Endothelial Nitric Oxide Synthase Mediates the First and Inducible Nitric Oxide Synthase Mediates the Second Window of Desflurane-Induced Preconditioning

Endothelial Nitric Oxide Synthase Mediates the First and Inducible Nitric Oxide Synthase Mediates the Second Window of Desflurane-Induced Preconditioning Andreas Redel, MD,*† Jan Stumpner, MD,* Thorsten M. Smul, MD,* Markus Lange, MD, PhD,* Virginija Jazbutyte, PhD,‡ Douglas G. Ridyard, BA, BS,§ Norbert Roewer, MD, PhD,† and Franz Kehl, MD, PhD储 Objectives: Nitric oxide synthases (NOSs) mediate the first window of anesthetic-induced preconditioning (APC). The authors tested the hypothesis that endothelial NOS (eNOS) mediates the first window and inducible NOS (iNOS) mediates the second window of APC. Design: Randomized, prospective, blinded laboratory investigation. Setting: Experimental laboratory. Participants: Mice. Interventions: Mice were subjected to a 45-minute coronary artery occlusion (CAO) and a 180-minute reperfusion. C57BL/6 mice received desflurane, 1.0 minimum alveolar concentration, for 30 minutes or 12, 24, 48, or 96 hours before CAO. In eNOSⴚ/ⴚ and iNOSⴚ/ⴚ mice, desflurane was given 30 minutes and 48 hours before CAO. In the control groups, no desflurane was administered. Myocardial infarct size (IS) was determined after staining with Evans blue and triphenyltetrazolium chloride. Measurements and Main Results: The second window of APC was detectable at 48 hours but not at 12, 24, and 96

hours after preconditioning. In the control groups, IS was not different among the wild-type (50 ⴞ 10%), eNOSⴚ/ⴚ (52 ⴞ 14%), and iNOSⴚ/ⴚ (46 ⴞ 10%) mice. The IS decreased significantly (p < 0.05) when desflurane was administered 30 minutes (10 ⴞ 6%) or 48 hours (16 ⴞ 7%) before CAO in wild-type mice, 48 hours (21 ⴞ 13%) before CAO in eNOSⴚ/ⴚ mice, and 30 minutes (13 ⴞ 6%) before CAO in iNOSⴚ/ⴚ mice. Desflurane given 30 minutes before CAO in eNOSⴚ/ⴚ mice (60 ⴞ 10%) and 48 hours before CAO in iNOSⴚ/ⴚ mice (48 ⴞ 21%) did not decrease the IS significantly compared with controls. Conclusions: Endothelial NOS and iNOS work independently to mediate the first and second windows of APC, respectively. Endothelial NOS is not necessary to trigger the second window of APC. © 2013 Elsevier Inc. All rights reserved.

A

induce the first window of preconditioning in vitro3 and in vivo, in rabbits,4 and in endothelial NO synthase (eNOS) genedeleted mice,5 several studies have not found an IS decrease by endogenous NO.4,6-8 Thus, it was concluded NO can induce, but is not necessary for, the first window of preconditioning.9 In addition, NO derived from constitutively expressed eNOS during or immediately after preconditioning serves as a trigger of the second window of ischemic preconditioning.10 Recently, the blocking of NOS with L-nitro-arginine methyl ester (LNAME) has been reported to abolish the first window of desflurane-induced APC.11 However, because L-NAME blocks all isoforms of NOS (ie, neuronal NOS [nNOS], inducible NOS [iNOS], and eNOS), it cannot be concluded from these studies which isoforms of NOS are crucial in the first window of APC. A pivotal step of the second window of APC12 and ischemic preconditioning13 is the increased production of NO. It is assumed that ischemic preconditioning triggers an eNOS-derived NO burst. This burst activates protein kinase-C⑀ and nuclear factor-␬B and leads to increased iNOS transcription. The resulting increase in the generation of NO provides myocardial protection during the ischemic challenge.14 Volatile anesthetics trigger NO production immediately after their administration,11,12 and the expression of iNOS and NOx levels are increased 24 hours after isoflurane administration.12 Thus, the second window of APC may be mediated by iNOS-derived NO similarly to ischemic preconditioning. In the present study, the authors characterized the time course of the second window of desflurane-induced preconditioning in mice. In addition, they used eNOS and iNOS gene-deleted mice to test the hypothesis that the first and second windows of APC are mediated by eNOS and iNOS, respectively.

