Nitric oxide mediates protective effect of endothelin receptor antagonism during myocardial ischemia and reperfusion

Endothelin (ET) receptor antagonism protects from ischemia-reperfusion injury. We hypothesized that the cardioprotective effect is related to nitric oxide (NO) bioavailability. Buffer-perfused rat and mouse hearts were subjected to ischemia and reperfusion. At the onset of ischemia, the rat hearts received vehicle, the dual endothelin type A/type B (ETA/ETB) receptor antagonist bosentan (10 microM), the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 microM), the combination of bosentan and L-NMMA or the combination of bosentan, L-NMMA, and the NO substrate L-arginine (1 mM). Hearts from wild-type and endothelial NO synthase (eNOS)-deficient mice received either vehicle or bosentan. Myocardial performance, endothelial function, NO outflow, and eNOS expression were monitored. Bosentan significantly improved myocardial function during reperfusion in rats and in wild-type mice, but not in eNOS-deficient mice. The functional protection afforded by bosentan was inhibited by L-NMMA, whereas it was restored by L-arginine. Myocardial expression of eNOS (immunoblotting) increased significantly in bosentan-treated rat hearts compared with vehicle hearts. Recovery of NO outflow during reperfusion was enhanced in the bosentan-treated rat heart. The endothelium-dependent vasodilator adenosine diphosphate increased coronary flow by 18 +/- 9% at the end of reperfusion in the bosentan group, whereas it reduced coronary flow by 7 +/- 5% in the vehicle group (P < 0.001). The response to the endothelium-independent dilator sodium nitroprusside was not different between the two groups. In conclusion, the dual ETA/ETB receptor antagonist bosentan preserved endothelial and cardiac contractile function during ischemia and reperfusion via a mechanism dependent on endothelial NO production.     

Triiodothyronine concomitantly inhibits calcium overload and postischemic myocardial stunning in diabetic rats.

Acute effects of triiodothyronine (T3) on postischemic myocardial stunning and intracellular Ca2+ contents were studied in the isolated working hearts of streptozotocin-induced diabetic rats and age-matched controls. After two weeks of diabetes, serum T3 and T4 levels were decreased to 62.5% and 33.9% of control values. Basal preischemic cardiac performance did not differ between diabetic and control rats. In contrast, during reperfusion after 20-min ischemia, diabetic rats exhibited an impaired recovery of heart rate (at 30-min reperfusion 57.5% of baseline vs. control 88.5%), left ventricular (LV) systolic pressure (44.1% vs. 89.5%), and cardiac work (23.1% vs. 66.0%). When 1 and 100 nM T3 was added before ischemia, heart rate was recovered to 77.2% and 81.8% of baseline, LV systolic pressure to 68.3% and 81.9%, and cardiac work to 50.8% and 59.0%, respectively. Diabetic rat hearts showed a higher Ca2+ content in the basal state and a further increase after reperfusion (4.96+/-1.17 vs. control 3.78+/-0.48 micromol/g, p<0.01). In diabetic hearts, H+ release was decreased after reperfusion (5.24+/-2.21 vs. 8.70+/-1.41 mmol/min/g, p<0.05). T3 administration caused a decrease in the postischemic Ca2+ accumulation (lnM T3 4.66+/-0.41 and 100 nM T3 3.58+/-0.36) and recovered the H+ release (lnM T3 16.2+/-3.9 and 100 nM T3 11.6+/-0.9). T3 did not alter myocardial O2 consumption. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning, and T3 protects the myocardial stunning possibly via inhibiting Ca2+ overload.     

The effect of a low molecular weight inhibitor of lipid peroxidation on ultrastructural alterations to ischemia-reperfusion in the isolated rat heart

