Ketamine preconditions isolated human right atrial myocardium: roles of adenosine triphosphate-sensitive potassium channels and adrenoceptors

BACKGROUND: The authors examined the effect of ketamine and its S(+) isomer on isolated human myocardium submitted to hypoxia-reoxygenation in vitro. METHODS: The authors studied isometric contraction of human right atrial trabeculae suspended in an oxygenated Tyrode's modified solution at 34 degrees C. Ten minutes before a 30-min hypoxic period followed by a 60-min reoxygenation, muscles were exposed for 15 min to racemic ketamine and its S(+) isomer at 10, 10, and 10 m alone or in the presence of 8.10 m 5-hydroxydecanoate, 10 m HMR 1098 (sarcolemmal adenosine triphosphate-sensitive potassium channel antagonist), 10 m phentolamine (alpha-adrenoceptor antagonist), and 10 m propranolol (beta-adrenoceptor antagonist). Force of contraction at the end of the 60-min reoxygenation period was compared between groups (mean +/- SD). RESULTS: Ketamine (10 m: 85 +/- 4%; 10 m: 95 +/- 10%; 10 m: 94 +/- 14% of baseline) and S(+)-ketamine (10-6 m: 85 +/- 4%; 10 m: 91 +/- 16%; 10 m: 93 +/- 14% of baseline) enhanced recovery of force of contraction at the end of the reoxygenation period as compared with the control group (47 +/- 10% of baseline; P < 0.001). Ketamine-induced preconditioning at 10 m was inhibited by 5-hydroxydecanoate (60 +/- 16%; P < 0.001), HMR 1098 (60 +/- 14%; P < 0.001), phentolamine (56 +/- 12%; P < 0.001), and propranolol (60 +/- 7%; P < 0.001). CONCLUSIONS: In vitro, ketamine preconditions isolated human myocardium, at least in part, via activation of adenosine triphosphate-sensitive potassium channels and stimulation of alpha- and beta-adrenergic receptors.     

Mechanisms of Desflurane-induced Preconditioning in Isolated Human Right Atria In Vitro

Background: The authors examined the role of adenosine triphosphate-sensitive potassium (KATP) channels, adenosine A1 receptor, and [alpha] and [beta] adrenoceptors in desflurane-induced preconditioning in human myocardium, in vitro.

Methods: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34[degrees]C; stimulation frequency, 1 Hz). Before a 30-min anoxic period, 3, 6, and 9% desflurane was administered during 15 min. Desflurane, 6%, was also administered in the presence of 10 [mu]m glibenclamide, a KATP channels antagonist; 10 [mu]m HMR 1098, a sarcolemmal KATP channel antagonist; 800 [mu]m 5-hydroxy-decanoate (5-HD), a mitochondrial KATP channel antagonist; 1 [mu]m phentolamine, an [alpha]-adrenoceptor antagonist; 1 [mu]m propranolol, a [beta]-adrenoceptor antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine (DPX), the adenosine A1 receptor antagonist. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- SD).

Results: Desflurane at 3% (95 +/- 13% of baseline), 6% (86 +/- 6% of baseline), and 9% (82 +/- 6% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (50 +/- 11% of baseline). Glibenclamide (60 +/- 12% of baseline), 5-HD (57 +/- 21% of baseline), DPX (63 +/- 19% of baseline), phentolamine (56 +/- 20% of baseline), and propranolol (63 +/- 13% of baseline) abolished desflurane-induced preconditioning. In contrast, HMR 1098 (85 +/- 12% of baseline) did not modify desflurane-induced preconditioning.     

Mechanisms of desflurane-induced preconditioning in isolated human right atria in vitro

BACKGROUND: The authors examined the role of adenosine triphosphate-sensitive potassium (K(ATP)) channels, adenosine A1 receptor, and alpha and beta adrenoceptors in desflurane-induced preconditioning in human myocardium, in vitro. METHODS: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34 degrees C; stimulation frequency, 1 Hz). Before a 30-min anoxic period, 3, 6, and 9% desflurane was administered during 15 min. Desflurane, 6%, was also administered in the presence of 10 microm glibenclamide, a K(ATP) channels antagonist; 10 microm HMR 1098, a sarcolemmal K(ATP) channel antagonist; 800 microm 5-hydroxy-decanoate (5-HD), a mitochondrial K(ATP) channel antagonist; 1 microm phentolamine, an alpha-adrenoceptor antagonist; 1 microm propranolol, a beta-adrenoceptor antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine (DPX), the adenosine A1 receptor antagonist. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- SD). RESULTS: Desflurane at 3% (95 +/- 13% of baseline), 6% (86 +/- 6% of baseline), and 9% (82 +/- 6% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (50 +/- 11% of baseline). Glibenclamide (60 +/- 12% of baseline), 5-HD (57 +/- 21% of baseline), DPX (63 +/- 19% of baseline), phentolamine (56 +/- 20% of baseline), and propranolol (63 +/- 13% of baseline) abolished desflurane-induced preconditioning. In contrast, HMR 1098 (85 +/- 12% of baseline) did not modify desflurane-induced preconditioning. CONCLUSIONS: In vitro, desflurane preconditions human myocardium against simulated ischemia through activation of mitochondrial K(ATP) channels, adenosine A1 receptor, and alpha and beta adrenoceptors.     

