Allopurinol-Enhanced Postischemic Recovery in the Isolated Pat Heart Involves Repletion of High-Energy Phosphates

The effects of allopurinol (AP) on functional and metabolic recovery of the isolated rat heart after global ischemia were studied. Hearts were subjected to aerobic perfusion (30 min), cardioplegic infusion (5 min), normothermic ischemia (37 min), and reperfusion (50 min) which was started with secondary cardioplegic infusion (10 min). AP was injected into rats (44 mg/kg body wt ip 2 h before heart excision) and added to cardioplegic solution (2 mM) prior and after ischemia. AP treatment significantly improved postischemic recovery of the function and reduced the leakage of lactate dehydrogenase from reperfused hearts. These beneficial effects were accompanied by a better preservation of tissue content of ATP, the total adenine nucleotides, phosphocreatine, and the total creatine at the end of reperfusion. Inhibition of xanthine oxidase by AP substantially decreased pre- and postischemic release of xanthine and uric acid and increased postischemic release of hypoxanthine into the coronary effluent. Despite this, AP treated hearts did not exhibit a reduction in hydroxyl radical adduct formation in the effluents at reperfusion assessed by the spin-trap measurements. The results suggest that AP may protect the heart from ischemia/reperfusion injury due to enhanced energy provision rather than by prevention of oxygen-derived free radical formation.     

An increase in the redox state during reperfusion contributes to the cardioprotective effect of GIK solution

This study aimed at determining whether glucose-insulin-potassium (GIK) solutions modify the NADH/NAD(+) ratio during postischemic reperfusion and whether their cardioprotective effect can be attributed to this change in part through reduction of the mitochondrial reactive oxygen species (ROS) production. The hearts of 72 rats were perfused with a buffer containing glucose (5.5 mM) and hexanoate (0.5 mM). They were maintained in normoxia for 30 min and then subjected to low-flow ischemia (0.5% of the preischemic coronary flow for 20 min) followed by reperfusion (45 min). From the beginning of ischemia, the perfusate was subjected to various changes: enrichment with GIK solution, enrichment with lactate (2 mM), enrichment with pyruvate (2 mM), enrichment with pyruvate (2 mM) plus ethanol (2 mM), or no change for the control group. Left ventricular developed pressure, heart rate, coronary flow, and oxygen consumption were monitored throughout. The lactate/pyruvate ratio of the coronary effluent, known to reflect the cytosolic NADH/NAD(+) ratio and the fructose-6-phosphate/dihydroxyacetone-phosphate (F6P/DHAP) ratio of the reperfused myocardium, were evaluated. Mitochondrial ROS production was also estimated. The GIK solution improved the recovery of mechanical function during reperfusion. This was associated with an enhanced cytosolic NADH/NAD(+) ratio and reduced mitochondrial ROS production. The cardioprotection was also observed when the hearts were perfused with fluids known to increase the cytosolic NADH/NAD(+) ratio (lactate, pyruvate plus ethanol) compared with the other fluids (control and pyruvate groups). The hearts with a high mechanical recovery also displayed a low F6P/DHAP ratio, suggesting that an accelerated glycolysis rate may be responsible for increased cytosolic NADH production. In conclusion, the cardioprotection induced by GIK solutions could occur through an increase in the cytosolic NADH/NAD(+) ratio, leading to a decrease in mitochondrial ROS production.     

The role of inhibition of NO formation in the metabolic recovery of ischemic rat heart by apelin-12

The effects of apelin-12, a 12 amino acid peptide (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A-12), on recovery of energy metabolism and cardiac function have been studied in isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose and subjected to global ischemia and reperfusion. Infusion of 140 μM A-12 before ischemia enhanced myocardial ATP, the total pool of adenine nucleotides (ΣAN = ATP+ADP+AMP) and the energy charge of cardiomyocytes ((ATP + 0.5ADP)/ΣAN) at the end of reperfusion compared with control (KB infusion) and decreased lactate content and lactate/pyruvate ratio in the reperfused myocardium up to the initial values. This was accompanied by improved recovery of coronary flow and cardiac function. Co-administration of A-12 and 100 μM L-NAME (an inhibitor of NO synthases) significantly attenuated the A-12 effects on metabolic and functional recovery of reperfused hearts. These results indicate involvement of NO in mechanisms of cardioprotection that are tightly associated with recovery of energy metabolism in the postischemic heart.     

