R56865 is antifibrillatory in reperfused ischemic guinea-pig hearts,even when given only during reperfusion

Summary R56865 was previously characterized as an inhibitor of Na and Ca overload that has beneficial effects during ischemia and reperfusion. An isolated guinea-pig heart preparation was subjected to 60 minutes of ischemia and 60 minutes of reperfusion. R56865 was given before ischemia and with the onset of reperfusion, applying different dosing schedules, including an initial loading dose. R56865 below 0.1 µmol/l had no cardiodepressant effects in normoxic hearts and at 0.1 µmol/l reduced left ventricular pressure marginally. The onset of ischemic contracture was delayed only at this concentration. R56865 given before ischemia potently inhibits delayed sustained fibrillation occurring during reperfusion in the concentration range between 0.01 µmol/l and 0.1 µmol/l. Analysis of cellular Na⁺, K⁺, and Ca²⁺ concentrations revealed that R56865 substantially improves the ionic homeostasis of myocardial cells. Most importantly, the compound also reduced the incidence of delayed sustained fibrillation when given at the onset of reperfusion. R56865 was most effective when fast equilibration of drug with tissue was achieved by giving an initial loading dose. In particular, the cellular Na⁺ and Ca²⁺ contents were improved using this dosing scheme. The results are compatible with the classification of R56865 as an inhibitor of Na⁺ and Ca²⁺ overload.     

Metabolic and Functional Consequences of Successive No-flow and Sustained Low-flow Ischaemia; aP MRS Study in Rat Hearts

Recently, a model of acute hibernation, based on successive no-flow and low-flow ischaemia in the isolated rabbit heart has been described. In the present study this model was used in isolated rat hearts.³¹P NMR was used to follow the time course of intracellular pH (pH_i) and high-energy phosphates; mechanical activity of the heart was assessed simultaneously. Control hearts were subjected to 180 min of low-flow ischaemia and 60 min of reperfusion (group A). In the acute hibernation group, low-flow was preceded by 5 min of no-flow ischaemia (group B). In group A contracture developed during low-flow. The time to onset of contracture was 51 min (range: 28 to 123 min). In group B, contracture did not occur during low-flow ischaemia (P<0.01); recovery of left ventricular developed pressure and end-diastolic pressure was significantly better during the first 15 min of reperfusion (P<0.05). In group A pH_idecreased from 7.06±0.04 to 6.64±0.14 during the first 30 min of low-flow. After contracture developed in this group two pH_ivalues were measured amounting to 6.33±0.15 and 6.86±0.05 at the end of low-flow. At the end of reperfusion pH_iwas 6.29±0.05 and 7.09±0.06. In group B, pH_idecreased from 7.08±0.03 to 6.55±0.03 during no-flow ischaemia. During low-flow ischaemia, pH_iincreased to 6.73±0.05 and remained constant. During reperfusion pH_irecovered to 7.06±0.03. In group A and B phosphocreatine (PCr) levels at the end of low-flow ischaemia amounted to 13±8% and 26±6% of pre-ischaemic levels, respectively. During reperfusion, PCr recovery was better in group B: 67±12%v23±11% (P<0.05). In group A and B, ATP levels at the end of low-flow ischaemia were 5±10% and 19±9%, respectively. The rate of ATP depletion during low-flow ischaemia was initially similar in both groups, but between 45 and 90 min ATP depletion still continued in group A, while this had leveled off in group B (P<0.01). During reperfusion no significant changes in ATP were observed. We propose that increased glucose transport and glycolytic flux are able to maintain ionic homeostasis and diastolic function when low-flow ischaemia is preceded by a short period of no-flow ischaemia.     

Predicting functional recovery from ischemia in the rat myocardium

Summary Depletion of high-energy phosphates, accumulation of inorganic phosphate and intracellular acidosis have each been proposed as important events in the transition from reversible to irreversible ischemic injury. To assess whether each variable is predictive of functional recovery on reperfusion, these were measured in the isolated isovolumic rat heart using³¹P NMR. Perfused hearts were subjected to either 10, 12 or 40 min of normothermic ischemia followed by 40 min of reperfusion. Hearts were then freeze-clamped for further analysis of phosphate metabolites by NMR and ion chromatography. High-energy phosphates, P_i, phosphomonoesters and pH were measured by³¹P NMR spectroscopy at 2 minute intervals. Heart rate and developed pressure were monitored simultaneously. All hearts undergoing 10 min of ischemia and 40% of hearts subjected to 12 min of ischemia demonstrated good functional recovery. The remainder of hearts ischemic for 12 min went into contracture on reperfusion with little return of function. Hearts subject to 40 min of ischemia went into ischemic contracture and showed no recovery on reperfusion. Intracellular pH, [ATP], and [P_i] measured prior to reperfusion did not predict the extent of recovery. However, phosphomonoesters were detected prior to reperfusion in all hearts that did not recover well, but were not observed in hearts that showed good mechanical recovery. Analysis of tissue extracts by³¹P NMR and ion chromatography indicated that the most prominent components of the phosphomonoesters were glucose 6-phosphate, -glycerol phosphate and AMP. In conclusion, of the various phosphorus metabolites that can be measured by³¹P NMR, only one group, the phosphomonoesters, was predictive of functional recovery.     

