Effects of acylcarnitine-transferase inhibitors on adenine nucleotide metabolism and ischemic tissue injury in isolated perfused rat heart

The ability of irreversible acylcarnitine-transferase inhibitors, sodium 2[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA) and 2-tetradecyl-glycidic acid (TDGA), to reduce myocardial ischemic injury was studied in Langendorff-perfused hearts exposed to ischemia (zero mmHg) followed by aerobic reperfusion (60 mmHg). Rat hearts were pretreated with either POCA (15 mg/kg) or TDGA (5 mg/kg) s.c. 120 min before the perfusion. Treated hearts showed a decreased release of creatine kinase and lactate on reperfusion after 30 min ischemia. POCA-treated hearts showed significantly higher ATP concentrations than control hearts on reperfusion. POCA also improved the maximum recovery of the pressure-rate product but with a significant delay. During the ischemic period, though, POCA decreased the ATP concentration at a rate three times that of controls during the first 10 min. No further reductions were observed for up to 30 min of ischemia. TDGA also showed a reduction of ATP. Thus, the observation that POCA stimulated ATP synthesis and reduced creatine kinase release on reperfusion after ischemia suggests that this agent provides some protection to the ischemic myocardium. However, during ischemia, it is likely that the depletion of ATP concentration induced by POCA resulted in delayed recovery of mechanical function on reperfusion.     

Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused ischemic hearts

Hydrogen peroxide (H2O2) has been implicated in cardiac damage due to ischemia and reperfusion. We adapted an electron microscopic, histochemical method for demonstrating H2O2 produced by isolated cells to isolated, buffer-perfused rabbit hearts. The method involves formation of an electron-dense precipitate when H2O2 reacts with cerium chloride (CeCl3). We perfused hearts retrograde via the aorta with well-oxygenated bicarbonate-buffered solution, followed by one in which bicarbonate was replaced with imidazole (IPSS) to prevent precipitation of bicarbonate and CeCl3. Some hearts were made globally ischemic (30 min, 37 degrees C), reperfused 5 min with well-oxygenated IPSS containing 1 mM CeCl3, then processed for electron microscopy. Others were perfused with IPSS containing catalase (300 U/ml) or albumin before ischemia and upon reperfusion, followed by CeCl3 administration. Nonischemic control hearts perfused with IPSS (+/- catalase) were also studied. Electron micrographs were assessed visually and by computer for precipitate localization and amount. There was abundant precipitate on the luminal face of the coronary vascular endothelium in ischemic-reperfused, cerium-treated hearts, including those treated with albumin. There was significantly less in reperfused catalase-treated or nonischemic control hearts. X-ray microbeam analysis of the endothelial precipitate indicated the presence of Ce. This appears to be the first visual demonstration of a CeCl3-H2O2-dependent reaction product in intact isolated ischemic hearts. The data indicate that at the time of reperfusion some H2O2 is accessible to the vascular space, and that its amount can be reduced by perfused catalase. Further modifications this technique may be useful for assessing the sites and pathways by which H2O2 is generated by hearts or other buffer-perfused organs subjected to stresses such as ischemia or hypoxia.     

A low concentration of nisoldipine reduces ischemic heart injury: enhanced reflow and recovery of contractile function without energy preservation during ischemia

Isolated working rat hearts which received no drug treatment had reduced ATP and creatine phosphate levels and increased lactate content during 20 min of ischemia. When subjected to 33 min of ischemia and 30 min of reperfusion, these hearts recovered low values of cardiac output (9.8 ml/min), heart rate, maximum developed pressure, pressure-rate product (72.9, 32.6, 27.5% of control, respectively), had low levels of tissue ATP, and reduced coronary flow upon reperfusion. Addition of nisoldipine (1 nM) 10 min before ischemia caused no decrease in cardiac output or heart rate, slightly decreased maximum developed pressure and pressure-rate product (93% of control), and did not reduce the degradation of ATP and creatine phosphate or the accumulation of lactate during 20 min of ischemia. When nisoldipine was included 10 min before ischemia, during ischemia (33 min) and reperfusion (30 min), however, the recovery of cardiac function and tissue ATP levels was significantly increased. This protective effect occurred when drug treated ischemic hearts were reperfused with control buffer, indicating residual effects. The beneficial effects of nisoldipine were not due to changes in afterload or preload (isolated perfused heart), collateral flow (zero flow model), energy preservation during ischemia (little contractile depression, ATP not enhanced during ischemia), or reduced lactate accumulation during ischemia. The beneficial effects were associated with increased coronary flow (31% higher than no drug) during reperfusion, indicating a reduction in the no-reflow phenomenon.     

