in primary murine cardiac cells (myocytes and non-myocytes). In vivo, myocardial HMGB1 expression and nitrotyrosine content (a marker of peroxynitrite generation) were determined following myocardial ischaemia and reperfusion in rats, whereas peroxynitrite formation was inhibited by two different peroxynitrite decomposition catalysts: 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III) (FeTPPS) or Mn(III)-tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). In all types of cells studied, peroxynitrite (100 μM) elicited significant necrosis, the loss of intracellular HMGB1, and its passive release into the medium. In vivo, myocardial ischaemia-reperfusion induced significant myocardial necrosis, cardiac nitrotyrosine formation, and marked overexpression of myocardial HMGB1. FeTPPS reduced nitrotyrosine, decreased infarct size, and suppressed HMGB1 overexpression, an effect that was similarly obtained with MnTBAP.. These findings indicate that peroxynitrite represents a key mediator of HMGB1 overexpression and release by cardiac cells and provide a novel mechanism linking myocardial oxidative/nitrosative stress with post-infarction myocardial inflammation.

Topics: Animals; Apoptosis; Cells, Cultured; HMGB1 Protein; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Myoblasts, Cardiac; Myocardial Infarction; Necrosis; Peroxynitrous Acid; Rats; Rats, Wistar; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Toll-Like Receptor 4; Tyrosine; Up-Regulation

Superoxide has been shown to play a major role in ventricular remodeling and arrhythmias after myocardial infarction. However, the source of increased myocardial superoxide production and the role of superoxide in sympathetic innervation remain to be further characterized. Male Wistar rats, after coronary artery ligation, were randomized to vehicle, allopurinol, or apocynin for 4weeks. To determine the role of peroxynitrite in sympathetic reinnervation, we also used 3-morpholinosydnonimine (a peroxynitrite generator). The postinfarction period was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine, xanthine oxidase activity, NADPH oxidase activity, and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Sympathetic hyperinnervation was blunted after administration of allopurinol. Arrhythmic scores in the allopurinol-treated infarcted rats were significantly lower than those in vehicle. For similar levels of ventricular remodeling, apocynin had no beneficial effects on oxidative stress, sympathetic hyperinnervation, or arrhythmia vulnerability. Allopurinol-treated hearts had significantly decreased nerve growth factor expression, which was substantially increased after coadministration of 3-morpholinosydnonimine. These results indicate that xanthine oxidase but not NADPH oxidase largely mediates superoxide production after myocardial infarction. Xanthine oxidase inhibition ameliorates sympathetic innervation and arrhythmias possibly via inhibition of the peroxynitrite-mediated nerve growth factor pathway.

Topics: Acetophenones; Allopurinol; Animals; Arrhythmias, Cardiac; Coronary Vessels; Ethidium; Male; Molsidomine; Myocardial Infarction; Myocardium; NADPH Oxidases; Nerve Growth Factor; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Wistar; Superoxides; Sympathetic Nervous System; Tyrosine; Xanthine Oxidase

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.

Topics: Animals; Cholesterol; Cholesterol, Dietary; Connexin 43; Hyperlipidemias; Ischemic Preconditioning, Myocardial; Male; Mitochondria, Heart; Models, Animal; Myocardial Infarction; Rats; Rats, Wistar; Sarcolemma; Superoxides; Tyrosine

Reactive oxygen and nitrogen species are critical in preconditioning (PC). We sought to determine the effect of N-2-mercaptopropionyl glycine (MPG) on infarct size and on the oxidative status. Rabbits were exposed to 30-minute regional ischemia of the heart, which was followed by 3-hour reperfusion: (1) a control group without further intervention, (2) a PC1 group that was subjected to one cycle of PC, (3) a PC4 group that was subjected to 4 cycles of PC, (4) an MPG group that was treated with MPG for 60 minutes, starting 10 minutes before reperfusion, (5) MPG-PC1, and (6) the MPG-PC4 groups that were treated with the same dose of MPG and with 1 or 4 cycles of PC, respectively. Blood samples were drawn and collected for metabonomic analysis. In another series of experiments, 6 groups respective to the described ones were subjected to 30-minute regional ischemia of the heart and 20 minutes of reperfusion, after which pieces of heart tissue were quickly excised for malondialdehyde, nitrotyrosine, and glutathione content assessment. All PC and MPG groups developed smaller infarct size compared with control (16.5% ± 3.9%, 13.7% ± 3.1%, 18.6% ± 5.0%, 9.7% ± 2.0%, 15.0% ± 2.8% vs. 48.05% ± 7.2%; P < 0.05). MPG did not prevent lipid peroxidation and nitrotyrosine formation but enhanced the glutathione content. PC and MPG induced similar nuclear magnetic resonance changes. Long MPG infusion reduces the infarct size without abolishing the effect of PC, providing novel insights into the activity of MPG in PC.

