Lynda M. Ludwig

Mechanisms of Cardioprotection by Volatile Anesthetics

A RAPIDLY growing body of evidence indicates that volatile anesthetics protect myocardium against reversible and irreversible ischemic injury. Identifying the mechanisms by which volatile agents mediate these antiischemic actions is the subject of intense research. This objective has been difficult to accomplish because volatile anesthetics also profoundly affect cardiovascular function. Volatile agents reduce arterial and coronary perfusion pressure, cause dose-related depression of myocardial contractility, produce coronary vasodilation, affect electrophysiologic function, and modify autonomic nervous system activity to varying degrees. Therefore, the antiischemic effects of volatile anesthetics may be mediated, at least in part, by favorable alterations in myocardial oxygen supply–demand relations, preservation of energy-dependent cellular functions, and increased coronary blood flow. However, it seems unlikely that changes in myocardial metabolism and coronary perfusion caused by volatile anesthetics are solely responsible for protection against ischemic damage. Instead, several endogenous signal transduction pathways, acting through the adenosine triphosphate (ATP)–sensitive potassium (KATP) channel and involving the generation of reactive oxygen species (ROS), have been implicated in mediating the antiischemic actions of volatile anesthetics. The experimental and clinical findings documenting the phenomenon of volatile anesthetic preconditioning against ischemic injury of myocardium are evaluated. Recent findings in vitro and in vivo that seek to define the intracellular mechanisms responsible for these beneficial actions are also summarized.

Mechanism of Preconditioning by Isoflurane in Rabbits: A Direct Role for Reactive Oxygen Species

Background Reactive oxygen species (ROS) contribute to myocardial protection during ischemic preconditioning, but the role of the ROS in protection against ischemic injury produced by volatile anesthetics has only recently been explored. We tested the hypothesis that ROS mediate isoflurane-induced preconditioning in vivo. Methods Pentobarbital-anesthetized rabbits were instrumented for measurement of hemodynamics and were subjected to a 30 min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive vehicle (0.9% saline), or the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptopropionyl glycine (2-MPG; 1 mg · kg−1· min−1), in the presence or absence of 1.0 minimum alveolar concentration (MAC) isoflurane. Isoflurane was administered for 30 min and then discontinued 15 min before coronary artery occlusion. A fluorescent probe for superoxide anion production (dihydroethidium, 2 mg) was administered in the absence of the volatile anesthetic or 5 min before exposure to isoflurane in 2 additional groups (n = 8). Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively. Results Isoflurane (P < 0.05) decreased infarct size to 24 ± 4% (mean ± SEM; n = 10) of the left ventricular area at risk compared with control experiments (43 ± 3%; n = 8). NAC (43 ± 3%; n = 7) and 2-MPG (42 ± 5%; n = 8) abolished this beneficial effect, but had no effect on myocardial infarct size (47 ± 3%; n = 8 and 46 ± 3; n = 7, respectively) when administered alone. Isoflurane increased superoxide anion production as compared with control experiments (28 ± 12 vs. −6 ± 9 fluorescence units;P < 0.05). Conclusions The results indicate that ROS produced following administration of isoflurane contribute to protection against myocardial infarction in vivo.

Mitochondrial adenosine triphosphate-regulated potassium channel opening acts as a trigger for isoflurane-induced preconditioning by generating reactive oxygen species

Background Whether the opening of mitochondrial adenosine triphosphate–regulated potassium (KATP) channels is a trigger or an end effector of anesthetic-induced preconditioning is unknown. We tested the hypothesis that the opening of mitochondrial KATP channels triggers isoflurane-induced preconditioning by generating reactive oxygen species (ROS) in vivo. Methods Pentobarbital-anesthetized rabbits were subjected to a 30-min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive a vehicle (0.9% saline) or the selective mitochondrial KATP channel blocker 5-hydroxydecanoate (5-HD) alone 10 min before or immediately after a 30-min exposure to 1.0 minimum alveolar concentration (MAC) isoflurane. In another series of experiments, the fluorescent probe dihydroethidium was used to assess superoxide anion production during administration of 5-HD or the ROS scavengers N-acetylcysteine or N-2-mercaptopropionyl glycine (2-MPG) in the presence or absence of 1.0 MAC isoflurane. Myocardial infarct size and superoxide anion production were measured using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively. Results Isoflurane (P < 0.05) decreased infarct size to 19 ± 3% (mean ± SEM) of the left ventricular area at risk as compared to the control (38 ± 4%). 5-HD administered before but not after isoflurane abolished this beneficial effect (37 ± 4% as compared to 24 ± 3%). 5-HD alone had no effect on infarct size (42 ± 3%). Isoflurane increased fluorescence intensity. Pretreatment with N-acetylcysteine, 2-MPG, or 5-HD before isoflurane abolished increases in fluorescence, but administration of 5-HD after isoflurane only partially attenuated increases in fluorescence produced by the volatile anesthetic agent. Conclusions The results indicate that mitochondrial KATP channel opening acts as a trigger for isoflurane-induced preconditioning by generating ROS in vivo.

