Selective mitochondrial adenosine triphosphate-sensitive potassium channel activation is sufficient to precondition human myocardium

OBJECTIVES: Recently, the mitochondrial adenosine triphosphate-sensitive potassium channel has been suggested to be the final common effector of myocardial preconditioning. The purpose of this study is to determine whether selective mitochondrial adenosine triphosphate-sensitive potassium channel activation alone can precondition human myocardium from an ischemia/reperfusion insult. METHODS: Isolated human right atrial trabeculae were placed in tissue baths, paced, and subjected to 30 minutes of normothermic hypoxia (ischemia) followed by 45 minutes of reoxygenation (reperfusion). Trabeculae were preconditioned with a selective mitochondrial adenosine triphosphate-sensitive potassium channel opener (diazoxide 30 micromol/L) or a nonselective purinergic agonist, adenosine (125 micromol/L), for 5 minutes (adenosine) followed by a 10-minute washout period. Developed force at end reperfusion (mean +/- standard error) was compared with baseline, and tissue creatine kinase and adenosine triphosphate levels were measured after ischemia/reperfusion. RESULTS: Trabeculae subjected to ischemia/reperfusion exhibited 30% +/- 2% of baseline developed force, whereas trabeculae subjected to selective adenosine triphosphate-sensitive potassium channel opening (diazoxide) and nonselective purinergic agonist (adenosine) recovered to 55% +/- 7% and 46% +/- 3% of baseline developed force, respectively. Tissue creatine kinase activity was preserved in both the diazoxide- and adenosine-treated trabeculae (5.4 +/- 12 and 5.4 +/- 14 micromol/L per gram wet tissue) compared with ischemia/reperfusion (1.8 +/- 0.2 U/mg wet tissue). Adenosine triphosphate levels at end reperfusion were also increased in the trabeculae treated with selective (diazoxide) and nonselective (adenosine) adenosine triphosphate-sensitive potassium channel opener (4.1 +/- 0.01 and 4. 4 +/- 0.2 micromol/L per gram wet tissue) compared with trabeculae subjected to ischemia/reperfusion (1.5 +/- 0.1 micromol/L per gram wet tissue). CONCLUSIONS: These results suggest that selective mitochondrial adenosine triphosphate-sensitive potassium channel activation preconditions human myocardium and the protection conferred is equal to that of adenosine preconditioning. Targeted openers of mitochondrial adenosine triphosphate- sensitive potassium channels promote constructive protection of myocellular energy levels, contractile function, and cellular viability in human myocardium after ischemia/reperfusion.     

Ischemic preconditioning with opening of mitochondrial adenosine triphosphate-sensitive potassium channels or Na/H exchange inhibition: which is the best protective strategy for heart transplants?

OBJECTIVE: This study was designed to compare ischemic preconditioning with opening of mitochondrial adenosine triphosphate-sensitive potassium channels and Na(+)/H(+) exchange inhibition in an isolated heart model of cold storage, simulating the situation of cardiac allografts. METHODS: Sixty-seven isolated isovolumic buffer-perfused rat hearts were arrested with and stored in Celsior solution (Imtix-Sangstat) at 4 degrees C for 4 hours before a 2-hour reperfusion. Group I hearts served as controls and were arrested with and stored in Celsior solution. In group II, hearts were preconditioned by two 5-minute episodes of global ischemia, each separated by 5 minutes of reperfusion before arrest with Celsior solution. Group III hearts were arrested with and stored in Celsior solution supplemented with 100 micromol/L of the mitochondrial adenosine triphosphate-sensitive potassium channel opener diazoxide. In group IV, hearts received an infusion of diazoxide (30 micromol/L) during the first 15 minutes of reperfusion. Group V hearts underwent a protocol combining both interventions used in groups III and IV. In group VI, hearts were arrested with and stored in Celsior solution supplemented with 1 micromol/L of the Na(+)/H(+) exchange inhibitor cariporide. Group VII hearts received an infusion of cariporide (1 micromol/L) during the first 15 minutes of reperfusion. In group VIII, hearts underwent a protocol combining both interventions used in groups VI and VII. Group IX hearts were ischemically preconditioned as in group II, and sustained Na(+)/H(+) exchange inhibition during both storage and early reperfusion was used as in group VIII. RESULTS: On the basis of comparisons of postischemic left ventricular contractility and diastolic function, coronary flow, total creatine kinase leakage, and myocardial water content, values indicative of improved protection were obtained by combining ischemic preconditioning with Na(+)/H(+) exchange inhibition by cariporide given during storage and initial reperfusion. The endothelium-dependent vasodilatory postischemic responses to 5-hydroxytryptamine or acetylcholine and endothelium-independent responses to papaverine were not affected by these interventions. CONCLUSIONS: These data suggest that cardioprotection conferred by the Na(+)/H(+) exchange inhibitor cariporide is additive to that of ischemic preconditioning and might effectively contribute to improve donor heart preservation during cardiac transplantation.     

