John G. Krolikowski

Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits.

Background: Brief episodes of ischemia during early reperfusion after coronary occlusion reduce the extent of myocardial infarction. Phosphatidylinositol-3-kinase (PI3K) signaling has been implicated in this “postconditioning” phenomenon. The authors tested the hypothesis that isoflurane produces cardioprotection during early reperfusion after myocardial ischemia by a PI3K-dependent mechanism. Methods: Pentobarbital-anesthetized rabbits (n = 80) subjected to a 30-min coronary occlusion followed by 3 h reperfusion were assigned to receive saline (control), three cycles of postconditioning ischemia (10 or 20 s each), isoflurane (0.5 or 1.0 minimum alveolar concentration), or the PI3K inhibitor wortmannin (0.6 mg/kg, intravenously) or its vehicle dimethyl sulfoxide. Additional groups of rabbits were exposed to combined postconditioning ischemia (10 s) and 0.5 minimum alveolar concentration isoflurane in the presence and absence of wortmannin. Phosphorylation of Akt, a downstream target of PI3K, was assessed by Western blotting. Results: Postconditioning ischemia for 20 s, but not 10 s, reduced infarct size (P < 0.05) (triphenyltetrazolium staining; 20 ± 3% and 34 ± 3% of the left ventricular area at risk, respectively) as compared with control (41 ± 2%). Exposure to 1.0, but not 0.5, minimum alveolar concentration isoflurane decreased infarct size (21 ± 2% and 43 ± 3%, respectively). Wortmannin abolished the protective effects of postconditioning (20 s) and 1.0 minimum alveolar concentration isoflurane. Combined postconditioning (10 s) and 0.5 minimum alveolar concentration isoflurane markedly reduced infarct size (17 ± 5%). This action was also abolished by wortmannin (44 ± 2%). Isoflurane (1.0 minimum alveolar concentration) increased Akt phosphorylation after ischemia (32 ± 6%), and this action was blocked by wortmannin. Conclusions: Isoflurane acts during early reperfusion after prolonged ischemia to salvage myocardium from infarction and reduces the threshold of ischemic postconditioning by activating PI3K.

Isoflurane produces delayed preconditioning against myocardial ischemia and reperfusion injury: role of cyclooxygenase-2.

BackgroundWhether volatile anesthetics produce a second window of preconditioning is unclear. The authors tested the hypothesis that isoflurane causes delayed preconditioning against infarction and, further, that cyclooxygenase (COX)-2 mediates this beneficial effect. MethodsRabbits (n = 43) were randomly assigned to receive 0.9% intravenous saline, the selective COX-2 inhibitor celecoxib (3 mg/kg intraperitoneal) five times over 2 days before coronary artery occlusion and reperfusion, or isoflurane (1.0 minimum alveolar concentration) 24 h before acute experimentation in the absence or presence of celecoxib pretreatment. Two additional groups of rabbits received a single dose of celecoxib either 30 min before or 21.5 h after administration of isoflurane. Rabbits were then instrumented for measurement of hemodynamics and underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Myocardial infarct size was measured using triphenyltetrazolium staining. Western immunoblotting to examine COX-1 and COX-2 protein expression was performed in rabbit hearts that had or had not been exposed to isoflurane. ResultsIsoflurane significantly (P < 0.05) reduced infarct size (22 ± 3% of the left ventricular area at risk) as compared with control (39 ± 2%). Celecoxib alone had no effect on infarct size (36 ± 4%) but abolished isoflurane-induced cardioprotection (36 ± 4%). A single dose of celecoxib administered 2.5 h before coronary occlusion and reperfusion also abolished the delayed protective effects of isoflurane (36 ± 4%), but celecoxib given 30 min before exposure to isoflurane had no effect (22 ± 4%). Isoflurane did not alter COX-1 and COX-2 protein expression. ConclusionsThe results indicate that the volatile anesthetic isoflurane produces a second window of preconditioning against myocardial ischemia and reperfusion injury. Furthermore, COX-2 is an important mediator of isoflurane-induced delayed preconditioning.