NESTHETIC-INDUCED PRECONDITIONING (APC) describes the phenomenon in which the administration of volatile anesthetics before sustained myocardial ischemia decreases the myocardial infarct size (IS).1 APC is characterized by 2 distinct periods of protection: the first window of APC lasts 2-4 hours after preconditioning, and the second window of APC occurs approximately 24 hours later and lasts up to 72 hours.2 However, the exact time course of the second window of preconditioning in mice has not been investigated. Furthermore, the underlying mechanisms of the first and second windows of preconditioning are understood incompletely. The role of nitric oxide (NO) in the first window of preconditioning remains controversial in the current literature. Although the administration of exogenous NO has been shown to

From the *Department of Anesthesia and Critical Care, University of Würzburg, Würzburg, Germany; †Department of Anesthesia, University of Regensburg, Regensburg, Germany; ‡Institute for Molecular and Translational Therapeutic Strategies, Hannover Medical School, Hannover, Germany; §Mucosal Inflammation Program, Department of Anesthesiology, School of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO; and 储Department of Anesthesiology and Critical Care, Klinikum, Karlsruhe, Germany. This work was supported in part by the Interdisciplinary Center for Clinical Research, Würzburg, Germany. F.K. received a speaker honorarium from Baxter (Unterschleißheim, Germany). Address reprint requests to Andreas Redel, MD, University of Regensburg, Klinik für Anästhesiologie, Franz-Joseph-Strauß-Allee 11, 93053 Regensburg, Germany. E-mail: [email protected] © 2013 Elsevier Inc. All rights reserved. 1053-0770/2703-0001$36.00/0 http://dx.doi.org/10.1053/j.jvca.2012.04.015 494

KEY WORDS: anesthetic-induced preconditioning, myocardial infarction, endothelial nitric oxide synthase, inducible nitric oxide synthase, mouse

Journal of Cardiothoracic and Vascular Anesthesia, Vol 27, No 3 (June), 2013: pp 494-501

