calcimycin and Myocardial-Ischemia

An inadequate platelet response to aspirin (ASA) has been identified in some patients under chronic ASA treatment. The aim of this study was to analyze if ASA-sensitive and ASA-resistant platelets have differences in their apoptotic capability. Clinically stable ischemic coronary patients who had been taking ASA (100 mg/d) for at least 9 months before inclusion were divided into ASA-resistant (n = 11) and ASA-sensitive (n = 13) groups as defined by the PFA-100 test. Platelets from ASA-sensitive patients showed higher expression of the proapoptotic proteins Bak and Bax than those from ASA-resistant patients, although only Bak protein remained different when the results were adjusted by age. In resting platelets, neither caspase-3 activity nor cytosolic cytochrome C levels were different between both experimental groups. Stimulation of platelets with calcium ionophore (10 nmol/L, A23187) increased caspase-3 activity (1.91-fold higher; P < 0.05) and cytosolic cytochrome C levels (1.84-fold higher; P < 0.05) to a higher degree in ASA-sensitive than in ASA-resistant platelets. In conclusion, ASA-sensitive platelets seem to be better prepared to undergo apoptosis during robust platelet activation.

Topics: Aged; Apoptosis; Apoptosis Regulatory Proteins; Aspirin; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blood Platelets; Calcimycin; Calcium Ionophores; Caspase 3; Drug Resistance; Electron Transport Complex IV; Female; Humans; Male; Myocardial Ischemia; Platelet Activation; Platelet Aggregation Inhibitors; Treatment Outcome

The aim of this study was to assess the effect of chronic administration of NCX4016 [2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester], a nitric oxide-releasing aspirin derivative on the consequences of coronary artery occlusion in streptozotocin-diabetic rats. Rats were made diabetic by injection of streptozotocin (60 mg kg(-1)) and received insulin (2.5 U kg(-1) s.c.) daily for 4 weeks. Animals received vehicle (1 ml kg(-1) polyethylene glycol), aspirin (65.2 mg kg(-1)), NCX4016 (60 mg kg(-1)), or (iv) NCX4016 (120 mg kg(-1)) orally, once daily for the last 5 days before coronary artery occlusion (CAO). One hour after the last dose, pentobarbital-anesthetized rats were subjected to CAO for 30 min followed by 120-min reperfusion. Neither drug significantly modified initial hemodynamics or plasma glucose levels compared with vehicle treatment in either nondiabetic or diabetic rats. Neither drug modified the total ventricular premature beat (VPB) count in normal animals, although NCX4016, but not aspirin, reduced the total VPB count and the incidence of ventricular tachycardia in diabetic rats. In nondiabetic animals, both aspirin and NCX4016 reduced infarct size. However, in diabetic rats, infarct size was reduced only by the larger dose of NCX4016 (120 mg kg(-1)) but not by aspirin or the lower dose of NCX4016. These results demonstrate that the cardioprotective effects of NCX4016 are reduced in the presence of diabetes compared with the effects seen in nondiabetic animals. In summary, the present study confirms the protective effect of NCX4016 against ischemia-reperfusion injury in the normal rat heart and demonstrates for the first time its protective effect in the heart of streptozotocin-diabetic rats.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Blood Pressure; Calcimycin; Diabetes Mellitus, Experimental; Heart Rate; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitric Oxide; Rats; Rats, Sprague-Dawley; Streptozocin