Effects of a Metalloproteinase that Truncates P-selectin Glycoprotein Ligand on Neutrophil-induced Cardiac Dysfunction in Ischemia/Reperfusion

This study was designed to test the effects of polymorphonuclear leukocytes (PMNs) in the presence and absence of a P-selectin blocker, mocarhagin, in provoking cardiac dysfunction in isolated perfused rat hearts following ischemia and reperfusion. Control rat hearts not subjected to ischemia were perfused without blood cells for 80 min. Additional control rat hearts were perfused with 100×10⁶PMNs in the presence and absence of 0.2μg/ml mocarhagin over a 5-min perfusion followed by a 45-min observation period. No significant reduction in coronary flow (CF), left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dtmax) was observed at the end of the observation period in any non-ischemic group. Similarly, global ischemia (I) for 20 min followed by 45 min of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of PMNs. I/R hearts perfused with PMNs exhibited decreases of 50–60% in all measurements of cardiac function (P<0.001). These PMN perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were attenuated markedly by the metalloproteinase, mocarhagin, which inhibits P-selectin-mediated cell adhesion by cleaving its high-affinity receptor, PSGL-1, present on neutrophils. These results provide evidence that neutrophils provoke post-reperfusion cardiac dysfunction, and that this may be largely due to P-selectin-induced adherence of neutrophils to the endothelium.     

Early in Reperfusion Following Myocardial Ischemia,Leukocyte Activation Is Necessary for Venular Adhesion But Not Capillary Retention

Objective : The pathobiology of leukocyte sequestration in the coronary microcircu-lation following ischemia is unclear. We examined the location(s) and persistence of leukocyte sequestration of unactivated and preactivated blood in the coronary microcirculation early during reperfusion following ischemia. Methods : Isolated rat hearts were subjected to 30 min of 37°C, no-flow ischemia. Hearts were initially reperfused with diluted whole blood containing fluorescent leukocytes (DWB*). At 5, 20, and 35 min of reperfusion (R), the deposition of leukocytes in the coronary capillaries and venules was observed directly using intravital fluorescence microscopy. Four groups were studied: a nonischemic control group (Gr I), and postischemic groups reperfused with DWB* treated with vehicle (Gr II) or preactivated with 10^?8 M N-formylmethionyl-leucyl-phenylala-nine (fMLP) (Gr III) or 10^?6 M fMLP (Gr IV). Results : At R5, postischemic reperfusion with unactivated blood caused a significant trapping of leukocytes in coronary capillaries (Gr I = 2.2 ± 0.4 versus Gr II = 5.6 ± 0.6 leukocytes per capillary field, P < 0.05). Hearts in Gr IV exhibited significantly greater leukocyte retention in capillaries compared to all other groups at R5 (R5, Gr IV = 8.8 leukocytes per capillary field, P < 0.05) and at R35. At R5, although more leukocytes were observed adhered to the venules in Gr II compared to Gr I, the difference was not statistically significant (Gr I = 1.7 ± 0.7 versus Gr II = 3.4 ± 0.5 leukocytes per 100 |μm venule, P = 0.23). DWB* preactivated with the lower concentration of fMLP (10^?8 M) resulted in a significant increase in venular leukocyte adhesion at R5 compared to Gr I and Gr II (Gr III 6.1 ± 0.5, P < 0.05). After 35 min of reperfusion, a greater percentage of leukocytes remained in the capillaries than in the venules. Conclusions : These direct observations suggest that early in reperfusion after ischemia, both leukocyte and endothelial activation are necessary for venular adhesion, but that ischemia-induced coronary microvascular alterations are sufficient to promote leukocyte retention in coronary capillaries. These results also indicate that during 35 min of reperfusion, the degree of leukocyte washout is greater in the venules than in the capillaries. These results suggest that the mechanisms contributing to leukocyte retention early in reperfusion following myocardial ischemia are, indeed, different in the capillaries and venules and that the mechanisms affecting retention in capillaries are more persistent than those in the venules.     