ROLE OF eNOS AND iNOS IN DESFLURANE-INDUCED PRECONDITIONING

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METHODS All experiments were performed in male mice (8-12 weeks old, weight 20-25 g). For the investigation of eNOS and iNOS in APC, the authors used eNOS⫺/⫺ mice15 (Harvard eNOS⫺/⫺ mice with the deletion of reduced nicotinamide adenine dinucleotide phosphate ribose and adenine binding sites) and iNOS⫺/⫺ mice (Department of Neurology, University of Würzburg, Würzburg, Germany).16 C57BL/6 mice (Charles River Laboratories, Sulzfeld, Germany) were used as wildtype (wt) controls. All mice were maintained in sterile microisolator cages under pathogen-free and controlled conditions (22°C, 55%-65% humidity, and 12-h light-dark cycle) and were allowed free access to tap water and a standard laboratory chow. All experimental procedures and protocols used in this investigation were reviewed and approved by the local animal care and use committee. Furthermore, all studies conformed to the Guiding Principles in the Care and Use of Animals of the American Physiological Society and were in accordance with the Guide for the Care and Use of Laboratory Animals.17 Hemodynamic measurements and ligation of the left anterior descending coronary artery (LAD) were performed as described previously.18 Briefly, after the induction of anesthesia with pentobarbital (60 mg/kg) and endotracheal intubation, mice were ventilated mechanically. A 3-lead needle-probe electrocardiograph was attached to the skin, and saline-filled polyethylene catheters were placed into the right common carotid artery for the measurement of mean arterial blood pressure and into the right jugular vein for continuous fluid administration (20 ␮L/g/h). After left thoracotomy, the LAD was exposed and a suture was passed around the LAD. Coronary artery occlusion (CAO) was achieved using the hanging-weight system as described previously.19 After the completion of surgical procedures, all animals were allowed a time-matched equilibration period before CAO. Preconditioning was achieved by the administration of desflurane (Suprane, Baxter, Unterschleißheim, Germany; 7.5 vol%20), 1.0 minimum alveolar concentration (MAC) for 15 minutes. Animals in the control groups received desflurane, 0.0 MAC. In the first subset of experiments, the authors characterized the second window of APC. C57BL/6 mice were assigned randomly to receive a memory period of 12, 24, 48, or 96 hours from preconditioning to CAO. For preconditioning, the mice were transferred into a cage prefilled with 50% room air, 50% oxygen, and desflurane, 1.0 MAC for 15 minutes. Eleven, 23, 47, or 95 hours after preconditioning, the mice were anesthetized with pentobarbital and the surgical procedure was performed as described above. To compare the magnitude of IS decrease in the 2 windows of preconditioning, additional mice were assigned to the first window of preconditioning with a memory period of 15 minutes. Based on previously published data, desflurane, 1.0 MAC, was administered for 15 minutes for preconditioning.21 Myocardial ischemia was induced by CAO for 45 minutes followed by 180 minutes of reperfusion in all animals. The experimental protocol of each group is illustrated in Figure 1. To investigate the role of eNOS and iNOS in the 2 windows of APC, wt, eNOS⫺/⫺, and iNOS⫺/⫺ mice were assigned randomly to the control, first window, or second window group. In animals in the control group, the surgical procedure was performed as described above, and after an equilibration period, the LAD was occluded for 45 minutes, followed by 180 minutes of reperfusion. Mice in the first window group received desflurane, 1.0 MAC for 15 minutes, at the end of the equilibration period. Fifteen minutes after the discontinuation of desflurane, the LAD was occluded for 45 minutes, followed by 180 minutes of reperfusion. Mice in the second window group were transferred into a cage prefilled with 50% room air, 50% oxygen, and desflurane, 1.0 MAC for 15 minutes. Forty-seven hours after preconditioning, the surgical procedure was performed as described above,

Fig 1. Experimental protocol. All animals were subjected to 45 minutes of coronary artery occlusion (CAO) and 180 minutes of reperfusion. Desflurane (DES), 1 minimum alveolar concentration, was administered for 15 minutes. The memory period varied from 15 minutes to 12, 24, 48, and 96 hours. Gene-targeted mice underwent control (CON) protocols and memory periods of 15 minutes and 48 hours. Note that the time course is not true to scale.

and after an equilibration period, the LAD was occluded for 45 minutes, followed by 180 minutes of reperfusion. The experimental protocols are illustrated in Figure 1. Myocardial IS and area at risk (AAR) were determined as described previously.18,22 Briefly, after reperfusion, the LAD was reoccluded and Evans blue (0.1 g/mL), 1 mL, was injected into the carotid artery. After an injection of a lethal dose of pentobarbital (150 ␮g/g intraperitoneally), the heart was removed, dissected, cut into transverse slices, and incubated with 2% triphenyltetrazolium chloride. The slices were then photographed and analyzed with picture analysis software. The infarct area, AAR and normal zone were quantified by an investigator blinded to the treatment protocol. The hemodynamic parameters, electrocardiogram, and body temperature were recorded as described previously.18 From published data obtained in the same experimental model,18,21,23,24 the authors expected a myocardial IS of 50% (infarct area/AAR). Power analysis showed that a group size of 6 was required to detect a decrease in IS from 50% to 25% with a power of 0.8 at an ␣ level of 0.05. Statistical analyses were performed separately for the experimental groups by analysis of variance, which were based on 2-tailed F tests for the comparison of components of the factors’ total deviation. Analysis for body weight, left ventricular weight, left ventricular weight/body weight, AAR, IS, IS/left ventricular weight, and AAR/left ventricular weight was performed using 1-way analysis of variance, including the factor treatment and post hoc Duncan test for significant main effects and interactions. Analyses of hemodynamic data were performed by analysis of variance for repeated measures, including the between-factor treatment and the within-factor time. The p values were adjusted for multiplicity. For any significant main effects or interactions, post hoc 1-way analyses of variance were conducted for each group and each time point. Statistical analysis of data was performed using SPSS 15.0 (SPSS, Inc, Chicago, IL). Changes in means were considered statistically significant at p ⬍ 0.05. Data are presented as mean ⫾ standard deviation. RESULTS