Topics: Animals; Antioxidants; Glutathione; Infusions, Intravenous; Ischemic Preconditioning, Myocardial; Lipid Peroxidation; Magnetic Resonance Spectroscopy; Male; Myocardial Infarction; Oxidative Stress; Rabbits; Time Factors; Tiopronin; Tyrosine

Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI, depending on its coupling status. MI was created in wild-type, iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared with other genotypes of mice 4 wk after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH(4)) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH(2)), BH(4), and BH(4)-to-BH(2) ratio in the infarcted but not sham-operated heart. The increase in BH(4)-to-BH(2) ratio was associated with inhibition of nitrotyrosine formation and an increase in nitrite plus nitrate. However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in wild-type, eNOS(-/-), and nNOS(-/-) but not iNOS(-/-) mice. N(ω)-nitro-L-arginine methyl ester abrogated sepiapterin-induced increase in nitrite plus nitrate and angiogenesis and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH(4) synthesis and increasing bioavailable nitric oxide predominantly derived from iNOS.

Topics: Administration, Oral; Angiogenesis Inducing Agents; Animals; Biopterins; Blood Pressure; Cardiotonic Agents; Disease Models, Animal; Enzyme Inhibitors; Fibrosis; GTP Cyclohydrolase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pterins; Recovery of Function; Stroke Volume; Time Factors; Tyrosine; Ultrasonography; Ventricular Function, Left

It is well known that insulin possesses a cardioprotective effect and that insulin resistance is closely related to cardiovascular diseases. Peroxynitrite (ONOO(-)) formation may trigger oxidative/nitrative stress and represent a major cytotoxic effect in heart diseases. This study was designed to investigate whether insulin attenuates ONOO(-) generation and oxidative/nitrative stress in acute myocardial ischemia/reperfusion (MI/R). Adult male rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. Rats randomly received vehicle, insulin, or insulin plus wortmannin. Arterial blood pressure and left ventricular pressure were monitored throughout the experiment. Insulin significantly improved cardiac functions and reduced myocardial infarction, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels following MI/R. Myocardial ONOO(-) formation was significantly attenuated after insulin treatment. Moreover, insulin resulted in a significant increase in Akt and endothelial nitric oxide (NO) synthase (eNOS) phosphorylation, NO production, and antioxidant capacity in ischemic/reperfused myocardial tissue. On the other hand, insulin markedly reduced MI/R-induced inducible NOS (iNOS) and gp91(phox) expression in cardiac tissue. Inhibition of insulin signaling with wortmannin not only blocked the cardioprotection of insulin but also markedly attenuated insulin-induced antioxidative/antinitrative effect. Furthermore, the suppression on ONOO(-) formation by either insulin or an ONOO(-) scavenger uric acid reduced myocardial infarct size in rats subjected to MI/R. We concluded that insulin exerts a cardioprotective effect against MI/R injury by blocking ONOO(-) formation. Increased physiological NO production (via eNOS phosphorylation) and superoxide anion reduction contribute to the antioxidative/antinitrative effect of insulin, which can be reversed by inhibiting phosphatidylinositol 3'-kinase. These results provide important novel information on the mechanisms of cardiovascular actions of insulin.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Creatine Kinase; Electrocardiography; Heart Function Tests; Hypoglycemic Agents; In Situ Nick-End Labeling; Insulin; L-Lactate Dehydrogenase; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Nitrogen Species; Reperfusion Injury; Tyrosine

