Failure of Isoflurane Cardiac Preconditioning in Obese Type 2 Diabetic Mice Involves Aberrant Regulation of MicroRNA-21, Endothelial Nitric-oxide Synthase, and Mitochondrial Complex I.

BACKGROUND: Diabetes impairs the cardioprotective effect of volatile anesthetics, yet the mechanisms are still murky. We examined the regulatory effect of isoflurane on microRNA-21, endothelial nitric-oxide synthase, and mitochondrial respiratory complex I in type 2 diabetic mice. METHODS: Myocardial ischemia/reperfusion injury was produced in obese type 2 diabetic (db/db) and C57BL/6 control mice ex vivo in the presence or absence of isoflurane administered before ischemia. Cardiac microRNA-21 was quantified by real-time quantitative reverse transcriptional-polymerase chain reaction. The dimers and monomers of endothelial nitric-oxide synthase were measured by Western blot analysis. Mitochondrial nicotinamide adenine dinucleotide fluorescence was determined in Langendorff-perfused hearts. RESULTS: Body weight and fasting blood glucose were greater in db/db than C57BL/6 mice. Isoflurane decreased left ventricular end-diastolic pressure from 35 ± 8 mmHg in control to 23 ± 9 mmHg (P = 0.019, n = 8 mice/group, mean ± SD) and elevated ±dP/dt 2 h after post-ischemic reperfusion in C57BL/6 mice. These beneficial effects of isoflurane were lost in db/db mice. Isoflurane elevated microRNA-21 and the ratio of endothelial nitric-oxide synthase dimers/monomers and decreased mitochondrial nicotinamide adenine dinucleotide levels 5 min after ischemia in C57BL/6 but not db/db mice. MicroRNA-21 knockout blocked these favorable effects of isoflurane, whereas endothelial nitric-oxide synthase knockout had no effect on the expression of microRNA-21 but blocked the inhibitory effect of isoflurane preconditioning on nicotinamide adenine dinucleotide. CONCLUSIONS: Failure of isoflurane cardiac preconditioning in obese type 2 diabetic db/db mice is associated with aberrant regulation of microRNA-21, endothelial nitric-oxide synthase, and mitochondrial respiratory complex I.

Alagebrium inhibits neointimal hyperplasia and restores distributions of wall shear stress by reducing downstream vascular resistance in obese and diabetic rats.

Mechanisms of restenosis in type 2 diabetes mellitus (T2DM) are incompletely elucidated, but advanced glycation end-product (AGE)-induced vascular remodeling likely contributes. We tested the hypothesis that AGE-related collagen cross-linking (ARCC) leads to increased downstream vascular resistance and altered in-stent hemodynamics, thereby promoting neointimal hyperplasia (NH) in T2DM. We proposed that decreasing ARCC with ALT-711 (Alagebrium) would mitigate this response. Abdominal aortic stents were implanted in Zucker lean (ZL), obese (ZO), and diabetic (ZD) rats. Blood flow, vessel diameter, and wall shear stress (WSS) were calculated after 21 days, and NH was quantified. Arterial segments (aorta, carotid, iliac, femoral, and arterioles) were harvested to detect ARCC and protein expression, including transforming growth factor-ß (TGF-ß) and receptor for AGEs (RAGE). Downstream resistance was elevated (60%), whereas flow and WSS were significantly decreased (44% and 56%) in ZD vs. ZL rats. NH was increased in ZO but not ZD rats. ALT-711 reduced ARCC and resistance (46%) in ZD rats while decreasing NH and producing similar in-stent WSS across groups. No consistent differences in RAGE or TGF-ß expression were observed in arterial segments. ALT-711 modified lectin-type oxidized LDL receptor 1 but not RAGE expression by cells on decellularized matrices. In conclusion, ALT-711 decreased ARCC, increased in-stent flow rate, and reduced NH in ZO and ZD rats through RAGE-independent pathways. The study supports an important role for AGE-induced remodeling within and downstream of stent implantation to promote enhanced NH in T2DM.

