1-3-dimethylthiourea and Myocardial-Ischemia

Heme oxygenase-1 (H(mox-1)) has been implicated in protection of cells against ischemia/reperfusion injury.. To examine the physiological role of H(mox-1), a line of heterozygous H(mox-1)-knockout mice was developed by targeted disruption of the mouse H(mox-1) gene. Transgene integration was confirmed and characterized at the protein level. A 40% reduction of H(mox-1) protein occurred in the hearts of H(mox-1)(+/)(-) mice compared with those of wild-type mice. Isolated mouse hearts from H(mox-1)(+/)(-) mice and wild-type controls perfused via the Langendorff mode were subjected to 30 minutes of ischemia followed by 120 minutes of reperfusion. The H(mox-1)(+/)(-) hearts displayed reduced ventricular recovery, increased creatine kinase release, and increased infarct size compared with those of wild-type controls, indicating that these H(mox-1)(+/)(-) hearts were more susceptible to ischemia/reperfusion injury than wild-type controls. These results also suggest that H(mox-1)(+/)(-) hearts are subjected to increased amounts of oxidative stress. Treatment with 2 different antioxidants, Trolox or N:-acetylcysteine, only partially rescued the H(mox-1)(+/)(-) hearts from ischemia/reperfusion injury. Preconditioning, which renders the heart tolerant to subsequent lethal ischemia/reperfusion, failed to adapt the hearts of the H(mox-1)(+/)(-) mice compared with wild-type hearts.. These results demonstrate that H(mox-1) plays a crucial role in ischemia/reperfusion injury not only by functioning as an intracellular antioxidant but also by inducing its own expression under stressful conditions such as preconditioning.

Topics: Acetylcysteine; Animals; Antioxidants; Chromans; Creatine Kinase; Disease Models, Animal; Gene Targeting; Heart; Heart Rate; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Heterozygote; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Malondialdehyde; Membrane Proteins; Mice; Mice, Transgenic; Myocardial Contraction; Myocardial Infarction; Myocardial Ischemia; Myocardium; Reperfusion Injury; Thiourea

Acute ischemia followed by prolonged reperfusion has been shown to induce cardiomyocyte apoptosis. In this report, we demonstrate that myocardial adaptation to ischemia induced by repeated cyclic episodes of short-term ischemia each followed by another short duration of reperfusion reduced cardiomyocyte apoptosis and DNA fragmentation. This was associated with the induction of the expression of Bcl-2 mRNA and translocation and activation of NF-kappaB. Another transcription factor, AP-1, remained unaffected by repeated ischemia and reperfusion, but exhibited significant upregulation by a single episode of 30 min ischemia followed by 2 h of reperfusion. This activation of AP-1 was inhibited by a scavenger of oxygen free radicals, DMTU. Thirty minutes ischemia and 120 min reperfusion downregulated the induction of the expression of Bcl-2 mRNA, but moderately activated NF-kappaB binding activity. This was associated with an increased number of apoptotic cells and DNA fragmentation in cardiomyocytes which were attenuated by DMTU. The results of this study indicate that Bcl-2, AP-1 and NF-kappaB differentially regulate cardiomyocyte apoptosis mediated by acute ischemia and prolonged reperfusion.

Topics: Animals; Apoptosis; Cell Nucleus; DNA; DNA Fragmentation; Gene Expression Regulation; Genes, bcl-2; Malondialdehyde; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; NF-kappa B; Rats; RNA, Messenger; Thiourea; Time Factors; Transcription Factor AP-1; Transcription Factors