Temporary blockade of contractility during reperfusion elicits a cardioprotective effect of the p38 MAP kinase inhibitor SB-203580

p38 MAP kinase activation is known to be deleterious not only to mitochondria but also to contractile function. Therefore, p38 MAP kinase inhibition therapy represents a promising approach in preventing reperfusion injury in the heart. However, reversal of p38 MAP kinase-mediated contractile dysfunction may disrupt the fragile sarcolemma of ischemic-reperfused myocytes. We, therefore, hypothesized that the beneficial effect of p38 MAP kinase inhibition during reperfusion can be enhanced when contractility is simultaneously blocked. Isolated and perfused rat hearts were paced at 330 rpm and subjected to 20 min of ischemia followed by reperfusion. p38 MAP kinase was activated after ischemia and early during reperfusion (<30 min). Treatment with the p38 MAP kinase inhibitor SB-203580 (10 microM) for 30 min during reperfusion, but not the c-Jun NH(2)-terminal kinase inhibitor SP-600125 (10 microM), improved contractility but increased creatine kinase release and infarct size. Cotreatment with SB-203580 and the contractile blocker 2,3-butanedione monoxime (BDM, 20 mM) or the ultra-short-acting beta-blocker esmorol (0.15 mM) for the first 30 min during reperfusion significantly reduced creatine kinase release and infarct size. In vitro mitochondrial ATP generation and myocardial ATP content were significantly increased in the heart cotreated with SB-203580 and BDM during reperfusion. Dystrophin was translocated from the sarcolemma during ischemia and reperfusion. SB-203580 increased accumulation of Evans blue dye in myocytes depleted of sarcolemmal dystrophin during reperfusion, whereas cotreatment with BDM facilitated restoration of sarcolemmal dystrophin and mitigated sarcolemmal damage after withdrawal of BDM. These results suggest that treatment with SB-203580 during reperfusion aggravates myocyte necrosis but concomitant blockade of contractile force unmasks cardioprotective effects of SB-203580.     

Ischemic preconditioning-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contraction-induced myocardial injury

Dystrophin is an integral membrane protein involved in the stabilization of the sarcolemmal membrane in cardiac muscle. We hypothesized that the loss of membrane dystrophin during ischemia and reperfusion is responsible for contractile force-induced myocardial injury and that cardioprotection afforded by ischemic preconditioning (IPC) is related to the preservation of membrane dystrophin. Isolated and perfused rat hearts were subjected to 30 min of global ischemia, followed by reperfusion with or without the contractile blocker 2,3-butanedione monoxime (BDM). IPC was introduced by three cycles of 5-min ischemia and 5-min reperfusion before the global ischemia. Dystrophin was distributed exclusively in the membrane of myocytes in the normally perfused heart but was redistributed to the myofibril fraction after 30 min of ischemia and was lost from both of these compartments during reperfusion in the presence or absence of BDM. The loss of dystrophin preceded uptake of the membrane-impermeable Evans blue dye by myocytes that occurred after the withdrawal of BDM and was associated with creatine kinase release and the development of contracture. Although IPC did not alter the redistribution of membrane dystrophin induced by 30 min of ischemia, it facilitated the restoration of membrane dystrophin during reperfusion. Also, myocyte necrosis was not observed when BDM was withdrawn after complete restoration of membrane dystrophin. These results demonstrate that IPC-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contractile force-induced myocardial injury and suggest that the cardioprotection conferred by IPC can be enhanced by the temporary blockade of contractile activity until restoration of membrane dystrophin during reperfusion.     