Isoflurane favorably modulates guanosine triphosphate cyclohydrolase-1 and endothelial nitric oxide synthase during myocardial ischemia and reperfusion injury in rats.

BACKGROUND: The authors investigated the hypothesis that isoflurane modulates nitric oxide (NO) synthesis and protection against myocardial infarction through time-dependent changes in expression of key NO regulatory proteins, guanosine triphosphate cyclohydrolase (GTPCH)-1, the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin and endothelial nitric oxide synthase (eNOS). METHODS: Myocardial infarct size, NO production (ozone-mediated chemiluminescence), GTPCH-1, and eNOS expression (real-time reverse transcriptase polymerase chain reaction and western blotting) were measured in male Wistar rats with or without anesthetic preconditioning (APC; 1.0 minimum alveolar concentration isoflurane for 30 min) and in the presence or absence of an inhibitor of GTPCH-1, 2,4-diamino-6-hydroxypyrimidine. RESULTS: NO2 production (158 ± 16 and 150 ± 13 pmol/mg protein at baseline in control and APC groups, respectively) was significantly (P < 0.05) increased 1.5 ± 0.1 and 1.4 ± 0.1 fold by APC (n = 4) at 60 and 90 min of reperfusion, respectively, concomitantly, with increased expression of GTPCH-1 (1.3 ± 0.3 fold; n = 5) and eNOS (1.3 ± 0.2 fold; n = 5). In contrast, total NO (NO2 and NO3) was decreased after reperfusion in control experiments. Myocardial infarct size was decreased (43 ± 2% of the area at risk for infarction; n = 6) by APC compared with control experiments (57 ± 1%; n = 6). 2, 4-Diamino-6-hydroxypyrimidine decreased total NO production at baseline (221 ± 25 and 175 ± 31 pmol/mg protein at baseline in control and APC groups, respectively), abolished isoflurane-induced increases in NO at reperfusion, and prevented reductions of myocardial infarct size by APC (60 ± 2%; n = 6). CONCLUSION: APC favorably modulated a NO biosynthetic pathway by up-regulating GTPCH-1 and eNOS, and this action contributed to protection of myocardium against ischemia and reperfusion injury.

MicroRNA-21 Mediates Isoflurane-induced Cardioprotection against Ischemia-Reperfusion Injury via Akt/Nitric Oxide Synthase/Mitochondrial Permeability Transition Pore Pathway.

BACKGROUND: The role of microRNA-21 in isoflurane-induced cardioprotection is unknown. The authors addressed this issue by using microRNA-21 knockout mice and explored the underlying mechanisms. METHODS: C57BL/6 and microRNA-21 knockout mice were echocardiographically examined. Mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in vivo or ex vivo in the presence or absence of 1.0 minimum alveolar concentration of isoflurane administered before ischemia. Cardiac Akt, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) proteins were determined by Western blot analysis. Opening of the mitochondrial permeability transition pore (mPTP) in cardiomyocytes was induced by photoexcitation-generated oxidative stress and detected by rapid dissipation of tetramethylrhodamine ethyl ester fluorescence using a confocal microscope. RESULTS: Genetic disruption of miR-21 gene did not alter phenotype of the left ventricle, baseline cardiac function, area at risk, and the ratios of phosphorylated-Akt/Akt, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS. Isoflurane decreased infarct size from 54 ± 10% in control to 36 ± 10% (P < 0.05, n = 8 mice per group), improved cardiac function after reperfusion, and increased the ratios of phosphorylated-Akt/AKT, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS in C57BL/6 mice subjected to ischemia-reperfusion injury. These beneficial effects of isoflurane were lost in microRNA-21 knockout mice. There were no significant differences in time of the mPTP opening induced by photoexcitation-generated oxidative stress in cardiomyocytes isolated between C57BL/6 and microRNA-21 knockout mice. Isoflurane significantly delayed mPTP opening in cardiomyocytes from C57BL/6 but not from microRNA-21 knockout mice. CONCLUSIONS: Isoflurane protects mouse hearts from ischemia-reperfusion injury by a microRNA-21-dependent mechanism. The Akt/NOS/mPTP pathway is involved in the microRNA-21-mediated protective effect of isoflurane.