Protective effects of hydrogen peroxide against ischemia/reperfusion injury in perfused rat hearts.

Among the several mechanisms proposed for ischemic preconditioning (IPC), generation of reactive oxygen species (ROS) is reported to be involved in the cardioprotective effects of IPC. The present study was designed to investigate whether repetitive exposure to hydrogen peroxide (H(2)O(2)) can protect the myocardium against subsequent ischemia/reperfusion injury, and whether the H(2)O(2)-induced cardioprotection is related to the preservation of energy metabolism. Langendorff-perfused rat hearts were exposed to two, 5 min episodes of IPC or to various concentrations of H(2)O(2) twice and then to 35 min global ischemia and 40 min reperfusion. Using (31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy, cardiac phosphocreatine (PCr) and ATP and intracellular pH (pH(i)) were monitored. IPC and the treatment with 2 micromol/L H(2)O(2) significantly improved the post-ischemic recovery of left ventricular developed pressure (LVDP) and the PCr and ATP compared with those of the control ischemia/reperfusion (LVDP: 36.9 +/-7.4% of baseline in control hearts, 84.0+/-3.5% in IPC, 65.4+/-3.8% in H(2)O(2); PCr: 51.1+/-5.3% in control hearts, 81.4+/-5.5% in IPC, 81.7+/-5.2% in H(2)O(2); ATP: 12.3+/-1.6% in control hearts; 30.0+/-2.8% in IPC, 28.6+/-2.3% in H(2)O(2), mean +/- SE, p<0.05). However, lower (0.5 micromol/L) or higher (10 micromol/L) concentration of H(2)O (2) had no effect. There were significant linear correlations between mean LVDP and high-energy metabolites after 40 min reperfusion in H(2)O(2)-treated hearts. In IPC-treated hearts, the mean LVDP was greater than that in the 2 micromol/L H(2)O(2)-treated hearts under similar levels of high-energy metabolites. IPC also ameliorated intracellular acidification (6.38+/-0.03 in control hearts, 6.65+/-0.04 in IPC, p<0.05), but treatment with H(2)O(2) did not affect pH(i) during ischemia (6.40+/-0.05 in H(2)O(2)). In conclusion, H(2)O(2) had protective effects against ischemia/reperfusion injury and the effects were related to the preservation of energy metabolism. IPC could have additional protective mechanisms that are associated with the amelioration of intracellular acidosis during ischemia.     

Trace amounts of albumin protect against ischemia and reperfusion injury in isolated rat hearts.

Albumin is used to provide colloid osmotic pressure in some resuscitation and organ preservation protocols. These solutions are expensive and carry the risks of using high concentrations of blood products. Used as a carrier of drugs and substrates, the concentration of albumin present in perfusates may be considerably lower in experimental ischemia. The present study examined if trace amounts of albumin (0.0004%) reduce injury from ischemia and reperfusion in isolated rat hearts. Hearts were perfused by the Langendorff technique (60 mmHg) with an intraventricular balloon. Zero-flow ischemia (20 min, 37 degrees C) was followed by reperfusion (35 min, 37 degrees C). Recovery of contractile function during reperfusion was significantly improved by the presence of fatty acid-free bovine serum albumin (BSA) (22 290+/-1280 mmHg/min, pressure-rate product) or rat serum albumin (RSA) (21 095+/-2836 mmHg/min) compared with Krebs-Henseleit buffer with no albumin (KHB) (9660+/-2324 mmHg/min). Release of lactate dehydrogenase activity, formation of tissue edema and accumulation of tissue malonyldialdehyde were significantly reduced in hearts receiving BSA or RSA compared with KHB alone. These parameters were not altered by the presence of albumin in non-ischemic control hearts or in the pre-ischemic values of the hearts subjected to ischemia and reperfusion. Development of ischemic contracture with an extended period of ischemia (27 min) was not altered by the presence of BSA, suggesting that protection observed with albumin occurred during reperfusion, rather than during ischemia. Reperfusion following 45 min of ischemia with bovine serum albumin resulted in similar myocardial injury to hearts that were reperfused following 20 min of ischemia without bovine serum albumin. Thus, trace amounts of albumin provide significant reduction in myocardial injury from ischemia and reperfusion, probably via antioxidant mechanisms.     