The role of neuronal nitric oxide synthase (nNOS) in cardiac ischemia-reperfusion (IR) and ischemia preconditioning (IP) is still controversial. Here, we focused on the possible roles of nNOS in cardiac IR and IP. Wild type C57BL/6 (WT) mice were subjected to coronary artery occlusion for 30 min followed by 24-h reperfusion (IR). Cardiac injury (infarct size and apoptotic cell number) was increased, associated with elevation of oxidative stress (lipid peroxidation) and nitrative stress (nitrotyrosine formation). A potent nNOS inhibitor, L-VNIO, and a superoxide dismutase mimetic and peroxynitrite scavenger, MnTBAP, significantly reduced IR-induced increases of oxidative/nitrative stress and cardiac injury. IR-induced cardiac injury in nNOS(-/-) (KO) mice was significantly lower than that in WT mice. MnTBAP markedly reduced IR-induced cardiac injury by suppression of oxidative/nitrative stress in KO mice. Cardiac IP was performed by three cycles of 5-min IR before 30-min ischemia followed by 24-h reperfusion. IP attenuated IR-induced cardiac injury in WT mice associated with reductions of oxidative/nitrative stress. IP-induced reduction of cardiac injury and oxidative/nitrative stress were eliminated by pretreatment with L-VNIO. In contrast with WT mice, IP had no protective effects in nNOS KO mice. In conclusion, nNOS played a dual role during cardiac IR and IP; nNOS exacerbated IR-induced injury by increasing oxidative/nitrative stress and contributed to IP-induced protection by inhibition of oxidative/nitrative stress.

Topics: Animals; Apoptosis; Enzyme Inhibitors; Free Radical Scavengers; Ischemic Preconditioning, Myocardial; Lipid Peroxidation; Metalloporphyrins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide Synthase Type I; Ornithine; Oxidative Stress; Reactive Nitrogen Species; Thiobarbituric Acid Reactive Substances; Tyrosine

The aim of the present study was to investigate if hyperlipidemia interferes with the infarct size-limiting effect of postconditioning and to study the involvement of peroxynitrite in this phenomenon. Rats were fed a 2% cholesterol-enriched or normal diet for 12 wk. Infarct size by triphenyltetrazolium chloride staining was measured in hearts isolated from both groups and subjected to 30 min coronary occlusion followed by 120 min reperfusion with or without the postconditioning protocol induced by six cycles of 10 s coronary occlusion and 10 s reperfusion at the onset of the reperfusion. Postconditioning significantly decreased infarct size in the normolipidemic but not in the hyperlipidemic group. Postconditioning increased cardiac 3-nitrotyrosine concentration (a marker for peroxynitrite formation) in the normal but not in the cholesterol-fed group when measured at the 5th min of reperfusion. Next, we tested if the postconditioning-induced acute increase in peroxynitrite is involved in the cardioprotection in normolipidemic animals in separate experiments. Postconditioning failed to decrease infarct size in the presence of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron [III] (20 mg/l) in normolipidemic animals. We conclude that an early increase in peroxynitrite after postconditioning plays a role in cardioprotection. Furthermore, hyperlipidemia blocks the cardioprotective effect of postconditioning at least in part via deterioration of the postconditioning-induced early increase in peroxynitrite formation.

Topics: Animals; Biomarkers; Cholesterol, Dietary; Disease Models, Animal; Hyperlipidemias; Male; Metalloporphyrins; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peroxynitrous Acid; Rats; Signal Transduction; Stress, Physiological; Tyrosine

We investigated the effects of co-administration of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor and angiotensin II type 1 receptor blocker (ARB) on nitric oxide (NO) bioavailability in genetically hyperlipidemic rabbits with our newly developed NO sensor. A total of 36 myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits equally derived (n=6 per group) were treated with 1) vehicle (control), 2) hydralazine (15 mg/kg/d), 3) the HMG-CoA reductase inhibitor pitavastatin (P: 0.5 mg/kg/d), 4) the ARB valsartan (V: 5 mg/kg/d), and 5) pitavastatin+valsartan (P+V) together without or 6) with N(G)-nitro-L-arginine methyl ester (L-NAME) for 8 weeks. After treatment, acetylcholine (ACh)-induced NO production was measured as a surrogate for endothelium protective function, and vascular peroxynitrite (a product of superoxide and NO) was measured for assessing dysfunctional endothelial NO synthase activity. Plaque area was quantified by histology as well as optical coherence tomography (OCT). Intra-aortic infusion of ACh produced an increase in plasma NO concentration, which was significantly greater with all drug treatments than with the control. P+V increased ACh-induced NO by 4.1 nmol/L significantly more than either P or V singly. The vascular peroxynitrite concentration was 1.6 pmol/mg protein in the control group and significantly less than those in the P- and V-monotherapy-groups. The lowest peroxynitrite concentration was observed in the P+V group (0.4 pmol/mg protein), which was significantly lower than those in the P- and the V-monotherapy-groups. OCT and histology of the thoracic aorta revealed that the plaque area decreased significantly more with the combination than with the monotherapy. In conclusion, the combined treatment with an HMG-CoA reductase inhibitor and an ARB may have additive protective effects on endothelial function as well as atherosclerotic change.