Genetically determined mitochondrial preservation and cardioprotection against myocardial ischemia-reperfusion injury in a consomic rat model.

Cardioprotection may be genome dependent. One example is the increased tolerance to cardiac ischemia-reperfusion (IR) in Brown Norway (BN) compared with Dahl salt-sensitive (SS) rats. By narrowing the genetic difference to chromosome 6 only, we found the consomic SS(6BN) to be similarly IR tolerant as BN. We hypothesized that better preserved mitochondrial structure and function are genetically determined and therefore critically linked to myocardial IR tolerance associated with BN chromosome 6. Langendorff-prepared BN, SS, and SS(6BN) rat hearts were subjected to IR, while corresponding controls were continuously perfused. Though largely equal in nonischemic controls, assessment of functional data and ventricular infarct size in IR experiments confirmed that BN and SS(6BN) have an equally higher tolerance to IR than SS hearts. This was complemented by equally better preserved mitochondrial structure, oxidative phosphorylation, and calcium retention capacity in BN and SS(6BN) vs. SS hearts. For the first time, our data indicate that SS(6BN) are as resistant to IR injury as BN hearts in mitochondrial and myocardial function and viability compared with SS hearts. These findings not only link myocardial and mitochondrial protection in a genetic model but also suggest that genetic information on rat chromosome 6 is critical for mitochondrial preservation and IR tolerance.

Cardioprotection during diabetes: the role of mitochondrial DNA.

BACKGROUND: Diabetes alters mitochondrial bioenergetics and consequently disrupts cardioprotective signaling. The authors investigated whether mitochondrial DNA (mtDNA) modulates anesthetic preconditioning (APC) and cardiac susceptibility to ischemia-reperfusion injury by using two strains of rats, both sharing nuclear genome of type 2 diabetes mellitus (T2DN) rats and having distinct mitochondrial genomes of Wistar and fawn-hooded hypertensive (FHH) rat strains (T2DN(mtWistar) and T2DN(mtFHH), respectively). METHODS: Myocardial infarct size was measured in Wistar, T2DN(mtWistar), and T2DN(mtFHH) rats with or without APC (1.4% isoflurane) in the presence or absence of antioxidant N-acetylcysteine. Flavoprotein fluorescence intensity, a marker of mitochondrial redox state, 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity, a marker of reactive oxygen species generation, and mitochondrial permeability transition pore opening were assessed in isolated rat ventricular cardiomyocytes with or without isoflurane (0.5 mmol/l). RESULTS: Myocardial infarct size was decreased by APC in Wistar and T2DN(mtWistar) rats (to 42 ± 6%, n = 8; and 44 ± 7%, n = 8; of risk area, respectively) compared with their respective controls (60 ± 3%, n = 6; and 59 ± 9%, n = 7), but not in T2DN(mtFHH) rats (60 ± 2%, n = 8). N-acetylcysteine applied during isoflurane treatment restored APC in T2DN(mtFHH) (39 ± 6%, n = 7; and 38 ± 5%, n = 7; 150 and 75 mg/kg N-acetylcysteine, respectively), but abolished protection in control rats (54 ± 8%, n = 6). Similar to the data on infarct size, APC delayed mitochondrial permeability transition pore opening in T2DN(mtWistar) but not in T2DN(mtFHH) cardiomyocytes. Isoflurane increased flavoprotein and 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity in all rat strains, with the greatest effect in T2DN(mtFHH) cardiomyocytes. CONCLUSION: Differences in the mitochondrial genome modulate isoflurane-induced generation of reactive oxygen species which translates into differential susceptibility to APC and ischemia-reperfusion injury in diabetic rats.

Apolipoprotein A-1 mimetic D-4F enhances isoflurane-induced eNOS signaling and cardioprotection during acute hyperglycemia.