Changes in work rate to oxygen consumption ratio during hypoxia and ischemia in immature and mature rabbit hearts

This study was designed to evaluate the relative response of myocardial efficiency to reduced oxygen supply (hypoxia and ischemia) in immature and mature isolated rabbit hearts. Hearts were subjected to either 15 min of hypoxia (60% or 30% O2) or reductions in coronary flow to 75%, 50%, 25%, and 15% of basal flow followed by 12 min of total global ischemia and 15 min of reperfusion. In order to examine changes in cardiac efficiency, we utilized the ratio of isovolumic contractile function (rate-pressure product) to myocardial oxygen consumption (RPP/MVO2). Under basal conditions, immature hearts displayed lower aortic pressure. RPP, coronary resistance and RPP/MVO2. Moderate hypoxia (60% O2) resulted in similar reductions in RPP and MVO2 in both age groups, with RPP/MVO2 remaining unchanged. During severe hypoxia, RPP/MVO2 increased significantly in mature hearts but not in immature hearts (P < 0.05). Underperfusion produced greater reductions in RPP and heart rate, whereas reperfusion after ischemia resulted in greater recovery of RPP, dP/dt and MVO2 in immature compared to mature hearts. When oxygen supply was limited by reductions in coronary perfusion. RPP/MVO2 tended to increase in mature hearts, whereas the ratio declined significantly in immature hearts. These data demonstrate that, in this model, a reduction in oxygen supply by hypoxia or hypoperfusion decreases efficiency in immature hearts, but increases efficiency in mature hearts under the same conditions.     

再灌注损伤抢救激酶对小鼠缺血后适应心肌再灌注损伤中的减轻作用

目的 研究缺血后适应(IPC)对离体小鼠心肌缺血再灌注(I/R)损伤的作用及其影响因素,探讨再灌注损伤抢救激酶在IPC心肌保护中的作用.方法 建立Langendofff小鼠心肌I/R模型,全心缺血30 min后分为6组[(1)对照组,(2)3次IPC组(采取缺血10 s及再灌注10 s的3次IPC周期),(3)6次IPC组(采取缺血10 s及再灌注10 s的6次IPC周期),(4)延迟IPC组(恢复再灌注1 min后进行IPC),(5)IPC+PD98059组,(6)I/R+PD98059组],随后再灌注2 h;观察IPC对心脏血流动力学、心肌酶的释放、心肌超氧化物歧化酶活性和丙二醛的含量、梗死心肌范围的影响以及与细胞外信号调节激酶(ERK1/2)、磷脂酰肌醇3激酶-蛋白激酶B表达水平的关系.结果 与对照组比较,3次IPC组和6次IPC组小鼠心脏血流动力学显著改善,心肌酶释放减少,心肌丙二醛减少、超氧化物歧化酶活性增加,心肌梗死范围减小.6次与3次IPC周期的保护作用相似.而IPC作用在恢复再灌注1 min后消失.3次IPC组和6次iPC组心肌的ERK1/2磷酸化水平显著增高,蛋白激酶B磷酸化水平无明显变化.PD98059显著抑制IPC所致的ERK1/2的磷酸化,并能消除IPC对心肌的保护作用.结论 IPC能有效地减轻离体小鼠心肌缺血再灌注损伤,增加IPC的周期数并没有扩大保护作用,延迟IPC没有产生类似的保护作用.ERK1/2细胞信号途径参与介导IPC对离体心脏缺血再灌注心肌的保护作用.     

Effect of classic preconditioning and diazoxide on endothelial function and O2- and NO generation in the post-ischemic guinea-pig heart