One hundred six (62 wt, 22 eNOS⫺/⫺, and 22 iNOS⫺/⫺) mice were included in the ischemia-reperfusion experiments to achieve 99 successful experiments. Two wt mice were excluded because of an AAR that was ⬍20% of the left ventricle and 5 mice were excluded because of cardiovascular failure during CAO or reperfusion (1 animal each in the wt 96-h group, the wt 12-h group, the wt second window group, the eNOS⫺/⫺ second window group, and the iNOS⫺/⫺ first window group).

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Table 1. Hemodynamics: Characterization of the Second Window of Anesthetic-Induced Preconditioning

HR (beats/min) CON 15 min 12 h 24 h 48 h 96 h MAP (mmHg) CON 15 min 12 h 24 h 48 h 96 h

BL

CAO

R60

R120

R180

453 ⫾ 40 447 ⫾ 45 444 ⫾ 34 439 ⫾ 45 426 ⫾ 40 470 ⫾ 38

417 ⫾ 38 433 ⫾ 43 459 ⫾ 47 457 ⫾ 65 427 ⫾ 36 451 ⫾ 38

429 ⫾ 47 420 ⫾ 38 400 ⫾ 18* 455 ⫾ 40 412 ⫾ 31 399 ⫾ 28*

405 ⫾ 34 443 ⫾ 40 442 ⫾ 52 453 ⫾ 31 409 ⫾ 45 420 ⫾ 52

432 ⫾ 25 433 ⫾ 34 447 ⫾ 63 482 ⫾ 25 436 ⫾ 52 418 ⫾ 49

63 ⫾ 9 64 ⫾ 7 68 ⫾ 7 65 ⫾ 9 64 ⫾ 7 66 ⫾ 7

53 ⫾ 4 58 ⫾ 7 66 ⫾ 7 64 ⫾ 9 60 ⫾ 7 59 ⫾ 9

52 ⫾ 7 58 ⫾ 2 71 ⫾ 9† 66 ⫾ 7 63 ⫾ 9 58 ⫾ 7

56 ⫾ 4 52 ⫾ 7 69 ⫾ 7† 61 ⫾ 4† 59 ⫾ 13 58 ⫾ 9

57 ⫾ 7 56 ⫾ 9 73 ⫾ 9† 58 ⫾ 7 59 ⫾ 11 54 ⫾ 9*

NOTE. Data are presented as mean ⫾ standard deviation. Abbreviations: BL, baseline; CAO, coronary artery occlusion; R60, 60 minutes after reperfusion; R120, 120 minutes after reperfusion; R180, 180 minutes after reperfusion; HR, heart rate; CON, control; MAP, mean arterial blood pressure. *Significantly different versus BL. †Significantly different versus CON.

In the experimental subgroups characterizing the second window of APC, heart rate and mean arterial blood pressure did not differ significantly among the groups at baseline (Table 1). In the experimental subgroups investigating the role of eNOS and iNOS in APC, mean arterial blood pressure was slightly, but not significantly, increased in the eNOS⫺/⫺ animals compared with the wt animals (Tables 2 and 3).15 There were no significant differences in heart rate among the groups throughout the experimental time course.

In the experimental subset characterizing the second window of APC, the AAR was not different among the groups (Table 4). The IS was 48 ⫾ 12% in the control group and decreased significantly (p ⬍ 0.05) when desflurane was administered up to 15 minutes (9 ⫾ 4%) or 48 hours (14 ⫾ 6%) before CAO (Fig 2). The application of desflurane 12 hours (42 ⫾ 17%), 24 hours (36 ⫾ 17%) or 96 hours (37 ⫾ 24%) before CAO did not decrease the myocardial IS significantly (Fig 2). In the experimental subset investigating the role of eNOS and iNOS in APC, the AAR was not different among the

Table 2. Hemodynamics: Role of Endothelial and Inducible Nitric Oxide Synthases in the First Window of Anesthetic-Induced Preconditioning