Preconditioning in Globally Ischemic Isolated Rat Hearts: Effect on Function and Metabolic Indices of Myocardial Damage

We assessed the effects of ischemic preconditioning on heart recovery and metabolic indices of damage following global ischemia and reperfusion, in relationship to post-ischemic lactate release. Three groups of Langendorff rat hearts were studied: (1) A control group of 40 min global ischemia and 45 min reperfusion; (2) preconditioning by 5 min global ischemia and 15 min reperfusion prior to sustained ischemia and reperfusion; (3) Preconditioning by three episodes of brief ischemia–reperfusion prior to sustained ischemia. Repetitive episodes of brief ischemia–reperfusion were associated with increased reactive hyperemia, decreased release of purines and prostaglandin 6-keto F_1α, lower tissue glycogen but no change in lactate washout. After 40 min ischemia, release of lactate was 173±17, 196±6 and 149±9μmol/g in groups 1, 2 and 3, respectively (P<0.01, group 2vgroup 3). Preconditioning had no effect on ischemic arrest but had divergent effects on the development and the magnitude of ischemic contracture: delay and attenuation in group 2 but enhanced onset in group 3. Preconditioning provided a dose-dependent protection from the increase in left ventricular end-diastolic pressure, reduced the reperfusion release of purine metabolites and of creatine kinase, but neither improved systolic function nor prevented arrhythmia. 6-Keto F_1αproduction was 87±13, 132±19 and 241±35 pmol/g in groups 1, 2, 3, respectively (P<0.01 group 1vgroup 3). We conclude that when subjected to prolonged global ischemia, preconditioned isolated rat hearts develop less post-ischemic contracture, lose less purine nucleosides and creatine kinase activity. In addition, preconditioning leads to increased production of prostacyclin. Differences among preconditioning protocols in lactate production seem to be related to the ischemic contracture development, but may not play an ultimate role in attenuation of myocardial damage or improvement of postischemic recovery.     

Effects of Protein Kinase C Inhibitors inIn Situand Isolated Ischemic Rabbit Myocardium

We tested the effects of the protein kinase C (PKC) inhibitors bisindolylmaleimide (1μ ) and chelerythrine (2μ ) on myocardial ischemia–reperfusion injury inin situand isolated perfused rabbit hearts. In non-ischemic isolated hearts, bisindolylmaleimide (1μ ) and chelerythrine (2μ ) blocked sn-1,2-dioctanoylglycerol (DOG)-induced coronary vasoconstriction by approximately 80%. Intact hearts were subjected to 45 min coronary artery occlusion and 2 h reperfusion. Infarct size, determined by triphenyltetrazolium chloride (TTC)-staining and expressed as percentage of risk area, was reduced approximately 50% by both bisindolylmaleimide (0.05 mg/kg, i.v.) and chelerythrine (0.1 mg/kg, i.v.) compared to vehicle treated controls. In contrast, a higher dose of chelerythrine (3.8 mg/kg, i.v.) did not significantly reduce infarct size. Isolated hearts were subjected to 45 min of global normothermic (37°C) ischemia and 60 min reperfusion. Control hearts exhibited 45±2% recovery of pre-ischemic left ventricular developed pressure (LVDP) compared to bisindolylmaleimide- (73±7%) and chelerythrine-treated hearts (70±11%). Bisindolylmaleimide and cherythrine reduced infarct size from a control value of 24±4 to 8±2 and 9±3%, respectively. Preconditioning isolated hearts with 5 min ischemia and 10 min reperfusion prior to prolonged ischemia reduced infarct size to 10.4±2.3%, an effect which was blocked by chelerythrine (22.5±4.2% infarct size). These results suggest that although PKC may play a role in ischemic preconditioning, PKC inhibitors can be cardioprotective during prolonged ischemia.     

The effect of magnesium added to secondary cardioplegia on postischemic myocardial metabolism and contractile function —a31P NMR spectroscopy and functional study in the isolated pig heart

Summary This study investigated whether increasing the magnesium concentration during secondary cardioplegia improves postischemic myocardial recovery. Twenty-four isolated pig hearts were divided into four groups. All hearts were initially subjected to control perfusion with modified Krebs-Henseleit solution for 30 min, followed by a single infusion of St. Thomas' solution #2. The hearts were then maintained without perfusion at 12°C for 4h. Following this hypothermic preservation, the hearts in group I were reperfused with modified Krebs-Henseleit solution for 50 min, while hearts in group II and III were reperfused with a secondary cardioplegic solution containing 16 or 0 mmol/L magnesium, respectively, for 20 min followed by 30 min of perfusion with modified Krebs-Henseleit solution. In group IV, the hearts were initially reperfused with Krebs-Henseleit solution containing 16 mmol/L potassium for 20 min, followed by 30 min of reperfusion with modified Krebs-Henseleit solution. The changes in high-energy phosphates and intracellular pH were monitored throughout the experiments using³¹P nuclear magnetic resonance (NMR) spectroscopy. Heart rate, left-ventricular systolic developed pressure, and rates of pressure increase and decrease were measured during control perfusion and reperfusion to calculate the percent contractile functional recovery. Needle biopsies for measurement of energy metabolites with high performance liquid chromatography were performed at the end of preservation and reperfusion to confirm the NMR measurements. All six hearts in group I showed significantly less recovery of contractile function during reperfusion when compared to the hearts in groups II, III, IV (p<0.05). There was no difference in either recovery of metabolism or mechanical function among the latter three groups of hearts. None of hearts in groups II, III, and IV showed ventricular fibrillation, which occurred in all six hearts of group I upon reperfusion. The results suggest that a short period of re-arrest perfusion following ischemia (secondary cardioplegia) improves postischemic contractile functional recovery and prevents reperfusion-induced ventricular fibrillation. Increased magnesium concentration in the secondary cardioplegia did not provide additional benefit to the ischemic myocardium, possibly due to the low permeability of the sarcolemmal membrane to magnesium.Supported by the Heart and Stroke Foundation of Ontario     