Topics: Acetylcholine; Angiotensin II Type 1 Receptor Blockers; Animals; Atherosclerosis; Biological Availability; Drug Therapy, Combination; Echocardiography; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipidemias; Myocardial Infarction; Nitric Oxide; Quinolines; Rabbits; Reactive Oxygen Species; Tetrazoles; Tyrosine; Valine; Valsartan

Oxidative stress plays an important role in the development of cardiac remodeling after myocardial infarction (MI), but the sources of oxidative stress remain unclear. We investigated the role of Nox2-containing reduced nicotinamide-adenine dinucleotide phosphate oxidase in the development of cardiac remodeling after MI. Adult Nox2(-/-) and matched wild-type (WT) mice were subjected to coronary artery ligation and studied 4 weeks later. Infarct size after MI was similar in Nox2(-/-) and WT mice. Nox2(-/-) mice exhibited significantly less left ventricular (LV) cavity dilatation and dysfunction after MI than WT mice (eg, echocardiographic LV end-diastolic volume: 75.7+/-5.8 versus 112.4+/-12.3 microL; ejection fraction: 41.6+/-3.7 versus 32.9+/-3.2%; both P<0.05). Similarly, in vivo LV systolic and diastolic functions were better preserved in Nox2(-/-) than WT mice (eg, LV dP/dt(max): 7969+/-385 versus 5746+/-234 mm Hg/s; LV end-diastolic pressure: 12.2+/-1.3 versus 18.0+/-1.8 mm Hg; both P<0.05). Nox2(-/-) mice exhibited less cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis; reduced increases in expression of connective tissue growth factor and procollagen 1 mRNA; and smaller increases in myocardial matrix metalloproteinase-2 activity than WT mice. These data suggest that the Nox2-containing reduced nicotinamide-adenine dinucleotide phosphate oxidase contributes significantly to the processes underlying adverse cardiac remodeling and contractile dysfunction post-MI.

Topics: Animals; Apoptosis; Cardiac Catheterization; Cardiomegaly; Echocardiography; Fibrosis; Matrix Metalloproteinase 2; Membrane Glycoproteins; Mice; Mice, Knockout; Myocardial Infarction; Myocardium; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; RNA, Messenger; Staining and Labeling; Survival Analysis; Tyrosine; Ventricular Remodeling

Previous experiments by our group have demonstrated a subacute increased contribution of nitric oxide (NO) to vasoreactivity after myocardial infarction in rats. However, the activation pattern of NO may be phasic after infarction and has been described to be strongly associated with superoxide production. Therefore, the present study evaluated the morphological distribution and time-dependent induction of NO and superoxide at the protein (via immunohistochemistry, chemiluminescence and spectrophotometry) and mRNA (via real-time RT-PCR) level after experimental induction of a myocardial infarction in rats. Myocardial infarction led to a modest but lasting upregulation of endothelial NO synthase (eNOS) in blood vessels and cardiomyocytes. In contrast, inducible NOS (iNOS) showed dramatic de novo formation 1 week after infarction, predominantly in the infarcted area and cardiomyocytes. In addition, a gradually increased myocardial production of superoxide was detected during remodelling, probably related by an upregulation of NADH oxidase in the peri-infarcted and remote myocardium. Furthermore, peroxynitrite formation was increased after myocardial infarction, indicating strong interaction between NO and superoxide. In conclusion, during the early remodelling phase after myocardial infarction mRNA concentrations and protein levels of eNOS and iNOS show an augmentation in a biphasic manner. Moreover, upregulation of NADH oxidase leads to increased presence of superoxide and peroxynitrite and thereby to a reduction of the bioavailability and compensatory effects of NO, as such influencing post-infarction remodelling.