OBJECTIVES: A hypothesis was tested that a reaction product between superoxide (O2-) and nitric oxide (NO) mediates post-ischemic coronary endothelial dysfunction that ischemic preconditioning (IPC) protects the endothelium by preventing post-ischemic cardiac O2- and/or NO formation, and that the opening of the mitochondrial ATP-dependent potassium channel (mKATP) plays a role in the mechanism of IPC. METHODS: Langendorff-perfused guinea-pig hearts were subjected either to 30 min global ischemia/30 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mKATP opener, diazoxide (0.5 microM). Coronary flow responses to acetylcholine (ACh) and nitroprusside were used as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of O2- and NO, and functional recoveries were followed during reperfusion. RESULTS: IR impaired the ACh response by approximately 60% and augmented cardiac O2- and NO outflow. Superoxide dismutase (150 U/ml) and NO synthase inhibitor, l-NMMA (100 microM) inhibited the burst of O2- and NO, respectively, and afforded partial preservation of the ACh response in IR hearts. NO scavenger, oxyhemoglobin (25 microM), afforded similar endothelial protection. IPC and diazoxide preconditioning attenuated post-ischemic burst of O2-, but not of NO, and afforded a complete endothelial protection. Diazoxide given after 30-min ischemia increased the O2- burst and was not protective. The effects of IPC and diazoxide preconditioning were not affected by HMR-1098 (25 microM), a selective blocker of plasmalemmal KATP, and were abolished by glibenclamide (0.6 microM) and 5-hydroxydecanoate (100 microM), a nonselective and selective mK(ATP) blocker, respectively. 5-Hydroxydecanoate produced similar effects, whether it was given as a continuous treatment or was washed out prior to IR. CONCLUSION: The results suggest that in guinea-pig heart: (i) a reaction product between O2- and NO mediates the post-ischemic endothelial dysfunction; (ii) the mK(ATP) opening serves as a trigger of the IPC and diazoxide protection; and (iii) the mK(ATP) opening protects the endothelium in the mechanism that involves the attenuation of the O2- burst at reperfusion.     

Angiotensin II subtype 1 (AT1) receptors contribute to ischemic contracture and regulate chemomechanical energy transduction in isolated transgenic rat (alphaMHC-hAT1)594-17 hearts

BACKGROUND: The role of AT1 receptors in myocardial ischemia/reperfusion injury is unclear. We, therefore, investigated the effects of the AT1 receptor antagonist irbesartan (Irb) in isolated hearts of selective myocardial AT1 overexpressing transgenic [transgenic(alphaMHC-hAT1)594-17] and Sprague-Dawley rats (SD) subjected to ischemia/reperfusion injury. METHODS AND RESULTS: Hearts of 4-week-old male SD or transgenic rats were isolated and perfused with Krebs-Henseleit buffer with or without 10 microM Irb in Langendorff mode. After 15 min of stabilization, pressure-volume curves were obtained and the hearts subjected to 20 min ischemia followed by 30 min reperfusion. A second set of pressure-volume curves was obtained thereafter. Left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), total coronary flow (CF) and oxygen consumption (MVO2) were recorded continuously. Myocardial efficiency was derived from the slope of relations of MVO2 to pressure/volume area. After 20 min ischemia, LVEDP was significantly higher in transgenic than in SD (35.7+/-1.8 vs. 29.2+/-1.0 mmHg, P<0.05) or Irb treated transgenic hearts (24.3+/-1.6 mmHg, P<0.05). Myocardial efficiency was increased by Irb before ischemia. Ischemia increased efficiency in SD but not in transgenic rats, Irb increased efficiency in transgenic hearts post-ischemia. CONCLUSION: Transgenic hearts developed ischemic contracture more rapidly than SD hearts as indicated by higher LVEDP during ischemia. This response was antagonized by Irb, indicating a role of AT1 receptors in ischemic contracture, AT1-receptors also appear to be involved in the control of myocardial efficiency.     

Separation of inherent diastolic myocardial fiber tension and coronary vascular erectile contributions to wall stiffness of rabbit hearts damaged by ischemia, hypoxia, calcium paradox and reperfusion

Ischemic myocardial contracture is exacerbated by reperfusion. This study examines the extent to which intensification of contracture by reperfusion is due to metabolic reoxygenation phenomena or hydraulic erectile contributions of coronary perfusion to left ventricular (LV) stiffness. Isolated rabbit hearts, with fluid-filled LV intraventricular baloons, were subjected either to: control aerobic perfusion; 30 or 60 min of global ischemia; 60 min of hypoxia with constant coronary flow; or 10 min of calcium-free perfusion to cause calcium paradox injury. During reperfusion with control perfusate isovolumic LV end diastolic pressure (LVEDP) was measured with constant coronary flow and during transient, 1 min, total global ischemia to measure the contribution of the coronary perfusion to LVEDP. In all injured groups LVEDP was increased compared to control hearts. The decrease in LVEDP during transient ischemia was greater in damaged hearts than in controls, demonstrating a greater contribution of coronary perfusion to LVEDP after injury. Only in the hypoxic hearts did diastolic fiber tension increase upon reperfusion. Inherent diastolic fiber tension decreased during 15 to 60 min of reperfusion in the ischemic and hypoxic injury groups, a trend which was masked by an increasing effect of coronary perfusion on LV chamber stiffness. During the reperfusion period enhancement of the erectile effect was more pronounced at higher preloads. Thus, reperfusion contracture was maintained both by changes in inherent fiber stiffness and by changes in the erectile effect. These contributions changed over time and varied with the type and severity of injury, but after all types of injury the erectile vascular effect made a greater contribution to diastolic chamber stiffness than inherent fiber tension.     

