Effects of adaptation to periodic hypoxia on bioelectric activity of cardiomyocytes of isolated heart in ischemia and reperfusion

Experiments on isolated Wistar rat heart perfused according to Langendorff showed that preliminary adaptation of rats to intermittent hypobaric hypoxia limited the fall of values of the resting potential and the amplitude and duration of action potential characteristic for ischemia. Under similar conditions, adaptation considerably reduced the increased time of impulse conduction along the myocardium. In reperfusion, the parameters enumerated restored much more efficiently in hearts from adapted animals than in controls. The role of these changes in the antiarrhythmic effect of adaptation to intermittent hypoxia is under discussion.     

Effects of adaptation to stress exposure and periodic hypoxia on bioelectric activity of cardiomyocytes of isolated heart in ischemia and reperfusion]

Action potential (AP) of cardiomyocytes was recorded in experiments on isolated perfused according to Langendorf rat hearts. The effect was estimated of preliminary adaptation to intermittent hypobaric hypoxia or to repeated short-term stress exposure on the resting potential (RP) and the amplitude and duration of action potential (APD) in global ischemia and reperfusion. It was shown that adaptation to hypoxia is more effective in prevention of ischemic fall of RP, AP and APD. In reperfusion, the parameters enumerated restored more quickly and efficiently in hearts from adapted to stress animals.     

Protective effect of antioxidant ionol in total ischemia of the heart

The effect of antioxidant ionol in total myocardial ischemia during 30 and 60 min, t = 20 degrees C and 37 degrees C was studied in 60 Wistar rats. The models of isolated perfused myocardium by Langendorf, Neely and that of heterotopic transplantation of myocardium to the rat peritoneal vessels were used. It has been shown that pretreatment of ionol (240 mg/kg before 24 h of ischemia) was accompanied by improvement of myocardial contractile and diastolic functions.     

异丙酚对离体大鼠心肌缺血、再灌注损伤后细胞凋亡及其机制研究

目的：观察异丙酚对离体大鼠心肌缺血/再灌注损伤的影响并从氧化应激和线粒体介导的凋亡方面探讨其作用机制。方法：应用Langendorff离体心脏灌注系统建立心肌缺血/再灌注损伤模型。40只SD大鼠随机分为正常对照组、缺血/再灌注模型（I/R）组、异丙酚15、30、60μmol.L-1组。除正常对照组外,各组分别平衡灌注20 min后,常温全心停灌25 min,再灌注30 min。Powerlab/8s仪记录各组平衡末、缺血前及再灌30 min时的各项心功能指标并测定冠脉流出液中乳酸脱氢酶（LDH）、肌酸激酶（CK）活性;检测心肌线粒体活力、膜肿胀度、锰超氧化物岐化酶（Mn-SOD）活性和丙二醛（MDA）含量;流式细胞仪检测心肌细胞凋亡;流式细胞术检测Bcl-2和Bax的表达,免疫组化法测定天冬氨酸特异的半胱氨酸蛋白酶（caspase）-3,9,8蛋白的表达。结果：与I/R组相比,异丙酚30、60μmol.L-1能明显改善缺血/再灌注后的心功能,减弱冠脉流出液中LDH、CK的活性（P〈0.05）;心肌线粒体活力有所恢复,膜肿胀度减轻,Mn-SOD活性升高,MDA生成明显减少（P〈0.05）,心肌细胞凋亡明显减少,Bcl-2表达上调,Bax表达下调,caspase-3,9阳性表达细胞数明显减少（P〈0.05）。结论：异丙酚明显减轻缺血/再灌注所致的心肌线粒体的过氧化损伤,抑制线粒体途径的凋亡,可能是其心肌保护作用机制之一。     

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 ischemia and reperfusion on antioxidant enzymes and mitochondrial inner membrane proteins in perfused rat heart

Experiments were performed to investigate the effects of 60 min severe global ischemia followed by 30 min reperfusion on the antioxidant enzymatic system in the isolated perfused rat heart. Ischemia induced a significant increase of cytoplasmic and mitochondrial selenium-dependent glutathione peroxidase (EC 1.11.1.9) activity. In reperfused hearts, only the mitochondrial form showed a further significant increase. Glutathione reductase (EC 1.6.4.2) was increased in ischemic hearts, whilst the reperfused hearts showed a decrease towards the level found in aerobic hearts. Mitochondrial superoxide dismutase (EC 1.15.1.1) activity was depressed in ischemic as well as in reperfused hearts, though the cytoplasmic form was unmodified. Catalase (EC 1.11.1.6), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutathione transferase (EC 2.5.1.18) activities were unchanged throughout the experiment. Ischemia and reperfusion induced a significant fall in tissue-reduced glutathione content concomitant with an increase of its oxidized form. We have also studied the mitochondrial inner membrane proteins for both molecular weight, with Coomassie blue, and thiol status, with monobromobimane stain, using a sodium dodecyl sulfate polyacrylamide gel electrophoresis technique. Neither ischemia nor reperfusion effected any relevant modification of the molecular weight of the mitochondrial inner-membrane proteins either in the presence or absence of a reducing agent. However, two of these proteins with an apparent molecular weight of 52 0000 and 12 000 showed a decrease in the monobromobimane stain, probably due to the oxidation of their thiol groups.     