Among factors underlying reperfusion injury are oxygen free radicals and Ca2+ influx via gated calcium channel or via Na+/H(+)-Na+/Ca2+ exchange which lead to calcium overload. The aim of the study was to ultrastructurally visualize the distribution of Ca2+ and to compare binding of calcium by the sarcolemma and calcium accumulation in mitochondria under therapy with an OH scavenger, dimethylthiourea (DMTU), Na+/H+ exchange inhibitor, amiloride, and calcium channel blocker, diltiazem, given alone or in combination to ischemic/reperfused hearts. Isolated working hearts subjected to 40 min ischemia and 30 min reperfusion were perfused with drugs added to the perfusate 15 min before ischemia and administered for the rest of the perfusion period. The cytochemical phosphate pyroantimonate method for localization of Ca2+ was used, and calcium distribution was analyzed with a computer image analyzer. All drugs given alone improved sarcolemmal ability to bind calcium. The best results were obtained with amiloride. All of the combined therapies gave even better results, but calcium accumulation in mitochondria diminished only with diltiazem therapy given alone or in combination with DMTU. Since the presence of Ca2+ deposits on the sarcolemma is believed to represent its normal function, and calcium sequestration by mitochondria reflects an increase in cytosolic calcium load, the lack of correlation between sarcolemmal and mitochondrial Ca2+ distribution might suggest impaired mechanisms of lowering cytoplasmic calcium or the existence of some mechanism other than Na+/Ca2+ exchange, mediated by activated Na+/H+ exchange.

Topics: Amiloride; Animals; Calcium; Calcium Channel Blockers; Diltiazem; Drug Therapy, Combination; Free Radical Scavengers; Heart; Hydroxyl Radical; In Vitro Techniques; Male; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Wistar; Sarcolemma; Sodium-Hydrogen Exchangers; Thiourea

It has been hypothesized that CoQ10 (CoQ) pretreatment protects myocardium from ischemia reperfusion (I/R) injury by its ability to increase aerobic energy production as well as its activity as an antioxidant. Isolated hearts from rats pretreated with either CoQ 20 mg/kg i.m. and 10 mg/kg i.p. or vehicle 24 and 2 h prior to the experiment, were subjected to 15 min of equilibration (EQ), 25 min of ischemia, and 40 min of reperfusion (RP). Developed pressure, +/-dp/dt, myocardial oxygen consumption, and myocardial aerobic efficiency (DP/MVO2) were measured. 31P NMR spectroscopy was used to determine ATP and PCr concentrations. Lucigenin-enhanced chemiluminescence of the coronary sinus effluent was utilized to determine oxidative stress through the protocol. CoQ pretreatment improved myocardial function after ischemia reperfusion. CoQ pretreatment improved tolerance to myocardial ischemia reperfusion injury by its ability to increase aerobic energy production, and by preserving myocardial aerobic efficiency during reperfusion. Furthermore, the oxidative burst during RP was diminished with CoQ. Similarly it was hypothesized that CoQ protected coronary vascular reactivity after I/R via an antioxidant mechanism. Utilizing a newly developed lyposomal CoQ preparation given i.v. 15 min prior to ischemia, ischemia reperfusion was carried out on Langendorff apparatus as previously described. Just prior to ischemia and after RP, hearts were challenged with bradykinin (BK) and sodium nitroprusside (SNP) and change in coronary flow was measured. CoQ pretreatment protected endothelial-dependent and endothelial-independent vasodilation after I/R. We conclude that CoQ pretreatment protects coronary vascular reactivity after I/R via OH radical scavenger action.

Topics: Adenosine Triphosphate; Aerobiosis; Animals; Antioxidants; Bradykinin; Catalase; Coenzymes; Coronary Vessels; Free Radical Scavengers; Heart; Luminescent Measurements; Magnetic Resonance Spectroscopy; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Nitroprusside; Oxidative Stress; Oxygen Consumption; Premedication; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Thiourea; Ubiquinone; Vasodilation