Glycolysis and glucose oxidation during reperfusion of ischemic hearts from diabetic rats

Stimulationg of glucose oxidation by dichloroacetate (DCA) treatment is beneficial during recovery of ischemic hearts from non-diabetic rats. We therfore determined whether DCA treatment of diabetic rat hearts (in which glucose use is extremely low), increases recovery of function of hearts reperfused following ischemia. Isolated working hearts from 6 week streptozotocindiabetic rats were perfused with 11 mM [2-³H/U-¹⁴C]glucose, 1.2 mM palmitate, 20 μU/ml insulin, and subjected to 30 min of no flow ischemia followed by 60 min reperfusion. Heart function (expressed as the product of heart rate and peak systolic pressure), prior to ischemia, was depressed in diabetic hearts compared to controls (HR × PSP × 10^?3 was 18.2 ± 1 and 24.3 ± 1 beats/mm Hg/min in diabetic and control hearts respectively) but recover to pre-ischemic levels following ischemia, whereas recovery of control of control hearts was significantly decreased (17.8 ± 1 and 11.9 ± 3 beats/mm Hg/min in diabetic and control hearts respectively). This enhanced recovery of diabetic rat hearts occurred even though glucose oxidation during reperfusion was significantly reduced as compared to controls (39 ± 6 and 208 ± 42 nmol/min/g dry wt, in diabetic and control hearts respectively). Glycolytic rate (³G₂O production) during reperfusion were similar in diabetic and control hearts (1623 ± 359 and 2071 ± 288 nmol/min/g dry wt, respectively). If DCA (1 mM) was added at reperfusion, hearts from control animals exhibited a significant improvement in function (HR × PSP × 10^? recovered to 20 ± 4 beats/mm Hg/min) that was accompanied by a 4-fold increase in glucose oxidation (from 208 ± 42 to 753 ± 111 nmol/min/g dry wt). DCA was without effect on functional recovery of diabetic rat hearts during reperfusion but did significantly increase glucose oxidation from 39 ± 6 to 179 ± 44 nmol/min/g dry wt). These data suggests that, unlike control hearts, low glucose oxidation rates are not an important factor in reperfusion recovery of previouskly ischemic diabetic rat hearts.     

In vitro study of functional parameters and cardiac arrhythmia in localized ischemia followed by reperfusion in two rat strains (Sprague Dawley and Wistar)

The cardiac parameters of two rat strains [Wistar (W) and Sprague Dawley (SD)] were compared during Langendorff perfusion. The values of coronary flow, heart rate, amplitude of contraction and the incidence of arrhythmias were studied during three 10 minutes periods: perfusion, ischemia by coronary artery occlusion and reperfusion. The values of heart rate and coronary flow of SD hearts are always higher than those of W hearts whatever the potassium (K+) concentration of perfusate (5.9 or 3 mM). Furthermore, with a high K+ perfusate (5.9 mM) and during ischemia. W rat hearts showed ventricular tachycardia periods which are never observed in SD rat hearts. It is concluded that W rat hearts present a higher sensitivity to the development of dysfunction than SD rat hearts.     