Acute hyperglycemia (AHG) decreases the availability of nitric oxide (NO) and impairs anesthetic preconditioning (APC)-elicited protection against myocardial infarction. We investigated whether D-4F, an apolipoprotein A-1 mimetic, rescues the myocardium by promoting APC-induced endothelial NO signaling during AHG. Myocardial infarct size was measured in mice in the absence or presence of APC [isoflurane (1.4%)] with or without AHG [dextrose (2 g/kg ip)] and D-4F (0.12 or 0.6 mg/kg ip). NO production, superoxide generation, protein compartmentalization, and posttranslational endothelial NO synthase (eNOS) modifications were assessed in human coronary artery endothelial cells cultured in 5.5 or 20 mM glucose with or without isoflurane (0.5 mM) in the presence or absence of D-4F (0.5 µg/ml). Myocardial infarct size was significantly decreased by APC (36 ± 3% of risk area) compared with control (54 ± 3%) in the absence, but not presence, of AHG (49 ± 4%). D-4F restored the cardioprotective effect of APC during AHG (36 ± 3% and 30 ± 3%, 0.12 and 0.6 mg/kg, respectively), although D-4F alone had no effect on infarct size (53 ± 3%). Isoflurane promoted caveolin-1 and eNOS compartmentalization within endothelial cell caveolae and eNOS dimerization, concomitant with increased NO production (411 ± 28 vs. 68 ± 10 pmol/mg protein in control). These actions were attenuated by AHG (NO production: 264 ± 18 pmol/mg protein). D-4F reduced superoxide generation and enhanced caveolin-1 and eNOS caveolar compartmentalization and posttranslational eNOS modifications, thus restoring NO production during isoflurane and AHG (418 ± 36 pmol/mg protein). In conclusion, D-4F restored the cardioprotective effect of APC during AHG, possibly by decreasing superoxide generation, which promoted isoflurane-induced eNOS signaling and NO biosynthesis.

Augmentation of Histone Deacetylase 6 Activity Impairs Mitochondrial Respiratory Complex I in Ischemic/Reperfused Diabetic Hearts.

BACKGROUND: Diabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor α (TNFα) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and ß-oxidation. Here we examined how HDAC6 regulates TNFα production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts. METHODS: HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNFα and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups. RESULTS: Myocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNFα levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNFα with an anti-TNFα monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNFα levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNFα levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSIONS: Augmenting HDAC6 activity inhibits mCI activity by increasing TNFα levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes.

Endothelial-cardiomyocyte crosstalk enhances pharmacological cardioprotection.

Endothelial cells (EC) serve a paracrine function to enhance signaling in cardiomyocytes (CM), and conversely, CM secrete factors that impact EC function. Understanding how EC interact with CM may be critically important in the context of ischemia-reperfusion injury, where EC might promote CM survival. We used isoflurane as a pharmacological stimulus to enhance EC protection of CM against hypoxia and reoxygenation injury. Triggering of intracellular signal transduction pathways culminating in the enhanced production of nitric oxide (NO) appears to be a central component of pharmacologically induced cardioprotection. Although the endothelium is well recognized as a regulator for vascular tone, little attention has been given to its potential importance in mediating cardioprotection. In the current investigation, EC-CM in co-culture were used to test the hypothesis that EC contribute to isoflurane-enhanced protection of CM against hypoxia and reoxygenation injury and that this protection depends on hypoxia-inducible factor (HIF1α) and NO. CM were protected against cell injury [lactate dehydrogenase (LDH) release] to a greater extent in the presence vs. absence of isoflurane-stimulated EC (1.7 ± 0.2 vs. 4.58 ± 0.8 fold change LDH release), and this protection was NO-dependent. Isoflurane enhanced release of NO in EC (1103 ± 58 vs. 702 ± 92 pmol/mg protein) and EC-CM in co-culture sustained NO release during reoxygenation. In contrast, lentiviral mediated HIF1α knockdown in EC decreased basal and isoflurane stimulated NO release in an eNOS dependent manner (517 ± 32 vs. 493 ± 38 pmol/mg protein) and prevented the sustained increase in NO during reoxygenation when co-cultured. Opening of mitochondrial permeability transition pore (mPTP), an index of mitochondrial integrity, was delayed in the presence vs. absence of EC (141 ± 2 vs. 128 ± 2.5 arbitrary mPTP opening time). Isoflurane stimulated an increase in HIF1α in EC but not in CM under normal oxygen tension (3.5 ± 0.1 vs. 0.79 ± 0.15 fold change density) and this action was blocked by pretreatment with the Mitogen-activated Protein/Extracellular Signal-regulated Kinase inhibitor U0126. Expression and nuclear translocation of HIF1α were confirmed by Western blot and immunofluorescence. Taken together, these data support the concept that EC are stimulated by isoflurane to produce important cardioprotective factors that may contribute to protection of myocardium during ischemia and reperfusion injury.