Reduced infarct size in the rabbit heartin vivo by ethylisopropyl-amiloride. A role for Na+/H+ exchange

Inhibition of Na⁺/H⁺ exchange with amiloride analogues has been shown to offer functional protection during ischemia and reperfusion and reduce infarct size in isolated rat hearts and intact pigs. The aim of the present study was to examine if pre- or postischemic treatment with ethylisopropylamiloride (EIPA), a selective Na⁺/H⁺ exchange inhibitor, could reduce infarct size in anin situ rabbit model of regional ischemia and reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of regional ischemia and 180 min of reperfusion. The risk zone was determined by fluorescent particles, and infarct size was determined by TTC staining. Preischemic treatment with EIPA (0.65 mg/kg) significantly reduced infarct size from 45.8±3.5% of the risk zone in the control group to 10.6±3.1% (p<0.01). EIPA-treatment during the first part of the reperfusion period did not reduced infarct size compared to controls (41.9±3.5%). We conclude that EIPA, when administered prior to ischemia, reduces infarct size in the rabbit heartin situ, a protection most likely due to inhibition of Na⁺/H⁺ exchange.     

Clinical L-Type CaChannel Blockade Prevents Ischemic Preconditioning of Human Myocardium

B. S. Cain, D. R. Meldrum, J. C. Cleveland, Jr, X. Meng, A. Banerjee and A. H. Harken. Clinical L-Type Ca²⁺Channel Blockade Prevents Ischemic Preconditioning of Human Myocardium. Journal of Molecular and Cellular Cardiology (1999) 31, 2191–2197. Although Ca²⁺channel blockers are commonly used to control both blood pressure and angina in patients with coronary artery disease, clinical trials have associated the use of -type Ca²⁺channel blockers with increased cardiovascular mortality. Recent evidence has implicated Ca²⁺entry through the -type Ca²⁺channel during transient ischemia as a proximal stimulus for ischemic preconditioning (IPC) in experimental animals. We therefore hypothesized that clinical -type Ca²⁺channel blockade prevents IPC in human myocardium. Human atrial trabeculae were suspended in organ baths, field simulated at 1 Hz, and force development was recorded. Following 90 min equilibration, trabeculae from control patients and patients taking -type Ca²⁺channel blockers were subjected to simulated ischemia/reperfusion (I/R: 45/120 min) with or without 5 min of simulated ischemia (IPC stimulus) prior to I/R. IPC increased post-ischemic developed force in control patients from 14.6±2.6 to 43.1±3.5% baseline developed force (%BDFP <0.05 I/R vs IPC). Whereas IPC failed to increase post-ischemic developed force in myocardium from patients taking -type Ca²⁺channel blockers (15.1±1.9 vs 16.6±1.7 %BDF,P >0.05 -type I/R v -type IPC). We conclude that: (1) atrial muscle can be preconditioned by transient ischemia; (2) atrial muscle from patients taking -type Ca²⁺channel blockers cannot be preconditioned by transient ischemia; and (3) the increased cardiovascular mortality historically associated with the use of Ca²channel blockers in patients with coronary artery disease may be, in part, due to the pharmacological inhibition of ischemic preconditioning.     

Effects of repeated brief episodes of ischemia and reperfusion in isolated perfused rat hearts

