Effect of ischemic preconditioning on free radical centers of the isolated rat heart during ischemia and early reperfusion

The effect of ischemic preconditioning on the free-radical state of isolated rat myocardium fixed by rapid freezing at the 25th min of normothermic total ischemia and the 3rd min of reperfusion was studied by the EPR method. It was shown that EPR spectra registered at -40 degrees C consist of two free-radical signals: of the semireduced forms of ubiquinone and flavine coynzymes. It was found that during ischemia and at the beginning of reperfusion, the preconditioning results in a narrowing of the spectra (as compared with control) due to an increase in the narrow ubisemiquinone EPR signal portion, and a decrease in the total concentration of free-radical centers: by 16% in the case of ischemia, and 23% in the case of reperfusion. It was concluded that in both cases the changes were due to a decrease in the concentration of myocardial flavosemiquinones as a result of ischemic preconditioning. We registered the microvawe power saturation curves for these two stages, which corresponded to control and ischemic preconditioning. In the case of ischemia these dependences had similar shapes; however, in the case of reperfusion they differ from each other due to changes in the relative intensities of the EPR signals from ubisemiquinone and flavosemiquinones in the integral myocardial free-radical spectra.     

Hydrogen peroxide mediates reperfusion injury in the isolated rat heart

In an isolated, normothermic rat heart model (Langendorff, 37 °C), dimethylthiourea (DMTU) infusion only during reperfusion reduced both injury and measurable hydrogen peroxide (H₂O₂) concentrations after global ischemia. Cardiac function was assessed by measurement of ventricular developed pressure (DP). H₂O₂ was assessed using H₂O₂ dependent aminotriazole inactivation of myocardial catalase. Depletion of xanthine oxidase by two methods (tungsten or allopurinol inhibition) also improved recovery of function and H₂O₂ production. The results indicate that XO derived H₂O₂ contributes to myocardial reperfusion injury.     

Detection of ferryl myoglobin in the isolated ischemic rat heart

Reflectance spectroscopy was utilized to monitor the oxidation states of myoglobin (Mb) in isolated, buffer-perfused rat hearts. Hearts were subjected to 30 min global, no-flow ischemia, followed by reperfusion under anoxic conditions. The addition of Na₂S to the buffer at reperfusion permitted the detection of ferryl myoglobin (MbIV) as its sulfmyoglobin derivative. The accumulation of MbIV was prevented by addition of ascorbic acid (1 mM), ergothioneine (2mM), or desferal (1mM) to the buffer prior to ischemia. Ascorbate and other agents have been previously shown to serve as one-electron reductants of MbIV. We propose that during the early phases of ischemia, deoxymyoglobin is oxidized to MbIV by residual H₂O₂. It also seems reasonable that the peroxidative activity of Mb(IV), during oxygenated reperfusion, might lead to cellular damage if this hypervalent form of Mb is not reduced.     

Hemodynamic action of calcitonin gene-related peptide in the isolated rat heart

The effects of calcitonin gene-related peptide (CGRP) on heart rate, coronary flow, pressure development, and time to ischemic contracture were studied in the isolated, perfused rat heart. A bolus of CGRP (2640 pmols) caused significant increases in heart rate and coronary flow; these effects were sustained for at least five minutes after injection. The increase in coronary flow was independent of heart rate, since CGRP caused an increase in coronary flow in non-beating (potassium-arrested) hearts. The dose-response of CGRP was studied using five doses (65, 218, 658, 1320 and 2640 pmols) given as bolus injections. Although the increase in heart rate was apparently dose-dependent, significant increases above baseline were observed only with the two highest doses. In contrast, coronary flow increased significantly above baseline with the injection of all but the lowest dose of CGRP. Ten minutes after injection of CGRP, all hearts were made ischemic. The time to onset of ischemic contracture was approximately 11 minutes for those hearts that received 65 pmols of CGRP; however, for those hearts receiving all other doses of CGRP, the time to onset of contracture was approximately 8 minutes. We conclude that CGRP significantly decreases the resistance of the coronary vascular bed, and that it may be an important regulator of regional blood flow in the heart.     