HR (beats/min) CON wt eNOS⫺/⫺ iNOS⫺/⫺ First window of APC wt eNOS⫺/⫺ iNOS⫺/⫺ MAP (mmHg) CON wt eNOS⫺/⫺ iNOS⫺/⫺ First window of APC wt eNOS⫺/⫺ iNOS⫺/⫺

BL

DES

MEM

CAO

R60

R120

R180

465 ⫾ 49 417 ⫾ 69 476 ⫾ 29

479 ⫾ 47 414 ⫾ 56 468 ⫾ 76

460 ⫾ 32 413 ⫾ 56 443 ⫾ 27

436 ⫾ 34 440 ⫾ 74 467 ⫾ 76

447 ⫾ 39 399 ⫾ 78 448 ⫾ 44

452 ⫾ 44 428 ⫾ 74 473 ⫾ 37

413 ⫾ 78 461 ⫾ 71 497 ⫾ 49

445 ⫾ 64 404 ⫾ 56 489 ⫾ 25

433 ⫾ 61 403 ⫾ 39 451 ⫾ 37

462 ⫾ 66 438 ⫾ 42 480 ⫾ 12

437 ⫾ 44 439 ⫾ 49 449 ⫾ 42

424 ⫾ 39 409 ⫾ 42 483 ⫾ 47

433 ⫾ 27 482 ⫾ 56 494 ⫾ 29

440 ⫾ 27 476 ⫾ 88 467 ⫾ 59

76 ⫾ 10 83 ⫾ 7 76 ⫾ 7

76 ⫾ 10 87 ⫾ 7 74 ⫾ 7

72 ⫾ 10 83 ⫾ 15 74 ⫾ 7

64 ⫾ 15 76 ⫾ 15 72 ⫾ 10

70 ⫾ 12 80 ⫾ 7 71 ⫾ 7

66 ⫾ 12 71 ⫾ 15 69 ⫾ 10

70 ⫾ 12 71 ⫾ 12 69 ⫾ 12

73 ⫾ 5 82 ⫾ 10 83 ⫾ 7

69 ⫾ 12 88 ⫾ 7 75 ⫾ 7

76 ⫾ 12 88 ⫾ 7 81 ⫾ 10

60 ⫾ 10 79 ⫾ 15 66 ⫾ 12

68 ⫾ 10 76 ⫾ 15 70 ⫾ 5

66 ⫾ 12 72 ⫾ 5 74 ⫾ 10

70 ⫾ 15 67 ⫾ 12 65 ⫾ 20

NOTE. Data are presented as mean ⫾ standard deviation. Abbreviations: BL, baseline; DES, desflurane; MEM, memory period; CAO, coronary artery occusion; R60, 60 minutes after reperfusion; R120, 120 minutes after reperfusion; R180, 180 minutes after reperfusion; HR, heart rate; CON, control; wt, wild type; eNOS _/_, endothelial nitric oxide synthase gene deletion; iNOS _/_, inductible nitric oxide synthase gene deletion; APC, anesthetic-induced preconditioning; MAP, mean arterial blood pressure.

ROLE OF eNOS AND iNOS IN DESFLURANE-INDUCED PRECONDITIONING

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Table 3. Hemodynamics: Role of Endothelial and Inducible Nitric Oxide Synthases in the Second Window of Anesthetic-Induced Preconditioning

HR (beats/min) CON wt eNOS⫺/⫺ iNOS⫺/⫺ Second window of APC wt eNOS⫺/⫺ iNOS⫺/⫺ MAP (mmHg) CON wt eNOS⫺/⫺ iNOS⫺/⫺ Second window of APC wt eNOS⫺/⫺ iNOS⫺/⫺