Summary The effects of ischemia and reperfusion on the coronary endothelium and myocardium as well as tolerance to ischemia/reperfusion injury were assessed using isolated retrogradely perfused rat hearts. Repeated brief episodes of myocardial ischemia followed by reperfusion is known to have a protective effect against subsequent myocardial infarction. However, no studies have been performed with perfusion in the absence of blood cells to determine the effect of repeated ischemia and reperfusion on the coronary endothelium and myocardium. Using the Langendorff perfusion technique, rat hearts were subjected to a 30-, 10-, 5-, or 2-min period of low-flow perfusion by reducing the coronary flow to 3ml/min followed by reperfusion at 20ml/min for the same period of time. Control perfusion was then performed at a constant flow rate of 20 ml/min for 60min. Acetylcholine-induced coronary vasodilation was significantly (P < 0.05) lower in hearts subjected to 30min of ischemia and 30min of reperfusion when compared with the control hearts. Myocardial creatinine kinase (CK) activity was significantly reduced (P < 0.01) in hearts subjected to ischemia and reperfusion for either 30, 10, or 5min. To assess the effect of repeated episodes of ischemia and reperfusion, the following protocols were used: a control study with constant perfusion for 60min (group A), 30min of ischemia and 30min of reperfusion (group B), three 10min episodes of ischemia and reperfusion (group C), six 5-min episodes of ischemia and reperfusion (group D), and 15 2-min episodes of ischemia and reperfusion (group E). Acetylcholine-induced coronary vasodilation was significantly inhibited in group B (80% ± 12%,P < 0.05) and group C (70% ± 13%,P < 0.01), but did not change significantly in either group D (123% ± 19%) or group E (142% ± 15%), compared with the control group (group A; 127% ± 15%, mean ± SEM). Nitroglycerin-induced coronary vasodilation was not altered by ischemia/reperfusion in any group. In contrast, myocardial CK activity was significantly lower in group B (3.6 ± 0.6IU/mg protein,P < 0.01), group C (3.2 ± 0.1IU/mg protein,P < 0.01), and group D (3.3 ± 0.2IU/mg protein,P < 0.01) than in group A (47 ± 6.7 IU/mg protein). The myocardial CK activity of group E was not significantly different from that of group A, but was significantly higher than in groups B, C, and D (P < 0.01). In isolated perfused rat hearts, both the coronary endothelium and myocardium are damaged by repeated episodes of ischemia and reperfusion. However, the coronary endothelium is more resistant to such damage than is the myocardium.     

The mechanism of protection from 5 (N-ethyl-N-isopropyl)amiloride differs from that of ischemic preconditioning in rabbit heart

We investigated the effects of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) on infarction in isolated rabbit hearts and cardiomyocytes. Thirty min of regional ischemia caused 29.6±2.8% of the risk zone to infarct in untreated Krebs buffer-perfused hearts. Treatment with EIPA (1 M) for 20 min starting either 15 min before ischemia or 15 min after the onset of ischemia significantly reduced infarction to 5.4±2.0% and 7.0±1.0%, respectively (p<0.01 versus untreated hearts). In both cases salvage was very similar to that seen with ischemic preconditioning (PC) (7.1±1.5% infarction). Unlike the case with ischemic preconditioning, however, protection from EIPA was not blocked by 50M polymyxin B, a PKC inhibitor, or 1M glibenclamide, a K_ATP channel blocker. Forty-five min of regional ischemia caused 51.0±2.9% infarction in untreated hearts. Ischemic preconditioning reduced infarction to 23.4±3.1% (p<0.001 versus untreated hearts). In these hearts with longer periods of ischemia pretreatment with EIPA reduced infarction similarly to 28.8±2.1% (p<0.01 versus untreated hearts). However, when EIPA was combined with ischemic PC, no further reduction in infarction was seen (23.8±3.5% infarction). To further elucidate the mechanism of EIPA's cardioprotective effect, this agent was also examined in isolated rabbit cardiomyocytes. Preconditioning caused a delay of about 30 min in the progressive increase in osmotic fragility that occurs during simulated ischemia. In contrast, EIPA had no effect on the time course of ischemia-induced osmotic fragility. Furthermore, EIPA treatment did not alter the salutary effect of ischemic preconditioning when the two were combined in this model. We conclude that Na⁺/H⁺ exchange inhibition limits myocardial infarction in the isolated rabbit heart by a mechanism which is quite different from that of ischemic preconditioning. Despite the apparently divergent mechanisms, EIPA's cardioprotective effect could not be added to that of ischemic or metabolic preconditioning in these models.This study was supported in part by grants from the National Institute of Health Heart, Lung and Blood Institute, HL-20648 and HL-50688     

Energy metabolism,intracellular Na+ and contractile function in isolated pig and rat hearts during cardioplegic ischemia and reperfusion:23Na- and31P-NMR studies