Suppression of ischemic and reperfusion ventricular arrhythmias by inhalational anesthetic-induced preconditioning in the rat heart

Inhalational anesthetic-induced preconditioning (APC) has been shown to reduce infarct size and attenuate contractile dysfunction caused by myocardial ischemia. Only a few studies have reported the effects of APC on arrhythmias during myocardial ischemia-reperfusion injury, focusing exclusively on reperfusion. Accordingly, the aim of the present study was to examine the influence of APC on ventricular arrhythmias evoked by regional no-flow ischemia. APC was induced in adult male Wistar rats by 12-min exposures to two different concentrations (0.5 and 1.0 MAC) of isoflurane followed by 30-min wash-out periods. Ventricular arrhythmias were assessed in the isolated perfused hearts during a 45-min regional ischemia and a subsequent 15-min reperfusion. Myocardial infarct size was determined after an additional 45 min of reperfusion. The incidence, severity and duration of ventricular arrhythmias during ischemia were markedly reduced by APC. The higher concentration of isoflurane had a larger effect on the incidence of ventricular fibrillation than the lower concentration. The incidence of ventricular tachycardia and reversible ventricular fibrillation during reperfusion was also significantly reduced by APC; the same was true for myocardial infarct size. In conclusion, we have shown that preconditioning with isoflurane confers profound protection against myocardial ischemia- and reperfusion-induced arrhythmias and lethal myocardial injury.     

Effects of synthetic antioxidant ionol on bioelectric activity of cardiomyocytes and arrhythmia in global ischemia and reperfusion of the isolated rat heart]

Isolated rat hearts were subjected to global ischemia (15 min) and reperfusion (20 min). Transmembrane potentials were recorded on the epicardial surface and contractile force was measured. Ischemia reduced the resting potential, the action potential (AP) amplitude and the AP duration (APD) in control animals. Pretreatment with synthetic antioxidant ionol (BHT, 50 mg/kg, per os) didn't influence the time course of changes in the resting potential and AP amplitude during ischemia, but significantly increased APD. In the pretreated group, 5 min after the aorta clamping, the APD at 50% and 90% levels of repolarization was 36% (p less than 0.05) and 13% (p less than 0.1) higher in comparison to the preischemic level and 10 min after clamping by 27% (p less than 0.1) and 29% (p less than 0.05), respectively. By the end of ischemia in the pretreated group, APD re-decreased almost to basal level, but in control group, it remained decreased. During reperfusion BHT improved the recovery of bioelectrical activity and the contractile function. The BHT 10-fold reduced the malignant arrhythmias duration and 2.5-fold the incidence of ventricular tachycardia and fibrillation during reperfusion. These results indicate that the induced by BHT increase in APD can contribute to the mechanism of BHT antiarrhythmic action.     

Naloxone reduces release of creatine kinase in the isolated ischemic rat heart

The effects of naloxone, propranolol, or both on the release of creatine kinase (CK) from the isolated ischemic rat heart were studied. Naloxone at concentrations of 1.1 and 3.6 mmole liter-1 in the perfusate at a rate of 1-2 ml min-1 reduced the release of CK from the isolated ischemic rat heart during myocardial ischemia in a dose-dependent manner. Propranolol at a concentration of 7 mumole liter-1 in the perfusate also reduced the release of CK. Addition of naloxone (1.1 mmole liter-1) to propranolol further reduced the release of CK. The effect of the joint administration of the two drugs seemed to be additive.     