Lidocaine and Mexiletine Inhibit Mitochondrial Oxidation in Rat Ventricular Myocytes

Background: Accumulating evidence suggests that mitochondrial rather than sarcolemmal adenosine triphosphate-sensitive K+ (KATP) channels may have an important role in the protection of myocardium during ischemia. Because both lidocaine and mexiletine are frequently used antiarrhythmic drugs during myocardial ischemia, it is important to investigate whether they affect mitochondrial KATP channel activities.

Methods: Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. The authors measured flavoprotein fluorescence to evaluate mitochondrial redox state. Lidocaine or mexiletine was applied after administration of diazoxide (25 [mu]m), a selective mitochondrial KATP channel opener. The redox signal was normalized to the baseline flavoprotein fluorescence obtained during exposure to 2,4-dinitrophenol, a protonophore that uncouples respiration from ATP synthesis and collapses the mitochondrial potential.

Results: Diazoxide-induced oxidation of flavoproteins and the redox changes were inhibited by 5-hydroxydecanoic acid, a selective mitochondrial KATP channel blocker, suggesting that flavoprotein fluorescence can be used as an index of mitochondrial oxidation mediated by mitochondrial KATP channels. Lidocaine (10-3 to 10 mm) and mexiletine (10-3 to 10 mm) reduced oxidation of the mitochondrial matrix in a dose-dependent manner with an EC50 of 98 +/- 63 [mu]m for lidocaine and 107 +/- 89 [mu]m for mexiletine.     

Diazoxide protects mitochondria from anoxic injury: implications for myopreservation

BACKGROUND: Heart muscle primarily relies on adenosine triphosphate produced by oxidative phosphorylation and is highly vulnerable to anoxic insult. Although a number of strategies aimed at improving myopreservation are available, no effective means of preserving mitochondrial energetics under conditions of anoxic injury have been developed. Openers of mitochondrial adenosine triphosphate-sensitive potassium channels have emerged as powerful cardioprotective agents presumably capable of maintaining mitochondrial function under metabolic stress. Here, we evaluated the ability of a prototype mitochondrial adenosine triphosphate-sensitive potassium channel opener, diazoxide, to preserve oxidative phosphorylation in mitochondria subjected to anoxia and reoxygenation. METHODS: Mitochondria were isolated from rat hearts and subjected to 20 minutes of anoxia, followed by reoxygenation. Mitochondrial respiration and oxidative phosphorylation, as well as mitochondrial integrity, were assessed by means of ion-selective minielectrodes, high-performance liquid chromatography, fluorometry, and electron microscopy. RESULTS: Anoxia-reoxygenation decreased the rate of adenosine diphosphate-stimulated oxygen consumption, inhibited adenosine triphosphate production, and disrupted mitochondrial integrity. On average, anoxic stress reduced adenosine diphosphate-stimulated respiration from 291 +/- 14 to 141 +/- 15 ng-atoms O(2). min(-1). mg(-1) protein and decreased the rate of adenosine triphosphate production from 752 +/- 14 to 414 +/- 34 nmol adenosine triphosphate. min(-1). mg(-1) protein. After anoxia, the majority (88%) of mitochondria was damaged or swollen and released adenylate kinase, a marker of mitochondrial integrity. Diazoxide (100 micromol/L), present throughout anoxia, preserved adenosine diphosphate-stimulated respiration at 255 +/- 7 ng-atoms O(2). min(-1). mg(-1) protein and adenosine triphosphate production at 640 +/- 39 nmol adenosine triphosphate. min(-1). mg(-1) protein. Diazoxide also protected mitochondrial structure from anoxia-mediated damage, so that after anoxic stress, 67% of mitochondria remained intact and adenylate kinase was confined to the mitochondria. CONCLUSIONS: The present study demonstrates that diazoxide diminishes anoxia-induced functional and structural deterioration of cardiac mitochondria. By protecting mitochondria and preserving myocardial energetics, diazoxide may be useful under conditions of reduced oxygen availability, including global surgical ischemia or storage of donor heart.     

Lidocaine and mexiletine inhibit mitochondrial oxidation in rat ventricular myocytes

BACKGROUND: Accumulating evidence suggests that mitochondrial rather than sarcolemmal adenosine triphosphate-sensitive K+ (K(ATP)) channels may have an important role in the protection of myocardium during ischemia. Because both lidocaine and mexiletine are frequently used antiarrhythmic drugs during myocardial ischemia, it is important to investigate whether they affect mitochondrial K(ATP) channel activities. METHODS: Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. The authors measured flavoprotein fluorescence to evaluate mitochondrial redox state. Lidocaine or mexiletine was applied after administration of diazoxide (25 microM), a selective mitochondrial K(ATP) channel opener. The redox signal was normalized to the baseline flavoprotein fluorescence obtained during exposure to 2,4-dinitrophenol, a protonophore that uncouples respiration from ATP synthesis and collapses the mitochondrial potential. RESULTS: Diazoxide-induced oxidation of flavoproteins and the redox changes were inhibited by 5-hydroxydecanoic acid, a selective mitochondrial K(ATP) channel blocker, suggesting that flavoprotein fluorescence can be used as an index of mitochondrial oxidation mediated by mitochondrial K(ATP) channels. Lidocaine (10(-3) to 10 mM) and mexiletine (10(-3) to 10 mM) reduced oxidation of the mitochondrial matrix in a dose-dependent manner with an EC50 of 98+/-63 microM for lidocaine and 107+/-89 microM for mexiletine. CONCLUSIONS: Both lidocaine and mexiletine reduced flavoprotein fluorescence induced by diazoxide in rat ventricular myocytes, indicating that these antiarrhythmic drugs may produce impairment of mitochondrial oxidation mediated by mitochondrial K(ATP) channels.     