The purpose of the study was to compare the role of Na ions in the damage caused by cardioplegic ischemia in fast (rat) and slow (pig) hearts. Changes in intracellular Na⁺ (Na⁺ _i), high energy phosphates, and contractile function were assessed during ischemia (36°C) and reperfusion in KCl-arrested perfused hearts using³¹P-NMR and shift reagent (DyTTHA^3–)-aided²³Na-NMR spectroscopy. In the pig hearts the rates of decrease of phosphocreatine (PCr), ATP and intracellular pH (pHi) were 3–4 times slower than the rates observed in the rat hearts. In the pig hearts PCr was observable (10%) during first 80 min of the ischemic period (90 min). Comparable decreases in ATP (32.0±6 vs. 38±15% of initial) and pH_i, (to 6.14±0.06 vs. 6.10±0.15) observed after 90 and 20 min ischemia in pig and rat hearts, respectively, were associated with a smaller Na⁺ _i increase in the pig hearts (to 175±31%) than in the rat hearts (to 368±62%). This Na⁺ increase in the rat hearts preceded development of ischemic contracture (41±6 mmHg at 23.6±0.7 min) while no contracture was observed in pig hearts. Reperfusion of the rat hearts (at 30 min ischemia) was followed by partial recovery of PCr (44±3%) and Na⁺ _i (234±69%) and poorer recovery of the pressure-rate product (PRP, 9±4%) and end-diastolic pressure (EDP, 72±5 mm Hg) compared to the pig hearts (PCr, 106±25%; Na⁺ _i, 82±17%; PRP, 24±3%; EDP, 4.6±2.5 mmHg). The loss of function in the pig hearts was reversed by increasing Ca⁺⁺ in the perfusate from 1 to 2.3 mM and resulted in a rise in both PRP and oxygen consumption rate, V(O₂), from 24±3.3 to 64.5±5.8% and from 55±10 to 74±10% of the control levels, respectively. The PRP/V(O₂) ratio was halved in the post-ischemic pig hearts and returned to the pre-ischemic level following Ca⁺⁺ stimulation. It is suggested that the higher stability of Na⁺ homeostasis to ischemic stress in the pig heart may result from: 1) a lower ratio of the rate of ATP hydrolysis to glycolytic ATP production; 2) differences in the kinetic properties of the Na⁺ transporters. Reduced Na⁺ accumulation during ischemia and reperfusion is benefical for metabolic and functional preservation of cardiomyocytes.     

Blockade of the Na+-Ca2+ exchanger is more efficient than blockade of the Na+-H+ exchanger for protection of the myocardium from lethal reperfusion injury

Since the Na⁺-H⁺ exchanger (NHE) is not the only pathway of Na⁺ influx into cardiomyocytes during ischemia/reperfusion, we hypothesized that blockade of Na⁺-Ca²⁺ exchanger (NCX) may be a more efficient strategy than is NHE inhibition for protecting the myocardium from infarction. To test this hypothesis, we compared KB-R7943 (KBR), a novel selective NCX blocker, with cariporide, a selective NHE blocker, with regard to their protective effects against infarction. In isolated rabbit hearts, infarction was induced by 30-min global ischemia/2-h reperfusion, and infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk (%IS/AR). Hearts received no drugs, or were infused with cariporide (1 M) for 10 min or KBR (0.3 or 10 M) for 5 min before ischemia or after the onset of reperfusion. Protein level of NCX was assessed by Western blotting. Cariporide infusion before ischemia significantly reduced %IS/AR from 63.9 ± 2.9% to 20.2 ± 3.0%, but its infusion upon reperfusion failed to achieve a significant protection (%IS/AR = 53.8 ± 4.6%). In contrast, KBR infusion similarly reduced infarct size both when infused before ischemia (%IS/AR = 33.3 ± 6.3% and 21.9 ± 4.7% by 0.3 and 10 M KBR, respectively) and when infused for only 5 min after reperfusion (%IS/AR = 35.3 ± 7.1% and 31.5 ± 2.1% by 0.3 and 10 M KBR, respectively). Protein levels of NCX after 30-min ischemia and 30-min ischemia/30-min reperfusion were similar to baseline values in both untreated controls and hearts treated with 0.3 M KBR upon reperfusion. These results suggest that lethal reperfusion injury is more efficiently suppressed by blockade of the NCX than by blockade of the NHE.     

Evidence for a reversible oxygen radical-mediated component of reperfusion injury: reduction by recombinant human superoxide dismutase administered at the time of reflow

It has been suggested that the beneficial effects of reperfusing ischemic myocardium might be in part reversed by the occurrence of "reperfusion injury." One possible mechanism could be the generation of oxygen free radicals. Superoxide dismutase enzymatically scavenges superoxide radicals by dismutation to hydrogen peroxide. This study tested the hypothesis that administration of recombinant human superoxide dismutase (h-SOD) at the time of reflow after a period of prolonged global ischemia would result in improved recovery of myocardial metabolism and function by preventing or reducing a potentially harmful component of reperfusion. We also sought to determine whether catalase, an enzymatic scavenger of hydrogen peroxide, was a necessary addition for optimal benefit. Langendorff perfused rabbit hearts were subjected to 30 min of normothermic (37 degrees C) total global ischemia. At the moment of reperfusion, 12 control hearts received a 10 ml bolus of normal perfusate followed by 15 min of reperfusion with normal perfusate (group I), 12 hearts received 60,000 IU of h-SOD as a bolus followed by a continuous infusion of 100 IU/ml for 15 min (group II), and 12 hearts received 60,000 IU of h-SOD and 60,000 IU of catalase as a bolus followed by 100 IU/ml of both enzymes for 15 min (group III). Myocardial ATP and phosphocreatine (PCr) content and intracellular pH during ischemia and reperfusion were continuously monitored with 31P nuclear magnetic resonance (NMR) spectroscopy. During 30 min of normothermic global ischemia intracellular pH dropped from 7.11-7.18 to 5.58-5.80 in all three groups of hearts. Likewise myocardial PCr content fell rapidly to 7% to 8% and ATP fell more slowly to 29% to 36% of preischemic control content. After 45 min of reperfusion PCr recovered to 65 +/- 5% of control in untreated (group I) hearts compared with 89 +/- 8% in h-SOD-treated (group II) hearts (p less than .01 vs group I) and with 83 +/- 6% of control in h-SOD/catalase-treated (group III) hearts (p less than .05 vs group I). Recovery of isovolumic left ventricular developed pressure was 68 +/- 5% of control in h-SOD-treated (group II) hearts and 66 +/- 6% of control in h-SOD/catalase-treated (group III) hearts after 45 min of reflow, compared with 48 +/- 6% of control in untreated (group I) hearts (p less than .005 for groups II and III vs group I). The NMR data confirmed equal depletion of ATP and PCr content in all three groups of hearts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Beneficial effects of adenosine triphosphate-sensitive K+ channel opener on liver ischemia/reperfusion injury