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.

Morphine enhances pharmacological preconditioning by isoflurane: Role of mitochondrial KATP channels and opioid receptors

Background Adenosine triphosphate–regulated potassium channels mediate protection against myocardial infarction produced by volatile anesthetics and opioids. We tested the hypothesis that morphine enhances the protective effect of isoflurane by activating mitochondrial adenosine triphosphate–regulated potassium channels and opioid receptors. Methods Barbiturate-anesthetized rats (n = 131) were instrumented for measurement of hemodynamics and subjected to a 30 min coronary artery occlusion followed by 2 h of reperfusion. Myocardial infarct size was determined using triphenyltetrazolium staining. Rats were randomly assigned to receive 0.9% saline, isoflurane (0.5 and 1.0 minimum alveolar concentration [MAC]), morphine (0.1 and 0.3 mg/kg), or morphine (0.3 mg/kg) plus isoflurane (1.0 MAC). Isoflurane was administered for 30 min and discontinued 15 min before coronary occlusion. In eight additional groups of experiments, rats received 5-hydroxydecanoic acid (5-HD; 10 mg/kg) or naloxone (6 mg/kg) in the presence or absence of isoflurane, morphine, and morphine plus isoflurane. Results Isoflurane (1.0 MAC) and morphine (0.3 mg/kg) reduced infarct size (41 ± 3%; n = 13 and 38 ± 2% of the area at risk; n = 10, respectively) as compared to control experiments (59 ± 2%; n = 10). Morphine plus isoflurane further decreased infarct size to 26 ± 3% (n = 11). 5-HD and naloxone alone did not affect infarct size, but abolished cardioprotection produced by isoflurane, morphine, and morphine plus isoflurane. Conclusions Combined administration of isoflurane and morphine enhances the protection against myocardial infarction to a greater extent than either drug alone. This beneficial effect is mediated by mitochondrial adenosine triphosphate–regulated potassium channels and opioid receptors in vivo.

Protein kinase C translocation and Src protein tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo : potential downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and reactive oxygen species

BackgroundThe authors tested the hypotheses that protein kinase C (PKC)–specific isoform translocation and Src protein tyrosine kinase (PTK) activation play important roles in isoflurane-induced preconditioning in vivo. MethodsRats (n = 125) instrumented for measurement of hemodynamics underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion and received 0.9% saline (control); PKC inhibitors chelerythrine (5 mg/kg), rottlerin (0.3 mg/kg), or PKC-&egr;V1–2 peptide (1 mg/kg); PTK inhibitors lavendustin A (1 mg/kg) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 1 mg/kg); mitochondrial adenosine triphosphate–sensitive potassium channel antagonist 5-hydroxydecanote (10 mg/kg); or reactive oxygen species scavenger N-acetylcysteine (150 mg/kg) in the absence and presence of a 30-min exposure to isoflurane (1.0 minimum alveolar concentration) in separate groups. Isoflurane was discontinued 15 min before coronary occlusion (memory period). Infarct size was determined using triphenyltetrazolium staining. Immunohistochemistry and confocal microscopic imaging were performed to examine PKC translocation in separate groups of rats. ResultsIsoflurane significantly (P < 0.05) reduced infarct size (40 ± 3% [n = 13]) as compared with control experiments (58 ± 2% [n = 12]). Chelerythrine, rottlerin, PKC-&egr;V1–2 peptide, lavendustin A, PP1, 5-hydroxydecanote, and N-acetylcysteine abolished the anti-ischemic actions of isoflurane (58 ± 2% [n = 8], 50 ± 3% [n = 9], 53 ± 2% [n = 9], 59 ± 3% [n = 6], 57 ± 3% [n = 7], 60 ± 3% [n = 7], and 53 ± 3% [n = 6], respectively). Isoflurane stimulated translocation of the &dgr; and &egr; isoforms of PKC to sarcolemmal and mitochondrial membranes, respectively. ConclusionsProtein kinase C-&dgr;, PKC-&egr;, and Src PTK mediate isoflurane-induced preconditioning in the intact rat heart. Opening of mitochondrial adenosine triphosphate–sensitive potassium channels and generation of reactive oxygen species are upstream events of PKC activation in this signal transduction process.

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.