Potassium-channel opener cardioplegia is superior to St. Thomas' solution in the intact animal.

BACKGROUND: In isolated hearts, the potassium-channel opener pinacidil is an effective cardioplegic agent. This study tested the hypothesis that pinacidil is superior to St. Thomas' solution in the more clinically relevant intact animal. METHODS: Sixteen pigs were placed on full cardiopulmonary bypass. Hearts underwent 2 hours of global ischemia (10 degrees to 15 degrees C). Either St. Thomas' or 100 micromol/L pinacidil was administered every 20 minutes (10 mL/kg). Preischemic and postreperfusion slopes of the preload-recruitable stroke work relationship were determined. Changes in myocardial adenine nucleotide levels and cellular ultrastructure were analyzed. RESULTS: Pinacidil cardioplegia resulted in an insignificant change in the slope of the preload-recruitable stroke work relationship (40.6+/-2.1 mm Hg/mm before ischemia and 36.5+/-3.7 mm Hg/mm after ischemia; p = 0.466). In contrast, St. Thomas' solution resulted in a significant decrease in the slope after reperfusion (34.3+/-5.5 mm Hg/mm and 13.5+/-2.3 mm Hg/mm; p = 0.003). Adenine nucleotide levels, myocardial tissue water, and ultrastructural changes were similar between groups. CONCLUSIONS: Pinacidil ameliorated myocardial stunning associated with traditional hyperkalemic cardioplegia without causing significant differences in cellular metabolism.     

Blood cardioplegia supplementation with the sodium-hydrogen ion exchange inhibitor cariporide to attenuate infarct size and coronary artery endothelial dysfunction after severe regional ischemia in a canine model

BACKGROUND: Activation of the sodium-hydrogen ion exchange mechanism results in accumulation of intracellular calcium through the sodium-calcium ion antiport mechanism. Administration of a sodium-hydrogen ion exchange inhibitor before or during ischemia attenuates myocardial ischemia and reperfusion injury. However, the cardioprotection exerted by sodium-hydrogen ion exchange inhibitors as adjuncts to cardioplegia without perioperative administration has not been tested in a model of surgical reperfusion of acute coronary occlusion with cardiopulmonary bypass. This study tested the hypothesis that sodium-hydrogen ion exchange inhibitor-supplemented blood cardioplegia would reduce postcardioplegia injury after severe regional ischemia. METHODS: In anesthetized open-chest dogs, the left anterior descending coronary artery was occluded for 75 minutes, after which total cardiopulmonary bypass was initiated. After crossclamping, cold (4 degrees C) antegrade blood cardioplegia was delivered every 20 minutes for a total of 60 minutes of cardioplegic arrest. In 8 dogs, the blood cardioplegic solution was unsupplemented (vehicle group), whereas in 8 others the solution was supplemented with the sodium-hydrogen ion exchange inhibitor cariporide (10 micro mol/L, cariporide group). RESULTS: In the in vitro studies, the direct effects of cariporide on neutrophil function were determined. Isolated canine neutrophils were stimulated by platelet activating factor. Cariporide attenuated superoxide anion production in a concentration-dependent manner, with no appreciable effect at 10 micro mol/L (the concentration used in blood cardioplegia) and a peak effect at 100 micro mol/L. In the in vivo cardiopulmonary bypass model, infarct size was significantly (P <.05) smaller in the cariporide group than in the vehicle group (22.4% +/- 3.5% vs 40.1% +/- 5.1% of area at risk), although there were no group differences in postischemic regional wall motion after 2 hours of reperfusion (0.1% +/- 0.9% vs -0.2% +/- 0.3% systolic shortening). Transmural myocardial edema in the area at risk was significantly decreased in the cariporide group (80.6% +/- 0.5%) relative to the vehicle group (83.1% +/- 0.6%). Myeloperoxidase activity in the area at risk, an index of neutrophil accumulation, was significantly lower in the cariporide group than in the vehicle group (4.7 +/- 0.9 absorbence units/[min. g tissue] vs 10.3 +/- 2.3 absorbence units/[min. g tissue]). In isolated postischemic left anterior descending coronary artery rings, maximum relaxation in response to the endothelium-dependent vasodilator acetylcholine was significantly greater in the cariporide group than in the vehicle group (77.5% +/- 7.4% vs 51.4% +/- 8.0%), whereas smooth muscle relaxation in response to nitroprusside was comparable between groups. CONCLUSION: In this canine model, supplementation of blood cardioplegia with cariporide, a sodium-hydrogen ion exchange inhibitor, reduced infarct size, attenuated neutrophil accumulation in the area at risk, and reduced postischemic coronary artery endothelial dysfunction without directly inhibiting neutrophil activity. Cariporide as an adjunct to blood cardioplegia without perioperative administration attenuated surgical ischemia-reperfusion injury in jeopardized myocardium.     