Mechanisms of Sevoflurane-induced Myocardial Preconditioning in Isolated Human Right Atria In Vitro

Background: The authors examined the role of adenosine triphosphate-sensitive potassium channels and adenosine A1 receptors in sevoflurane-induced preconditioning on isolated human myocardium.

Methods: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34[degrees]C; stimulation frequency, 1 Hz). In all groups, a 30-min hypoxic period was followed by 60 min of reoxygenation. Seven minutes before hypoxia reoxygenation, muscles were exposed to 4 min of hypoxia and 7 min of reoxygenation or 15 min of sevoflurane at concentrations of 1, 2, and 3%. In separate groups, sevoflurane 2% was administered in the presence of 10 [mu]m HMR 1098, a sarcolemmal adenosine triphosphate-sensitive potassium channel antagonist; 800 [mu]m 5-hydroxy-decanoate, a mitochondrial adenosine triphosphate-sensitive potassium channel antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist. Recovery of force at the end of the 60-min reoxygenation period was compared between groups (mean +/- SD).

Results: Hypoxic preconditioning (90 +/- 4% of baseline) and sevoflurane 1% (82 +/- 3% of baseline), 2% (92 +/- 5% of baseline), and 3% (85 +/- 7% of baseline) enhanced the recovery of force after 60 min of reoxygenation compared with the control groups (52 +/- 9% of baseline). This effect was abolished in the presence of 5-hydroxy-decanoate (55 +/- 14% of baseline) and 8-cyclopentyl-1,3-dipropylxanthine (58 +/- 16% of baseline) but was attenuated in the presence of HMR 1098 (73 +/- 10% of baseline).     

Mechanisms of sevoflurane-induced myocardial preconditioning in isolated human right atria in vitro

BACKGROUND: The authors examined the role of adenosine triphosphate-sensitive potassium channels and adenosine A(1) receptors in sevoflurane-induced preconditioning on isolated human myocardium. METHODS: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34 degrees C; stimulation frequency, 1 Hz). In all groups, a 30-min hypoxic period was followed by 60 min of reoxygenation. Seven minutes before hypoxia reoxygenation, muscles were exposed to 4 min of hypoxia and 7 min of reoxygenation or 15 min of sevoflurane at concentrations of 1, 2, and 3%. In separate groups, sevoflurane 2% was administered in the presence of 10 microm HMR 1098, a sarcolemmal adenosine triphosphate-sensitive potassium channel antagonist; 800 microm 5-hydroxy-decanoate, a mitochondrial adenosine triphosphate-sensitive potassium channel antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A(1) receptor antagonist. Recovery of force at the end of the 60-min reoxygenation period was compared between groups (mean +/- SD). RESULTS: Hypoxic preconditioning (90 +/- 4% of baseline) and sevoflurane 1% (82 +/- 3% of baseline), 2% (92 +/- 5% of baseline), and 3% (85 +/- 7% of baseline) enhanced the recovery of force after 60 min of reoxygenation compared with the control groups (52 +/- 9% of baseline). This effect was abolished in the presence of 5-hydroxy-decanoate (55 +/- 14% of baseline) and 8-cyclopentyl-1,3-dipropylxanthine (58 +/- 16% of baseline) but was attenuated in the presence of HMR 1098 (73 +/- 10% of baseline). CONCLUSIONS: In vitro, sevoflurane preconditions human myocardium against hypoxia through activation of adenosine triphosphate-sensitive potassium channels and stimulation of adenosine A(1) receptors.     

Sarcolemmal and Mitochondrial Adenosine Triphosphate- dependent Potassium Channels: Mechanism of Desflurane-induced Cardioprotection

Background: Volatile anesthetic-induced preconditioning is mediated by adenosine triphosphate-dependent potassium (KATP) channels; however, the subcellular location of these channels is unknown. The authors tested the hypothesis that desflurane reduces experimental myocardial infarct size by activation of specific sarcolemmal and mitochondrial KATP channels.

Methods: Barbiturate-anesthetized dogs (n = 88) were acutely instrumented for measurement of aortic and left ventricular pressures. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle (0.9% saline) or the nonselective KATP channel antagonist glyburide (0.1 mg/kg intravenously) in the presence or absence of 1 minimum alveolar concentration desflurane. In four additional groups, dogs received 45-min intracoronary infusions of the selective sarcolemmal (HMR 1098; 1 [mu]g [middle dot] kg-1 [middle dot] min-1) or mitochondrial (5-hydroxydecanoate [5-HD]; 150 [mu]g [middle dot] kg-1 [middle dot] min-1) KATP channel antagonists in the presence or absence of desflurane. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively.

Results: Desflurane significantly (P < 0.05) decreased infarct size to 10 +/- 2% (mean +/- SEM) of the area at risk as compared with control experiments (25 +/- 3% of area at risk). This beneficial effect of desflurane was abolished by glyburide (25 +/- 2% of area at risk). Glyburide (24 +/- 2%), HMR 1098 (21 +/- 4%), and 5-HD (24 +/- 2% of area at risk) alone had no effects on myocardial infarct size. HMR 1098 and 5-HD abolished the protective effects of desflurane (19 +/- 3% and 22 +/- 2% of area at risk, respectively).     