Cardiac Effects of ST‐6, a Novel Cyclohexane Dicarboximide Derivative

Na⁺ channel blockade is thought to be involved in the cardioprotection against ischemia/reperfusion injury. We synthesized various cyclohexane dicarboximides and examined their cardioprotective actions. Some of these derivatives had local anesthetic action and were capable of enhancing post‐hypoxic contractile recovery of the isolated perfused rat heart. Among them, 2‐[4‐[4‐(4‐chlorophenyl)‐4‐hydroxy‐1‐piperidinyl]butyl]hexahydro‐1H‐isoindol‐1,3(2H)‐dione hydrochloride (ST‐6) was most effective in the enhancement of post‐hypoxic contractile recovery of isolated perfused rat hearts subjected to 20‐min hypoxia and 45‐min reoxygenation. This enhanced recovery by 30 mg/min of ST‐6 was associated with attenuation of Na⁺, but not of Ca²⁺, accumulation during ischemia and prevention of creatine kinase release from the heart during reperfusion. When hearts subjected to 30‐min ischemia followed by 60‐min reperfusion were pretreated with 30 μM ST‐6, the post‐ischemic contractile recovery was enhanced and ischemia‐induced accumulation of Na⁺, as well as reperfusion‐induced accumulation of Na⁺ and Ca²⁺, was attenuated. Also the reperfusion‐induced release of creatine kinase was reduced, while restoration of myocardial high‐energy phosphates was enhanced during reperfusion. Na⁺ channel blockade by ST‐6, as assessed by the depression of the Vmax of the action potential, was similar to that produced by flecainide but more pronounced than with either lidocaine or disopyramide. ST‐6, 1, or 2 mg/kg i.v. or 10 mg/kg i.p., abolished ventricular fibrillation induced by 4 min of ischemia and subsequent 4 min of reperfusion in rats. The prevention of ventricular fibrillation by the continuous injection of 0.2 mg/kg per min ST‐6 from the first min after ischemia to the end of reperfusion was similar in degree to that produced by 0.1 mg/kg/min lidocaine or 0.5 mg/kg/min diltiazem. The former treatment elicited a transient decrease in the systemic blood pressure in anesthetized rats during ischemia, whereas treatment with the latter did not reduce systemic blood pressure. These findings suggest that ST‐6 may have cardioprotective effects in ischemia/reperfusion injury.     

七氟烷预处理改善离体大鼠心脏的再灌注心律失常

目的在Langendorff离体灌注模型研究七氟烷预处理对再灌注心律失常的影响。方法取SD大鼠心脏建立Langendorff灌注模型,随机分入以下三组：（1）对照组;（2）缺血再灌注组;（3）七氟烷预处理组（3％七氟烷预处理15分钟）。记录各组的血流动力学、心电图,测量冠脉流出液肌钙蛋白I水平,测定细胞内钙离子和活性氧水平。结果七氟烷预处理能显著增加缺血再灌注损伤后左室发展压,左室内压上升／下降速率和心率,降低左室舒张末期压力,减少冠脉流出液肌钙蛋白I水平（P均〈O．05）。在再灌注心律失常方面,与缺血再灌注组相比,七氟烷预处理能显著减少室性早搏个数[从182（133）次／分降至83（52）次／分],缩短室速[41（45）s降至20（22）s]和室颤[从22（43）s降至0（0）s]的发作时程,减少室颤发生率（从80％降至10％）,并降低再灌注心律失常评分[从4（0）降至2（O）]（P均〈0.05）。七氟烷预处理还能降低心肌细胞内钙离子和活性氧水平（P均〈0.05）。结论七氟烷预处理对缺血再灌注损伤心脏起保护作用,能改善离体大鼠心脏的再灌注心律失常。     

Myocardial stretch induced by increased left ventricular diastolic pressure preconditions isolated perfused hearts of normotensive and spontaneously hypertensive rats