Opening of mitochondrial ATP-sensitive potassium channels enhances cardioplegic protection

BACKGROUND: Mitochondrial and sarcolemmal ATP-sensitive potassium channels have been implicated in cardioprotection; however, the role of these channels in magnesium-supplemented potassium (K/Mg) cardioplegia during ischemia or reperfusion is unknown. METHODS: Rabbit hearts (n = 76) were used for Langendorff perfusion. Sham hearts were perfused for 180 minutes. Global ischemia hearts received 30 minutes of global ischemia and 120 minutes of reperfusion. K/Mg hearts received cardioplegia before ischemia. The role of ATP-sensitive potassium channels in K/Mg cardioprotection during ischemia and reperfusion was investigated, separately using the selective mitochondrial ATP sensitive potassium and channel blocker, 5-hydroxydecanoate, and the selective sarcolemmal ATP-sensitive potassium channel blocker HMR1883. Separate studies were performed using the selective mitochondrial ATP-sensitive potassium channel opener, diazoxide, and the nonselective ATP-sensitive potassium channel opener pinacidil. RESULTS: Infarct size was 1.9%+/-0.4% in sham, 3.7%+/-0.5% in K/Mg, and 27.8%+/-2.4% in global ischemia hearts (p < 0.05 versus K/Mg). Left ventricular peak-developed pressure (percent of equilibrium) at the end of 120 minutes of reperfusion was 91%+/-6% in sham, 92% +/-2% in K/Mg, and 47%+/-6% in global ischemia (p < 0.05 versus K/Mg). Blockade of sarcolemmal ATP-sensitive potassium channels in K/Mg hearts had no effect on infarct size or left ventricular peak-developed pressure. However, blockade of mitochondrial ATP-sensitive potassium channels before ischemia significantly increased infarct size to 23%+/-2% in K/Mg hearts (p < 0.05 versus K/Mg; no statistical significance [NS] as compared to global ischemia) and significantly decreased left ventricular peak-developed pressure to 69%+/-4% (p < 0.05 versus K/Mg). Diazoxide when added to K/Mg cardioplegia significantly decreased infarct size to 1.5%+/-0.4% (p < 0.05 versus K/Mg). CONCLUSIONS: The cardioprotection afforded by K/Mg cardioplegia is modulated by mitochondrial ATP-sensitive potassium channels. Diazoxide when added to K/Mg cardioplegia significantly reduces infarct size, suggesting that the opening of mitochondrial ATP-sensitive potassium channels with K/Mg cardioplegic protection would allow for enhanced myocardial protection in cardiac operations.     

Cariporide is cardioprotective after iatrogenic ventricular fibrillation in the intact swine heart

BACKGROUND: We sought to introduce sodium-hydrogen exchange inhibition as prophylaxis against the development of ventricular dysfunction in the setting of implantable cardioverter defibrillator insertion in high-risk patients. Cariporide, shown to be safe in humans, was used to reproduce previous results in our laboratory that demonstrated that sodium-hydrogen exchange inhibition preserves left ventricular (LV) function after ventricular fibrillation (VF) and reperfusion. METHODS: Twelve pigs (weight, 35 to 55 kg) were divided into two groups of six. Baseline ventricular function studies were based on echocardiography, conductance, aortic flow, and LV pressure. Animals were given vehicle (control) or cariporide (3 mg/kg intravenously). Ten minutes later, hearts underwent 80 seconds of VF. After reperfusion for 40 minutes, function studies were repeated. RESULTS: Postmortem examination included measuring passive pressure-volume curves and myocardial water content. Systolic indices, including preload recruitable stroke work and ejection fraction, were significantly depressed from baseline after VF and reperfusion for control animals (preload recruitable stroke work, 30.13 +/- 0.59 [standard error of the mean] versus 43.85 +/- 2.60 mm Hg; ejection fraction, 25.7% +/- 2.4% versus 33.5% +/- 3.0%) but not for those in the cariporide group (preload recruitable stroke work, 38.36 +/- 1.87 versus 40.86 +/- 1.45 mm Hg; ejection fraction, 33.9% +/- 3.5% versus 32.8% +/- 3.9%). In vivo diastolic indices demonstrated trends toward diminished ventricular compliance in control animals but not in the cariporide group after VF and reperfusion. Control animals had significantly increased postmortem LV stiffness, myocardial water content, and normalized LV mass. CONCLUSIONS: Cariporide preserves LV function after 80 seconds of VF and 40 minutes of reperfusion. Cariporide may prove useful in patients with severe LV dysfunction undergoing VF for implantable cardioverter defibrillator testing.     

A new role for cardioplegic buffering: should acidosis or calcium accumulation be counteracted to salvage jeopardized hearts?