Sarcolemmal and mitochondrial adenosine triphosphate- dependent potassium channels: mechanism of desflurane-induced cardioprotection

BACKGROUND: Volatile anesthetic-induced preconditioning is mediated by adenosine triphosphate-dependent potassium (KATP) channels; however, the subcellular location of these channels is unknown. The authors tested the hypothesis that desflurane reduces experimental myocardial infarct size by activation of specific sarcolemmal and mitochondrial KATP channels. METHODS: Barbiturate-anesthetized dogs (n = 88) were acutely instrumented for measurement of aortic and left ventricular pressures. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle (0.9% saline) or the nonselective KATP channel antagonist glyburide (0.1 mg/kg intravenously) in the presence or absence of 1 minimum alveolar concentration desflurane. In four additional groups, dogs received 45-min intracoronary infusions of the selective sarcolemmal (HMR 1098; 1 microg. kg-1. min-1) or mitochondrial (5-hydroxydecanoate [5-HD]; 150 microg. kg-1. min-1) KATP channel antagonists in the presence or absence of desflurane. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively. RESULTS: Desflurane significantly (P < 0.05) decreased infarct size to 10 +/- 2% (mean +/- SEM) of the area at risk as compared with control experiments (25 +/- 3% of area at risk). This beneficial effect of desflurane was abolished by glyburide (25 +/- 2% of area at risk). Glyburide (24 +/- 2%), HMR 1098 (21 +/- 4%), and 5-HD (24 +/- 2% of area at risk) alone had no effects on myocardial infarct size. HMR 1098 and 5-HD abolished the protective effects of desflurane (19 +/- 3% and 22 +/- 2% of area at risk, respectively). CONCLUSION: Desflurane reduces myocardial infarct size in vivo, and the results further suggest that both sarcolemmal and mitochondrial KATP channels could be involved.     

Hypoxic preconditioning in coronary microarteries: role of EDHF and K+ channel openers

BACKGROUND: Hypoxic preconditioning may provide a useful method of myocardial protection in cardiac operations. The present study was designed to investigate the possible mechanisms of preconditioning regarding endothelium-derived hyperpolarizing factor (EDHF) and the effect of a potassium channel opener KRN4884 on the porcine coronary microartery in mimicking hypoxic preconditioning. METHODS: Porcine coronary microartery rings (diameter 200 to 500 microm) studied in a myograph were divided into seven groups: (1) control group; (2) hypoxia-reoxygenation group (hypoxia for 60 minutes followed by reoxygenation for 30 minutes); (3) preconditioning group (hypoxia for 5 minutes followed by reoxygenation for 10 minutes before hypoxia reoxygenation); (4) KRN4884 pretreatment group (KRN4884 was added into the myograph chamber 20 minutes before hypoxia reoxygenation); (5) 5-hydroxydecanoate + KRN group (5-hydroxydecanoate was given 20 minutes before KRN4884 pretreatetment); (6) glibenclamide (GBC) + KRN group (GBC was added 20 minutes before KRN4884 pretreatment); and (7) endothelium denuded group (the endothelium was removed). The endothelium-derived hyperpolarizing factor-mediated relaxation to bradykinin was studied in the rings precontracted with U46619 in the presence of N(omega)-nitro-L-arginine and indomethacin. RESULTS: The maximal relaxation induced by bradykinin was reduced in hypoxia reoxygenation (40.7% +/- 2.8% vs 66.9% +/- 2.5% in control, p = 0.000). This reduced relaxation was recovered in either preconditioning (64.6% +/- 4.6%, p = 0.002), or KRN4884 pretreatment (67.1% +/- 3.6%, p = 0.000). The 5-hydroxydecanoate, but not GBC pretreatment abolished the effect of KRN44884 pretreatment (67.1% +/- 3.6% vs 42.9% +/- 3%, p = 0.001). CONCLUSIONS: Hypoxia reoxygenation reduces the relaxation mediated by endothelium-derived hyperpolarizing factor in the coronary microartery. This function can be restored by either hypoxic preconditioning or the K(ATP) channel opener KRN4884, and therefore K(ATP) channel openers may provide similar effect as preconditioning. The mechanism is mainly related to the mitochondrial ATP-sensitive K+ channels.     

Prevention of Isoflurane-induced Preconditioning by 5-Hydroxydecanoate and Gadolinium: Possible Involvement of Mitochondrial Adenosine Triphosphate-sensitive Potassium and Stretch-activated Channels

Background: Both mitochondrial adenosine triphosphate-sensitive potassium (MKATP) channels (selectively blocked by 5-hydroxydecanoate) and stretch-activated channels (blocked by gadolinium) have been involved in the mechanism of ischemic preconditioning. Isoflurane can reproduce the protection afforded by ischemic preconditioning. We sought to determine whether isoflurane-induced preconditioning may involve MKATP and stretch-activated channels.