Objective: The aim of our study was to determine whether myocardial stretch (non-ischemic stress) could precondition isolated perfused hearts of both normotensive Wister-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).Methods: The perfused hearts in Langendorff mode were subjected to 30 min of global no-flow ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) were measured. In the control group, LVEDP was set at 10 mmHg. In the stretch group, LVEDP was increased to 30 or 60 mmHg for 5 min before 30 min of ischemia. In the ischemic preconditioning group, the hearts were exposed to two cycles of a 5-min period of ischemia before 30 min of ischemia. Myocardial lactate contents were measured at the baseline and at the end of the 60 mmHg stretch.Results: Hemodynamic parameters of LVDP and LVEDP at 30 min of reperfusion improved in the stretch group (LVEDP at 60 mmHg) and the ischemic preconditioning group. Coronary flow did not decrease during the stretch. Recovery of the coronary flow during reperfusion was better in the stretch and ischemic preconditioning groups. Postischemic contractile function was better in WKY rats than in SHR. Myocardial lactate contents at the end of 60 mmHg stretch were negligible. Conclusions: Myocardial stretch induced by increasing LVEDP preconditioned isolated perfused hearts of both WKY rats and SHR, via mechanisms not involving myocardial ischemia during stretch.     

PI 3-kinase regulates the mitochondrial transition pore in controlled reperfusion and postconditioning

OBJECTIVE: We investigated whether phosphatidylinositol 3-kinase (PI3K) might regulate mitochondrial permeability transition pore (mPTP) opening in hearts reperfused with either low pressure or postconditioning. METHODS: Male Wistar rat hearts (n=72) were perfused according to the Langendorff technique, exposed to 30 min of ischemia, and assigned to one of the following groups: (1) reperfusion with normal pressure (NP; 100 cm H2O), (2) reperfusion with low pressure (LP; 70 cm H2O), or reperfusion with postconditioning, i.e. 3 episodes of 30 s reperfusion followed by 30 s of ischemia (PostC). Hearts received either the PI3K inhibitors wortmannin or LY294002, or vehicle at the onset of the 60 min reperfusion. Postischemic functional recovery was assessed by rate-pressure product (RPP), and irreversible injury by lactate dehydrogenase (LDH), creatine kinase (CK) and troponin I (TnI) release. Mitochondria were isolated from the reperfused myocardium, and Ca2+-induced mPTP opening was measured using a potentiometric method. RESULTS: Functional recovery was significantly improved in LP and PostC hearts with RPP averaging 13,880+/-810 (LP) and 17,130+/-900 mm Hgxbeats/min (PostC) versus 6450+/-500 mm Hgxbeats/min in NP hearts (p<0.01). LDH release averaged 230+/-30 and 145+/-15 IU/h/g of myocardial tissue in LP and PostC versus 340+/-10 IU/h/g in NP (p<0.05). Wortmannin and LY294002 prevented both RPP improvement and decrease in LDH, CK, and TnI release in LP and PostC groups. The Ca2+ load required to induce mPTP opening averaged 58+/-3 and 52+/-1 nmol/mg mitochondrial proteins in LP and PostC groups, respectively, versus 35+/-4 nmol/mg in the NP group (p<0.01). Wortmannin and LY294002 prevented the beneficial effect in both the LP and PostC groups. CONCLUSION: These results suggest that PI3K regulates the opening of the mitochondrial permeability transition pore in rat hearts reperfused with low pressure or postconditioning.     

Effects of endothelin-1 in the isolated heart in ischemia/reperfusion and hypoxia/reoxygenation injury.

The effects of the vasoconstrictor peptide endothelin-1 were examined in the isolated heart during hypoxia, reoxygenation and reperfusion. Isovolumic rat hearts were perfused with Krebs-Henseleit buffer at constant pressure. Cumulative dose-response curves were obtained for endothelin-1 boluses of 0.04 to 400 pmol in five groups of hearts. Coronary flow declined with increasing dosages and was almost abolished at 400 pmol in control hearts. In hearts subjected to mild hypoxia (perfusate PO2 approximately 150 mmHg), the constrictor effect of endothelin-1 was attenuated at moderate dose compared to control hearts (4 vs. 16% flow reduction at 40 pmol; P less than 0.05). The constrictor effect was unaltered in hearts subjected to either 60 min of severe hypoxia (PO2 approximately 35 mmHg) followed by reoxygenation or to 10 min of total ischemia followed by reperfusion (stunning). When hearts were reperfused following 30 min of total ischemia (irreversible injury), the constrictor response to endothelin-1 was potentiated compared to control (e.g. 36 vs. 16% flow reduction at 40 pmol; P less than 0.05). We conclude that endothelin-1 is a potent coronary constrictor in hypoxic, reoxygenated and reperfused heart. The constrictor effect is attenuated during hypoxia, most likely due to the presence of counteracting vasodilator metabolites. During reperfusion, the constrictor effect is unchanged in stunned myocardium, but is augmented in irreversibly injured heart, due to either increased endothelin-1 binding sites or loss of counteracting vasodilator mechanisms such as prostaglandins and/or endothelium-derived relaxing factor.     