Decreased tetrahydrobiopterin and disrupted association of Hsp90 with eNOS by hyperglycemia impair myocardial ischemic preconditioning.

Cardioprotection by ischemic preconditioning (IPC) is impaired during hyperglycemia, but the mechanisms underlying this phenomenon are poorly understood. This study investigated the role of hyperglycemia to adversely modulate tetrahydrobiopterin (BH(4)) and heat shock protein 90 (Hsp90) during cardioprotection by IPC. Rabbits or mice underwent 30 min of coronary occlusion followed by reperfusion with or without IPC in the presence or absence of hyperglycemia. IPC significantly (P < 0.05) decreased myocardial infarct size (46 ± 1 to 19 ± 2% of the area at risk in control and IPC rabbits, respectively) and increased BH(4) concentrations (HPLC; 7.6 ± 0.2 to 10.2 ± 0.3 pmol/mg protein, respectively), Hsp90-endothelial nitric oxide synthase (eNOS) association (coimmunoprecipitation and Western blotting in mice; 4.0 ± 0.3 to 5.4 ± 0.1, respectively), and the ratio of phosphorylated eNOS/total eNOS. These beneficial actions of IPC on infarct size, BH(4), Hsp90/eNOS, and phosphorylated eNOS were eliminated by hyperglycemia. Pretreatment of animals with the Hsp90 inhibitor geldanamycin (0.6 mg/kg) or the BH(4) synthesis inhibitor diamino-6-hydroxypyrimidine (1.0 g/kg) also eliminated cardioprotection produced by IPC. In contrast, the BH(4) precursor sepiapterin (2 mg/kg iv) restored the beneficial effects of IPC on myocardial BH(4) concentrations, eNOS dimerization, and infarct size during hyperglycemia. A-23871 increased Hsp90-eNOS association (0.33 ± 0.06 to 0.59 ± 0.3) and nitric oxide production (184 ± 17%) in human coronary artery endothelial cells cultured in normal (5.5 mM) but not high (20 mM) glucose media. These data indicate that hyperglycemia eliminates protection by IPC via decreases in myocardial BH(4) concentration and disruption of the association of Hsp90 with eNOS. The results suggest that eNOS dysregulation may be a central mechanism of impaired cardioprotection during hyperglycemia.

Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: implications for preconditioning.

BACKGROUND: Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, the authors investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, the authors investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles. METHODS: Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2 h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined. RESULTS: Isoflurane reduced myocardial infarct size (40 ± 9% = mean ± SD) compared with control experiments (60 ± 4%). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60 ± 9%). Isoflurane enhanced ROS generation in submitochondrial particles with nicotinamide adenine dinucleotide (reduced form), but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity. CONCLUSIONS: The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.

Isoflurane postconditioning protects against reperfusion injury by preventing mitochondrial permeability transition by an endothelial nitric oxide synthase-dependent mechanism.