Increase in free intracellular calcium [Ca 2+]i plays a crucial role in cardiomyocyte ischemic injury. Here we demonstrate that overexpression of the sarcoplasmic-reticulum stress-protein Grp94 reduces myocyte necrosis due to calcium overload or simulated ischemia. Selective three- to eightfold Grp94 increase, with no change in Grp78 or calreticulin amount, was achieved by stable transfection of skeletal C2C12 and cardiac H9c2 muscle cells. After exposure to the calcium ionophore A23187, LDH release from five different Grp94-overexpressing clones of either C2C12 and H9c2 origin was significantly lower than that of control ones and [Ca 2+]i increase was significantly delayed. The number of necrotic cells, evaluated by propidium iodide uptake, was reduced when cells from the Grp94-overexpressing H9c2 clone were exposed to conditions simulating ischemia. Experiments performed in neonatal rat cardiomyocytes co-transfected with grp94 and the green fluorescent protein (GFP) cDNAs validated the protective effect of Grp94 overexpression. A lower percentage of propidium-iodide positive/GFP-fluorescent myocytes co-expressing exogenous Grp94, with respect to myocytes expressing GFP alone, was observed after exposure to either A23187 (6.6% vs. 14.0%, respectively) or simulated ischemia (8.5% vs. 17.7%, respectively). In conclusion, the selective increase in Grp94 protects cardiomyocytes from both ischemia and calcium overload counteracting [Ca 2+]i elevations.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Cell Line; Endoplasmic Reticulum Chaperone BiP; Homeostasis; HSP70 Heat-Shock Proteins; Ionophores; Membrane Proteins; Mice; Myocardial Ischemia; Myocytes, Cardiac; Necrosis; Rats; Transfection

1. We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys-LT) generated upon neutrophil-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. 2. Hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) purified human neutrophils (PMNL), and challenged with 0.5 microM A-23187 for 30 min. Coronary perfusion pressure (CPP) and left-ventricular end-diastolic pressure (LVEDP) were monitored. Cys-LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL, adhesion to the coronary endothelium. 3. Basal CPP and LVEDP values averaged 45 +/- 1.4 mmHg and 5 +/- 0.1 mmHg, respectively; A-23187 triggered an increase in CPP (134 +/- 9 mmHg, at 30 min) which was significantly attenuated by pretreatment with L-arginine, 100 microM (90 +/- 3 mmHg, at 30 min). Pretreatment with NG-monomethyl-L-arginine, 10 microM (L-NMMA), induced a marked increase in CPP (290 +/- 40 mmHg, at 20 min) and in LVEDP (47 +/- 16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by L-arginine, 100 microM, (CPP 115 +/- 10 mmHg, LVEDP 6 +/- 1.1 mmHg, at 30 min). 4. The increase in CPP was accompanied by the release of cys-LT in the circulating buffer, which was reduced significantly by L-arginine. Pretreatment with L-NMMA, caused a marked rise in cys-LT concentrations which was prevented by L-arginine. 5. Neither L-arginine nor L-NMMA affected directly the A-23187-induced arachidonic acid (AA) metabolism in isolated PMNL alone. 6. Pretreatment with L-NMMA caused a prompt drop in myeloperoxidase (MPO), activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by L-arginine. 7. This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys-LT by preventing PMNL adhesion to the coronary intima.

Topics: Animals; Calcimycin; Enzyme Inhibitors; Heart; Hemodynamics; Humans; In Vitro Techniques; Leukotrienes; Male; Myocardial Ischemia; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Perfusion; Rabbits

Perfusion of the isolated rabbit heart with 5 x 10(6) human polymorphonuclear leukocytes, under recirculating conditions (50 ml), and challenge with A-23187 (0.5 microM) caused an increase in coronary perfusion pressure (from a prechallenge value of 46 +/- 1.1 to 176.2 +/- 29.7 mm Hg, 30 min after challenge, n = 6-4), which was linearly correlated (P < .006) with formation of cysteinyl leukotrienes (29.7 +/- 7.3 pmol/ml, 30 min after challenge). Pretreatment with the leukotriene synthesis inhibitor BAY X1005 (1 microM) (n = 6) resulted in significant protection against the increase in coronary perfusion pressure (76.7 +/- 12.8 mm Hg, 30 min after challenge) and in almost complete inhibition of sulfidopeptide leukotriene synthesis (3.2 +/- 1.7 pmol/ml, 30 min after challenge). In in vivo experiments, ligation of the left anterior descending coronary artery in the rabbit (n = 10) resulted in acute myocardial infarction marked by a mortality rate of 60% compared with sham-operated animals (n = 10). Intravenous treatment of the rabbits with BAY X1005 (10 mg/kg/h, for 2 h) (n = 10) markedly reduced the mortality rate (20%), protected the rabbits against the marked electrocardiogram derangement and abolished the significant increase in plasma creatine phosphokinase activity and cardiac tissue myeloperoxidase activity induced by coronary artery ligation. BAY X1005 exerts a significant cardioprotection and suggests that specific leukotriene synthesis inhibitors may lead to innovative therapy in myocardial ischemia.