Methods: Anesthetized open-chest rabbits underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Before this, rabbits were randomized into one of six groups and underwent a treatment period consisting of either no intervention for 40 min (control group; n = 9) or 15 min of isoflurane inhalation (1.1% end tidal) followed by a 15-min washout period (isoflurane group; n = 9). The two groups received an intravenous bolus dose of either 5-hydroxydecanoate (5 mg/kg) or gadolinium (40 [mu]mol/kg) before coronary occlusion and reperfusion (5-hydroxydecanoate, n = 9; gadolinium, n = 7). Two additional groups received 5-hydroxydecanoate or gadolinium before isoflurane exposure (isoflurane-5-hydroxydecanoate, n = 10; isoflurane-gadolinium, n = 8). Area at risk and infarct size were assessed by blue dye injection and tetrazolium chloride staining.

Results: Area at risk was comparable among the six groups (29 +/- 7, 30 +/- 5, 27 +/- 6, 35 +/- 7, 31 +/- 7, and 27 +/- 4% of the left ventricle in the control, isoflurane, isoflurane-5-hydroxydecanoate, 5-hydroxydecanoate, isoflurane-gadolinium, and gadolinium groups, respectively). Infarct size averaged 60 +/- 20% (SD) in untreated controls versus 54 +/- 27 and 65 +/- 15% of the risk zone in 5-hydroxydecanoate- and gadolinium-treated controls (P = nonsignificant). In contrast, infarct size in the isoflurane group was significantly reduced to 26 +/- 11% of the risk zone (P < 0.05 vs. control). Both 5-hydroxydecanoate and gadolinium prevented this attenuation: infarct size averaged 68 +/- 23 and 56 +/- 21% of risk zone in the isoflurane-5-hydroxydecanoate and isoflurane-gadolinium groups, respectively (P = nonsignificant vs. control).     

The inotropic and lusitropic effects of ketamine in isolated human atrial myocardium: the effect of adrenoceptor blockade

We studied the direct myocardial effects of racemic ketamine, in the presence of alpha- and beta-adrenoceptor blockade, on isolated human right atrial myocardium. Isometric force of contraction (FoC), its first derivative with time (+dF/dt), the contraction relaxation coupling parameter R2 = (+dF/dt) / (-dF/dt), and time to half relaxation (T(1/2)) were recorded before and after addition of 10(-6), 10(-5) and 10(-4) M racemic ketamine alone and in the presence of alpha-adrenoceptor blockade (phentolamine 10(-6) M) and beta-adrenoceptor blockade (propranolol at 10(-6) M). Ketamine had a moderate positive inotropic effect at 10(-5) M (FoC, 104% +/- 5% of baseline value; P = 0.03) and 10(-4) M (FoC, 107% +/- 11% of baseline value; P = 0.09). Racemic ketamine had a negative inotropic effect in the presence of propranolol (FoC, ketamine 10(-6) M, 77% +/- 11%; ketamine 10(-5) M, 63% +/- 16%; ketamine 10(-4) M, 62% +/- 17% of baseline; P < 0.001) but not phentolamine (FoC, ketamine at 10(-6) M, 94% +/- 6%; ketamine 10(-5) M, 96% +/- 5%; and ketamine 10(-4) M, 98% +/- 15% of baseline). Ketamine decreased T(1/2) (ketamine 10(-5) M, 94% +/- 3% of baseline value; P < 0.001 and ketamine 10(-4) M, 90% +/- 9% of baseline value; P = 0.007) but did not modify R2. In human right atrial myocardium, racemic ketamine induced a moderate positive inotropic effect and hastened isometric relaxation. In the presence of beta-adrenoceptor blockade it induced a direct negative inotropic effect.     

Signal Transduction of Opioid-induced Cardioprotection in Ischemia-Reperfusion

Background: Morphine reduces myocardial ischemia-reperfusion injury in vivo and in vitro. The authors tried to determine the role of opioid [delta]1 receptors, oxygen radicals, and adenosine triphosphate-sensitive potassium (KATP) channels in mediating this effect.

Methods: Chick cardiomyocytes were studied in a flow-through chamber while pH, flow rate, oxygen, and carbon dioxide tension were controlled. Cell viability was quantified by nuclear stain propidium iodide, and oxygen radicals were quantified using molecular probe 2',7'-dichlorofluorescin diacetate.

Results: Morphine (1 [mu]m) or the selective [delta]-opioid receptor agonist BW373U86 (10 pm) given for 10 min before 1 h of ischemia and 3 h of reoxygenation reduced cell death (31 +/- 5%, n = 6, and 28 +/- 5%, n = 6 [P < 0.05], respectively, 53 +/- 6%, n = 6, in controls) and generated oxygen radicals before ischemia (724 +/- 53, n = 8, and 742 +/- 75, n = 8 [P < 0.05], respectively, vs. 384 +/- 42, n = 6, in controls, arbitrary units). The protection of morphine was abolished by naloxone, or the selective [delta]1-opioid receptor antagonist 7-benzylidenenaltrexone. Reduction in cell death and increase in oxygen radicals with BW373U86 were blocked by the selective mitochondrial KATP channel antagonist 5-hydroxydecanoate or diethyldithiocarbamic acid (1000 [mu]m), which inhibited conversion of O2- to H2O2. The increase in oxygen radicals was abolished by the mitochondrial electron transport inhibitor myxothiazol. Reduction in cell death was associated with attenuated oxidant stress at reperfusion.     