The effects of H290/51, a novel indenoindole derivative inhibitor of lipid peroxidation, on ultrastructural changes during cardiac ischemia-reperfusion injury were investigated. Langendorff-perfused rat hearts were exposed to 30 minutes of global ischemia followed by 20 minutes of reperfusion: Group A: Control hearts with standard buffer perfusion with vehicle added. Group B: H290/51 (10(-6) mol/l) added to buffer throughout stabilisation and reperfusion. In an additional Group C, where hearts were given H290/51, but not subjected to ischemia, the ultrastructure was preserved till the end of reperfusion. Absolute volumes and calculated volume fractions (Vv) of tissue and subcellular components were assessed with quantitative stereologic morphometry. After ischemia the increase in volume of extracellular interstitium was inhibited by H290/51 (247 +/- 80 vs. 159 +/- 50 microl, mean +/- SD, groups A and B, respectively, p<0.05). The Vv (interstitium/myocard) was higher in control hearts (0.318 +/- 0.062 vs. 0.206 +/- 0.067, p<0.05). Vv (cell edema/myocyte) was higher in the control group (0.144 +/- 0.07 vs. 0.083 +/- 0.033, p<0.05). Vv (myocyte/myocard) was higher in group B after ischemia than in the control group (0.622 +/- 0.071 vs. 0.707 +/- 0.052, p<0.05). The decreased Vv (capillary/myocard) after ischemia was inhibited by H290/51. After reperfusion there was no difference between groups. Treatment with H290/51 reduced edema and ensured better preserved sarcolemmal membrane structure during ischemia. The effect was no longer present after reperfusion.     

Nitric oxide — a retrograde messenger for carbon monoxide signaling in ischemic heart

To examine the intracellular signaling mechanism of NO in ischemic myocardium, isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine in the presence or absence of 650 M of protoporphyrin, a heme oxygenase inhibitor for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor placed into the right atrium. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. Induction for the expression of heme oxygenase was studied by Northern hybridization. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [³H] myoinositol and [¹⁴C] arachidonic acid. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. Protoporphyrin modulated the effects of L-arginine. cGMP, which was remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The NO-mediated augmentation of cGMP was reduced by protoporphyrin suggesting that part of the effects may be mediated by CO generated through the heme oxygenase pathway. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [³H] inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [¹⁴C] arachidonic acid resulted in modest increases in [¹⁴C] diacylglycerol and [¹⁴C] phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfision the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. The results suggests for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. This signaling appears to be on- and off- nature, and linked with SOD content of the tissue. The signaling is transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. Our results also suggest that NO activates heme oxygenase which further stimulates the production of cGMP presumably by CO signaling. Thus, NO not only potentiates cGMP mediated intracellular signaling, it also functions as a retrograde messenger for CO signaling in heart.     

A novel water-soluble and cell-permeable calpain inhibitor protects myocardial and mitochondrial function in postischemic reperfusion

The effects of the novel calpain inhibitor A-705239 were studied in isolated perfused rabbit hearts subjected to 45 min of global ischemia, followed by 60 min of reperfusion. During 15 min of perfusion the inhibitor accumulated in myocardial tissue up to 16 times the concentration in the perfusate. Almost complete recovery and survival of heart function (90%) was seen with an inhibitor concentration of 10(-8) M in the perfusion fluid when the compound was administered prior to ischemia. Left ventricular pressure amplitude and coronary flow showed significantly higher values during reperfusion in the presence of the inhibitor. A-705239 significantly reduced the release of creatine kinase, from 166+/-49 U/l in untreated hearts to 44+/-10 U/l, and diminished the release of lactate dehydrogenase from 118+/-20 U/l in untreated hearts to 63+/-4 U/l. Mitochondrial dysfunction following ischemia and reperfusion was markedly attenuated by the inhibitor. Thus, the state 3 respiration rate only decreased to 4.2 in contrast to 2.6 nmol O2/(min x mg s.w.) in untreated hearts, reflecting a reduced damage of oxidative phosphorylation. Furthermore, in the presence of the inhibitor the inner mitochondrial membranes became less permeable as indicated by a smaller leak respiration. The excellent properties of A-705239 should make this compound a valuable tool for further pharmacological studies.     

Mechanism of decreased adenosine protection in reperfusion injury of aging rats

The purpose of this study was to determine whether the protective effects of adenosine on myocardial ischemia-reperfusion injury are altered with age, and if so, to clarify the mechanisms that underlie this change related to nitric oxide (NO) derived from the vascular endothelium. Isolated perfused rat hearts were exposed to 30 min of ischemia and 60 min of reperfusion. In the adult hearts, administration of adenosine (5 micromol/l) stimulated NO release (1. 06 +/- 0.19 nmol. min(-1). g(-1), P < 0.01 vs. vehicle), increased coronary flow, improved cardiac functional recovery (left ventricular developed pressure 79 +/- 3.8 vs. 57 +/- 3.1 mmHg in vehicle, P < 0.001; maximal rate of left ventricular pressure development 2,385 +/- 103 vs. 1,780 +/- 96 in vehicle, P < 0.001), and reduced myocardial creatine kinase loss (95 +/- 3.9 vs. 159 +/- 4.6 U/100 mg protein, P < 0.01). In aged hearts, adenosine-stimulated NO release was markedly reduced (+0.42 +/- 0.12 nmol. min(-1). g(-1) vs. vehicle), and the cardioprotective effects of adenosine were also attenuated. Inhibition of NO production in the adult hearts significantly decreased the cardioprotective effects of adenosine, whereas supplementation of NO in the aged hearts significantly enhanced the cardioprotective effects of adenosine. The results show that the protective effects of adenosine on myocardial ischemia-reperfusion injury are markedly diminished in aged animals, and that the loss in NO release in response to adenosine may be at least partially responsible for this age-related alteration.     