We previously showed that generation of reactive oxygen species during myocardial ischemia and reperfusion stimulates cardiac sympathetic afferent nerve endings. We hypothesized that, in this feline model of brief ischemia and reperfusion, HO. is produced during ischemia and the rate and concentration of production of HO.during reperfusion is dependent on the duration of myocardial ischemia. Therefore, we evaluated the time dependency of production of HO. during reperfusion after 2, 5, and 10 min of reversible occlusion of the left anterior descending (LAD) coronary artery to induce ischemia in cats (n = 10). Blood samples collected from the coronary vein at 0.25, 1, 2, and 4 min after 2 min of ischemia revealed net cumulative rate of production of p-, m-, and o-tyrosine of 99 +/- 31, 10 +/- 5.1, and 0.8 +/- 0.2 nmol.min-1.g-1, respectively. After 5 min of ischemia, net cumulative rates of production of p-, m-, and o-tyrosine during reperfusion were 177 +/- 63, 74 +/- 26, and 1.6 +/- 0.8 nmol.min-1.g-1, respectively, whereas after 10 min of ischemia production rates were 153 +/- 42, 78 +/- 29, and 2.1 +/- 0.5 nmol.min-1.g-1, respectively. The highest rate of production of tyrosines was observed immediately after ischemia, perhaps indicating a washout of HO.-derived products that had accumulated in the myocardium during ischemia. To evaluate production of HO. during ischemia, deoxygenated saline (PO2 10 +/- 0.9 mmHg) containing phenylalanine was perfused into the ischemic coronary vascular bed through a cannula placed in the LAD (n = 16). Perfusate was collected from the coronary vein during the 10 min of ischemia. Net production of HO. during ischemia, measured by the production of p-, m-, and o-tyrosine, was 82 +/- 11, 6.6 +/- 0.4, and 1.7 +/- 0.3 nmol.min-1.g-1, respectively. Pretreatment with deferoxamine (10 mg/kg, n = 7) or dimethylthiourea (10 mg/kg, n = 6) decreased net production of HO. during ischemia and reperfusion. These results demonstrate that HO. is produced during brief ischemia and reperfusion, with the greatest amount being produced immediately after ischemia. Additionally, we show that the duration of brief ischemia determines the rate of production of HO. during reperfusion.

Topics: Animals; Cats; Deferoxamine; Female; Free Radical Scavengers; Heart Ventricles; Hydroxyl Radical; Lactic Acid; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Organ Size; Siderophores; Thiourea

The relative contribution of oxygen free radicals, disturbances in calcium homeostasis and Na+/H+ exchange in the development of injury in the ischemic/reperfused heart was investigated. The study was designed to assess whether these factors initiate independent mechanisms of injury or, alternatively, they share a common mechanism of toxicity.. Isolated working rat hearts were subjected to different periods (30-55 min) of global ischemia and then were reperfused for 30 min. We compared the effects of oxygen radical scavengers (10 mM dimethylthiourea, DMTU and 0.6 mM desferrioxamine), inhibitors of Na+/H+ exchange (0.15 mM amiloride and 15 microM dimethylamiloride, DMA) and of 0.1 microM diltiazem, which was used to limit calcium overload, given alone or in combination, on the rate of myocardial injury development (recovery of hemodynamic function, LDH release, incidence of severe arrhythmias and structural integrity of cardiomyocytes were estimated at reperfusion following different periods of ischemia).. All interventions studied, when given alone, provided nearly equivalent cardioprotection. DMTU or desferrioxamine when applied in combination with diltiazem provided additive cardioprotection, relatively limited, however, as compared to the remarkable cardioprotection achieved by DMTU or desferrioxamine in combination with amiloride.. All mechanisms studied may contribute in an equal manner to the rate of injury development in the ischemic/reperfused heart. The oxygen free radicals-induced myocardial injury may be partially attributed to some disturbance in intracellular calcium homeostasis, possibly calcium overload, whereas the damaging effect of the Na+/H+ exchange activated upon reperfusion is probably largely related to some other mechanism.

Topics: Amiloride; Animals; Diltiazem; Dose-Response Relationship, Drug; Drug Therapy, Combination; Endocardium; In Vitro Techniques; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Rats; Rats, Wistar; Thiourea

Activation of cardiac sympathetic afferents leads to excitatory cardiovascular reflexes and pain during myocardial ischemia. We hypothesized that cardiac sympathetic afferents are activated by reactive oxygen species produced during ischemia and reperfusion. Single-unit nerve activity of 55 afferents was recorded from the left paravertebral sympathetic chain (T1-T4) in cats anesthetized with alpha-chloralose. Receptive fields of all afferents were located on the right or left ventricle. Mechanical and chemical sensitivities of each afferent ending were evaluated by von Frey hairs, cardiac distension, and local application of bradykinin (BK, 142 pmol) or H2O2 (7.5-15 mumol) to the receptive field. Thirty-one afferents (56%) were responsive to bradykinin (BK), H2O2, and ischemia (2 or 10 min). Deferoxamine (Def, 10-100 mg/kg), dimethylthiourea (DMTU, 10-100 mg/kg), or iron-loaded Def (10 mg/kg) were employed to evaluate the role of H2O2 and hydroxyl radicals (.OH) in activating these afferents (10A delta and 21C fibers) during ischemia and reperfusion. Treatment with the nonspecific scavenger DMTU (n = 10) significantly diminished the increase in discharge activity evoked by ischemia and reperfusion. Treatment with Def also significantly attenuated the responses during ischemia and reperfusion. Thus reactive oxygen species, particularly .OH, activate a group of cardiac sympathetic A delta- and C-fiber afferents during myocardial ischemia and reperfusion and may play an important role in mediating cardiovascular sympathetic reflex responses and/or pain transmission.