Prevention of isoflurane-induced preconditioning by 5-hydroxydecanoate and gadolinium: possible involvement of mitochondrial adenosine triphosphate-sensitive potassium and stretch-activated channels

BACKGROUND: Both mitochondrial adenosine triphosphate-sensitive potassium (MKATP) channels (selectively blocked by 5-hydroxydecanoate) and stretch-activated channels (blocked by gadolinium) have been involved in the mechanism of ischemic preconditioning. Isoflurane can reproduce the protection afforded by ischemic preconditioning. We sought to determine whether isoflurane-induced preconditioning may involve MKATP and stretch-activated channels. METHODS: Anesthetized open-chest rabbits underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Before this, rabbits were randomized into one of six groups and underwent a treatment period consisting of either no intervention for 40 min (control group; n = 9) or 15 min of isoflurane inhalation (1.1% end tidal) followed by a 15-min washout period (isoflurane group; n = 9). The two groups received an intravenous bolus dose of either 5-hydroxydecanoate (5 mg/kg) or gadolinium (40 micromol/kg) before coronary occlusion and reperfusion (5-hydroxydecanoate, n = 9; gadolinium, n = 7). Two additional groups received 5-hydroxydecanoate or gadolinium before isoflurane exposure (isoflurane-5-hydroxydecanoate, n = 10; isoflurane-gadolinium, n = 8). Area at risk and infarct size were assessed by blue dye injection and tetrazolium chloride staining. RESULTS: Area at risk was comparable among the six groups (29 +/- 7, 30 +/- 5, 27 +/- 6, 35 +/- 7, 31 +/- 7, and 27 +/- 4% of the left ventricle in the control, isoflurane, isoflurane-5-hydroxydecanoate, 5-hydroxydecanoate, isoflurane-gadolinium, and gadolinium groups, respectively). Infarct size averaged 60 +/- 20% (SD) in untreated controls versus 54 +/- 27 and 65 +/- 15% of the risk zone in 5-hydroxydecanoate- and gadolinium-treated controls (P = nonsignificant). In contrast, infarct size in the isoflurane group was significantly reduced to 26 +/- 11% of the risk zone (P < 0.05 vs.control). Both 5-hydroxydecanoate and gadolinium prevented this attenuation: infarct size averaged 68 +/- 23 and 56 +/- 21% of risk zone in the isoflurane-5-hydroxydecanoate and isoflurane-gadolinium groups, respectively (P = nonsignificant vs.control). CONCLUSION: 5-Hydroxydecanoate and gadolinium inhibited pharmacologic preconditioning by isoflurane. This result suggests that MKATP channels and mechanogated channels are probably involved in this protective mechanism.     

Halothane Inhibits Contraction and Action Potential Duration to a Greater Extent in Subendocardial than Subepicardial Myocytes from the Rat Left Ventricle

Background : Halothane inhibits the 4-aminopyridine-sensitive transient outward K+ current (Ito), which in many species, including humans, plays an important role in determining action potential duration. As Ito is greater in the ventricular subepicardium than subendocardium, halothane may have differential effects on action potential duration and, therefore, contraction in cells isolated from these two regions.

Methods : Myocytes were isolated from the subendocardium and subepicardium of the rat left ventricle. Myocytes from each region were electrically stimulated at 1 Hz to measure contractions and action potentials and exposed to 0.6 mm halothane (approximately 2 x minimum alveolar concentration50 for the rat) for 1 min. The time from the peak of the action potential to repolarization at 0 and -50 mV was measured to assess the effects of halothane on action potential duration.

Results : Halothane inhibited contraction to a significantly (P = 0.002) greater extent in subendocardial myocytes than in subepicardial myocytes: the amplitude of contraction during control conditions was 3.6 +/- 0.4 [mu]m and 3.2 +/- 0.7 [mu]m in subendocardial and subepicardial cells, respectively, and this was reduced to 1.1 +/- 0.2 [mu]m (29 +/- 2% of control, P < 0.0001, n = 10) and 1.4 +/- 0.3 [mu]m (46 +/- 3% of control, P = 0.007, n = 7), respectively, after a 1-min exposure to 0.6 mm halothane. Control action potential duration (at -50 mV) was 67 +/- 10 and 28 +/- 4 ms in subendocardial and subepicardial myocytes, respectively, and these values were reduced to 39 +/- 6 ms (58 +/- 3% of control, P < 0.001) and 20 +/- 3 ms (73 +/- 5% of control, P = 0.009) by halothane, respectively.     

The role of mitochondrial and sarcolemmal K(ATP) channels in canine ethanol-induced preconditioning in vivo