BL

CAO

R60

R120

R180

465 ⫾ 49 417 ⫾ 69 476 ⫾ 29

436 ⫾ 34 440 ⫾ 74 467 ⫾ 76

447 ⫾ 39 399 ⫾ 78 448 ⫾ 44

452 ⫾ 44 428 ⫾ 74 473 ⫾ 37

413 ⫾ 81 461 ⫾ 71 497 ⫾ 49

422 ⫾ 51 426 ⫾ 42 465 ⫾ 47

420 ⫾ 47 445 ⫾ 74 494 ⫾ 51

417 ⫾ 47 438 ⫾ 37 464 ⫾ 39

404 ⫾ 54 446 ⫾ 49 477 ⫾ 37

438 ⫾ 69 472 ⫾ 51 502 ⫾ 64

76 ⫾ 10 83 ⫾ 7 76 ⫾ 7

64 ⫾ 15 76 ⫾ 15 72 ⫾ 10

70 ⫾ 12 80 ⫾ 7 71 ⫾ 7

66 ⫾ 12 71 ⫾ 15 69 ⫾ 10

70 ⫾ 12 71 ⫾ 12 69 ⫾ 12

68 ⫾ 5 83 ⫾ 12 72 ⫾ 15

63 ⫾ 5 72 ⫾ 7 65 ⫾ 22

67 ⫾ 7 79 ⫾ 10 76 ⫾ 10

61 ⫾ 10 71 ⫾ 15 71 ⫾ 10

64 ⫾ 10 71 ⫾ 10 67 ⫾ 5

NOTE. Data are presented as mean ⫾ standard deviation. Abbreviations: BL, baseline; CAO, coronary artery occusion; R60, 60 minutes after reperfusion; R120, 120 minutes after reperfusion; R180, 180 minutes after reperfusion; HR, heart rate; CON, control; wt, wild type; eNOS⫺/⫺, endothelial nitric oxide synthase gene deletion; iNOS⫺/⫺, inductible nitric oxide synthase gene deletion; APC, anesthetic-induced preconditioning; MAP, mean arterial blood pressure.

groups (Table 5). In the control groups, myocardial IS was not different in the eNOS⫺/⫺ (52 ⫾ 14%) and iNOS⫺/⫺ (46 ⫾ 10%) animals compared with the wt animals (50 ⫾ 10%; Fig 3). Myocardial IS decreased significantly (p ⬍ 0.05) when desflurane was administered in the wt animals 15 minutes (10 ⫾ 6%) or 48 hours (16 ⫾ 7%), in eNOS⫺/⫺ mice 48 hours (21 ⫾ 13%), and in iNOS⫺/⫺ mice 15 minutes (13 ⫾ 6%) before CAO, respectively (Fig 3). The application of desflurane 15 minutes before CAO in the eNOS⫺/⫺ mice (60 ⫾ 10%) and 48 hours before CAO in the iNOS⫺/⫺ mice (48 ⫾ 21%) did not decrease myocardial IS significantly compared with the respective control groups (Fig 3). DISCUSSION

In the present study, the authors characterized the time course of the second window of desflurane-induced preconditioning in mice and characterized the role of eNOS and iNOS in the first and second windows of desflurane-induced preconditioning. Volatile anesthetics induce a second window of preconditioning in different species, including rabbits25-27 and rats.28 In mice, an IS

decrease was documented when isoflurane was administered 24 hours before CAO.29 However, the time course of the second window of preconditioning in mice has not been characterized. In the present study, to determine the onset of the second window, the authors chose memory periods of 12 and 24 hours, and to determine the duration of the second window, periods of 48 and 96 hours were chosen. Twentyfour hours after preconditioning, the authors observed an insignificantly decreased IS. Forty-eight hours after preconditioning, the IS decreased significantly, indicating that the second window of desflurane-induced preconditioning commenced 24 to 48 hours after preconditioning. Ninety-six hours after preconditioning, the second window of desflurane-induced preconditioning was not detectable. Thus, in mice, the second window of desflurane-induced preconditioning occurred 48 to 72 hours after preconditioning in mice. Numerous studies using pharmacologic NOS inhibitors have investigated the role of NOSs in the 2 windows of APC. The unselective pharmacologic inhibition of all NOS isoforms, but not the selective inhibition of iNOS and nNOS, abolished the first window of isoflurane-induced preconditioning in rabbits.30 Thus, the authors concluded that eNOS mediates the first window of APC in rabbits. The present data showed an abrogated

Table 4. Body Weight and Planimetry: Characterization of the Second Window of Anesthetic-Induced Preconditioning

CON 15 min 12 h 24 h 48 h 96 h

n

BW (g)