Topics: Animals; Arachidonate 5-Lipoxygenase; Calcimycin; Coronary Vessels; Creatine Kinase; Cysteine; Electrocardiography; Endothelium, Vascular; Enzyme Activation; Heart; Heart Diseases; Humans; In Vitro Techniques; L-Lactate Dehydrogenase; Leukotrienes; Lipoxygenase Inhibitors; Male; Myocardial Ischemia; Myocardium; Neutrophils; Peroxidase; Quinolines; Rabbits

Reperfusion of globally ischemic rat hearts in vitro causes release of inositol(1,4,5) trisphosphate (Ins(1,4,5)P3) which is associated with the development of reperfusion arrhythmias. Both of these responses require the presence of a receptor agonist, either norepinephrine or thrombin, and both responses are inhibited by the aminoglycoside, gentamicin and the polyamine, spermine. In the current study, the role of Ins(1,4,5)P3 in the development of arrhythmias under ischemic conditions was addressed. Arrhythmias [ventricular premature beats, ventricular tachycardia and ventricular fibrillation (VF)] occurring over 25 min subsequent to coronary artery ligation were shown to be independent of endogenous norepinephrine or adrenergic receptor stimulation but were effectively inhibited by gentamicin (0.15-1.5 mM, 95% VF in controls compared with 0% VF, at 1.5 mM, P < 0.01) and spermine (5 mM, 40% VF, P < 0.01). Depletion of Ca2+ stores, including Ins(1,4,5)P3-sensitive Ca2+ stores, with thapsigargin (300 nM) reduced the incidence of ischemic arrhythmias (40% VF, P < 0.01). [3H]-Inositol-labeled right atria incubated under conditions of simulated ischemia retained the ability to respond to norepinephrine by releasing inositol phosphates. Under ischemic conditions, gentamicin (1.5 mM) caused a reduction in [3H]Ins(1,4,5)P3 without any effect on the other inositol phosphates. Similar effects of gentamicin were observed under ischemic conditions in the absence of norepinephrine (95 +/- 8 cpm/mg, mean +/- S.E.M., n = 4, v 29 +/- 4, P < 0.0] for 1.5 mM gentamicin). Agonist independent release of [3H]Ins(1,4,5)P3 under ischemic conditions required extracellular Ca2+ suggesting the operation of a Ca(2+)-activated phospholipase C. In agreement with this, release of [3H]Ins(1,4,5)P3 could be initiated by Ca2+ overload under normoxic conditions and this was inhibited by gentamicin. These findings show that Ca2+ overload can enhance release of Ins(1,4,5)P3 under ischemic conditions and provide evidence that this release is involved in the genesis of arrhythmias under these conditions.

Topics: Animals; Arrhythmias, Cardiac; Calcimycin; Calcium; Gentamicins; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionophores; Male; Myocardial Ischemia; Norepinephrine; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic

Selectins are important adhesion molecules which utilize a carbohydrate ligand such as sialyl Lewisx (SLex). Our objective was to study the effects of a liposome-conjugated SLex (Lipo-SLex) in myocardial ischaemia (MI) and reperfusion (R) injury in order to further clarify the actions of this carbohydrate.. We studied the efficacy of Lipo-SLex in a feline model of MI (90 min) and R (270 min) injury in vivo. Lipo-SLex (400 micrograms SLex/kg, iv) was administered intravenously 10 min prior to R. We also utilized an in vitro system of neutrophil adherence to thrombin-stimulated coronary endothelium to validate the efficacy of Lipo-SLex.. Lipo-SLex significantly attenuated myocardial necrosis (8.6 +/- 1.2 vs. 29.5 +/- 3.1% of area-at-risk, P < 0.01) and plasma creatine kinase activities (P < 0.01) compared to vehicle (liposome alone). Moreover, endothelium-dependent relaxation to acetylcholine and A23187 in ischaemic-reperfused coronary rings obtained from cats treated with Lipo-SLex was significantly preserved compared to cats given liposomes without SLex (P < 0.01). After reperfusion, ex vivo PMN adherence to ischaemic-reperfused coronary endothelium was significantly increased in vehicle-treated cats, however, this was significantly attenuated in Lipo-SLex-treated cats (82 +/- 7 vs. 28 +/- 3 PMNs/mm2, P < 0.01). Myeloperoxidase activity in the ischaemic myocardium, a marker of PMN accumulation, was also significantly attenuated in Lipo-SLex-treated cats compared to liposomes without SLex (P < 0.01).. Liposome-conjugated SLex-oligosaccharide attenuates myocardial necrosis and preserves coronary endothelial function following MI/R in vivo. The mechanism appears to be mediated by inhibition of the initial PMN-endothelial interaction and eventual accumulation into the ischaemic cardiac tissue. The liposome-SLex complex may be an efficient drug formulation for acute inflammatory diseases.

Topics: Acetylcholine; Animals; Calcimycin; Cats; Cell Adhesion; Creatine Kinase; Drug Carriers; Endothelium, Vascular; In Vitro Techniques; Liposomes; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Neutrophils; Oligosaccharides; Peroxidase; Sialyl Lewis X Antigen; Thrombin; Vasodilation

This study was done to determine whether abnormal receptor-dependent release of endothelium-derived relaxing factor (EDRF) might be caused by G-protein dysfunction. Dogs were exposed to global myocardial ischemia (45 minutes, induced by aortic cross-clamping) followed by reperfusion (60 minutes) while on cardiopulmonary bypass, and coronary arteries were then studied in vitro in organ chamber experiments. After reperfusion, endothelium-dependent relaxation to the receptor-dependent agonists adenosine diphosphate and acetyl-choline was significantly impaired as well as to sodium fluoride, which acts on a pertussis toxin-sensitive G-protein. In contrast, endothelium-dependent relaxations to the receptor-independent agonists A23187 and phospholipase C were normal. Furthermore, endothelium-dependent relaxation to poly-L-arginine (molecular weight, 139,200), which appears to induce endothelium-dependent relaxation of the canine coronary artery by a nonnitric oxide pathway, was unaffected by ischemia and reperfusion. These experiments suggest that global myocardial ischemia and reperfusion selectively impair receptor-mediated release of EDRF (nitric oxide) but that the ability of the endothelial cell to produce EDRF or generate endothelium-dependent relaxation to nonnitric oxide-dependent agonists remains intact. We hypothesize that coronary reperfusion injury leads to G-protein dysfunction in the endothelium.

Topics: Acetylcholine; Adenosine Diphosphate; Aluminum Chloride; Aluminum Compounds; Animals; Calcimycin; Cardiopulmonary Bypass; Chlorides; Coronary Vessels; Dinoprost; Dogs; Endothelium, Vascular; Female; GTP-Binding Proteins; Isoproterenol; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Nitric Oxide; Peptides; Sodium Fluoride; Type C Phospholipases