Chronic consumption of small doses of ethanol protects myocardium from ischemic injury. We tested the hypothesis that mitochondrial and sarcolemmal adenosine triphosphate-dependent potassium (K(ATP)) channels mediate these beneficial effects. Dogs (n = 76) were fed with ethanol (1.5 g/kg) or water mixed with dry food bid for 6 or 12 wk, fasted overnight before experimentation, and instrumented for measurement of hemodynamics. Dogs received intracoronary saline (vehicle), 5-hydroxydecanoate (a mitochondrial K(ATP) channel antagonist; 6.75 mg/kg over 45 min), or HMR-1098 (a sarcolemmal K(ATP) channel antagonist; 45 microg/kg over 45 min) and were subjected to a 60 min coronary artery occlusion followed by 3 h of reperfusion. A final group of dogs was pretreated with ethanol and chow for 6 wk before occlusion and reperfusion. Myocardial infarct size and transmural coronary collateral blood flow were measured with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. The area at risk of infarction was similar between groups. A 12-wk pretreatment with ethanol significantly reduced infarct size to 13% +/- 2% (mean +/- SEM; n = 8) of the area at risk compared with control experiments (25% +/- 2%; n = 8), but a 6-wk pretreatment did not (21% +/- 2%; n = 8). 5-hydroxydecanoate and HMR-1098 abolished the protective effects of 12-wk ethanol pretreatment (24% +/- 2% and 29% +/- 3%, respectively; n = 8 for each group) but had no effect in dogs that did not receive ethanol (22% +/- 2% and 23% +/- 4%, respectively; n = 8 for each group). No differences in hemodynamics or transmural coronary collateral blood flow were observed between the groups. The results indicate that mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning in dogs independent of alterations in systemic hemodynamics or coronary collateral blood flow. IMPLICATIONS: Mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning independent of alterations in systemic hemodynamics or coronary collateral perfusion in vivo.     

Myocardial preconditioning against ischemia-induced apoptosis and necrosis in man

BACKGROUND: Ischemic preconditioning (IPC) protects against apoptosis and necrosis but the contribution of the two forms of cell death and whether the beneficial effects are mediated by similar or different signal transduction pathways remains unclear. Here we have investigated the effect of IPC on the type of cell death in the human heart and whether the inhibition of apoptosis and necrosis by IPC requires the opening of mitoK(ATP) channels and the activation of PKC and p38MAPK. MATERIALS AND METHODS AND RESULTS: Free-hand tissue sections (n = 6/group) obtained from the right atrium of patients at the time of coronary bypass surgery were subjected to 90-min simulated ischemia followed by 120-min reoxygenation (SI/R) with or without IPC (5 min SI/5 min R) prior to SI/R. IPC reduced apoptosis from 30.0 +/- 3.8 to 11.0 +/- 1.5% (P < 0.05) by TUNEL technique and necrosis from 11.6 +/- 2.4 to 4.2 +/- 1.7% (P < 0.05) by propidium iodide staining. When inhibitors of mitoKATP channels (1 mm 5-hydroxydecanoate), PKC (10 microm chelerythrine), and p38MAPK (10 microm SB203580) were added for 10 min before SI, the protection against necrosis was abolished. However, whereas 5-hydroxydecanoate and chelerythrine also abolished the protection of IPC against apoptosis, SB203580 did not. The activation of mitoKATP channels (100 microm diazoxide), PKC (1 microm PMA), and p38MAPK (1 nm anisomycin) were a mirror image of the findings with blockers. CONCLUSIONS: IPC protects the human myocardium against both apoptosis and necrosis. The anti-necrotic effect is mediated by the opening of mitoKATP channels and activation of PKC and p38MAPK; however, the anti-apoptotic effect requires opening of the mitoKATP channels and PKC activation but is p38MAPK-independent.     

Signal transduction of opioid-induced cardioprotection in ischemia-reperfusion

BACKGROUND: Morphine reduces myocardial ischemia-reperfusion injury in vivo and in vitro. The authors tried to determine the role of opioid delta1 receptors, oxygen radicals, and adenosine triphosphate-sensitive potassium (KATP) channels in mediating this effect. METHODS: Chick cardiomyocytes were studied in a flow-through chamber while pH, flow rate, oxygen, and carbon dioxide tension were controlled. Cell viability was quantified by nuclear stain propidium iodide, and oxygen radicals were quantified using molecular probe 2',7'-dichlorofluorescin diacetate. RESULTS: Morphine (1 microM) or the selective delta-opioid receptor agonist BW373U86 (10 pM) given for 10 min before 1 h of ischemia and 3 h of reoxygenation reduced cell death (31 +/- 5%, n = 6, and 28 +/- 5%, n = 6 [P < 0.05], respectively, 53 +/- 6%, n = 6, in controls) and generated oxygen radicals before ischemia (724 +/- 53, n = 8, and 742 +/- 75, n = 8 [P < 0.05], respectively, vs. 384 +/- 42, n = 6, in controls, arbitrary units). The protection of morphine was abolished by naloxone, or the selective delta1-opioid receptor antagonist 7-benzylidenenaltrexone. Reduction in cell death and increase in oxygen radicals with BW373U86 were blocked by the selective mitochondrial KATP channel antagonist 5-hydroxydecanoate or diethyldithiocarbamic acid (1,000 microM), which inhibited conversion of O2- to H2O2. The increase in oxygen radicals was abolished by the mitochondrial electron transport inhibitor myxothiazoL Reduction in cell death was associated with attenuated oxidant stress at reperfusion. CONCLUSION: Stimulation of delta1-opioid receptors generates oxygen radicals via mitochondrial KATP channels. This signaling pathway attenuates oxidant stress and cell death in cardiomyocytes.     

Halothane inhibits contraction and action potential duration to a greater extent in subendocardial than subepicardial myocytes from the rat left ventricle.