Chronic Hyperglycemia Attenuates Coronary Collateral Development and Impairs Proliferative Properties of Myocardial Interstitial Fluid by Production of Angiostatin

Background—Development of coronary collateral vessels is impaired in patients with diabetes mellitus. We tested the hypothesis that hyperglycemia alone attenuates collateral development and abolishes proliferative properties of myocardial interstitial fluid (MIF) by enhancing expression of matrix metalloproteinases (MMP) and angiostatin. Methods and Results—Chronically instrumented dogs were randomly assigned to receive an infusion of normal saline (control; n=9) or 70% dextrose in water to increase blood glucose to 350 to 400 mg/dL for 8 h/d (hyperglycemia; n=7) in the presence or absence (sham; n=9) of brief (2 minutes), repetitive coronary artery occlusions (1/h; 8/d for 21 days). Collateral perfusion increased to 41±11% and 49±6% of normal zone flow in control dogs on days 14 and 21 (P <0.05) but remained unchanged over 21 days in hyperglycemic and sham dogs (12±3% and 13±3%, respectively). A progressive reduction of the postocclusive peak reactive hyperemic response was also observed in control dogs (16±1 to 10±1 Hz · 102 on days 1 and 21, respectively) but not in hyperglycemic (17±2 to 20±2) or sham (17±2 to 16±1) dogs. Endothelial cell tube formation was produced by MIF obtained from control dogs but not hyperglycemic or sham dogs. Coincubation of MIF from hyperglycemic dogs with an angiostatin antibody restored endothelial cell tube formation. MMP-9 activity and expression of angiostatin were increased in dogs receiving exogenous glucose compared with controls Conclusions—Chronic hyperglycemia abolishes development of coronary collateral vessels by increasing MMP-9 activity and angiostatin expression in dogs.

Delta opioid agonists and volatile anesthetics facilitate cardioprotection via potentiation of KATP channel opening

Opioids and volatile anesthetics produce marked cardioprotective effects against myocardial infarction via the activation of ATP‐sensitive potassium (KATP) channels, however, the effect of combined treatment with both drugs is unknown. We examined the hypothesis that opioids and volatile anesthetics potentiate cardiac KATP channel opening, thereby enhancing cardioprotection. Rats were treated with the delta opioid agonists, TAN‐67 or BW373U86, or isoflurane, together or alone with and without diazoxide, a mitochondrial KATP channel opener. Glibenclamide, a non‐selective KATP channel blocker, was used to further characterize the signaling mechanism involved. Myocardial infarct size (IS) was determined by tetrazolium staining and was expressed as a percent of the area at risk (AAR). High doses of TAN‐67 (10 mg/kg), diazoxide (10 mg/kg), and isoflurane (1 MAC) produced a significant reduction in IS compared with the control group (30±3%, 36±5%, and 42±2 vs. 58±2%, respectively), whereas lower doses of the drugs had no effect except for the low dose of isoflurane (0.5 MAC). The combination of TAN‐67 and diazoxide or isoflurane and diazoxide resulted in a marked reduction in IS compared with controls in the presence of high (9±3% and 14±3%) and low (17±7% and 31±7%) dose combinations, respectively. The combination of TAN‐67 or BW373U86 and isoflurane also caused a striking reduction in IS/AAR (16±7% and 7±2%, respectively). To date, this is the first demonstration that opioids and volatile anesthetics work in conjunction to confer protection against myocardial infarction through potentiation of cardiac KATP channel opening.

Preconditioning by isoflurane is mediated by reactive oxygen species generated from mitochondrial electron transport chain complex III.

Reactive oxygen species (ROS) mediate volatile anesthetic preconditioning. We tested the hypothesis that isoflurane (ISO) generates ROS from electron transport chain complexes I and III. Rabbits (n = 55) underwent 30 min coronary artery occlusion followed by 3 h reperfusion and received 0.9% saline, the complex I inhibitor diphenyleneiodonium (DPI; 1.5 mg/kg bolus followed by 1.5 mg/kg over 1 h), or the complex III inhibitor myxothiazol (MYX; 0.1 mg/kg bolus followed by 0.3 mg/kg over 1 h) in the absence and presence of 1.0 minimum alveolar concentration ISO. ISO was administered for 30 min and discontinued 15 min before coronary occlusion. Infarct size and ROS production (n = 32) were determined using triphenyltetrazolium staining and ethidium-DNA fluorescence, respectively. Adenosine triphosphate (ATP) synthesis in mitochondria obtained from rabbit hearts (n = 24) subjected to drug interventions was measured by luciferin-luciferase luminometry. ISO significantly (P < 0.05) reduced infarct size (19% ± 4%) as compared with control (39% ± 4%). MYX (35% ± 4%), but not DPI (24% ± 2%), abolished this protection. ISO increased ethidium-DNA fluorescence (83 ± 11 U) as compared with control (40 ± 12 U). MYX (35 ± 3 U), but not DPI (78 ± 9 U), abolished ROS generation. DPI and MYX selectively reduced complex I- and complex III-mediated ATP synthesis, respectively. ROS generated from electron transport chain complex III mediate ISO-induced cardioprotection.