OBJECTIVES: Thirty minutes of unprotected ischemia produced a jeopardized heart that was treated with a blood cardioplegic solution containing the natural erythrocyte and protein buffers. Cardioplegic pH was changed to 7.7 (buffered) or 7.2 (nonbuffered), and this was tested alone and after pretreatment with Na(+)-H(+) exchange blockade (cariporide) to define their protective effects. METHODS: Twenty-four Yorkshire-Duroc pigs (27-34.5 kg) underwent 30 minutes of normothermic global ischemia, followed by 30 minutes of aortic clamping during protection with buffered (n = 12) or nonbuffered (n = 12) glutamate-aspartate-enriched blood cardioplegic solution. Twelve hearts (6 buffered and 6 nonbuffered) were pretreated with intravenous cariporide (5 mg/kg) 15 minutes before ischemia. RESULTS: Severe and comparable left ventricle dysfunction followed buffered or nonbuffered cardioplegia: Preload recruitable stroke work recovered to 56% +/- 21% and 45% +/- 20% of baseline levels; creatine kinase MB, conjugated dienes, and myeloperoxidase activity markedly increased; moderate myocardial edema occurred; and endothelin-1 increased 2-fold more than baseline values. Cariporide pretreatment caused a similar return of preload recruitable stroke work to 86% +/- 9% and 90% +/- 6% after buffered or nonbuffered cardioplegia (P <.05 vs nonpretreated groups), allowed only minor creatine kinase MB and conjugated diene changes, and reduced endothelin-1 release 3-fold compared with hearts without sodium-hydrogen exchange blockage. CONCLUSIONS: The severe ischemia-reperfusion injury of 30 minutes of normothermic ischemia is not altered by an acidic or alkalotic pH cardioplegic solution. Correction of damage is achieved by adding Na(+)-H(+) exchange blocker therapy before treatment with buffered and nonbuffered solutions; thus, sodium-hydrogen exchange inhibition plays a more vital role in recovery than pH management.     

Myocyte and endothelial effects of preconditioning the jeopardized heart by inhibiting Na/H exchange

OBJECTIVES: The preconditioning effects of the adjunctive, cardiac-specific sodium-hydrogen ion exchange inhibitor cariporide (cariporide mesilate, HOE 642) were studied in hearts subjected to 30 minutes of normothermic ischemia and reperfusion to assess myocardial and endothelial changes. METHODS: Sixteen Yorkshire-Duroc pigs (27-34 kg) receiving cardiopulmonary bypass underwent either cardiopulmonary bypass alone (control, n = 4) or 30 minutes of normothermic ischemia, followed by 30 minutes of blood reperfusion (n = 12). Six hearts were treated with 5 mg/kg cariporide administered intravenously 15 minutes before ischemia. RESULTS: Cardiopulmonary bypass alone caused no changes. Conversely, 30 minutes of global normothermic ischemia caused 33% mortality and, in survivors, depression of left ventricular function to 22% +/- 6% of baseline preload recruitable stroke work and increased creatine kinase MB by 406% (88 +/- 13 U/L), conjugated dienes by 17% (161 +/- 0.2 AU/mL), and myeloperoxidase activity by 297% (0.036 +/- 0.005 U/g). Myocardial edema developed (3.5% water gain). Coronary sinus endothelin 1 increased by 111% (2.05 +/- 0.38 pg/mL), and nitric oxide production decreased by 10%. These adverse effects were limited by pretreatment with cariporide, which allowed complete survival and restored preload recruitable stroke work to 78% +/- 11%. Measurements of creatine kinase MB, conjugated dienes, myeloperoxidase, water, and endothelin 1 returned to baseline values, and nitric oxide production was accentuated 3-fold. CONCLUSIONS: These observations show that adjunctive pretreatment with cariporide delays myocardial and endothelial injury during ischemia and reperfusion, limits oxygen-derived radical injury, restores function, reduces edema, and preserves endothelin and nitric oxide balance at normal values. The myeloperoxidase changes show that less white blood cell adherence supports reduced reperfusion endothelial damage.     

Cardioprotective effects of propofol and sevoflurane in ischemic and reperfused rat hearts: role of K(ATP) channels and interaction with the sodium-hydrogen exchange inhibitor HOE 642 (cariporide).

BACKGROUND: Sodium ion-hydrogen ion (Na(+)-H(+)) exchange inhibitors are effective cardioprotective agents. The N(+)-H(+) exchange inhibitor HOE 642 (cariporide) has undergone clinical trials in acute coronary syndromes, including bypass surgery. Propofol and sevoflurane are also cardioprotective via unknown mechanisms. The authors investigated the interaction between propofol and HOE 642 in the ischemic reperfused rat heart and studied the role of adenosine triphosphate-sensitive potassium (K(ATP)) channels in the myocardial protection associated with propofol and sevoflurane. METHODS: Isolated rat hearts were perfused by the Langendorff method at a constant flow rate, and left ventricular function and coronary pressures were assessed using standard methods. Energy metabolites were also determined. To assess the role of K(ATP) channels, hearts were pretreated with the K(ATP) blocker glyburide (10 microM). Hearts were then exposed to either control buffer or buffer containing HOE 642 (5 microM), propofol (35 microM), sevoflurane (2.15 vol%), the K(ATP) opener pinacidil (1 microM), or the combination of propofol and HOE 642. Each heart was then subjected to 1 h of global ischemia followed by 1 h of reperfusion. RESULTS: Hearts treated with propofol, sevoflurane, pinacidil, or HOE 642 showed significantly higher recovery of left ventricular developed pressure and reduced end-diastolic pressures compared with controls. The combination of propofol and HOE 642 provided superior protection toward the end of the reperfusion period. Propofol, sevoflurane, and HOE 642 also attenuated the onset and magnitude of ischemic contracture and preserved high-energy phosphates (HEPs) compared with controls. Glyburide attenuated the cardioprotective effects of sevoflurane and abolished the protection observed with pinacidil. In contrast, glyburide had no effect on the cardioprotection associated with propofol treatment. CONCLUSION: HOE 642, propofol, and sevoflurane provide cardioprotection via different mechanisms. These distinct mechanisms may allow for the additive and superior protection observed with the combination of these anesthetics and HOE 642.     