葛根素对离体大鼠缺血/复灌心脏的保护作用及其作用机制

目的：探讨葛根素（puerarin,Pue）预处理抗心肌缺血／复灌（ischemia／reperfusion,I／R）损伤是否与线粒体渗透性转换孔和／或线粒体ATP敏感性钾通道有关。方法：采用离体大鼠心脏Leangendorff灌流方法,全心停灌30min,复灌120min复制I／R模型。测定心室力学指标和复灌各时间点冠脉流出液中乳酸脱氢酶（LDH）含量。实验结束测定心肌组织formazan量的变化。结果：与单纯I／R组相比,Pue（0．24mmol／L,5min）预处理明显提高心肌细胞的formazan含量,降低复灌期间冠脉流出液中LDH含量,明显促进左室发展压、左心室内压最大上升和下降速率、心率与发展压乘积和左室舒张末压力的恢复,缓解冠脉流量的减少。线粒体渗透性转换孔开放剂苍术苷（20μmol／L。复灌前给药20min）和线粒体ATP敏感性钾通道抑制剂5-羟基癸酸（100μmol／L,缺血前给药20min）能明显减弱Pue的保护作用。结论：在大鼠离体心脏灌流模型上,Pue预处理具有抗心脏缺血／复灌损伤的作用,这种保护作用可能与其抑制线粒体渗透性转换孔的开放和促进线粒体ATP敏感性钾通道的开放有关。     

Effects of endotoxin on neutrophil-mediated ischemia/reperfusion injury in the rat heart in vivo

We have previously shown that a nonlethal dose of lipopolysaccharide (LPS) decreases L-selectin expression of neutrophils (PMNs), thereby preventing PMN-mediated reperfusion injury in the isolated heart. In the present study we determined whether or not that dose of LPS would protect hearts during in vivo ischemia and reperfusion by preventing PMN-induced reperfusion injury. Rats receiving saline vehicle showed marked myocardial injury (necrotic area/area at risk = 82%+/-2%) and significant depression in left ventricular function as assessed in the isolated isovolumic heart preparation at constant flow rates of 5, 10, 15, and 20 ml/min. The administration of LPS (100 microg/kg body wt) 7 hr prior to ischemia resulted in a reduction in myocardial damage (necrotic area/area at risk = 42%+/-3%) and preservation of function. Myocardial function was similar to that of sham ischemic saline- and LPS-treated rats. Moreover, PMN infiltration as determined by histology was quantitatively more severe in hearts of saline-treated rats than in hearts of LPS-treated rats. Isolated hearts from vehicle- and LPS-treated animals undergoing sham ischemia in vivo recovered to the same extent after in vitro ischemia/reperfusion, suggesting that LPS did not induce protection by altering intrinsic properties of the heart. Our results indicate that LPS-induced protection of the heart from in vivo PMN-mediated ischemia/reperfusion injury may be due to decreased L-selectin expression of PMNs in LPS-treated animals.     