The effect of zinc on reperfusion arrhythmias in the isolated perfused rat heart

Considerable evidence suggests that free radicals engendered by redox-active metals, particularly iron and copper, are causative agents in reperfusion injury following ischemia. This study demonstrates that perfusion of the isolated rat heart with a buffer containing zinc, a non-redox active metal similar to copper in its coordination chemistry, inhibits the development of ventricular arrhythmias during reperfusion. Zinc was employed as the bishistidine complex, Zn--His2, to maintain solubility and permeability. Zn--His2 exerted an antiarrhythmic activity as hearts spent a longer time in normal sinus rhythm and a shorter time in ventricular fibrillation during reperfusion following 10 min of regional ischemia. However, Zn--His2 also produced a negative inotropic and chronotropic effect, evident during equilibration and ischemia. In the course of experiments which began in Israel and continued in the U.S. it was necessary to use two different sources of rats. Hearts from the two sources manifested different sensitivities to the concentrations of Zn--His2, although their physiological effects were similar. Differential activity responses were noted for antiarrhythmic activity, negative inotropic and chronotropic properties, and toxicity. In both groups of untreated hearts the incidence of ventricular fibrillation after ischemia was 100%. Ventricular fibrillation was reduced to 17% at 37.5 microM Zn--His2 in the U.S.-bred rat hearts and to 9% at 200 microM Zn--His2 in those from Israel. These changes in Zn--His2 treated animals were accompanied by a decrease in lactate dehydrogenase release from the myocardium during reperfusion. None of the protective effects was due to histidine alone. These results indicate that zinc prevents ventricular arrhythmias during reperfusion following regional ischemia and may prevent membrane damage, possibly, by reduction of free radical formation.     

Mechanism of action of arachidonic acid in the isolated perfused rat heart

The purpose of this study was to elucidate the mechanism of action of arachidonic acid in the isolated rat heart perfused with Krebs solution at a constant flow. Administration of arachidonic acid, 3.3-33 nmol, into the heart caused a small transient increase followed by a pronounced decrease in coronary perfusion pressure and increased myocardial tension, heart rate, and the output of prostaglandins (6-keto-PGF1 alpha, PGE2, and PGF2 alpha). Administration of structurally similar fatty acids, dihomo-gamma-linolenic acid, and 8,14,17-eicosatrienoic acid, produced vasoconstriction and decreased myocardial tension without affecting heart rate or the output of prostaglandins. Infusion of PGI2, PGF2 alpha, or PGE2 produced coronary vasodilation and increased myocardial tension, whereas PGF2 alpha increased heart rate, an effect which was not prevented by propranolol. Indomethacin blocked the effect of arachidonic acid on myocardial tension and heart rate, but only reduced the duration of coronary vasodilation. The initial component of arachidonic acid induced coronary vasodilation which was unaffected by indomethacin and also remained unaltered during the infusion of three structurally dissimilar lipoxygenase inhibitors, eicosatetraynoic acid, nordihydroguaiaretic acid, and 1-phenyl-3-pyrazolidone. Indomethacin did not alter the effects of the exogenously administered prostaglandins on perfusion pressure or myocardial tension; however, it blocked the effect of PGF2 alpha on heart rate. The effect of arachidonic acid or PGF2 alpha to increase heart rate was not blocked by thromboxane synthetase inhibitors, imidazole, or OKY-1581. We conclude that the cardiac effects of arachidonic acid are mediated primarily through its conversion to cyclooxygenase products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian rhythm disorders and dynamic changes of energy metabolism in rat heart muscle cells

The functional states of pro- and antioxidant systems in blood and heart muscle cells in rats with long-term emotional stress have been studied. It has been shown that daily rhythm disorders produce psycho-emotional stress in animals and that, this is accompanied by quantitative changes in physiological parameters and hormones in the blood. In the present study, it was observed that such stress increased lipid peroxidation in blood and heart muscle cells. Also, activities of antioxidant enzymes, superoxide dismutase, and catalase were diminished, indicating deterioration of the antioxidant system. In addition, there were decreased activities of mitochondrial enzymes participating in energy metabolism, indicating decreased energy levels in heart muscle cells. These results suggest the likelihood that emotional stress is a key factor that can cause a whole range of diseases of the cardiovascular system.     

Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury

During postischemic reperfusion, free radicals are produced and have deleterious effects in isolated rat hearts. We investigated whether melatonin (MEL) reduces the production of hydroxyl radical (*OH) in the effluent and aids in recovery of left ventricular (LV) function. Hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. Salicylic acid (SAL) was used as the probe for *OH, and its derivatives 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were quantified using HPLC. In addition, thiobarbituric acid reactive substances (TBARS) in the myocardium was measured. Plateaus in the measurement of 2,5- and 2,3-DHBA were seen from 3 to 8 min after reperfusion in each group. The group that received 100 microM MEL+ SAL had significantly reduced amounts of 2,5- and 2,3-DHBA by multiple folds, compared to the SAL group. TBARS was significantly decreased in the 100 microM MEL group (1.20+/-0.36 vs 1.85+/-0.10 micromol/g of drug-free group, p<0.001). More importantly, the 100 microM MEL group significantly recovered in LV function (LV developed pressure, +dp/dt, and -dp/dt; 63.0%, 60.3%, and 59.4% in the 100 microM MEL group; 30.2%, 29.7%, and 31.5% in the drug-free group, respectively; p<0.05). Duration of ventricular tachycardia or ventricular fibrillation significantly decreased in the 100 microM MEL group (100 microM MEL, 159+/-67 sec; drug-free, 1244+/-233 sec; p<0.05). As a result of scavenging *OH and reducing the extent of lipid peroxidation, MEL is an effective agent for protection against postischemic reperfusion injury.     