间歇性低压低氧方式对发育大鼠心脏缺血/再灌注损伤的影响

本研究旨在探讨两种不同形式的间歇性低压低氧（intermittent hypobaric hypoxia,IHH）对发育大鼠心脏缺血,再灌注损伤的影响。雄性Sprague-Dawley（SD）新生大鼠72只,随机分为三组：对照组、IHH3000in组（IHH3000）、IHH5000m组（IHH5000）。低氧组大鼠出生后立即于低压氧舱分别接受28d、42d和56d（海拔5000m、每天6h：海拔3000m、每天5h）的低压低氧处理。应用Langendorff离体心脏灌流技术,给予心脏缺血（停灌30min）／再灌注（复灌60min）处理,分别在缺血前5min及复灌后l、5、10、20、30、60min记录心功能和冠状动脉流量变化,并测定乳酸脱氢酶（1actate dehydrogenase,LDH）活性。实验结束时测定心脏重量。结果显示：（1）IHH3000组大鼠体重增长与对照组无明显差异;IHH5000组大鼠体重增长明显慢于对照组及IHH3000组大鼠（P〈0．01）。（2）IHH3000组人鼠表现明显的心脏保护效应。与对照组相比较,在心脏停灌,再灌注60min时,心功能（LVDP、±LVdp／drmax）恢复增强（P〈0．05）、LDH活性降低（P〈0．05）、冠状动脉流量增多（P〈0．05）;心脏重量与对照组大鼠无差异;IHH42d处理的大鼠心功能恢复明显好于IHH28d处理的大鼠（P〈0．05）。（3）IHH5000组大鼠表现出明显的心脏损伤效应,各项心功能指标（LVDP、±LVdp／dtmax）的恢复均低于对照组（P〈0．05）,复灌过程中LDH活性明显高于相应对照组（P〈0．05）,右心室重量明显高于对照组大鼠（P〈0．05）。结果表明,适当的IHH增强发育大鼠心脏对缺血,再灌注损伤的抵抗能力;间歇性低氧方式是影响其心脏保护作用的重要因素。     

Effects of L-carnitine on mechanical recovery of isolated rat hearts in relation to the perfusion with glucose and palmitate

The purpose of this study was to investigate the effects of L-carnitine on the hemodynamic parameters of Langendorff hearts. Isolated rat hearts were perfused with various solutions containing high or low concentrations of fatty acids, additional glucose or no glucose, and L-carnitine or no L-carnitine. The most interesting part of the experiments was the behaviour of the hearts in the reperfusion period after no-flow ischemia of 20 min. The results were: (1) With glucose and high fatty acid concentrations the hearts showed an improved recovery of the left ventricular functions in the reperfusion period compared with low fatty acid concentrations. Without glucose the left ventricular pressure is much lower in the reperfusion period. (2) Addition of L-carnitine improved the recovery of the ischemically damaged hearts. This improvement is especially impressive at low fatty acid concentrations. L-carnitine addition at high fatty acid concentrations but without glucose strongly improved reperfusion behaviour. (3) The coronary flow is increased by 2 experimental conditions: (i) perfusion at low levels of fatty acids, carnitine and with glucose and (ii) high levels of fatty acids and carnitine but without glucose. These findings suggest that supplementation of L-carnitine has a beneficial effect on the isolated heart under various conditions, and possibly on specific human heart diseases.     

Energy metabolism and mechanical recovery after cardioplegia in moderately hypertrophiedrats

It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions.Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25øC with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by³¹ P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia.Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8 ± 4.9 mol/g dry wt in the control group and to 46.3 ± 2.8 mol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts.Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.     

Protection of the reperfused heart by L-propionylcarnitine.

The effects of L-propionylcarnitine on mechanical function, creatine phosphate and ATP content, and lactate dehydrogenase leakage were studied in isolated perfused rat hearts exposed to global no-flow ischemia for 30 min followed by reperfusion for 20 min. Five and 10 mM L-propionylcarnitine resulted in a 100% recovery of left ventricular-developed pressure, whereas the recovery was only 40% in the hearts perfused without this agent. Ischemia-reperfusion caused a 85% loss of creatine phosphate and a 77% loss of ATP, which was prevented by 10 mM L-propionylcarnitine. Five millimolar L-propionylcarnitine protected the heart from the loss of creatine phosphate but not from the loss of ATP. Ten millimolar L-propionylcarnitine failed to improve the postischemic left ventricular-developed pressure, when it was added to the perfusate only after ischemia. L-propionylcarnitine alleviated the decrease of coronary flow in the reperfused hearts. Lactate dehydrogenase leakage was aggravated in the beginning of the reperfusion period by 10 mM L-propionylcarnitine. This adverse effect was, however, transient. L-Propionylcarnitine provides protection for the postischemic reperfused heart in a dose-dependent manner. The optimal time for administration is before the ischemic insult. High doses of this compound may perturb cell membrane integrity. Moreover, the present data point to an intracellular, metabolic, and perhaps anaplerotic mechanism of action of L-propionylcarnitine in cardiac ischemia-reperfusion injury.     

Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron - providing protection to the ischemic heart via preconditioning (PC). PC was accomplished by subjecting isolated rat hearts to three episodes of 2 min ischemia separated by 3 min of reperfusion. Prolonged ischemia followed the PC phase. PC hearts (group I) were compared to hearts subjected to normal perfusion (group II, no ischemia) and to ischemia without PC (group III). Group I showed a marked improvement in the recovery of hemodynamic function vs. group III. Biochemical parameters further substantiated the PC protection provided to group I against prolonged ischemia. Correspondingly, group I presented markedly lower re-distribution and mobilization of iron and copper into the coronary flow, following prolonged ischemia, as evinced from the decrease in total levels, and in the 'free' fraction of iron and copper. During the PC phase no loss of cardiac function was observed. A small wave of re-distribution and mobilization of iron (typically less than 4-8% of the value of 35 min ischemia) was recorded. The cellular content of ferritin (Ft) measured in the heart was significantly higher in group I than in group III (0.90 and 0.54 microg/mg, respectively). Also, iron-saturation of Ft was significantly lower for PC hearts, compared to both groups II and III (0.22 vs. 0.32 and 0.31 microg/mg, for 35 min ischemia, respectively). These findings are in accord with the proposal that intracellular re-distribution and mobilization of small levels of iron, during PC, cause rapid accumulation of ferritin - the major iron-storage protein. It is proposed that iron play a dual role: (i) It serves as a signaling pathway for the accumulation of Ft following the PC phase. This iron is not involved in cardiac injury, but rather prepares the heart against future high levels of 'free' iron, thus reducing the degree of myocardial damage after prolonged ischemia. (ii) High levels of iron (and copper) are mobilized following prolonged ischemia and cause tissue damage.     

Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release

We investigated the contribution of dexamethasone treatment on the recovery of postischemic cardiac function and the development of reperfusion-induced arrhythmias in ischemic/reperfused isolated rat hearts. Rats were treated with 2 mg/kg of intraperitoneal injection of dexamethasone, and 24 hours later, hearts were isolated according to the 'working' mode, perfused, and subjected to 30 min global ischemia followed by 120 min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 60 min and 120 min reperfusion, 2 mg/kg of dexamethasone significantly improved the postischemic recovery of aortic flow and left ventricular developed pressure from their control values of 10.7 +/- 0.3 ml/min and 10.5 +/- 0.3 kPa to 22.2 +/- 0.3 ml/min (p < 0.05) and 14.3 +/- 0.5 kPa (p < 0.05), 19.3 +/- 0.3 ml/min (p < 0.05) and 12.3 +/- 0.5 kPa (p < 0.05), respectively. Heart rate and coronary flow did not show a significant change in postischemic recovery after 60 or 120 min reperfusion. In rats treated with 0.5 mg/kg of actinomycin D injected i.v., one hour before the dexamethasone injection, suppressed the dexamethasone-induced cardiac protection. Electrocardiograms were monitored to determine the incidence of reperfusion-induced ventricular fibrillation. Dexamethasone pretreatment significantly reduces the occurrence of ventricular fibrillation. Cytochrome c release was also observed in the cytoplasm. The results suggest that the inhibition of cytochrome c release is involved in the dexamethasone-induced cardiac protection.     