尾加压素Ⅱ对正常及缺血-再灌注离体大鼠心脏的影响

在正常Langendorff灌流与缺血-再灌注(停灌20 min-复灌20 min)离体大鼠心脏模型,观察尾加压素Ⅱ(urotensin Ⅱ,UⅡ)对冠脉流量、心功能和心肌代谢的影响以及心肌UⅡ受体的功能,以探讨UⅡ的心脏效应。对正常心脏给予0.1、1和10 nmol/L UⅡ各5 min,然后换洗5 min,对停灌缺血-再灌注心脏在再灌注期给予1或10nmol/L UⅡ。监测心率、左室内压和左室内压升降的最大变化率等心功能指标,计算冠脉流量,测定冠脉流出液中总蛋白和肌红蛋白含量以及乳酸脱氢酶(LDH)活性。灌流结束后,测定心肌丙二醛(MDA)含量和质膜UⅡ结合位点(放射性配基结合法)。结果如下:(1)正常心脏灌流UⅡ后,冠脉流量和心功能呈浓度依赖下降,换洗后没有完全恢复。心肌蛋白、肌红蛋白和LDH漏出随UⅡ浓度的增加而增加,换洗后迅速减少。UⅡ组心肌MDA含量与对照组差异无显著性。(2)缺血-再灌注后,冠脉流量显著减少,心功能显著抑制,再灌注期心肌蛋白、肌红蛋白和LDH明显漏出;给予UⅡ后,上述变化增强,且高浓度组更强,与对照组差异有显著性(P<<0.01),再灌注后心肌MDA含量亦显著高于对照(P<0.01)。(3)缺血-再灌注心肌质膜UⅡ受体的B_(max)显著高于正常对照心肌(14.65±1.78vs20.53±1.98 fmol/mg pr,P<0.01),Kd值变化无统计学意义。上述结果表明,在正常     

Effects of polyamines on apoptosis induced by simulated ischemia/reperfusion injury in cultured neonatal rat cardiomyocytes

We incubated neonatal Sprague-Dawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re-incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases-3 and -9 and Bcl-2 and the release of cytochrome c by Western blotting, and the intracellular free-calcium concentration ([Ca2+]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 micromol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases-3 and -9 and cytochrome c release, up-regulated Bcl-2 expression and decreased [Ca2+]i. However, pretreatment with 10 micromol/l putrescine had the opposite effects. Evidence for this "double-edged" effect of polyamines on apoptosis in I/R-injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury.     

Beneficial effects of different flavonoids, on functional recovery after ischemia and reperfusion in isolated rat heart

Three newly synthesised lipid peroxidation inhibitors (7, 11, 14) were evaluated for their effects on myocardial functional recovery during reperfusion after 30 min global ischemia in isolated rat hearts. The flavonoid compounds (7, 11, 14, rutin) reduce ischemia/reperfusion-induced cardiac dysfunction.     

Contradictory effects of superoxide dismutase after global or regional ischemia in the isolated rat heart

The effect of superoxide dismutase was investigated in two different models of ischemia and reperfusion in the isolated rat heart: global and regional ischemia. The results of this comparison show that reperfusion arrhythmias after 10 and 15 min of regional ischemia, induced by occlusion of the left coronary artery, can be prevented by SOD confirming the results of other investigators. Paradoxically SOD was without effect after 10 min of global ischemia, obtained by stopping coronary flow completely. After 15 min of global ischemia, SOD induced ventricle fibrillation. Apparently the effect of SOD depends on the model of ischemia and reperfusion that is used.     

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.     

Polymerized placenta hemoglobin attenuates ischemia/reperfusion injury and restores the nitroso-redox balance in isolated rat heart

Ischemia/reperfusion (I/R) injury mainly caused by oxidative stress plays a major role in cardiac damage. The extent of the I/R injury is also an important factor that determines the function of a transplanted heart. This study first examined whether hemoglobin-based oxygen carriers (HBOCs) could protect isolated rat heart from I/R injury and then elucidated the underlying mechanism. Using the Langendorff model, isolated Sprague–Dawley rat hearts were arrested and stored at 4°C for 8 h and then reperfused for 2 h. Compared with St. Thomas' solution (STS) and rat self blood in STS, polymerized placenta hemoglobin (PolyPHb) in STS greatly improved heart contraction and decreased infarction size. The extent of myocardial apoptosis was also significantly decreased, which was related to reduced iNOS-derived nitric oxide production, increased protein ratio of Bcl-2/Bax, and reduced caspase-3 activity and cleavage level. Furthermore, PolyPHb in STS did not increase malondialdehyde, peroxynitrite, or mitochondrial hydrogen peroxide formation, but greatly elevated superoxide dismutase activity and preserved mitochondrial ATP synthesis, which served to maintain redox homeostasis in I/R heart. In conclusion, our results demonstrate that HBOCs protected isolated heart from I/R injury and this protection was associated with attenuation of NO-mediated myocardial apoptosis and restoration of the nitroso-redox balance.     