Hyperglycemia Prevents Isoflurane-induced Preconditioning against Myocardial Infarction

Background Volatile anesthetics stimulate but hyperglycemia attenuates activity of mitochondrial adenosine triphosphate–regulated potassium channels. The authors tested the hypothesis that acute hyperglycemia interferes with isoflurane-induced preconditioning in vivo. Methods Barbiturate-anesthetized dogs (n = 79) were instrumented for measurement of hemodynamics. Myocardial infarct size and collateral blood flow were assessed with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Dogs were randomly assigned to receive an infusion of normal saline (normoglycemic controls) or 15% dextrose in water to increase blood glucose concentrations to 300 or 600 mg/dl in the absence or presence of isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) in separate experimental groups. Isoflurane was discontinued, and blood glucose concentrations were allowed to return to baseline values before left anterior descending coronary artery occlusion. Results Myocardial infarct size was 26 ± 1% of the left ventricular area at risk in control experiments. Isoflurane reduced infarct size (15 ± 2 and 13 ± 1% during 0.5 and 1.0 MAC, respectively). Hyperglycemia alone did not alter infarct size (26 ± 2 and 33 ± 4% during 300 and 600 mg/dl, respectively). Moderate hyperglycemia blocked the protective effects of 0.5 MAC (25 ± 2%) but not 1.0 MAC isoflurane (13 ± 2%). In contrast, severe hyperglycemia prevented reductions of infarct size during both 0.5 MAC (29 ± 3%) and 1.0 MAC isoflurane (28 ± 4%). Conclusions Acute hyperglycemia attenuates reductions in myocardial infarct size produced by isoflurane in dogs.

Is isoflurane-induced preconditioning dose related?

Background Volatile anesthetics precondition against myocardial infarction, but it is unknown whether this beneficial action is threshold- or dose-dependent. The authors tested the hypothesis that isoflurane decreases myocardial infarct size in a dose-dependent fashion in vivo. Methods Barbiturate-anesthetized dogs (n = 40) were instrumented for measurement of systemic hemodynamics including aortic and left ventricular pressures and rate of increase of left ventricular pressure. Dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion and were randomly assigned to receive either 0.0, 0.25, 0.5, 1.0, or 1.25 minimum alveolar concentration (MAC) isoflurane in separate groups. Isoflurane was administered for 30 min and discontinued 30 min before left anterior descending coronary artery occlusion. Results Infarct size (triphenyltetrazolium staining) was 29 ± 2% of the area at risk in control experiments (0.0 MAC). Isoflurane produced significant (P < 0.05) reductions of infarct size (17 ± 3, 13 ± 1, 14 ± 2, and 11 ± 1% of the area at risk during 0.25, 0.5, 1.0, and 1.25 MAC, respectively). Infarct size was inversely related to coronary collateral blood flow (radioactive microspheres) in control experiments and during low (0.25 or 0.5 MAC) but not higher concentrations of isoflurane. Isoflurane shifted the linear regression relation between infarct size and collateral perfusion downward (indicating cardioprotection) in a dose-dependent fashion. Conclusions Concentrations of isoflurane as low as 0.25 MAC are sufficient to precondition myocardium against infarction. High concentrations of isoflurane may have greater efficacy to protect myocardium during conditions of low coronary collateral blood flow.

Inhibition of mitochondrial permeability transition enhances isoflurane-induced cardioprotection during early reperfusion: the role of mitochondrial KATP channels.

Inhibition of the mitochondrial permeability transition pore (mPTP) mediates the protective effects of brief, repetitive ischemic episodes during early reperfusion after prolonged coronary artery occlusion. Brief exposure to isoflurane immediately before and during early reperfusion also produces cardioprotection, but whether mPTP is involved in this beneficial effect is unknown. We tested the hypothesis that mPTP mediates isoflurane-induced postconditioning and also examined the role of mitochondrial KATP (mKATP) channels in this process. Rabbits (n = 102) subjected to a 30-min coronary occlusion followed by 3 h reperfusion received 0.9% saline (control), isoflurane (0.5 or 1.0 MAC) administered for 3 min before and 2 min after reperfusion, or the mPTP inhibitor cyclosporin A (CsA, 5 or 10 mg/kg) in the presence or absence of the mPTP opener atractyloside (5 mg/kg) or the selective mKATP channel antagonist 5-hydroxydecanoate (5-HD; 10 mg/kg). Other rabbits received 0.5 MAC isoflurane plus 5 mg/kg CsA in the presence and absence of atractyloside or 5-HD. Isoflurane (1.0 but not 0.5 MAC) and CsA (10 but not 5 mg/kg) reduced (P < 0.05) infarct size (21% ± 4%, 44% ± 6%, 24% ± 3%, and 43% ± 6%, respectively, mean ± sd of left ventricular area at risk; triphenyltetrazolium staining) as compared with control (42% ± 7%). Isoflurane (0.5 MAC) plus CsA (5 mg/kg) was also protective (27% ± 4%). Neither atractyloside nor 5-HD alone affected infarct size, but these drugs abolished protection by 1.0 MAC isoflurane, 10 mg/kg CsA, and 0.5 MAC isoflurane plus 5 mg/kg CsA. The results indicate that mPTP inhibition enhances, whereas opening abolishes, isoflurane-induced postconditioning. This isoflurane-induced inhibition of mitochondrial permeability transition is dependent on activation of mitochondrial KATP channels in vivo.