AIM: To investigate the effect of diazoxide administration on liver ischemia/reperfusion injury.METHODS: Wistar male rats underwent partial liver ischemia performed by clamping the pedicle from the medium and left anterior lateral segments for 1 h under mechanical ventilation. They were divided into 3 groups: Control Group, rats submitted to liver manipulation, Saline Group, rats received saline, and Diazoxide Group, rats received intravenous injection diazoxide (3.5 mg/kg) 15 min before liver reperfusion. 4 h and 24 h after reperfusion, blood was collected for determination of aspartate transaminase (AST), alanine transaminase (ALT), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), nitrite/nitrate, creatinine and tumor growth factor-β1 (TGF-β1). Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidase (MPO) were also determined.RESULTS: Four hours after reperfusion the diazoxide group presented with significant reduction of AST (2009 ± 257 U/L vs 3523 ± 424 U/L, P = 0.005); ALT (1794 ± 295 U/L vs 3316 ± 413 U/L, P = 0.005); TNF-α (17 ± 9 pg/mL vs 152 ± 43 pg/mL, P = 0.013; IL-6 (62 ± 18 pg/mL vs 281 ± 92 pg/mL); IL-10 (40 ± 9 pg/mL vs 78 ± 10 pg/mL P = 0.03), and nitrite/nitrate (3.8 ± 0.9 μmol/L vs 10.2 ± 2.4 μmol/L, P = 0.025) when compared to the saline group. A significant reduction in liver mitochondrial dysfunction was observed in the diazoxide group compared to the saline group (P < 0.05). No differences in liver MDA content, serum creatinine, pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion the diazoxide group showed a reduction of AST (495 ± 78 U/L vs 978 ± 192 U/L, P = 0.032); ALT (335 ± 59 U/L vs 742 ± 182 U/L, P = 0.048), and TGF-β1 (11 ± 1 ng/mL vs 17 ± 0.5 ng/mL, P = 0.004) serum levels when compared to the saline group. The control group did not present alterations when compared to the diazoxide and saline groups.CONCLUSION: Diazoxide maintains liver mitochondrial function, increases liver tolerance to ischemia/reperfusion injury, and reduces the systemic inflammatory response. These effects require further evaluation for using in a clinical setting.     

Captopril improves recovery of adenosine triphosphate during reperfusion of the ischemic isolated rat heart; a 31-phosphorusnuclear magnetic resonance study

Summary The effect of captopril on energy-rich phosphates and pH during normothermic ischemic arrest, hypothermic cardioplegic arrest and subsequent reperfusion was investigated in the isolated rat heart using 31P-nuclear magnetic resonance. The hearts remained in the probe during all perfusion procedures and captopril (80 ml·l^–1) treatment was started directly after cannulation.After normothermic ischemic arrest (15 min), the ATP content of captopril-treated hearts was not significantly different from that of untreated hearts (53±9% and 52±8%, respectively). Accumulation of inorganic phosphate at the end of ischemia was significantly less in treated hearts, suggesting a higher end-ischemic nucleotide content in treated hearts.Hypothermic cardioplegic arrest (St. Thomas' Hospital solution, 4°C) lasted for 3 h at 10°C. Adenosine triphosphate in treated hearts was significantly lower at the end of ischemia; 36±6% compared to 53±9% for untreated hearts.Adenosine triphosphate in untreated hearts recovered to 76±9% after normothermic ischemia and to 72±7% after hypothermic ischemia at the end of 30 min reperfusion. Captopril significantly improved adenosine triphosphate recovery in both treated groups; 89±4% after normothermic and 83±4% hypothermic ischemia.We conclude that captopril has a beneficial effect on recovery of adenosine triphosphate both after normothermic and after hypothermic ischemia.     