Targeted deletion of A(3) adenosine receptors improves tolerance to ischemia-reperfusion injury in mouse myocardium

A(3) adenosine receptors (A(3)ARs) have been implicated in regulating mast cell function and in cardioprotection during ischemia-reperfusion injury. The physiological role of A(3)ARs is unclear due to the lack of widely available selective antagonists. Therefore, we examined mice with targeted gene deletion of the A(3)AR together with pharmacological studies to determine the role of A(3)ARs in myocardial ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in isovolumic Langendorff hearts from A(3)AR(-/-) and wild-type (A(3)AR(+/+)) mice. Loss of contractile function during ischemia was unchanged, but recovery of developed pressure in hearts after reperfusion was improved in A(3)AR(-/-) compared with wild-type hearts (80 +/- 3 vs. 51 +/- 3% at 30 min). Tissue viability assessed by efflux of lactate dehydrogenase was also improved in A(3)AR(-/-) hearts (4.5 +/- 1 vs. 7.5 +/- 1 U/g). The adenosine receptor antagonist BW-A1433 (50 microM) decreased functional recovery following ischemia in A(3)AR(-/-) but not in wild-type hearts. We also examined myocardial infarct size using an intact model with 30-min left anterior descending coronary artery occlusion and 24-h reperfusion. Infarct size was reduced by over 60% in A(3)AR(-/-) hearts. In summary, targeted deletion of the A(3)AR improved functional recovery and tissue viability during reperfusion following ischemia. These data suggest that activation of A(3)ARs contributes to myocardial injury in this setting in the rodent. Since A(3)ARs are thought to be present on resident mast cells in the rodent myocardium, we speculate that A(3)ARs may have proinflammatory actions that mediate the deleterious effects of A(3)AR activation during ischemia-reperfusion injury.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Reduction of asymmetric dimethylarginine involved in the cardioprotective effect of losartan in spontaneously hypertensive rats

Previous studies have indicated that nitric oxide synthase (NOS) inhibitors can induce an increase of blood pressure and exacerbate myocardial injury induced by ischemia and reperfusion, whereas angiotensin II receptor antagonists protect the myocardium against injury induced by ischemia and reperfusion. Isolated hearts from male spontaneously hypertensive rats (SHR) or male Wistar-Kyoto rats (WKY) were subjected to 20 min global ischemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dP/dt(max)) were recorded, and serum concentrations of asymmetric dimethylarginine (ADMA) and NO and the release of creatine kinase in coronary effluent were measured. The level of ADMA was significantly increased and the concentration of NO was decreased in SHR. Ischemia and reperfusion significantly inhibited the recovery of cardiac function and increased the release of creatine kinase, and ischemia and reperfusion-induced myocardial injury in SHR was aggravated compared with WKY. Vasodilation responses to acetylcholine of aortic rings were decreased in SHR. Treatment with losartan (30 mg/kg) for 14 days significantly lowered blood pressure, elevated the plasma level of NO, and decreased the plasma concentration of ADMA in SHR. Treatment with losartan significantly improved endothelium-dependent relaxation and cardiac function during ischemia and reperfusion in SHR. Exogenous ADMA also aggravated myocardial injury induced by ischemia and reperfusion in isolated perfused heart of WKY, as shown by increasing creatine kinase release and decreasing cardiac function. The present results suggest that the protective effect of losartan on myocardial injury induced by ischemia and reperfusion is related to the reduction of ADMA levels.     