Time related changes in calcium handling in the isolated ischemic and reperfused rat heart

The main aim of this study was to assess the kinetics of intracellular free calcium (Ca²⁺ _i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca²⁺ _i transient. Changes in mechanical performance and degradation of membrane phospholipids – estimated by tissue arachidonic acid content – were correlated with Ca²⁺ _i levels of the heart. The fluorescence ratio technique was applied to estimate Ca²⁺ _i. The disappearance of mechanical activity of the heart preceded that of the Ca²⁺ _i transient in the first 2 min of ischemia. The slope of upstroke of the Ca²⁺ _i transient, reflecting Ca²⁺ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca²⁺ sequestration, expressed a significant prolongation (105 ± 17 vs. 149 ± 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca²⁺ _i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca²⁺ _i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca²⁺ _i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca²⁺ _i handling and sustained depression of mechanical performance.     

血红素氧合酶-1在离体大鼠心肌缺血预适应中的作用

目的:分别观察给予HO-1诱导剂和抑制剂对心肌相对缺血再灌注损伤和缺血预适应的影响,探讨HO-1在缺血预适应中的作用.方法:实验动物随机分为对照组(CN)、缺血/再灌损伤组(I/R)、缺血预适应 缺血/再灌损伤组(PC)、HO-1诱导剂 缺血/再灌损伤组(HM)、HO-1抑制剂 缺血预适应组(ZP).心肌缺血/再灌损伤采用相对缺血/再灌损伤模型,缺血预适应则为相对缺血5min恢复5min,反复2次.测定心功能、MDA及HO-1活性变化.结果:HM组HO-1活性升高,心功能恢复率均显著高于IR组(P＜0.01),MDA含量显著低于IR组(P＜0.05).ZP组活性降低,心功能恢复率显著低于PC组(P＜0.05),MDA含量显著高于PC组(P＜0 05).结论:HO-1是缺血预适应释放的内源性活性物质之一.     

The role of NO in ischemia/reperfusion injury in isolated rat heart

Nitric oxide (NO) is an important regulator of myocardial function and vascular tone under physiological conditions. However, its role in the pathological situations, such as myocardial ischemia is not unequivocal, and both positive and negative effects have been demonstrated in different experimental settings including human pathology. The aim of the study was to investigate the role of NO in the rat hearts adapted and non-adapted to ischemia. Isolated Langendorff-perfused hearts were subjected to test ischemic (TI) challenge induced by 25 min global ischemia followed by 35 min reperfusion. Short-term adaptation to ischemia (ischemic preconditioning, IP) was evoked by 2 cycles of 5 min ischemia and 5 min reperfusion, before TI. Recovery of function at the end of reperfusion and reperfusion-induced arrhythmias served as the end-points of injury. Coronary flow (CF), left ventricular developed pressure (LVDP), and dP/dt(max) (index of contraction) were measured at the end of stabilization and throughout the remainder of the protocol until the end of reperfusion. The role of NO was investigated by subjecting the hearts to 15 min perfusion with NO synthase (NOS) inhibitor L-NAME (100 mmol/l), prior to sustained ischemia. At the end of reperfusion, LVDP in the controls recovered to 29.0 +/- 3.9 % of baseline value, whereas preconditioned hearts showed a significantly increased recovery (LVDP 66.4 +/- 5.7 %, p < 0.05). Recovery of both CF and dP/dt(max) after TI was also significantly higher in the adapted hearts (101.5 +/- 5.8 % and 83.64 +/- 3.92 % ) as compared with the controls (71.9 +/- 6.3 % and 35.7 +/- 4.87 %, respectively, p < 0.05). NOS inhibition improved contractile recovery in the non-adapted group (LVDP 53.8 +/- 3.1 %; dP/dt(max) 67.5 +/- 5.92 %) and increased CF to 82.4 +/- 5.2 %. In contrast, in the adapted group, it abolished the protective effect of IP (LVDP 31.8 +/- 3.1 %; CF 70.3 +/- 3.4 % and dP/dt(max) 43.25 +/- 2.19 %). Control group exhibited 100 % occurrence of ventricular tachycardia (VT), 57 % incidence of ventricular fibrillation (VF) - 21 % of them was sustained VF (SVF); application of L-NAME attenuated reperfusion arrhythmias (VT 70 %, VF 20 %, SVF 0 %). Adaptation by IP also reduced arrhythmias, however, L-NAME in the preconditioned hearts increased the incidence of arrhythmias (VT 100 %, VF 58 %, SVF 17 %). In conclusion: our results indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion (I/R) alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts. On the other hand, blockade of cardioprotective effect of IP by L-NAME points out to a dual role of NO in the heart: a negative role in the non-adapted myocardium subjected to I/R, and a positive one, due to its involvement in the mechanisms of protection triggered by short-term cardiac adaptation by preconditioning.     