Effect of reperfusion on the cardiac acetylcholine and norepinephrine contents in rat hearts

The effects of ischemia, reperfusion and hypoxia on the cardiac acetylcholine, choline, norepinephrine and cyclic AMP contents were investigated in isolated, spontaneously beating rat hearts perfused under constant pressure (100 cm H2O) with Krebs-Henseleit solution gassed with 95% O2-5% CO2. Acetylcholine, choline and norepinephrine were determined by high performance liquid chromatography with electrochemical detection. Cyclic AMP was determined by radioimmunoassay. One min reperfusion following 15 min ischemia (termination of perfusion) caused a significant decrease in both cardiac acetylcholine (P less than 0.05) and norepinephrine (P less than 0.01) contents, but had no significant effect on the cardiac norepinephrine/acetylcholine content ratio, or choline or cyclic AMP content. By contrast, 16 min ischemia did not significantly affect the cardiac acetylcholine, norepinephrine, choline or cyclic AMP content. Also, 16 min hypoxia (perfusion with Krebs Henseleit solution gassed with 95% N2 5% CO2) decreased the cardiac norepinephrine content significantly (P less than 0.01) and norepinephrine/acetylcholine content ratio slightly but not significantly. However, hypoxia had no significant effect on the cardiac acetylcholine, choline or cyclic AMP content. Pre-treatment with 10 microns atropine sulfate prevented the decrease in the cardiac acetylcholine content caused by reperfusion but caused a significant depletion in the cardiac norepinephrine content in the control (P less than 0.01) and ischemia (P less than 0.05) groups and a significant decrease in the norepinephrine/acetylcholine content ratio in all three groups (all, P less than 0.05). Extending the reperfusion period to 5 and 10 min following 15 min ischemia also caused a significant decrease in both cardiac acetylcholine and norepinephrine contents compared with the control groups. However, no significant difference in these contents was found between 1 min reperfusion group and 5 or 10 min reperfusion group. Twenty or 25 min ischemia alone did not significantly affect these contents. These findings suggest that reperfusion disturbs both the sympathetic and parasympathetic nervous systems in the heart and that pre-treatment with atropine adversely affects the balance of the autonomic nervous system.     

A sphingosine kinase 1 mutation sensitizes the myocardium to ischemia/reperfusion injury

OBJECTIVE: Sphingosine kinase (SphK) is a key enzyme in the synthesis of sphingosine 1-phosphate (S1P), a bioactive sphingolipid. SphK is involved in ischemic preconditioning (IPC). To date no studies in genetically altered animals have examined the role of SphK1 in myocardial ischemia/reperfusion (IR) injury and IPC. METHODS AND RESULTS: Wild-type and SphK1 null mouse hearts were subjected to IR (50 min global ischemia and 40 min reperfusion) in a Langendorff apparatus. IPC consisted of 2 min of global ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), +/-dP/dtmax, and LV end-diastolic pressure (EDP) between SphK1 mutant and wild-type (WT) mouse hearts. In the mutants, total SphK enzyme activity was reduced by 44% and S1P levels were decreased by 41%. SphK1 null hearts subjected to IR exhibited more cardiac damage compared with WT: LVDP and +/-dP/dtmax decreased, LVEDP increased, and infarct size increased (n=6, P<0.05). Apoptosis was markedly enhanced in SphK1 mutant IR mouse hearts. IPC was cardioprotective in WT hearts, but this protection appeared to be ineffective in SphK1 null hearts. There was no change in infarct size in the IPC+IR group compared to the IR group in the null hearts (50.1+/-5.0% vs 45.0+/-3.8%, n=6, P=NS). IPC remained ineffective in the null hearts even when the index ischemia time was shortened by 10 min. CONCLUSIONS: Deletion of the SphK1 gene sensitizes the myocardium to IR injury and appears to impair the protective effect of IPC. These data provide the first genetic evidence that the SphK1-S1P pathway is a critical mediator of IPC and cell survival.     

Effects of substrates on tissue metabolic changes in the isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion.