Topics: Animals; Enzyme Activation; Immunohistochemistry; Kinetics; Male; Myocardial Infarction; Myocardium; NADH, NADPH Oxidoreductases; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Peroxynitrous Acid; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxides; Time Factors; Tyrosine

Several clinical studies have demonstrated that levels of adiponectin are significantly reduced in patients with type 2 diabetes and that adiponectin levels are inversely related to the risk of myocardial ischemia. The present study was designed to determine the mechanism by which adiponectin exerts its protective effects against myocardial ischemia/reperfusion.. Adiponectin-/- or wild-type mice were subjected to 30 minutes of myocardial ischemia followed by 3 hours or 24 hours (infarct size and cardiac function) of reperfusion. Myocardial infarct size and apoptosis, production of peroxynitrite, nitric oxide (NO) and superoxide, and inducible NO synthase (iNOS) and gp91(phox) protein expression were compared. Myocardial apoptosis and infarct size were markedly enhanced in adiponectin-/- mice (P<0.01). Formation of NO, superoxide, and their cytotoxic reaction product, peroxynitrite, were all significantly higher in cardiac tissue obtained from adiponectin-/- than from wild-type mice (P<0.01). Moreover, myocardial ischemia/reperfusion-induced iNOS and gp91(phox) protein expression was further enhanced, but endothelial NOS phosphorylation was reduced in cardiac tissue from adiponectin-/- mice. Administration of the globular domain of adiponectin 10 minutes before reperfusion reduced myocardial ischemia/reperfusion-induced iNOS/gp91(phox) protein expression, decreased NO/superoxide production, blocked peroxynitrite formation, and reversed proapoptotic and infarct-enlargement effects observed in adiponectin-/- mice.. The present study demonstrates that adiponectin is a natural molecule that protects hearts from ischemia/reperfusion injury by inhibition of iNOS and nicotinamide adenine dinucleotide phosphate-oxidase protein expression and resultant oxidative/nitrative stress.

Topics: Adiponectin; Animals; Apoptosis; Cardiotonic Agents; Cells, Cultured; Male; Membrane Glycoproteins; Mice; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; NADPH Oxidase 2; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxynitrous Acid; Reactive Nitrogen Species; Superoxides; Tumor Necrosis Factor-alpha; Tyrosine; Up-Regulation

Ischemic preconditioning (IPC) strongly protects against ischemia-reperfusion injury; however, its effect on subsequent myocardial oxygenation is unknown. Therefore, we determine in an in vivo mouse model of regional ischemia and reperfusion (I/R) if IPC attenuates postischemic myocardial hyperoxygenation and decreases formation of reactive oxygen/nitrogen species (ROS/RNS), with preservation of mitochondrial function. The following five groups of mice were studied: sham, control (I/R), ischemic preconditioning (IPC + I/R, 3 cycles of 5 min coronary occlusion/5 min reperfusion) and IPC + I/R N(G)-nitro-L-arginine methyl ester treated, and IPC + I/R eNOS knockout mice. I/R and IPC + I/R mice were subjected to 30 min regional ischemia followed by 60 min reperfusion. Myocardial Po(2) and redox state were monitored by electron paramagnetic resonance spectroscopy. In the IPC + I/R, but not the I/R group, regional blood flow was increased after reperfusion. Po(2) upon reperfusion increased significantly above preischemic values in I/R but not in IPC + I/R mice. Tissue redox state was measured from the reduction rate of a spin probe, and this rate was 60% higher in IPC than in non-IPC hearts. Activities of NADH dehydrogenase (NADH-DH) and cytochrome c oxidase (CcO) were reduced in I/R mice after 60 min reperfusion but conserved in IPC + I/R mice compared with sham. There were no differences in NADH-DH and CcO expression in I/R and IPC + I/R groups compared with sham. After 60 min reperfusion, strong nitrotyrosine formation was observed in I/R mice, but only weak staining was observed in IPC + I/R mice. Thus IPC markedly attenuates postischemic myocardial hyperoxygenation with less ROS/RNS generation and preservation of mitochondrial O(2) metabolism because of conserved NADH-DH and CcO activities.