LV (mg)

LV/BW (%)

AAR (mg)

IS (mg)

IS/LV (%)

AAR/LV (%)

6 6 6 6 6 6

22.7 ⫾ 1.3 23.0 ⫾ 1.6 22.5 ⫾ 1.3 22.3 ⫾ 0.5 22.0 ⫾ 0.9 22.8 ⫾ 1.1

61.0 ⫾ 5.6 66.0 ⫾ 7.4 58.5 ⫾ 2.7 62.8 ⫾ 3.1 64.0 ⫾ 3.8 65.3 ⫾ 1.2

0.27 ⫾ 0.05 0.29 ⫾ 0.02 0.26 ⫾ 0.02 0.28 ⫾ 0.02 0.29 ⫾ 0.02 0.29 ⫾ 0.02

19.7 ⫾ 8.3 17.9 ⫾ 5.8 17.9 ⫾ 3.6 19.1 ⫾ 5.6 23.3 ⫾ 13.4 21.5 ⫾ 2.5

9.1 ⫾ 3.6 1.6 ⫾ 0.9* 7.5 ⫾ 3.2 7.3 ⫾ 5.2 3.4 ⫾ 2.5* 9.0 ⫾ 4.7

14.9 ⫾ 5.8 2.5 ⫾ 1.8* 12.9 ⫾ 4.9 11.8 ⫾ 8.3 5.3 ⫾ 3.6* 14.1 ⫾ 8.1

32.4 ⫾ 12.9 27.3 ⫾ 8.5 30.6 ⫾ 6.1 30.8 ⫾ 9.6 36.3 ⫾ 19.9 33.3 ⫾ 5.2

NOTE. Data are presented as mean ⫾ standard deviation. Abbreviations: BW, body weight; LV, left ventricle;AAR, area at risk; IS, infarct size; CON, control. *Significantly (p ⬍ 0.05) different from CON.

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REDEL ET AL

Fig 2. Time course of the second window of desflurane-induced preconditioning. Myocardial infarct size (IS)/area at risk (AAR) in wildtype mice decreased significantly (*p < 0.05) when the memory period was 15 minutes or 48 hours, but not when the memory period was 12, 24, or 96 hours. Thus, in mice, the first window of anesthetic-induced preconditioning is detectable at a memory period of 15 minutes, and the second window at a memory period of 48 hours. Data are presented as mean ⴞ standard deviation (n ⴝ 6 in all groups).

protection during the first window of APC in eNOS genedeleted mice, confirming the protective role of eNOS in the first window of APC. Instantaneously available eNOS-derived NO is crucial not only for the first window of APC, but also in anesthetic-induced postconditioning.31,32 Contrary to the authors’ results using volatile anesthetics, ischemic preconditioning elicited the first window of preconditioning in eNOS⫺/⫺ mice.5,33 Ischemic preconditioning induces the first window of preconditioning by signal transduction cascades independent of eNOS, whereas eNOS-derived NO needs to be available for volatile anesthetics to induce this window of preconditioning. In addition to previously published data, the authors provide evidence that iNOS-derived NO is not crucial for the first window of APC because the first window of APC protection is preserved in iNOS gene-deleted mice. Pharmacologic studies addressing the role of NOS isoforms in the second window of APC have yielded contradicting results.11,12,28,34 In a study performed in rabbits, the

Fig 3. Role of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in the first and second windows of desflurane-induced preconditioning. In the control (CON) groups, myocardial infarct size (IS) did not differ in eNOSⴚ/ⴚ and iNOSⴚ/ⴚ mice compared with wild-type (wt) mice. Infarct size decreased significantly (*p < 0.05) in the first (memory period 15 minutes) and second (memory period 48 hours) windows of desflurane-induced preconditioning in the wild-type mice. In eNOSⴚ/ⴚ mice, the first window of desflurane-induced preconditioning was abolished, whereas the second window was detectable. In iNOSⴚ/ⴚ mice, the first window of desflurane-induced preconditioning was detectable, whereas the second window was abolished. Data are presented as mean ⴞ standard deviation (n ⴝ 7 in all groups). AAR, area at risk.