Trolox C, a lipid-soluble membrane protective agent, attenuates myocardial injury from ischemia and reperfusion

The lipophilic antioxidant Trolox C, a vitamin E analog, was administered to isolated, buffer-perfused rabbit hearts subjected to 25 min of global stop-flow ischemia and 30 min of reperfusion. In six hearts, Trolox C (200 microM) was infused for 15 min immediately prior to ischemia and for the first 15 min of reperfusion. Six control hearts received only vehicle. Gas chromatography analysis confirmed that effective myocardial levels of Trolox were attained. At 30 min reperfusion, the recovery of left ventricular developed pressure was 56 +/- 3% of baseline in control hearts versus 70 +/- 4% in Trolox-treated hearts (p < .01). There was also significant improvement in recovery of Trolox-treated hearts in diastolic pressure and both maximum and minimum values of the first derivative of left ventricular pressure (dP/dt). Creatine phosphokinase release into the coronary effluent at 30 min of reperfusion was 16.5 +/- 8.4 IU/min in untreated and 6.3 +/- 1.0 IU/min (p < .05) in Trolox-treated hearts. Thus Trolox C, a lipophilic antioxidant, attenuated myocardial injury during stop-flow ischemia and reperfusion.     

Effects of genetic deletion of angiotensin-(1-7) receptor Mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart

In this study we investigated the role of Mas on cardiac function during ischemia/reperfusion in isolated perfused mouse heart. Following a stabilization period of 30 min, hearts from WT and Mas KO mice were subjected to global ischemia. After 20 min of ischemia, the flow was restarted and the hearts were reperfused for 30 min. An additional group of WT mice was perfused with solution containing the Ang-(1-7) receptor Mas antagonist A-779. Isolated heart of Mas KO and WT treated with A-779 presented an increase in the perfusion pressure in the baseline period. This difference increased with 5 min of reperfusion reaching similar values to baseline period at the end of the reperfusion. Isolated hearts of Mas KO and WT treated with A-779 also presented a decreased systolic tension, +/-dT/dt, and HR. Upon global ischemia WT hearts showed a significant decrease in systolic tension and an increase in diastolic tension. During reperfusion an increase in systolic and diastolic tension was observed in WT mice. Deletion or blockade of Mas markedly attenuated these changes in isolated hearts. These results indicate that Mas plays an important role in cardiac function during ischemia/reperfusion which is in keeping with the cardiac and coronary effects previously described for Ang-(1-7).     

The mitochondrial permeability transition pore and the Ca2+-activated K+ channel contribute to the cardioprotection conferred by tumor necrosis factor-alpha

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) is known to trigger cardioprotection and it can activate multiple downstream signaling cascades. However, it is not known whether the mitochondrial permeability transition pore and the Ca(2+)-activated K(+) channel (K(Ca) channel) are involved in the TNF-alpha-induced cardioprotection. In the present study, we examined whether TNF-alpha inhibits pore opening and activates the K(Ca) channel in the cardioprotection. In isolated rat hearts subjected to 30 min of regional ischemia and 120 min of reperfusion, pretreatment with 10 U/ml TNF-alpha for 7 min followed by 10 min washout improved the recovery of rate-pressure product (RPP=left ventricular developed pressure x heart rate) and coronary flow (CF) during reperfusion, and reduced the infarct size and release of lactate dehydrogenase (LDH). Administration of 20 micromol/L atractyloside, a pore opener, for the last 5 min of ischemia and first 15 min of reperfusion, and pretreatment with 1 micromol/L paxilline, an inhibitor of the K(Ca) channel, for 5 min before ischemia, attenuated the recovery of RPP and CF, and the reductions of infarct size and release of LDH induced by TNF-alpha. On the other hand, administration of 10 micromol/L NS 1619, an opener of the K(Ca) channel, for 10 min before ischemia, decreased the infarct size and LDH release, and improved contractile functions and CF; these effects were attenuated by atractyloside. Pretreatment with 0.2 micromol/L cyclosporin A for the last 5 min of ischemia and first 15 min of reperfusion showed similar effects to those of TNF-alpha, and they were not attenuated by paxilline. In mitochondria isolated from hearts pretreated with 10 U/ml TNF-alpha for 7 min, a significant inhibition of Ca(2+)-induced swelling was observed. Furthermore, paxilline attenuated the inhibition of Ca(2+)-induced mitochondrial swelling by TNF-alpha. These findings indicate that TNF-alpha protects the myocardium against ischemia and reperfusion injury by inhibiting mitochondrial permeability transition pore opening as well as activating K(Ca) channels, probably the mitochondrial K(Ca) channel, which is upstream from the pore.     