BACKGROUND: The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. The authors addressed this issue using eNOS mice. METHODS: In vivo or Langendorff-perfused mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in the presence and absence of postconditioning produced with isoflurane 5 min before and 3 min after reperfusion. Ca+-induced mitochondrial permeability transition (MPT) pore opening was assessed in isolated mitochondria. Echocardiography was used to evaluate ventricular function. RESULTS: Postconditioning with 0.5, 1.0, and 1.5 minimum alveolar concentrations of isoflurane decreased infarct size from 56 +/- 10% (n = 10) in control to 48 +/- 10%, 41 +/- 8% (n = 8, P < 0.05), and 38 +/- 10% (n = 8, P < 0.05), respectively, and improved cardiac function in wild-type mice. Improvement in cardiac function by IsoPC was blocked by N-nitro-L-arginine methyl ester (a nonselective nitric oxide synthase inhibitor) administered either before ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca+ loading than did controls (78 +/- 29 microm vs. 40 +/- 25 microm CaCl2 per milligram of protein, n = 10, P < 0.05) to open the MPT pore. Hearts from eNOS mice displayed no marked differences in infarct size, cardiac function, and sensitivity of MPT pore to Ca+, compared with wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function, or the amount of Ca+ necessary to open the MPT pore in mitochondria isolated from the eNOS hearts compared with control hearts. CONCLUSIONS: IsoPC protects mouse hearts from reperfusion injury by preventing MPT pore opening in an eNOS-dependent manner. Nitric oxide functions as both a trigger and a mediator of cardioprotection produced by IsoPC.

Hyperglycemia adversely modulates endothelial nitric oxide synthase during anesthetic preconditioning through tetrahydrobiopterin- and heat shock protein 90-mediated mechanisms.

BACKGROUND: Endothelial nitric oxide synthase activity is regulated by (6R-)5,6,7,8-tetrahydrobiopterin (BH4) and heat shock protein 90. The authors tested the hypothesis that hyperglycemia abolishes anesthetic preconditioning (APC) through BH4- and heat shock protein 90-dependent pathways. METHODS: Myocardial infarct size was measured in rabbits in the absence or presence of APC (30 min of isoflurane), with or without hyperglycemia, and in the presence or absence of the BH4 precursor sepiapterin. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells cultured in normal (5.5 mm) or high (20 mm) glucose conditions, with or without sepiapterin (10 or 100 microm). RESULTS: APC decreased myocardial infarct size compared with control experiments (26 +/- 6% vs. 46 +/- 3%, respectively; P < 0.05), and this action was blocked by hyperglycemia (43 +/- 4%). Sepiapterin alone had no effect on infarct size (46 +/- 3%) but restored APC during hyperglycemia (21 +/- 3%). The beneficial actions of sepiapterin to restore APC were blocked by the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester (47 +/- 2%) and the BH4 synthesis inhibitor N-acetylserotonin (46 +/- 3%). Isoflurane increased nitric oxide production to 177 +/- 13% of baseline, and this action was attenuated by high glucose concentrations (125 +/- 6%). Isoflurane increased, whereas high glucose attenuated intracellular BH4/7,8-dihydrobiopterin (BH2) (high performance liquid chromatography), heat shock protein 90-endothelial nitric oxide synthase colocalization (confocal microscopy) and endothelial nitric oxide synthase activation (immunoblotting). Sepiapterin increased BH4/BH2 and dose-dependently restored nitric oxide production during hyperglycemic conditions (149 +/- 12% and 175 +/- 9%; 10 and 100 microm, respectively). CONCLUSION: The results indicate that tetrahydrobiopterin and heat shock protein 90-regulated endothelial nitric oxide synthase activity play a central role in cardioprotection that is favorably modulated by volatile anesthetics and dysregulated by hyperglycemia. Enhancing the production of BH4 may represent a potential therapeutic strategy.

Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

BACKGROUND: Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. METHODS: Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. RESULTS: APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. CONCLUSION: The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.

Continuous perioperative insulin infusion decreases major cardiovascular events in patients undergoing vascular surgery: a prospective, randomized trial.