Topics: Animals; Coronary Vessels; Cytochromes c; Ischemic Preconditioning, Myocardial; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Myocardial Infarction; Myocardium; NADH Dehydrogenase; Nitric Oxide Synthase Type III; Oxidation-Reduction; Oxidative Stress; Oxygen Consumption; Random Allocation; Reactive Oxygen Species; Regional Blood Flow; Tyrosine

Myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, and myocyte death. Intracellular Ca(2+) overload with reduced activity of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a critical mechanism of this injury. Although upregulation of SERCA function is well documented to improve postischemic cardiac function, there are conflicting reports where pharmacological inhibition of SERCA improved postischemic function. SERCA2a is the primary cardiac isoform regulating intracellular Ca(2+) homeostasis; however, SERCA1a has been shown to substitute SERCA2a with faster Ca(2+) transport kinetics. Therefore, to further address this issue and to evaluate whether SERCA1a expression could improve postischemic cardiac function and myocardial salvage, in vitro and in vivo myocardial I/R studies were performed on SERCA1a transgenic (SERCA1a(+/+)) and nontransgenic (NTG) mice. Langendorff-perfused hearts were subjected to 30 min of global ischemia followed by reperfusion. Baseline preischemic coronary flow and left ventricular developed pressure were significantly greater in SERCA1a(+/+) mice compared with NTG mice. Independent of reperfusion-induced oxidative stress, SERCA1a(+/+) hearts demonstrated greatly improved postischemic (45 min) contractile recovery with less persistent arrhythmias compared with NTG hearts. Morphometry showed better-preserved myocardial structure with less infarction, and electron microscopy demonstrated better-preserved myofibrillar and mitochondrial ultrastructure in SERCA1a(+/+) hearts. Importantly, intraischemic Ca(2+) levels were significantly lower in SERCA1a(+/+) hearts. The cardioprotective effect of SERCA1a was also observed during in vivo regional I/R with reduced myocardial infarct size after 24 h of reperfusion. Thus SERCA1a(+/+) hearts were markedly protected against I/R injury, suggesting that expression of SERCA 1a isoform reduces postischemic Ca(2+) overload and thus provides potent myocardial protection.

Topics: Animals; Arrhythmias, Cardiac; Calcium; Coronary Circulation; Disease Models, Animal; Electron Spin Resonance Spectroscopy; Fluorescent Dyes; Free Radicals; Heterocyclic Compounds, 3-Ring; Immunohistochemistry; Isoenzymes; Mice; Mice, Transgenic; Mitochondria, Heart; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; Time Factors; Tyrosine; Ventricular Function, Left; Ventricular Pressure

Hyperlipidemia attenuates the cardioprotective effect of preconditioning via unknown mechanisms. We have reported previously that in normolipidemic rats, preconditioning decreased ischemia-induced activation and release of myocardial matrix metalloproteinase (MMP)-2 into the coronary perfusate. Here, we investigated whether hyperlipidemia interferes with the cardioprotective effect of preconditioning through modulation of MMP-2. Hearts isolated from male Wistar rats fed 2% cholesterol-enriched or control chow for 9 weeks were subjected to a preconditioning protocol (three intermittent periods of ischemia/reperfusion of 5-min duration each) or a time-matched nonpreconditioning protocol. This was followed by a test ischemia/reperfusion (30-min ischemia and 120-min reperfusion) in both groups. Preconditioning decreased infarct size in the control but not the cholesterol-fed group. Cardioprotection in the preconditioned control group but not in the cholesterol-fed group was associated with an 18 +/- 3% (p < 0.05) inhibition of test ischemia/reperfusion-induced activation and release of myocardial MMP-2 into the perfusate. Myocardial protein levels of tissue inhibitors of MMPs [tissue inhibitor of metalloproteinases (TIMP)-2 and TIMP-4] were not changed in either group. A reduction of infarct size in nonpreconditioned hearts from both control and cholesterol-fed group was produced by the MMP inhibitor ilomastat at 0.25 microM, a concentration producing MMP-2 inhibition comparable with that of preconditioning in the control group. We conclude that hyperlipidemia blocks preconditioning-induced cardioprotection, hyperlipidemia abolishes preconditioning-induced inhibition of myocardial MMP-2 activation and release, preconditioning-induced inhibition of MMP-2 activation and release is not mediated by TIMPs, and pharmacological inhibition of MMPs produces cardioprotection in both normal and hyperlipidemic rats.