In Langendorff-perfused rat hearts, the perfusion pressure was reduced from 100 cm H2O to 20 cm H2O for 30 minutes to produce a model of global ischemia with a residual oxygen uptake. The release of lactate dehydrogenase (LDH) and the occurrence of ventricular arrhythmias during reperfusion were dependent on the substrate. Glucose-perfused hearts had the highest rates of glycolytic ATP production (2.5 mumol/g per min) during ischemia with normal contents of tissue cyclic adenosine 3',5'-monophosphate (cAMP) and, during reperfusion, the release of LDH was lowest and severe ventricular arrhythmias did not occur. In pyruvate-perfused hearts, glycolysis was inhibited during ischemia, the rate of production of glycolytic ATP was only 0.5 mumol/g per min. and tissue cAMP doubled; during reperfusion, LDH release was 14-fold higher and ventricular arrhythmias were more severe. Total tissue contents of ATP and phosphocreatine were similar in glucose- and in pyruvate-perfused hearts. In hearts perfused with acetate, there was virtually no glycolytic ATP synthesized during the last 5 minutes of ischemia and cAMP increased further. Acetate- and palmitate-perfused hearts showed greatest release of LDH and had severest arrhythmias during reperfusion, suggesting that it was the metabolic and not the detergent effects of palmitate that were operating. Lipolysis was not a major factor in the cause of reperfusion LDH release. A role of glycolytic ATP in the maintenance of membrane integrity is postulated.     

Induction of ischemic tolerance in rat liver via reduced nicotinamide adenine dinucleotide phosphate oxidase in Kupffer cells

AIM To elucidate the mechanisms of hepatocyte preconditioning by H2O2 to better understand the pathophysiology of ischemic preconditioning.METHODS The in vitro effect of H2O2 pretreatment was investigated in rat isolated hepatocytes subjected to anoxia/reoxygenation. Cell viability was assessed with propidium iodide fluorometry. In other experiments, rat livers were excised and subjected to warm ischemia/reperfusion in an isolated perfused liver system to determine leakage of liver enzymes. Preconditioning was performed by H2O2 perfusion, or by stopping the perfusion for 10 min followed by 10 min of reperfusion.To inhibit Kupffer cell function or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,gadolinium chloride was injected prior to liver excision, or diphenyleneiodonium, an inhibitor of NADPH oxidase, was added to the perfusate, respectively. Histological detection of o~gen radical formation in Kupffer cells was performed by perfusion with nitro blue tetrazolium.RESULTS Anoxia/reoxygenation decreased hepatocyte viability compared to the controls. Pretreatment with H2O2 did not improve such hepatocyte injury. In liver perfusion experiments, however, H2O2 preconditioning reduced warm ischemia/reperfusion injury, which was reversed by inhibition of Kupffer cell function or NADPH oxidase. Histological examination revealed that H2O2 preconditioning induced oxygen radical formation in Kupffer cells. NADPH oxidase inhibition also reversed hepatoprotection by ischemic preconditioning.CONCLUSION H2O2 preconditioning protects hepatocytes against warm ischemia/reperfusion injury via NADPH oxidase in Kupffer cells, and not directly. NADPH oxidase also mediates hepatoprotection by ischemic preconditioning.     

UTP减轻大鼠心脏缺血/再灌注损伤的作用

目的:探讨P2Y受体激动剂尿苷三磷酸(UTP)对于大鼠心脏缺血/再灌注损伤(I/RI)的延迟性拮抗作用。方法:24只SD大鼠随机分为4组:实验对照组、UTP组、UTP+苏拉明(suramin,SRM)组及SRM组。所有大鼠尾静脉给药24 h后,建立Langendorff离体心脏灌流模型。平衡10 min后全心停灌,25 min后复灌,持续再灌40 min。观察心脏再灌注前后血流动力学指标及心肌超微结构,记录心脏表面心电图计算心律失常的发生率,收集冠脉流出液,用全自动生化分析仪测量乳酸脱氢酶(LDH)的水平。结果:复灌后第5 min和25 min时,UTP组的左室发展压(LVDP)、左室压力微分(±dp/dtmax)恢复率均优于实验对照组(P0.05,P0.01);冠脉流出液中LDH的水平明显值降低(P0.01);复灌第5~15 min和第25~35min时的心律失常的发生频率均显著下降(P0.05,P0.01);心肌超微结构的损伤减轻。而以SRM与UTP同时作用后,UTP对心脏的保护作用则被取消。SRM组与实验对照组相比各项指标无明显变化。结论:UTP预处理可对心脏I/RI产生延迟性拮抗作用;而P2Y受体拮抗剂SRM可取消这种作用,表明UTP对心脏的保护作用是通过P2Y受体介导的。