Noble Gases Without Anesthetic Properties Protect Myocardium Against Infarction by Activating Prosurvival Signaling Kinases and Inhibiting Mitochondrial Permeability Transition In Vivo

BACKGROUND:The anesthetic noble gas, xenon, produces cardioprotection. We hypothesized that other noble gases without anesthetic properties [helium (He), neon (Ne), argon (Ar)] also produce cardioprotection, and further hypothesized that this beneficial effect is mediated by activation of prosurvival signaling kinases [including phosphatidylinositol-3-kinase, extracellular signal-regulated kinase, and 70-kDa ribosomal protein s6 kinase] and inhibition of mitochondrial permeability transition pore (mPTP) opening in vivo. METHODS:Rabbits (n = 98) instrumented for hemodynamic measurement and subjected to a 30-min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), three cycles of 70% He-, Ne-, or Ar-30% O2 administered for 5 min interspersed with 5 min of 70% N2–30% O2 before LAD occlusion, or three cycles of brief (5 min) ischemia interspersed with 5 min reperfusion before prolonged LAD occlusion and reperfusion (ischemic preconditioning). Additional groups of rabbits received selective inhibitors of phosphatidylinositol-3-kinase (wortmannin; 0.6 mg/kg), extracellular signal-regulated kinase (PD 098059; 2 mg/kg), or 70-kDa ribosomal protein s6 kinase (rapamycin; 0.25 mg/kg) or mPTP opener atractyloside (5 mg/kg) in the absence or presence of He pretreatment. RESULTS:He, Ne, Ar, and ischemic preconditioning significantly (P < 0.05) reduced myocardial infarct size [23% ± 4%, 20% ± 3%, 22% ± 2%, 17% ± 3% of the left ventricular area at risk (mean ± sd); triphenyltetrazolium chloride staining] versus control (45% ± 5%). Wortmannin, PD 098059, rapamycin, and atractyloside alone did not affect infarct size, but these drugs abolished He-induced cardioprotection. CONCLUSIONS:The results indicate that noble gases without anesthetic properties produce cardioprotection by activating prosurvival signaling kinases and inhibiting mPTP opening in rabbits.

Morphine Enhances Isoflurane-Induced Postconditioning Against Myocardial Infarction: The Role of Phosphatidylinositol-3-Kinase and Opioid Receptors in Rabbits

Isoflurane reduces myocardial infarct size during early reperfusion by activating phosphatidylinositol-3-kinase (PI3K) signaling. We tested the hypothesis that this cardioprotection against reperfusion injury is enhanced by morphine and that a decrease in apoptosis plays a role in preservation of myocardial viability. Rabbits (n = 108) instrumented for hemodynamic measurement and subjected to a 30-min coronary occlusion followed by 3 h reperfusion received 0.9% saline, the selective PI3K inhibitor wortmannin (0.6 mg/kg), or the nonselective opioid antagonist naloxone (6 mg/kg) before coronary occlusion in the presence or absence of isoflurane (0.5 or 1.0 MAC), morphine (0.05 or 0.1 mg/kg), or their combination administered for 3 min before and 2 min after reperfusion. Infarct size was determined using triphenyltetrazolium staining and apoptosis assessed using cytochrome c translocation and Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) staining of left ventricular myocardium in situ. Isoflurane (1.0 but not 0.5 MAC) and morphine (0.1 but not 0.05 mg/kg) reduced (P < 0.05) infarct size (mean ± sd 21% ± 4%, 44% ± 6%, 19% ± 4%, and 41% ± 6% of left ventricular area at risk, respectively) as compared with control (41% ± 4%). The combination of 0.5 MAC isoflurane and 0.05 mg/kg morphine also decreased infarct size (18% ± 9%). Wortmannin and naloxone alone did not affect infarct size but blocked the protection produced by isoflurane, morphine, and their combination. Isoflurane and morphine reduced cytochrome c translocation and TUNEL staining. The results indicate that morphine enhances isoflurane-induced postconditioning by activating PI3K and opioid receptors in vivo. A reduction in apoptotic cell death contributes to preservation of myocardial integrity during postconditioning by isoflurane.

Intravenous emulsified halogenated anesthetics produce acute and delayed preconditioning against myocardial infarction in rabbits.