Topics: Animals; Blotting, Western; Cholesterol, Dietary; Diet; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydroxamic Acids; Hyperlipidemias; Indoles; Ischemic Preconditioning, Myocardial; L-Lactate Dehydrogenase; Male; Matrix Metalloproteinase Inhibitors; Myocardial Infarction; Myocardium; Protease Inhibitors; Rats; Rats, Wistar; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinase-4; Tissue Inhibitor of Metalloproteinases; Tyrosine

The present study provides evidence that inducible nitric-oxide synthase (iNOS)-mediated nitrative stress plays a pivotal role in chronic beta-adrenergic receptor (AR) stimulation-induced cardiac damage. In mice, 14 days of isoproterenol (ISO) stimulation via an osmotic minipump induced an up-regulation of iNOS as evidenced by increases in mRNA, protein expression, and immunochemical staining of myocardial iNOS. Serum level of C-reactive protein, an inflammatory mediator, was also markedly increased. Under chronic ISO stimulation, the up-regulated iNOS produced a significantly increased amount of nitric oxide (NO) and its byproduct, peroxynitrite, in the circulation and heart and subsequently resulted in an accelerated myocardial apoptosis. Forty-minute myocardial ischemia (MI) and 24-h reperfusion (R) further increased NO production and peroxynitrite formation and resulted in an enlarged infarct size in mice receiving chronic ISO stimulation. However, the treatment with a selective iNOS inhibitor [N-(3-(aminomethyl) benzyl)acetamidine] (1400W) or the use of a genetic modified animal (iNOS-knockout mice) markedly reduced iNOS-mediated production of NO and formation of peroxynitrite and consequently significantly decreased myocardial apoptosis and infarct size, showing a crucial link between iNOS-mediated nitrative stress and myocardial injury. In conclusion, chronic beta-AR stimulation up-regulates iNOS expression and increases NO production in the heart, which subsequently markedly enhances formation of reactive nitrogen species/peroxynitrite in the heart, thereby eliciting myocardial apoptosis and potentiating MI/R injury.

Topics: Adrenergic beta-Agonists; Animals; Apoptosis; Blotting, Western; C-Reactive Protein; Heart; Immunohistochemistry; In Situ Nick-End Labeling; Isoproterenol; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; RNA; Stress, Physiological; Tyrosine

We investigated the role of inducible nitric oxide synthase (iNOS) on ischemic myocardial damage and angiogenic process in genetically deficient iNOS (iNOS(-/-)) mice and wild-type littermates (iNOS(+/+)), with and without streptozotocin-induced (70 mg/kg intravenously) diabetes. After ischemia (25 min) and reperfusion (120 min), both iNOS(+/+) and iNOS(-/-) diabetic mice (blood glucose 22 mmol/l) had myocardial infarct size greater than their respective nondiabetic littermates (P < 0.01). Myocardial infarct size (P < 0.05), apoptotic index (P < 0.005), and tissue levels of tumor necrosis factor (P < 0.01), interleukin-6 (P < 0.01), and interleukin-18 (P < 0.01) were higher in nondiabetic iNOS(-/-) mice compared with nondiabetic iNOS(+/+) mice. As compared with diabetic iNOS(-/-) mice, diabetic iNOS(+/+) mice showed a greater infarct size (P < 0.01) associated with the highest tissue levels of nitrotyrosine and proinflammatory cytokines, as well as apoptosis. The beneficial role of iNOS in modulating defensive responses against ischemia/reperfusion injury seems to be abolished in diabetic mice.

Topics: Animals; Blood Glucose; Blood Pressure; Diabetes Mellitus, Experimental; Hyperglycemia; Insulin; Interleukins; Mice; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Reference Values; RNA; Streptozocin; Tyrosine

The role of nitric oxide (NO) generated by the inducible NO synthase (iNOS) during myocardial ischemia and reperfusion is not understood. We investigated the role of iNOS during early reperfusion damage induced in genetically deficient iNOS (iNOS-/-) mice and wild-type littermates. In wild-type mice, ischemia (60 min) and reperfusion (60 min) induced an elevation in serum levels of creatine phosphokinase and myocardial injury characterized by the presence of scattered apoptotic myocytes and mild neutrophil infiltration. Northern blot analysis showed increased expression of iNOS, whose activity was markedly elevated after reperfusion. Immunohistochemistry showed staining for nitrotyrosine; Western blot analysis showed elevated expression of heat shock protein 70 (HSP70), a putative cardioprotective mediator. Plasma levels of nitrite and nitrate, tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-10 were also increased. These events were preceded by degradation of inhibitor kappaBalpha (IkappaBalpha), activation of IkappaB kinase complex (IKK) and c-Jun-NH2-terminal kinase (JNK), and subsequently activation of nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1) as early as 15 min after reperfusion. In contrast, iNOS-/- mice experienced 35% mortality after reperfusion. The extensive myocardial injury was associated with marked apoptosis and infiltration of neutrophils whereas expression of HSP70 was less pronounced. Nitrotyrosine formation and plasma levels of nitrite and nitrate were undetectable. TNF-alpha and IL-6 were increased and IL-10 was reduced in earlier stages of reperfusion. Activation of IKK and JNK and binding activity of NF-kappaB and AP-1 were significantly reduced. Thus, we conclude that iNOS plays a beneficial role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction.