BACKGROUND: A growing body of evidence suggests that hyperglycemia is an independent predictor of increased cardiovascular risk. Aggressive glycemic control in the intensive care decreases mortality. The benefit of glycemic control in noncardiac surgery is unknown. METHODS: In a single-center, prospective, unblinded, active-control study, 236 patients were randomly assigned to continuous insulin infusion (target glucose 100-150 mg/dl) or to a standard intermittent insulin bolus (treat glucose > 150 mg/dl) in patients undergoing peripheral vascular bypass, abdominal aortic aneurysm repair, or below- or above-knee amputation. The treatments began at the start of surgery and continued for 48 h. The primary endpoint was a composite of all-cause death, myocardial infarction, and acute congestive heart failure. The secondary endpoints were blood glucose concentrations, rates of hypoglycemia (< 60 mg/dl) and hyperglycemia (> 150 mg/dl), graft failure or reintervention, wound infection, acute renal insufficiency, and duration of stay. RESULTS: The groups were well balanced for baseline characteristics, except for older age in the intervention group. There was a significant reduction in primary endpoint (3.5%) in the intervention group compared with the control group (12.3%) (relative risk, 0.29; 95% confidence interval, 0.10-0.83; P = 0.013). The secondary endpoints were similar. Hypoglycemia occurred in 8.8% of the intervention group compared with 4.1% of the control group (P = 0.14). Multivariate analysis demonstrated that continuous insulin infusion was a negative independent predictor (odds ratio, 0.28; 95% confidence interval, 0.09-0.87; P = 0.027), whereas previous coronary artery disease was a positive predictor of adverse events. CONCLUSION: Continuous insulin infusion reduces perioperative myocardial infarction after vascular surgery.

Simvastatin restores ischemic preconditioning in the presence of hyperglycemia through a nitric oxide-mediated mechanism.

BACKGROUND: A growing body of evidence indicates that statins decrease perioperative cardiovascular risk and that these drugs may be particularly efficacious in diabetes. Diabetes and hyperglycemia abolish the cardioprotective effects of ischemic preconditioning (IPC). The authors tested the hypothesis that simvastatin restores the beneficial effects of IPC during hyperglycemia through a nitric oxide-mediated mechanism. METHODS: Myocardial infarct size was measured in dogs (n = 76) subjected to coronary artery occlusion and reperfusion in the presence or absence of hyperglycemia (300 mg/dl) with or without IPC in separate groups. Additional dogs received simvastatin (20 mg orally daily for 3 days) in the presence or absence of IPC and hyperglycemia. Other dogs were pretreated with N-nitro-l-arginine methyl ester (30 mg intracoronary) with or without IPC, hyperglycemia, and simvastatin. RESULTS: Ischemic preconditioning significantly (P < 0.05) reduced infarct size (n = 7, 7 +/- 2%) as compared with control (n = 7, 29 +/- 3%). Hyperglycemia (n = 7), simvastatin (n = 7), N-nitro-l-arginine methyl ester alone (n = 7), and simvastatin with hyperglycemia (n = 6) did not alter infarct size. Hyperglycemia (n = 7, 24 +/- 2%), but not N-nitro-l-arginine methyl ester (n = 5, 10 +/- 1%), blocked the protective effects of IPC. Simvastatin restored the protective effects of IPC in the presence of hyperglycemia (n = 7, 14 +/- 1%), and this beneficial action was blocked by N-nitro-l-arginine methyl ester (n = 7, 29 +/- 4%). CONCLUSIONS: The results indicate that simvastatin restored the cardioprotective effects of IPC during hyperglycemia by nitric oxide-mediated signaling. The results also suggest that enhanced cardioprotective signaling could be a mechanism for statin-induced decreases in perioperative cardiovascular risk.

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.

ACC/AHA 2006 guideline update on perioperative cardiovascular evaluation for noncardiac surgery: focused update on perioperative beta-blocker therapy--a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2002 Guidelines on Perioperative Cardiovascular Evaluation for Noncardiac Surgery).

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