Lipid Peroxidation, Tissue Necrosis, and Metabolic and Mechanical Recovery of Isolated Reperfused Rat Heart as a Function of Increasing Ischemia

Isolated Langendorff-perfused rat hearts, after 30 min of preperfusion, were submitted to increasing times of global normothermic ischemia (1, 2, 5, 10, 20 and 30 min) or to the same times of ischemia followed by 30 min of reperfusion. Analysis of malondialdehyde, ascorbic acid, oxypurines, nucleosides, nicotinic coen-zymes and high-energy phosphates was carried out by HPLC on neutralized perchloric acid extracts of freeze-clamped tissues. In addition, maximum rate of intra-ventricular pressure development and cardiac output of malondialdehyde, lactate dehydrogenase, oxypurines and nucleosides were monitored during both preperfusion and reperfusion. Besides decreasing energy metabolites and nicotinic coenzyme pool, prolonged ischemia produced oxidation of significant amounts of hypoxanthine and xanthine to uric acid and generation of detectable levels of malondialdehyde (0.002 μmollg dry weight). After oxygen and substrate readmission, tissue and perfusate malondialdehyde increased only if previous ischemia was longer than 5 min, while lactate dehydrogenase was detected in perfusate of reperfused hearts following 10, 20, and 30 min of ischemia. Highest values of tissue malondialdehyde and total malondialdehyde output were recorded in reperfused hearts subjected to 30 min of ischemia (0.043 μmol/g dry weight and 0.069 μmol/ 30 min/g dry weight, respectively). Since tissue malondialdehyde was observed without detectable lactate dehydrogenase release in perfusate, it might be stated that malondialdehyde generation (i.e., lipid peroxidation) temporally preceded lactate dehydrogenase release (i.e., tissue necrosis). In reperfused hearts, evaluation of myocardial energy state and of mechanical recovery allowed us to determine times of ischemia beyond which reperfusion did not positively affect these metabolic and functional parameters. Main findings are that, under these experimental conditions, lipid peroxidation might be the cause and not the consequence of tissue necrosis and that duration of ischemia might be the factor deciding effectiveness of reperfusion.     

Real-time monitoring of nitric oxide in ischemic myocardium using an NO-selective electrode calibrated by electron spin resonance

Using a Langendorff-perfused rat heart preparation and selective electrodes, we determined nitric oxide (NO) and oxygen levels in cardiac tissue. An NO-selective electrode that was calibrated by electron spin resonance (ESR) spectroscopy was inserted into the middle of the myocardium in the left ventricle. Simultaneously, we used an O2-selective electrode to measure the partial pressure of oxygen (pO2) in the perfusate, Krebs-Henseleit (K-H) solution, that was ejected from the heart. After 30 min of aerobic control perfusion, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Under ischemic conditions, with a gradually decreasing pO2, NO detected by an NO-sensitive electrode within the myocardium was gradually increased. The maximum concentration increases in NO and decreases in pO2 during global ischemia were +10.200 +/- 1.223 microM and -58.608 +/- 4.123 mmHg, respectively. NO and pO2 levels both recovered to pre-ischemia baseline values when perfusion was restarted after global ischemia (reperfusion). The presence of Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mM), a NOS inhibitor, prevented ischemia/reperfusion-induced changes in NO. This study shows that an NO-selective electrode that is calibrated by ESR can provide accurate, real-time monitoring of cardiac NO in normal and ischemic myocardium.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium

Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium, which generates oxyradicals and depletes myocardial high-energy phosphates. The glycolytic metabolite pyruvate, but not its reduced congener lactate, increases phosphorylation potential and detoxifies oxyradicals in ischemic and postischemic myocardium. This study tested the hypothesis that pyruvate mitigates oxidative stress and preserves the energy state in cardioplegically arrested myocardium. In situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate and then reperfused for 3 min with cardioplegia-free blood. Glutathione (GSH), glutathione disulfide (GSSG), and energy metabolites [phosphocreatine (PCr), creatine (Cr), P(i)] were measured in myocardium, which was snap frozen at 45 min arrest and 3 min reperfusion to determine antioxidant GSH redox state (GSH/GSSG) and PCr phosphorylation potential {[PCr]/([Cr][P(i)])}. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate cardioplegia lowered 8-isoprostane release approximately 40% during arrest versus control and lactate cardioplegia. Lactate and pyruvate cardioplegia dampened (P < 0.05 vs. control) the surge of 8-isoprostane release following reperfusion. Pyruvate doubled GSH/GSSG versus lactate cardioplegia during arrest, but GSH/GSSG fell in all three groups after reperfusion. Myocardial [PCr]/([Cr][P(i)]) was maintained in all three groups during arrest. Pyruvate cardioplegia doubled [PCr]/([Cr][P(i)]) versus control and lactate cardioplegia after reperfusion. Pyruvate cardioplegia mitigates oxidative stress during cardioplegic arrest and enhances myocardial energy state on reperfusion.     