Topics: Animals; Bradykinin; Cats; Deferoxamine; Female; Free Radical Scavengers; Heart Conduction System; Hydrogen Peroxide; Hydroxyl Radical; Iron; Male; Myocardial Ischemia; Myocardial Reperfusion; Neurons, Afferent; Sympathetic Nervous System; Thiourea

The role of oxygen-derived free radicals in reperfusion arrhythmias was investigated in open-chest anesthetized dogs. The left anterior descending coronary artery was cannulated and perfused by an arterial bypass shunt. Ischemia was produced for 15 min by shunt occlusion and retrograde diversion of collateral blood flow. Dogs (n = 12) were treated with saline, N-(2-mercaptopropionyl)glycine (50 mg/kg), deferoxamine (10 mg/kg), superoxide dismutase (15,000 U/kg) plus catalase (55,000 U/kg), or dimethylthiourea (500 mg/kg). All agents were infused intravenously for 1 h starting 30 min before occlusion and continuing for 5 min of reperfusion. There were no differences in mean arterial blood pressure, heart rate, antegrade coronary flow, retrograde coronary flow, or size of the risk region among the five treatment groups. None of the dogs developed ventricular fibrillation during occlusion, whereas 88% of the 60 dogs fibrillated upon reperfusion. The antioxidant interventions did not alter the incidence of reperfusion-induced ventricular fibrillation compared with the saline-treated controls. The results suggest that free radicals do not play a role in lethal canine reperfusion arrhythmias.

Topics: Animals; Antioxidants; Arrhythmias, Cardiac; Blood Pressure; Catalase; Coronary Circulation; Coronary Vessels; Deferoxamine; Dogs; Electrocardiography; Female; Free Radicals; Heart Rate; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Superoxide Dismutase; Thiourea; Time Factors; Tiopronin; Ventricular Fibrillation

Ischaemic preconditioning may be mediated by oxygen free radicals generated during preconditioning. Conflicting results have been reported regarding the effect of superoxide dismutase (SOD) in attenuating the cardioprotective effect of preconditioning. The aim of the study was to reconcile this conflict by examining the effect of three different oxyradical scavengers on the infarct size limiting effect of preconditioning.. Anaesthetised open chest rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. In the preconditioning groups, rabbits were subjected to a single 5 min occlusion and 5 min reperfusion before 30 min sustained ischaemia. In these groups, the oxyradical scavengers SOD (15,000 U.kg-1), N-2-mercaptopropionyl glycine (MPG, 20 mg.kg-1), and dimethylthiourea (DMTU, 500 mg.kg-1), or placebo saline, were infused before and during preconditioning. In the non-preconditioning groups, these agents were given in the same time frame before 30 min of ischaemia. After 2 d reperfusion, infarct size was measured microscopically.. In the saline treated controls, preconditioning markedly limited microscopical infarct size (percent of area at risk): 13(SEM 3)% (n = 9) v 49(9)% (n = 8), p < 0.05. Treatment of the preconditioning groups with SOD or MPG attenuated this cardioprotection [infarct size 31(5)% (n = 11) and 42(8)% (n = 11), respectively, p < 0.05 v the saline treated preconditioning group], but treatment with DMTU did not [infarct size 23(6)% (n = 11), p = NS v the saline treated preconditioning group]. In the non-preconditioning groups, none of the treatments modified infarct size: 50(9)% (n = 7), 56(5)% (n = 8), and 61(6)%, (n = 8), respectively, p = NS v saline treated control.. Cardioprotection by preconditioning is mediated, at least in part, by oxyradicals which are scavenged by SOD or MPG in rabbits.

Topics: Animals; Free Radicals; Male; Myocardial Infarction; Myocardial Ischemia; Myocardium; Rabbits; Superoxide Dismutase; Thiourea; Tiopronin