Opening of mitochondrial potassium channels: a new target for graft preservation strategies?

BACKGROUND: This study was designed to assess the protective effects of the mitochondrial adenosine triphosphate-sensitive potassium channel (KATP) opener diazoxide as an additive to heart preservation solution. METHODS: Forty isolated isovolumic buffer-perfused rat hearts were divided into four groups. Groups I and III hearts were arrested with and cold-stored in Celsior solution for 4 hr and 10 hr, respectively. In Groups II and IV, hearts underwent a protocol similar to that used in Group I and III, respectively, except that Celsior was supplemented with 100 micromol/L of diazoxide. RESULTS: The protective effects of diazoxide were primarily manifest as a better preservation of diastolic function and a reduction of myocardial edema. The improvement of postischemic systolic function was observed only after prolonged exposure to diazoxide in Group IV, compared with Group III. The endothelium-dependent and endothelium-independent coronary flow postischemic responses were not affected by the supplementation of Celsior with diazoxide. CONCLUSIONS: Pharmacologic activation of mitochondrial KATP channels seems to be an effective means of improving preservation of cold-stored hearts, which is consistent with the presumed role of these channels as end effectors of the cardioprotective preconditioning pathway.     

The preischemic combination of the sodium-hydrogen exchanger inhibitor cariporide and the adenosine agonist AMP579 acts additively to reduce porcine myocardial infarct size

BACKGROUND: We tested whether the combination of two known cardioprotective agents, the type-1 sodium-hydrogen exchanger inhibitor cariporide plus the adenosine A(1)/A(2a) receptor agonist AMP579 ([1S-[1a,2b,3b, 4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino]-[(3)H]-imidazo[4,5-b]pyridyl-3-yl]cyclopentane carboxamide), acted additively to reduce myocardial infarct size. STUDY DESIGN: Pigs underwent 1 hour of coronary artery occlusion and 3 hours reperfusion. Vehicle-treated controls were compared with animals treated before ischemia with low-dose and high-dose cariporide and AMP579, and low-dose cariporide plus AMP579. The effects of both agents, alone and in combination, were also tested in isolated porcine polymorphonuclear neutrophils (PMNs). The PMN respiratory burst was induced with phorbol 12-myristate 13-acetate and quantified by the increase in 2',7'-dichlorofluorescein fluorescence, measured by flow cytometry. RESULTS: Infarct size in the control pigs was 65 +/- 1% of the area at risk. Cariporide dose-dependently reduced infarct size to 39 +/- 2% and 24 +/- 3% in the low- and high-dose groups, respectively. Infarct size was 54 +/- 3% in the low-dose AMP579 group and 47 +/- 3% with high dose. The combination of low doses of cariporide and AMP579 reduced infarction to 25 +/- 6% of the area at risk. In the PMN studies, cariporide and AMP579 alone had no effect on 2',7'-dichlorofluorescein fluorescence, but the combination of the two agents reduced the PMN 2',7'-dichlorofluorescein increase to 79 +/- 5% of the vehicle control response. CONCLUSIONS: The preischemic combination of low doses of cariporide and AMP579 decreased myocardial infarct size more than either agent used alone in low concentration, indicating an additive effect of the two agents. Given the effects that cariporide plus AMP579 combination exerted on PMN activity, it appears that this combination has the potential to reduce PMN-mediated effects during myocardial reperfusion.     

Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CA1 region subjected to transient ischemia

BACKGROUND: Excessive extracellular glutamate produced by cerebral ischemia has been proposed to initiate the cascade toward neuronal cell death. Changes in extracellular glutamate concentration are closely linked to changes in intracellular calcium ion concentration. Dantrolene inhibits calcium release from intracellular calcium stores. In this study, the authors investigated the effects of dantrolene on extracellular glutamate accumulation and neuronal degeneration in a rat model of transient global forebrain ischemia. METHODS: Male Wistar rats weighing 230-290 g were anesthetized with halothane in nitrous oxide-oxygen and were subjected to 10 min of transient forebrain ischemia using a four-vessel occlusion technique. Fifteen minutes before ischemic injury, dantrolene sodium (5 mm), dimethyl sulfoxide as a vehicle for dantrolene, or artificial cerebrospinal fluid as a control was intracerebroventricularly administered (n = 8 in each group). In the hippocampal CA1 subfield, the extracellular glutamate concentration in vivo was measured during the periischemic period with a microdialysis biosensor, and the number of intact neurons was evaluated on day 7 after reperfusion. RESULTS: Both dantrolene and dimethyl sulfoxide significantly reduced the ischemia-induced increase in glutamate concentration to a similar extent, i.e., by 53 and 51%, respectively, compared with artificial cerebrospinal fluid (P < 0.01). The number of intact hippocampal CA1 neurons (mean +/- SD; cells/mm) in dantrolene-treated rats (78 +/- 21) was significantly higher than that in artificial cerebrospinal fluid- (35 +/- 14; P < 0.001) and dimethyl sulfoxide-treated (56 +/- 11; P < 0.05) animals. Dimethyl sulfoxide also significantly increased the number of preserved neurons in comparison with artificial cerebrospinal fluid (P < 0.05). CONCLUSIONS: Intracerebroventricular dantrolene prevents delayed neuronal loss in the rat hippocampal CA1 region subjected to transient ischemia; however, this neuroprotection cannot be accounted for only by the reduced concentrations of extracellular glutamate during ischemia.     