Effect of the He-Ne laser irradiation on resistance of the isolated heart to the ischemic and reperfusion injury

The aim of this work was to investigate the myocardial protection against ischemia/reperfusion using low level laser irradiation (LLLI). It has been shown that pulse pressure was higher in the period of post-ischemic reperfusion as compared with the control group. It provided a better restoration of myocardial contractility as well as increasing of coronary flow in the reperfusion period. The amount of ventricular rhythm disorder episodes decreased. These effects of laser application were registered in conditions of coronary flow reduction less than 50%. One of the suggested mechanisms of laser effect is an ATP-sensitive channel activation.     

白细胞介素-2对心脏节律的作用及其机制的研究

目的：探讨白细胞介素－2（1L－2）对心脏节律作用及其可能机制。方法：采用体外培养乳鼠心肌细胞模型和离体人鼠灌流心脏模型,观察培养的心肌细胞搏动频率和离体心脏心率及节律。结果：①2.5-200u/ml的IL－2呈浓度依赖性地降低心肌细胞的搏动频率。②50u/ml的IL-2明显增加离体心脏心率和室性早搏个数。③propranolol预处理可取消50u/mlIL-2的离体心脏作用。④热失活IL－2对培养的心肌细胞搏动频率和离体心脏心率和心律都无显著作用。结论：IL－2可直接制培养心肌自律性,其对离体心脏的正性变时和致心律失常作用可能由内源性作茶酚胺介导。     

Inhibition of ubiquinone synthesis in isolated rat heart under an ischemic condition

1. The biosynthesis of ubiquinone (UQ) in isolated rat heart under ischemic and hypoxic conditions was investigated. 2. Under ischemic perfusion, a greater amount of biosynthetic intermediates, 3-nonaprenyl and 3-decaprenyl-4-hydroxybenzoate (PPHBs) was accumulated and a smaller amount of UQ-9 and -10 was synthesized when compared with normal conditions. 3. The accumulation of PPHBs was observed without forming UQs during anaerobic perfusion. 4. Hydroxylation which is the following reaction of PPHBs for the biosynthesis of UQ in rat heart, was proceeded by the monooxygenase(s) depending upon the oxygen concentrations.     

血红素氧合酶-1 mRNA在离体大鼠心肌缺血预处理中的变化

目的:观察血红素氧合酶-1(HO-1)mRNA在缺血预适应中的变化.方法:实验动物随机分为对照组(CN)、缺血/再灌损伤组(I/R)、缺血预适应 缺血/再灌损伤组(PC).结果:PC组的心功能恢复率高于I/R组(P<0.05),MDA含量低于I/R组(P<0.05),HO-1 mRNA又高于I/R组(P<0.05).结论:HO-1mRNA表达上调与缺血预适应保护缺血/再灌注损伤心肌有关.