Phospholipase D signaling in ischemic heart.

Phospholipase D (PLD) activity was found to be present in the membrane fraction of rat myocardial cells by in vitro assays (36.7 +/- 4.1 nmol/mg protein per h against 1-palmitoyl-2-arachidonoyl- phosphatidylcholine) and demonstrated in intact cells by the specific transphosphatidylation reaction (in the presence of 0.02% ethanol) quantitated using n-[1-14C]butanol (201.16 +/- 7.1 pmol/min per g dry weight in the whole heart). Both methods showed a significant increase in PLD activity (by 62 and 44%, respectively) in hearts subjected to reversible (30 min) global normothermic ischemia followed by reperfusion (30 min). In hearts prelabeled with [1-14C]arachidonic acid, ischemia/reperfusion induced a significant increase in the amount of radiolabel incorporated into phosphatidic acid (PtdOH) (by 49.6%) and diacylglycerol (DG) (by 259%). DG kinase inhibition by 100 microM dioctanoylethylene glycol did not affect the ischemia/reperfusion DG and PtdOH levels while PtdOH phosphohydrolase inhibition with 40 microM propranolol produced a further increase in PtdOH (to 2.36-fold the baseline level) and a reduction in DG (to only 145% over the baseline levels). Put together, all these results suggest an activation of PLD during myocardial ischemia/reperfusion generating intracellular PtdOH, part of which is converted by PtdOH phosphohydrolase to DG. We further investigated the possible pathophysiological significance of the observed PLD activation. Stimulation of PLD with sodium oleate (20 microM) induced a significant improvement of functional recovery of ischemic hearts during reperfusion (as monitored by coronary flow and left intraventricular pressure measurements) and an attenuation of cellular injury as expressed by lactate dehydrogenase and creatine kinase release in the coronary effluent during reperfusion. These results suggest a PLD-mediated signaling in the ischemic heart which may benefit functional recovery during reperfusion.     

Substrate-dependent proton load and recovery of stunned hearts during pyruvate dehydrogenase stimulation

Stimulation of pyruvate dehydrogenase (PDH) improves functional recovery of postischemic hearts. This study examined the potential for a mechanism mediated by substrate-dependent proton production and intracellular pH. After 20 min of ischemia, isolated rabbit hearts were reperfused with or without 5 mM dichloroacetate (DCA) in the presence of either 5 mM glucose, 5 mM glucose + 2.5 mM lactate, or 5 mM glucose + 2.5 mM pyruvate. DCA inhibits PDH kinase, increasing the proportion of dephosphorylated, active PDH. Unlike pyruvate or glucose alone, lactate + glucose did not support the effects of DCA on the recovery of rate-pressure product (RPP) (without DCA, RPP = 14,000 +/- 1,200, n = 6; with DCA, RPP = 13,700 +/- 1,800, n = 9). Intracellular pH, from (31)P nuclear magnetic resonance spectra, returned to normal within 2.1 min of reperfusion with all substrates except for lactate + glucose + DCA or lactate + DCA, which delayed pH recovery for up to 12 min (at 2.1 min pH = 6. 00 +/- 0.08, lactate + glucose + DCA; pH = 6.27 +/- 0.34, for lactate + DCA). Hearts were also reperfused after 10 min of ischemia with 0.5 mM palmitate + 5 mM DCA and either 2.5 mM pyruvate or 2.5 mM lactate. Again, intracellular pH recovery was delayed in the presence of lactate. PDH activation in the presence of lactate also decreased coupling of oxidative metabolism to mechanical work. These findings have implications for therapeutic use of stimulated carbohydrate oxidation in stunned hearts.     

C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.