Effects of Dantrolene on Extracellular Glutamate Concentration and Neuronal Death in the Rat Hippocampal CA1 Region Subjected to Transient Ischemia

Background: Excessive extracellular glutamate produced by cerebral ischemia has been proposed to initiate the cascade toward neuronal cell death. Changes in extracellular glutamate concentration are closely linked to changes in intracellular calcium ion concentration. Dantrolene inhibits calcium release from intracellular calcium stores. In this study, the authors investigated the effects of dantrolene on extracellular glutamate accumulation and neuronal degeneration in a rat model of transient global forebrain ischemia.

Methods: Male Wistar rats weighing 230-290 g were anesthetized with halothane in nitrous oxide-oxygen and were subjected to 10 min of transient forebrain ischemia using a four-vessel occlusion technique. Fifteen minutes before ischemic injury, dantrolene sodium (5 mm), dimethyl sulfoxide as a vehicle for dantrolene, or artificial cerebrospinal fluid as a control was intracerebroventricularly administered (n = 8 in each group). In the hippocampal CA1 subfield, the extracellular glutamate concentration in vivo was measured during the periischemic period with a microdialysis biosensor, and the number of intact neurons was evaluated on day 7 after reperfusion.

Results: Both dantrolene and dimethyl sulfoxide significantly reduced the ischemia-induced increase in glutamate concentration to a similar extent, i.e., by 53 and 51%, respectively, compared with artificial cerebrospinal fluid (P < 0.01). The number of intact hippocampal CA1 neurons (mean +/- SD; cells/mm) in dantrolene-treated rats (78 +/- 21) was significantly higher than that in artificial cerebrospinal fluid- (35 +/- 14;P < 0.001) and dimethyl sulfoxide-treated (56 +/- 11;P < 0.05) animals. Dimethyl sulfoxide also significantly increased the number of preserved neurons in comparison with artificial cerebrospinal fluid (P < 0.05).     

Enhanced cardioprotection of the rat heart during hypothermic storage with combined Na+ - H+ exchange inhibition and ATP-dependent potassium channel activation.

BACKGROUND: We investigated the ability of mitochondrial adenosine triphosphate-dependent potassium-channel activation to augment the protection of Na(+)-H(+) exchanger inhibition in isolated working rat hearts after 6 hours of hypothermic storage in an extracellular-based cardioplegic solution. METHODS: We treated hearts with the potassium-channel openers diazoxide (100 micromol/liter) or BMS-180448 (10 micromol/liter) or with the Na(+)-H(+) exchanger inhibitor cariporide (10 micromol/liter). Cariporide also was administered in combination with either diazoxide or BMS-180448 in 2 other treatment groups. All hearts were arrested and stored at 2 to 3 degrees C. After storage, we reperfused hearts for 10 minutes before performing work for a further 15 minutes, and then we measured and assessed cardiac function using a 2-way analysis of variance model. RESULTS: Neither diazoxide nor BMS-180448 significantly improved recovery of cardiac output. Cariporide therapy significantly improved cardiac output compared with control. However, we obtained the greatest recovery of cardiac output when we combined cariporide with either diazoxide or BMS-180448. CONCLUSIONS: Cariporide is more cardioprotective than the potassium-channel openers diazoxide and BMS-180448 after prolonged hypothermic storage. Co-administration of diazoxide or BMS-180448 with cariporide results in additive cardioprotection, with significantly improved cardiac function when compared with either treatment given alone. Such a combination could be used to improve the functional recovery of hearts stored for cardiac transplantation.     