Background:Preconditioning against myocardial infarction by volatile anesthetics is well known. The authors tested the hypothesis that new emulsified formulations of halogenated anesthetics administered intravenously reduce myocardial infarct size when administered either 1 or 24 h before prolonged ischemia and reperfusion. Methods:Pentobarbital-anesthetized rabbits (n = 39) were instrumented for measurement of hemodynamics and randomly assigned to receive intravenous saline (control), lipid vehicle, or infusions (3.5 ml · kg−1 · h−1 for 30 min) of emulsified isoflurane (6.9%), enflurane (7.1%), or sevoflurane (7.5%). Infusions were discontinued 30 min before a 30-min coronary occlusion and 3 h of reperfusion. In three additional groups, conscious rabbits (n = 21) received saline, lipid vehicle, or emulsified sevoflurane (7.5%) infusions (3.5 ml · kg−1 · h−1 for 30 min) 24 h before ischemia and reperfusion. Infarct size was determined using triphenyltetrazolium staining. Results:Lipid vehicle produced transient increases in heart rate, whereas emulsified volatile anesthetics had no effect on hemodynamics before coronary occlusion. Lipid vehicle did not affect infarct size (38 ± 2% of the area at risk; mean ± SEM) as compared with saline control (41 ± 4%). In contrast, emulsified isoflurane, enflurane, and sevoflurane reduced infarct size (20 ± 3%, 20 ± 3%, and 21 ± 2% of the area at risk, respectively; P < 0.05). Administration of lipid vehicle or emulsified sevoflurane did not produce sedation or respiratory depression in conscious rabbits. Emulsified sevoflurane (18 ± 2%) but not lipid vehicle (44 ± 2%) reduced infarct size as compared with control in delayed preconditioning experiments. Conclusions:Intravenous emulsified halogenated anesthetics produce acute and delayed preconditioning against myocardial infarction.

Role of endothelial nitric oxide synthase as a trigger and mediator of isoflurane-induced delayed preconditioning in rabbit myocardium.

Background:Isoflurane produces delayed preconditioning in vivo. The authors tested the hypothesis that endothelial, inducible, or neuronal nitric oxide synthase (NOS) is a trigger or mediator of this protective effect. Methods:In the absence or presence of exposure to isoflurane (1.0 minimum alveolar concentration) 24 h before experimentation, pentobarbital-anesthetized rabbits (n = 128) instrumented for hemodynamic measurement received 0.9% saline (control), the nonselective NOS inhibitor N-nitro-l-arginine methyl ester (10 mg/kg), one of two of the selective inducible NOS antagonists aminoguanidine (300 mg/kg) or 1400W (0.5 mg/kg), or the selective neuronal NOS inhibitor 7-nitroindazole (50 mg/kg) administered before exposure to isoflurane (trigger; day 1) or left anterior descending coronary artery occlusion (mediator; day 2). All rabbits underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Tissue samples for reverse-transcription polymerase chain reaction and immunohistochemistry were also obtained in the presence or absence of N-nitro-l-arginine methyl ester with or without isoflurane pretreatment. Results:Isoflurane significantly (P < 0.05) reduced infarct size (23 ± 5% [mean ± SD] of the left ventricular area at risk; triphenyltetrazolium chloride staining) as compared with control (42 ± 7%). N-nitro-l-arginine methyl ester administered before isoflurane or coronary occlusion abolished protection (49 ± 7 and 43 ± 10%, respectively). Aminoguanidine, 1400W, and 7-nitroindazole did not alter infarct size or affect isoflurane-induced delayed preconditioning. Isoflurane increased endothelial but not inducible NOS messenger RNA transcription and protein translation immediately and 24 h after administration of the volatile agent. Pretreatment with N-nitro-l-arginine methyl ester attenuated isoflurane-induced increases in endothelial NOS expression. Conclusions:The results suggest that endothelial NOS but not inducible or neuronal NOS is a trigger and mediator of delayed preconditioning by isoflurane in vivo.

Role of Erk1/2, p70s6K, and eNOS in isoflurane-induced cardioprotection during early reperfusion in vivo.