BACKGROUND: Halothane inhibits the 4-aminopyridine-sensitive transient outward K(+) current (I(to)) which in many species, including humans, plays an important role in determining action potential duration. As I(to) is greater in the ventricular subepicardium than subendocardium, halothane may have differential effects on action potential duration and, therefore, contraction in cells isolated from these two regions. METHODS: Myocytes were isolated from the subendocardium and subepicardium of the rat left ventricle. Myocytes from each region were electrically stimulated at 1 Hz to measure contractions and action potentials and exposed to 0.6 mm halothane (approximately 2 x minimum alveolar concentration(50) for the rat) for 1 min. The time from the peak of the action potential to repolarization at 0 and -50 mV was measured to assess the effects of halothane on action potential duration. RESULTS: Halothane inhibited contraction to a significantly (P = 0.002) greater extent in subendocardial myocytes than in subepicardial myocytes: the amplitude of contraction during control conditions was 3.6 +/- 0.4 microm and 3.2 +/- 0.7 microm in subendocardial and subepicardial cells, respectively, and this was reduced to 1.1 +/- 0.2 microm (29 +/- 2% of control, P < 0.0001, n = 10) and 1.4 +/- 0.3 microm (46 +/- 3% of control, P = 0.007, n = 7), respectively, after a 1-min exposure to 0.6 mm halothane. Control action potential duration (at -50 mV) was 67 +/- 10 and 28 +/- 4 ms in subendocardial and subepicardial myocytes, respectively, and these values were reduced to 39 +/- 6 ms (58 +/- 3% of control, P < 0.001) and 20 +/- 3 ms (73 +/- 5% of control, P = 0.009) by halothane, respectively. CONCLUSIONS: Action potential duration was reduced to a greater extent in subendocardial than subepicardial myocytes, which would contribute to the greater negative inotropic effect of halothane in the subendocardium. Furthermore, the transmural difference in action potential duration was reduced by halothane, which could contribute to its arrhythmogenic properties.     

Long-term administration of nicorandil abolishes ischemic and pharmacologic preconditioning of the human myocardium: role of mitochondrial adenosine triphosphate-dependent potassium channels

BACKGROUND: Acute administration of mitochondrial adenosine triphosphate-dependent potassium channel openers preconditions the heart, but whether their long-term administration induces a permanent state of protection is unknown. These studies investigate the effect of long-term treatment with the mitochondrial adenosine triphosphate-dependent potassium channel opener nicorandil on the response of the human myocardium to ischemia and preconditioning. METHODS: Right atrial tissue obtained from patients regularly treated with or without nicorandil (mean of 20 mg/d for 18.6 +/- 2.5 months) and undergoing cardiac surgery was sliced and equilibrated for 30 minutes and then subjected to 90 minutes of simulated ischemia, followed by 120 minutes of reoxygenation. In study 1 the following groups were studied to investigate the effect of nicorandil on the susceptibility of the myocardium to ischemia and on the protective effect of ischemic and pharmacologic preconditioning: (1) aerobic control; (2) simulated ischemia and reoxygenation alone; (3) ischemic preconditioning with 5 minutes of simulated ischemia and 5 minutes of reoxygenation; and (4) phenylephrine (0.1 micromol/L) for 5 minutes and 5 minutes' washout before simulated ischemia and reoxygenation. In study 2 the following groups were studied to investigate the effect of nicorandil on the responsiveness of mitochondrial adenosine triphosphate-dependent potassium channels: (1) aerobic control; (2) simulated ischemia and reoxygenation; (3) ischemic preconditioning; (4) diazoxide (100 micromol/L) for 10 minutes before simulated ischemia and reoxygenation, and (5) 5-hydroxydecanoate (1 mmol/L) for 10 minutes before simulated ischemia and reoxygenation. In study 3 the following groups were included to investigate the effect of the long-term administration of nicorandil on the kinase pathway involved in preconditioning: (1) aerobic control; (2) simulated ischemia and reoxygenation alone; (3) ischemic preconditioning; (4) phorbol 12-myristate 13-acetate (1 micromol/L), a protein kinase C activator, for 10 minutes before simulated ischemia and reoxygenation; and (5) anisomycin (1 nmol/L), a p38 mitogen-activated protein kinase activator, for 10 minutes before simulated ischemia and reoxygenation. At the end of each protocol, the leakage of creatine kinase (in units per gram wet weight) and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide into insoluble formazan dye (in millimoles per gram wet weight) were measured. RESULTS: In study 1 the leakage of creatine kinase and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide induced by simulated ischemia and reoxygenation were similar in the groups with or without nicorandil (creatine kinase, 3.4 +/- 0.1 and 3.5 +/- 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 74.6 +/- 3.9 and 67.9 +/- 7.3, respectively; P >.2 in each instance). Ischemic preconditioning and pharmacologic preconditioning protected the myocardium from patients without nicorandil (creatine kinase, 2.3 +/- 0.1 and 2.4 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 131.4 +/- 4.9 and 128.4 +/- 5.6, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) but not the myocardium from patients receiving nicorandil (creatine kinase, 3.3 +/- 0.1 and 3.3 +/- 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 89.7 +/- 6.5 and 86.4 +/- 5.2, respectively; P >.2 vs simulated ischemia and reoxygenation alone in each instance). In study 2 the administration of diazoxide had identical protection to that of ischemic preconditioning in the myocardium of patients not receiving nicorandil (creatine kinase, 2.1 +/- 0.2 and 2.3 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 141.4 +/- 7.4 and 131.4 +/- 4.9, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) but failed to precondition the myocardium from patients treated with nicorandil (creatine kinase, 3.3 +/- 0.2 and 3.4 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 90.1 +/- 7.2 and 86.4 +/- 5.2, respectively; P > 0.2 vs simulated ischemia and reoxygenation alone in each instance). In study 3 phorbol 12-myristate 13-acetate or anisomycin given for 10 minutes before simulated ischemia and reoxygenation afforded similar protection to that of ischemic preconditioning in the myocardium from patients with (creatine kinase, 1.5 +/- 0.3 and 1.4 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 147.0 +/- 4.9 and 160.0 +/- 16.1, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) and without nicorandil (creatine kinase, 1.7 +/- 0.4 and 1.4 +/- 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 160.3 +/- 13.6 and 158.3 +/- 11.8, respectively; P <.001 vs simulated ischemia and reoxygenation alone in each instance). CONCLUSION: The myocardium of patients chronically treated with nicorandil cannot be preconditioned either by ischemia or pharmacologically, and this is because of unresponsive mitochondrial adenosine triphosphate-dependent potassium channels. However, protection can be obtained by protein kinase C and p38 mitogen-activated protein kinase activation, which are downstream of mitochondrial adenosine triphosphate-dependent potassium channels in the signaling transduction pathway of preconditioning.     