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.     

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial reperfusion injury in isolated cold-arrested rat hearts.

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate+bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 +/- 24%; p less than .05) than in groups A (42 +/- 40%), C (47 +/- 36%), D (44 +/- 33%) and E (58 +/- 25%). Likewise, percent recovery of the double product (heart rate x systolic left ventricular pressure) was better in group B (104 +/- 18%; p less than .05) than in the other groups (A: 47 +/- 37%, C: 49 +/- 36%, D: 50 +/- 35%, E: 69 +/- 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.     

Effects of cold cardioplegia on pH, Na, and Ca in newborn rabbit hearts

Many studies suggest myocardial ischemia-reperfusion (I/R) injury results largely from cytosolic proton (H(i))-stimulated increases in cytosolic Na (Na(i)), which cause Na/Ca exchange-mediated increases in cytosolic Ca concentration ([Ca]i). Because cold, crystalloid cardioplegia (CCC) limits [H]i, we tested the hypothesis that in newborn hearts, CCC diminishes H(i), Na(i), and Ca(i) accumulation during I/R to limit injury. NMR measured intracellular pH (pH(i)), Na(i), [Ca]i, and ATP in isolated Langendorff-perfused newborn rabbit hearts. The control ischemia protocol was 30 min for baseline perfusion, 40 min for global ischemia, and 40 min for reperfusion, all at 37 degrees C. CCC protocols were the same, except that ice-cold CCC was infused for 5 min before ischemia and heart temperature was lowered to 12 degrees C during ischemia. Normal potassium CCC solution (NKCCC) was identical to the control perfusate, except for temperature; the high potassium (HKCCC) was identical to NKCCC, except that an additional 11 mmol/l KCl was substituted isosmotically for NaCl. NKCCC and HKCCC were not significantly different for any measurement. The following were different (P < 0.05). End-ischemia pH(i) was higher in the CCC than in the control group. Similarly, CCC limited increases in Na(i) during I/R. End-ischemia Na(i) values (in meq/kg dry wt) were 115 +/- 16 in the control group, 49 +/- 13 in the NKCCC group, and 37 +/- 12 in the HKCCC group. CCC also improved [Ca]i recovery during reperfusion. After 40 min of reperfusion, [Ca](i) values (in nmol/l) were 302 +/- 50 in the control group, 145 +/- 13 in the NKCCC group, and 182 +/- 19 in the HKCCC group. CCC limited ATP depletion during ischemia and improved recovery of ATP and left ventricular developed pressure and decreased creatine kinase release during reperfusion. Surprisingly, CCC did not significantly limit [Ca]i during ischemia. The latter is explained as the result of Ca release from intracellular buffers on cooling.     

The effect of captopril on nitric oxide formation and on generation of radical forms of mitochondrial respiratory chain compounds in ischemic rat heart.

The increase of radical forms of mitochondrial respiratory chain compounds (MRCC) is an indicator of an increased risk of the formation of oxygen radicals. Using electron paramagnetic resonance (EPR), we found an increase of signals corresponding to ubisemichinone radical (.QH) and ironsulfur proteins radical forms (-FeS) of these respiratory chain compounds during ischemia in the isolated perfused rat heart (.QH increased from 1.51 to 3.08, .FeS1 from 1.14 to 2.65 arbitrary units). During the 5-min reperfusion, the signals returned to normoxic levels. In isolated mitochondria exposed to anoxia and reoxygenation the radical forms of .QH and FeS2 changed in a similar manner as in the intact heart. A combination of in vivo captopril treatment and in vitro L-arginine administration significantly decreased the levels of MRCC radicals in the isolated myocardium (.QH from 2.61 to 1.72 and .FeS, from 1.82 to 0.46 under normoxia; .QH from 4.35 to 2.66 and .FeS1 from 1.93 to 1.35 during ischemia). This decrease in MRCC radical forms was associated with increased NO levels in the perfusate, determined as NO2- / NO3-, as well as tissue NO levels determined using EPR as the dinitrosyl iron complex (DNIC). These results provide new information about the cardioprotective effects of ACE inhibitors and L-arginine.     

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.     

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

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