Impact of intestinal ischemia/reperfusion and lymph drainage on distant organs in rats

AIM:To investigate the impact of intestinal ischemia/reperfusion(I/R) injury and lymph drainage on distant organs in rats.METHODS:Thirty-two Sprague-Dawley male rats,weighing 280-320 g,were randomly divided into blank,sham,I/R,and ischemia/reperfusion and drainage(I/R + D) groups(n = 8).All rats were subjected to 60 min ischemia by clamping the superior mesenteric artery,followed by 120 min reperfusion.The rats in the I/R + D group received intestinal lymph drainage for 180 min.In the sham group,the abdominal cavity was opened for 180 min,but the rats received no treatment.The blank group served as a normal and untreated control.A chromogenic limulus assay kit was used for quantita-tive detection of serum endotoxin.The serum concentrations of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1β,soluble cell adhesion molecules(sICAM-1),and high mobility group protein box 1(HMGB1) were determined with an enzyme-linked immunosorbent assay kit.Histological evaluations of the intestine,liver,kidney,and lung were performed by hematoxylin and eosin staining and immunohistochemistry.HMGB1 protein expression was assayed by western blot analysis.RESULTS:The serum levels of endotoxin and HMGB1 in the I/R and I/R + D groups were significantly higher than those in the sham group(endotoxin,I/R and I/R + D vs sham:0.033 ± 0.004 EU/mL,0.024 ± 0.003 EU/mL vs 0.017 ± 0.009 EU/mL,respectively,P 0.05;HMGB1,I/R and I/R + D vs sham:5.473 ± 0.963 EU/mL,4.906 ± 0.552 EU/mL vs 0.476 ± 0.406 EU/mL,respectively,P 0.05).In addition,endotoxin and HMGB1 were significantly lower in the I/R + D group compared to the I/R group(P 0.05).The serum inflammatory factors IL-6,IL-1β,and sICAM-1 in the I/R and I/R + D groups were significantly higher than those in the sham group(IL-6,I/R and I/R + D vs sham:41.773 ± 9.753 pg/mL,19.204 ± 4.136 pg/mL vs 11.566 ± 2.973 pg/mL,respectively,P 0.05;IL-1β,I/R and I/R + D vs sham:144.646 ± 29.378 pg/mL,65.829 ± 10.888 pg/mL vs 38.178 ± 7.157 pg/mL,respectively,P 0.05;sICAM-1,I/R and I/R + D vs sham:97.360 ± 12.714 ng/mL,48.401 ± 6.547 ng/mL vs 33.073 ± 5.957 ng/mL,respectively;P 0.05).The serum TNF-α in the I/R group were significantly higher than in the sham group(45.863 ± 11.553 pg/mL vs 18.863 ± 6.679 pg/mL,respectively,P 0.05).These factors were significantly lower in the I/R + D group compared to the I/R group(P 0.05).The HMGB1 immunohistochemical staining results showed no staining or apparent injury in the blank group,and slight staining at the top of the microvillus was detected in the sham group.In the I/R group,both the top of villi and the basement membrane were stained for HMGB1 in most areas,and injury in the I/R + D group was less than that in the I/R group.HMGB1 expression in the liver,kidney,and lung of rats in the I/R + D group was significantly lower than the rats in the I/R group(P 0.05).CONCLUSION:Lymph drainage could block the "gutlymph" pathway,improve intestinal barrier function,and attenuate distant organ injury incurred by intestinal I/R.     

The Protective Effect of Heat Stress Against Reperfusion Arrhythmias in the Rat

Heat stress provides protection against mechanical dysfunction and myocardial necrosis after prolonged ischemia. We have investigated whether this protection extends to reperfusion arrhythmias occurring after a short (non-lethal) ischemic insult. Anaesthetized open chest rats were subjected to a 5-min occlusion of the left coronary artery. The incidence and duration of reperfusion arrhythmias and the duration of sinus rhythm were assessed in the first 5 min of reperfusion. Prior heat stress led to a reduction in the incidence (100-63%, P 0.05) and duration (66.2 ± 15.8 to 9.4 ± 2.9s, P 0.05) of ventricular tachycardia and a non-significant reduction in the incidence (76-50%) and duration (74.3 ± 23.4 to 42.9 ± 24.4s, P = 0.09) of ventricular fibrillation. This resulted in a significant increase in the duration of sinus rhythm (142.1 ± 27.6 to 216.7 ± 24.8s, P 0.05) and reduction in arrhythmia score (P 0.05) in heat stressed rats compared with controls. This protection against reperfusion arrhythmias was associated with a two-fold increase in endogenous catalase activity and expression of the inducible heat stress protein HSP 70. Inhibition of catalase with pre-administered 3-amino triazole resulted in a paradoxical protection in both sham and heat stress hearts. We conclude that heat stress leads to protection against reperfusion arrhythmias; however, we have been unable to resolve whether the changes in catalase activity of HSP expression are the mediators of the demonstrated cardioprotection.