Topics: Animals; Apoptosis; Cardiotonic Agents; Cytokines; HSP70 Heat-Shock Proteins; JNK Mitogen-Activated Protein Kinases; Kinetics; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Models, Biological; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neutrophil Infiltration; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Signal Transduction; Transcription Factor AP-1; Tyrosine

Inducible nitric oxide synthase (iNOS) is expressed in the myocardium after myocardial infarction (MI) and in heart failure. Its pathophysiological role in these conditions, however, is not clear. We hypothesized that increased NO production from iNOS expression causes myocardial dysfunction and results in higher mortality after MI.. MI was induced by left coronary artery ligation in iNOS(-/-) mutant and wild-type mice. Mortality was followed up for 30 days. MI resulted in a significant increase in mortality in both iNOS(-/-) and wild-type mice compared with sham operation (P<0.01). Mortality was significantly decreased and LV myocardial contractility was increased, however, in iNOS(-/-) mice compared with the wild-type mice (P<0.05). Five days after MI, myocardial iNOS mRNA expression, plasma nitrate and nitrite concentrations, and myocardial and plasma nitrotyrosine levels were significantly increased in wild-type compared with iNOS(-/-) mutant mice (P<0.05). Both basal LV +dP/dt and its response to dobutamine were significantly increased in iNOS(-/-) compared with the wild-type mice (P<0.05).. Increased NO production from iNOS expression contributes to myocardial dysfunction and mortality after MI in mice.

Topics: Animals; Female; Gene Expression Regulation, Enzymologic; Genotype; Heart Ventricles; Hemodynamics; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Myocardial Contraction; Myocardial Infarction; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; RNA, Messenger; Survival Rate; Time Factors; Tyrosine

Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) attenuate angiotensin II-induced cellular signaling. Because angiotensin II is involved in left ventricular (LV) remodeling after myocardial infarction (MI), we examined the effects of statin treatment in an experimental model of chronic heart failure after MI.. Rats with extensive MI were treated with placebo or cerivastatin (0.3 mg/kg per day) as a dietary supplement or via gavage for 11 weeks starting on the 7th postoperative day. Infarct size and cholesterol levels were similar among all groups. LV cavity area, an index of LV dilatation, was reduced in MI rats on cerivastatin compared with placebo. LV end-diastolic pressure was increased in MI rats on placebo (24.1+/-4.1 mm Hg versus sham: 5.1+/-0.3 mm Hg; P<0.01), and it was significantly reduced by cerivastatin treatment (13.7+/-2.7 mm Hg; P<0.05 versus placebo). Cerivastatin partially normalized LV dP/dt(max) and dP/dt(min), indices of LV systolic and diastolic function, which were significantly reduced in MI rats on placebo. Improvement of LV function by cerivastatin was accompanied by a reduced expression of collagen type I and beta-myosin heavy chain. LV endothelial nitric oxide synthase was increased, whereas the nitrotyrosine protein level was decreased in MI rats by cerivastatin treatment.. Cerivastatin improved LV remodeling and function in rats with heart failure. This effect was associated with an attenuated LV expression of fetal myosin heavy chain isoenzymes and collagen I. Statin treatment may retard the progression of chronic heart failure.

Topics: Animals; Blotting, Northern; Blotting, Western; Collagen; Gene Expression Regulation; Heart Failure; Heart Ventricles; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Male; Myocardial Contraction; Myocardial Infarction; Myosin Heavy Chains; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Pyridines; Rats; Rats, Wistar; RNA, Messenger; Tyrosine

Topics: Animals; Blood Physiological Phenomena; Cats; Crystalloid Solutions; Cytokines; Glutathione; Heart; Heart Diseases; Heart Ventricles; Humans; Isotonic Solutions; Myocardial Infarction; Nitrates; Nitric Oxide; Perfusion; Plasma Substitutes; Superoxides; Tyrosine