Recovery of the failing canine heart with biventricular support in a previously fatal experimental model

Prolonged normothermic ischemia in the canine model is generally fatal with standard resuscitative techniques. To determine whether such myocardial injury is recoverable with biventricular support, we subjected 10 dogs to 45 minutes of ischemia at 37 degrees C. After ischemia, the animals were supported for 24 hours with biventricular assist with the centrifugal pump. During early reperfusion, none of the hearts could sustain a stable rhythm or blood pressure. Myocardial adenosine triphosphate concentration, expressed as micromoles per gram of heart protein, was dramatically reduced from a control of 31.5 +/- 2.4 to 14.6 +/- 2.9 (p less than 0.01 versus control), a 54% reduction. Ultrastructural analysis did not reveal the explosive cell swelling of irreversible cell injury. After 12 hours of biventricular assist, developed pressure partially recovered to 60.0 +/- 10 mm Hg (p less than 0.01 versus control) and maximal positive dP/dt measured 2,649 +/- 412 mm Hg/sec (p less than 0.01 versus control). Adenosine triphosphate concentration increased to 25.2 +/- 5.5 (p less than 0.01 versus control). Electron microscopic examination showed less chromatin clumping, no further mitochondrial distortion, and more abundant glycogen. After 24 hours of biventricular assist, cardiac output in the seven dogs successfully weaned from biventricular assist measured 3.6 +/- 0.6 L/min, developed pressure recovered to 76.3 +/- 8.9 mm Hg, and its first derivative recovered to 4,282 +/- 585 mm Hg/sec (all measurements not significant compared with control). Examination by an electron microscope revealed no severe mitochondrial injury.     

Cardioprotective Effects of Propofol and Sevoflurane in Ischemic and Reperfused Rat Hearts: Role of KATP Channels and Interaction with the Sodium-Hydrogen Exchange Inhibitor HOE 642 (Cariporide)

Background: Sodium ion-hydrogen ion (Na+-H+) exchange inhibitors are effective cardioprotective agents. The N+-H+ exchange inhibitor HOE 642 (cariporide) has undergone clinical trials in acute coronary syndromes, including bypass surgery. Propofol and sevoflurane are also cardioprotective via unknown mechanisms. The authors investigated the interaction between propofol and HOE 642 in the ischemic reperfused rat heart and studied the role of adenosine triphosphate-sensitive potassium (KATP) channels in the myocardial protection associated with propofol and sevoflurane.

Methods: Isolated rat hearts were perfused by the Langendorff method at a constant flow rate, and left ventricular function and coronary pressures were assessed using standard methods. Energy metabolites were also determined. To assess the role of KATP channels, hearts were pretreated with the KATP blocker glyburide (10 [mu]M). Hearts were then exposed to either control buffer or buffer containing HOE 642 (5 [mu]M), propofol (35 [mu]M), sevoflurane (2.15 vol%), the KATP opener pinacidil (1 [mu]M), or the combination of propofol and HOE 642. Each heart was then subjected to 1 h of global ischemia followed by 1 h of reperfusion.

Results: Hearts treated with propofol, sevoflurane, pinacidil, or HOE 642 showed significantly higher recovery of left ventricular developed pressure and reduced end-diastolic pressures compared with controls. The combination of propofol and HOE 642 provided superior protection toward the end of the reperfusion period. Propofol, sevoflurane, and HOE 642 also attenuated the onset and magnitude of ischemic contracture and preserved high-energy phosphates (HEPs) compared with controls. Glyburide attenuated the cardioprotective effects of sevoflurane and abolished the protection observed with pinacidil. In contrast, glyburide had no effect on the cardioprotection associated with propofol treatment.     

Isoflurane sensitizes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to pinacidil

BACKGROUND: Cardioprotective effects of isoflurane are partially mediated by the sarcolemmal adenosine triphosphate-sensitive potassium (sarcK ATP ) channel. The authors tested the hypothesis that isoflurane sensitizes sarcK ATP channels to a potassium channel opener, pinacidil, adenosine- and phospholipid-mediated pathways. METHODS: Activation by pinacidil of the K ATP current (I KATP ) was monitored in guinea pig ventricular myocytes at 0.5 and 5 mm intracellular ATP in the whole cell configuration of the patch clamp technique. The sensitization effect was evaluated by pretreating each myocyte with isoflurane (0.57 +/- 0.04 mm) before application of pinacidil (5 micro m) in the continued presence of the anesthetic. To investigate whether intracellular signaling pathways may be involved in isoflurane sensitization, the authors used the adenosine receptor antagonist theophylline (100 micro m) and the phosphatidylinositol kinase inhibitor wortmannin (100 micro m). RESULTS: The density of pinacidil-activated I KATP was higher at 0.5 mm ATP (20.7 +/- 3.2 pA/pF) than at 5 mm ATP (2.0 +/- 0.3 pA/pF). At 0.5 mm ATP, pretreatment with isoflurane caused an increase in density of pinacidil-activated I KATP (42.4 +/- 6.2 pA/pF) and accelerated the rate of current activation (from 5.4 +/- 1.2 to 39.0 +/- 7.9 pA. pF(-1). min(-1) ). Theophylline attenuated current activation by pinacidil (9.4 +/- 3.9 pA/pF) and abolished the sensitization effect of isoflurane on I KATP (10.0 +/- 2.5 pA/pF). Wortmannin did not alter pinacidil activation of I KATP (13.2 +/- 1.7 pA/pF) but prevented sensitization by isoflurane (15.8 +/- 4.5 pA/pF). CONCLUSIONS: These results suggest that isoflurane increases sensitivity of cardiac sarcK ATP channels to the potassium channel opener pinacidil. Blockade of adenosine receptors or phosphatidylinositol kinases abolishes the sensitization effect, suggesting that the adenosine and phospholipid signaling pathways may be involved in the actions by isoflurane.