Sevoflurane Confers Additional Cardioprotection after Ischemic Late Preconditioning in Rabbits

Background: Sevoflurane exerts cardioprotective effects that mimic the early ischemic preconditioning phenomenon (EPC) by activating adenosine triphosphate-sensitive potassium (KATP) channels. Ischemic late preconditioning (LPC) is an important cardioprotective mechanism in patients with coronary artery disease. The authors investigated whether the combination of LPC and sevoflurane-induced preconditioning results in enhanced cardioprotection and whether opening of KATP channels plays a role in this new setting.

Methods: Seventy-three rabbits were instrumented with a coronary artery occluder. After recovery for 10 days, they were subjected to 30 min of coronary artery occlusion and 120 min of reperfusion (I/R). Controls (n = 14) were not preconditioned. LPC was induced in conscious animals by a 5-min period of coronary artery occlusion 24 h before I/R (LPC, n = 15). Additional EPC was induced by a 5-min period of myocardial ischemia 10 min before I/R (LPC+EPC, n = 9). Animals of the sevoflurane (SEVO) groups inhaled 1 minimum alveolar concentration of sevoflurane for 5 min at 10 min before I/R with (LPC+SEVO, n = 10) or without (SEVO, n = 15) additional LPC. The KATP channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg) was given intravenously 10 min before sevoflurane administration (LPC+SEVO+5-HD, n = 10).

Results: Infarct size of the area at risk (triphenyltetrazolium staining) was reduced from 45 +/- 16% (mean+/-SD, control) to 27 +/- 11% by LPC (P < 0.001) and to 27 +/- 17% by sevoflurane (P = 0.001). Additional sevoflurane administration after LPC led to a further infarct size reduction to 14 +/- 8% (LPC+SEVO, P = 0.003 vs. LPC; P = 0.032 vs. SEVO), similar to the combination of LPC and EPC (12 +/- 8%; P = 0.55 vs. LPC+SEVO). Cardioprotection induced by LPC+SEVO was abolished by 5-HD (LPC+SEVO+5-HD, 41 +/- 19%, P = 0.001 vs. LPC+SEVO).     

Contractile recovery of heart muscle after hypothermic hypoxia is improved by nicorandil via mitochondrial K(ATP) channels.

BACKGROUND: The ATP-sensitive potassium channel (K(ATP)) opener nicorandil used instead of potassium in hypothermic cardioplegia significantly improves preservation of cardiac function and energetics in the in situ heart preparation. The present study, therefore, examines the effect of nicorandil at different temperatures and the role of sarcolemmal and mitochondrial K(ATP) channels under ex vivo conditions using contractile force (CF) and action potential duration (APD) as end points. METHODS: Guinea-pig papillary muscles at 37, 27, or 22 degrees C (1Hz) were exposed to nicorandil 0.2-1.1 mM. The contributions of K(ATP) channel subtypes in cardioprotection were examined using mitochondrial (mito) (0.1 mM) or non-selective (1.0 mM) concentrations of nicorandil, mito K(ATP) blocker 5-hydroxyl decanoate (5HD, 300 microM) or sarcolemmal (sarc) K(ATP) blocker HMR1098 (30 microM) before and during 140 min of hypothermic (22 degrees C) glucose-free hypoxia. RESULTS: Nicorandil >0.5 mM shortened the APD, and this was abolished by hypothermia and HMR1098 but not by 5HD. Nicorandil in both tested concentrations preserved contractile force after hypoxia-reoxygenation significantly better than control (73.7+/-4.4% and 75.8+/-3.9% vs 40.6+/-2.6%, n=6 in each group). Protection was blocked by 5HD but not by HMR1098. 5HD and HMR1098 alone did not change recovery of contractile force compared to control. CONCLUSION: Shortening of APD and activation of sarc K(ATP) by nicorandil were not related to myocardial protection. Thus, the mito K(ATP) seems to play a significant role in cardioprotection compared to the sarc K(ATP) also when substrate depletion and hypoxia are combined with hypothermia.