白细胞介素—2对离体大鼠心脏的作用及其机理

The purpose of the present study was to explore the biological effects and mechanism of interleukin-2 (IL-2) on the isolated rat heart. The results showed that hrIL-2 increased the number of premature ventricular contraction, heart rate, left ventricular developed pressure, left ventricular end-diastolic pressure and coronary flow in the isolated perfused rat heart. Heat inactivated hrIL-2 had no effect on the heart. Pretreatment with ryanodine canceled the positive effects of hrIL-2 on left ventricular developed pressure, left ventricular end-diastolic pressure and coronary flow but had no effects on arrhythmogenesis and tachycardia by hrIL-2. Pretreatment with nifedipine or low extracellular calcium abolished the arrhythmogenic effect of hrIL-2 and attenuated partially the augment of heart rate, left ventricular developed pressure, left ventricular end-diastolic pressure and coronary flow. It suggests that the cardiac activity of hrIL-2 depended on the integrity of its spatial structure and transmembrane influx Ca2+ and intracellularly stored Ca2+ were involved in the cardiac activity of hrIL-2.     

海州香薷总黄酮对大鼠离体心脏缺血/再灌注损伤的保护作用

目的：探讨海州香薷总黄酮（TrES）预处理对大鼠离体心脏缺血/再灌注损伤的保护作用及其可能机制。方法：应用Lgendorff心脏灌流系统建立离体大鼠心脏缺血/再灌注模型,采用全心停灌的处理方法,平衡后,停止灌流30min,再灌注120min作为缺sk/再灌注过程。设立正常对照组,模型对照组,药物预处理组（1,10,100tμg/mlTrES）,利用RM6240BD型多道生理信号采集处理系统实时监测左心室血流动力学各项指标,用TIE染色法测定心肌梗死面积,测定再灌注期间冠脉流出液中乳酸脱氢酶（LDH）含量,以及在520mm处测定由200pmol/LCaC12引起心肌线粒体渗透性转换孔的开放情况。结果：海州香薷总黄酮预处理可以明显改善缺血/再灌注后所引起的左心室收缩功能下降、心肌梗死面积增加的现象、降低复灌期间冠脉流出液中LDH的含量以及能够明显降低由CaC12引起的线粒体在520am处吸光度值,且上述作用具有剂量依赖性。结论：海州香薷总黄酮能够对抗大鼠心肌缺血再灌注损伤,且具有剂量依赖性,其心脏保护机制与抑制线粒体渗透性转换孔（MPTP）的开放有关。     

Contractile properties and creatine kinase activity of myofilaments following ischemia and reperfusion of the rat heart

After prolonged ischemia followed by reperfusion of the isolated rat heart, irreversible heart failure is associated with creatine kinase leakage from the cells. The possible implications of MM creatine kinase leakage from myofibrillar compartments on the contractile properties of ventricular muscle have been studied in control versus ischemic hearts. Total creatine kinase activity decreased in ischemic cells while creatine kinase and ATPase activities were not modified in isolated myofibrils. The efficiency of creatine kinase and phosphocreatine in the relaxation of rigor tension in skinned ventricular preparations was not changed after ischemia. Furthermore, neither the pCa/tension relationship nor the rate of tension development following length changes were modified by ischemia. These results show that the contractile properties of myofilaments as well as the functional coupling between myosin ATPase and creatine kinase are preserved in ischemic hearts suffering irreversible contractile failure.     

大鼠心脏缺血-再灌注损伤对心肌L-Arg/NO途径的影响

为探讨大鼠心脏缺血-再灌注损伤(IRI)期间一氧化氮(NO)生成增加的环节和过程。本实验用离体灌流大鼠心脏,预灌流15 min,停灌45 min,取30 ml KH 液循环灌流15 min,观察冠脉流出液中细胞胞浆酶(LDH)、蛋白质、肌红蛋白漏出量和NO     

Influence of global ischemia on the sarcolemmal ATPases in the rat heart

To elucidate the effect of global ischemia on the energy utilizing processes, regarding the molecular principles, the kinetic and thermodynamic properties of the sarcolemmal ATPases were investigated in the rat heart. The activation energy for hydrolysis of ATP during ischemia was higher when the reaction was catalyzed by Ca-ATPase or Mg-ATPase. For the Na,K-ATPase reaction, no changes in the activation energy were observed. With respect to the enzyme kinetics, ischemia in a timedependent manner induced important alterations in K_M and V_max values of Na,K-ATPase, Ca-ATPase and Mg-ATPase. The V_max value decreased significantly already after 15 min of ischemia, and it also remained low after 30, 45 and 60 min for all 3 enzymes. The significant diminution of K_M values occurred later in the 30th min for Ca-ATPase, in the 45th min for Na,K-ATPase. The observed drop in K_M indicates the increase in the affinity of the enzymes to substrate, suggesting thus the adaptation to ischemic conditions on the molecular level. This effect could be attributed to some conformational changes of the protein molecule in the vicinity of the ATP-binding site developing after longer duration of ischemia.