PurposeAdministration of isoflurane during early reperfusion after prolonged coronary artery occlusion decreases myocardial infarct size by activating phosphatidylinositol-3-kinase (PI3K) signal transduction. The extracellular signal-related kinases (Erk1/2) represent a redundant mechanism by which signaling elements downstream from PI3K, including 70-kDA ribosomal protein s6 kinase (p70s6K) and endothelial nitric oxide synthase (eNOS), may be activated to reduce reperfusion injury. We tested the hypothesis Erk1/2, p70s6K, and eNOS mediate isoflurane-induced postconditioning in rabbit myocardiumin vivo.MethodsBarbiturate-anesthetized rabbits (n = 78) instrumented for measurement of systemic hemodynamics were subjected to a 30-min coronary occlusion followed by three hours reperfusion. Rabbits were randomly assigned to receive 0.9% saline (control), the Erk1/2 inhibitor PD 098059 (2 mg·kg-1), the p70s6K inhibitor rapamycin (0.25 mg·kg-1), the nonselective nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME; 10 mg·kg-1), the selective inducible NOS antagonist aminoguanidine hydrochloride (AG, 300 mg·kg-1), or the selective neuronal NOS inhibitor 7-nitroindazole (7-NI, 50 mg·kg-1) in the presence or absence of 1.0 minimum alveolar concentration isoflurane administered for three minutes before and two minutes after reperfusion.ResultsBrief exposure to 1.0 minimum alveolar concentration isoflurane reduced (P < 0.05) infarct size (21 ± 4% [mean ± SD] of left ventricle area at risk, respectively; triphenyltetrazolium staining) as compared to control (41 ± 5%). PD 098059, rapamycin, and L-NAME, but not AG nor 7-NI, abolished the protection produced by isoflurane.ConclusionThe results suggest that the protective effects of isoflurane against infarction during early reperfusion are mediated by Erk1/2, p70s6K, and eNOSin vivo.RésuméObjectifĽadministration ďisoflurane pendant la reperfusion précoce qui suit une occlusion prolongée de ľartère coronaire diminue la taille de ľinfarctus myocardique en activant la transduction du signal de la phosphatidylinositol-3-kinase (PI3K). Les kinases extracellulaires reliées au signal (Erk1/2) représentent un mécanisme redondant par lequel le signalement des éléments en aval à partir de PI3K, incluant la s6 kinase de protéines ribosomales 70-kDA (p70s6K) et ľoxyde nitrique synthase endothéliale (eNOS), peuvent être activés pour réduire la lésion de reperfusion. Nous avons testé ľhypothèse que les Erk1/2, p70s6K et eNOS assuraient la médiation du postconditionnement induit par ľisoflurane dans des myocardes de lapin in vivo.MéthodeDes lapins anesthésiés aux barbituriques (n = 78), instrumentés pour la mesure de ľhémodynamique générale, ont été soumis à une occlusion coronaire de 30 min, suivie de trois heures de reperfusion. Répartis au hasard, ils ont reçu une solution salée à 0,9 % (témoin), ľinhibiteur de Erk1/2, PD 098059 (2 mg·kg-1), ľinhibiteur de p70s6K, la rapamycine (0,25 mg·kg-1), ľinhibiteur non sélectif de ľoxyde nitrique synthase (NOS) ľester méthylique N-nitro-L-arginine (L-NAME; 10 mg·kg-1), ľantagoniste sélectif de la NOS inductible, le chlorhydrate ďaminoguanidine (AG, 300 mg·kg-1) ou ľinhibiteur sélectif de NOS neuronal, le 7-nitro-indazole (7-NI, 50 mg·kg-1) en présence ou non ďune concentration alvéolaire minimale de 1,0 ďisoflurane administrée pendant trois minutes avant et deux minutes après la reperfusion.RésultatsĽexposition brève à une concentration alvéolaire minimale de 1,0 ďisoflurane a réduit (P < 0,05) la taille de ľinfarctus (21 ± 4 % [moyenne ± ET] de ľaire du ventricule gauche à risque, respectivement; coloration au triphényltétrazolium) comparativement au témoin (41 ± 5 %). Les PD 098059, rapamycine et L-NAME, mais non les AG ou 7-NI, ont aboli la protection produite par ľisoflurane.ConclusionLe résultat suggère que les effets protecteurs de ľisoflurane contre ľinfarctus pendant la reperfusion précoce dépendent de la médiation des Erk1/2, p70s6K et eNOS in vivo.Objectif Ľadministration ďisoflurane pendant la reperfusion precoce qui suit une occlusion prolongee de ľartere coronaire diminue la taille de ľinfarctus myocardique en activant la transduction du signal de la phosphatidylinositol-3-kinase (PI3K). Les kinases extracellulaires reliees au signal (Erk1/2) representent un mecanisme redondant par lequel le signalement des elements en aval a partir de PI3K, incluant la s6 kinase de proteines ribosomales 70-kDA (p70s6K) et ľoxyde nitrique synthase endotheliale (eNOS), peuvent etre actives pour reduire la lesion de reperfusion. Nous avons teste ľhypothese que les Erk1/2, p70s6K et eNOS assuraient la mediation du postconditionnement induit par ľisoflurane dans des myocardes de lapin in vivo.