Repetitive, brief periods of ischemia and reperfusion ("preconditioning") increase the resistance of myocardial tissue to subsequent prolonged ischemic episodes and limit infarct size. We investigated whether preconditioning also protects against coronary endothelial dysfunction induced by ischemia and reperfusion.. Experiments were performed in four groups of rats (n = 8 in each group): group 1 rats underwent sham surgery, group 2 rats were subjected to 20 minutes of left coronary artery occlusion without reperfusion, group 3 rats underwent 20 minutes of occlusion followed by 1 hour of reperfusion, and group 4 rats (preconditioning group) underwent the same protocol as group 3 rats, preceded by three cycles of 5 minutes of ischemia and 5 minutes of reperfusion. At the end of the experiments, coronary segments (internal diameter, 250 to 300 microns) were removed distal to the occlusion site and mounted in wire myographs for isometric tension recording. Relaxations induced by increasing concentrations of acetylcholine, the calcium ionophore A23187, or the nitric oxide (NO) donor SIN-1 were determined in arteries precontracted by serotonin. Basal NO release was estimated by measuring contractions to NG-nitro L-arginine methyl ester (L-NAME). In addition, we determined the effect of preconditioning on infarct size in two additional groups that were subjected to the same protocols as those of groups 3 and 4. In those animals, area at risk (India ink injection) and infarct size (triphenyltetrazolium stain) were determined by computerized analysis of enlarged sections after video acquisition. Preconditioning markedly limited infarct size (percent of area at risk: controls, 57 +/- 2; preconditioning, 2.2 +/- 0.6; P < .01). Ischemia (without or with reperfusion) or preconditioning did not affect the coronary responses to L-NAME, serotonin, A23187, or SIN-1. Ischemia without reperfusion did not modify the relaxations to acetylcholine (maximal relaxation: sham, 58 +/- 4%; ischemia, 56 +/- 7%; P = NS). In contrast, ischemia followed by reperfusion markedly impaired the response to acetylcholine (26 +/- 6%; P < .01 versus sham). This impaired response was restored by preconditioning (maximal relaxation: 59 +/- 9%; P = NS versus sham; P < .01 versus ischemia/reperfusion).. In addition to protecting myocardial cells, preconditioning also protects coronary endothelial cells against ischemia/reperfusion injury.

Topics: Acetylcholine; Animals; Arginine; Calcimycin; Coronary Vessels; Endothelium, Vascular; Male; Molsidomine; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Wistar; Serotonin; Vasodilator Agents

Myocardial dysfunction after cardiac operations might be influenced by altered myocardial perfusion in the postoperative period. To investigate possible alterations in vascular reactivity, in vitro coronary microvascular responses were examined after ischemic cardioplegia with use of a porcine model of cardiopulmonary bypass. Since myocardial perfusion is primarily regulated by arteries less than 200 microns in diameter, these vascular segments were examined. After 1 hour of ischemic arrest with cold crystalloid cardioplegia and 1 hour of reperfusion, microvessels (100 to 190 microns in diameter) were pressurized in a no-flow state, preconstricted by 30% to 60% of the baseline diameter with acetylcholine, and examined with video microscopic imaging and electronic dimension analysis. Endothelium-dependent relaxations to bradykinin (55% +/- 13% versus 99% +/- 1% = maximum relaxation of the preconstricted diameter in cardioplegia-reperfusion vessels versus control vessels, respectively; p < 0.05) and the calcium ionophore A 23187 (33% +/- 6% versus 90% +/- 4%; p < 0.05) were markedly impaired while endothelium-independent relaxation to sodium nitroprusside was similar to control value. After 1 hour of ischemic cardioplegia without reperfusion, endothelium-dependent relaxation was only slightly affected. Transmission electron microscopy showed minimal endothelial damage after ischemic cardioplegia and reperfusion. These findings have important implications regarding coronary spasm and cardiac dysfunction after cardiac operations.

Topics: Acetylcholine; Aminoquinolines; Animals; Bradykinin; Calcimycin; Coronary Vessels; Disease Models, Animal; Endothelium, Vascular; Evaluation Studies as Topic; Heart Arrest, Induced; Hyperthermia, Induced; Indomethacin; Microcirculation; Microscopy, Electron, Scanning Transmission; Myocardial Ischemia; Nitroprusside; Potassium; Saponins; SRS-A; Swine; Vasodilation