second window of isoflurane-induced preconditioning was abolished when all NOS isoforms were inhibited by LNAME before preconditioning or CAO.34 Pharmacologic iNOS or nNOS inhibition did not affect preconditioning in this study. The authors concluded that eNOS alone triggers and mediates the second window of isoflurane-induced preconditioning in rabbits. However, a selective pharmacologic eNOS inhibitor to confirm this interpretation is not available. In contrast to this study, studies performed in rats12 and mice28 have provided evidence that iNOS expression is increased after isoflurane-induced preconditioning and that the second window of APC is eliminated by pharmacologic

Table 5. Body Weight and Planimetry: Role of Nitric Oxide Synthase in Anesthetic-Induced Preconditioning

CON wt eNOS⫺/⫺ iNOS⫺/⫺ First window of APC wt eNOS⫺/⫺ iNOS⫺/⫺ Second window of APC wt eNOS⫺/⫺ iNOS⫺/⫺

n

BW (g)

LV (mg)

LV/BW (%)

AAR (mg)

IS (mg)

IS/LV (%)

AAR/LV (%)

7 7 7

24.6 ⫾ 1.5 23.6 ⫾ 2.9 24.7 ⫾ 1.7

73.0 ⫾ 12.3 62.0 ⫾ 5.9 76.6 ⫾ 10.5

0.30 ⫾ 0.05 0.27 ⫾ 0.02 0.31 ⫾ 0.05

27.7 ⫾ 10.5 25.4 ⫾ 5.9 28.3 ⫾ 5.4

13.6 ⫾ 6.1 13.6 ⫾ 5.6 13.2 ⫾ 4.2

18.3 ⫾ 5.4 21.6 ⫾ 8.6 17.3 ⫾ 5.9

37.3 ⫾ 9.8 40.7 ⫾ 7.4 37.3 ⫾ 8.6

7 7 7

24.1 ⫾ 2.2 22.6 ⫾ 1.7 24.6 ⫾ 1.2

70.6 ⫾ 10.8 67.9 ⫾ 6.9 78.0 ⫾ 15.2

0.29 ⫾ 0.02 0.30 ⫾ 0.02 0.32 ⫾ 0.05

27.9 ⫾ 7.6 25.9 ⫾ 8.6 29.2 ⫾ 6.1

2.8 ⫾ 1.7* 15.3 ⫾ 4.7 3.6 ⫾ 1.5*

4.0 ⫾ 2.2* 22.6 ⫾ 6.4 4.6 ⫾ 2.0*

39.7 ⫾ 10.1 37.9 ⫾ 10.5 38.6 ⫾ 10.5

7 7 7

22.9 ⫾ 1.0 24.1 ⫾ 0.7 24.7 ⫾ 1.5

65.1 ⫾ 3.9 69.4 ⫾ 7.4 76.3 ⫾ 6.6

0.29 ⫾ 0.02 0.29 ⫾ 0.02 0.31 ⫾ 0.05

24.8 ⫾ 13.7 28.5 ⫾ 4.7 29.6 ⫾ 10.0

4.2 ⫾ 3.2* 6.1 ⫾ 4.2* 14.3 ⫾ 7.6

6.4 ⫾ 4.7* 8.4 ⫾ 5.2* 18.9 ⫾ 10.0

38.0 ⫾ 20.3 41.2 ⫾ 6.6 39.4 ⫾ 14.7

NOTE. Data are presented as mean ⫾ standard deviation. Abbreviations: BW, body weight; LV, left ventricle; AAR, area at risk; IS, infarct size; CON, control; wt, wild type; eNOS⫺/⫺, endothelial nitric oxide synthase gene deletion; iNOS⫺/⫺, inductible nitric oxide synthase gene deletion; APC, anesthetic-induced preconditioning. *Significantly (p ⬍ 0.05) different from CON.

ROLE OF eNOS AND iNOS IN DESFLURANE-INDUCED PRECONDITIONING

iNOS inhibition. Species differences and the limited selectivity of pharmacologic NOS inhibitors35 may account for this discrepancy. To dispel any concerns regarding limited drug selectivity, the authors used gene-deleted animals to investigate the role of eNOS and iNOS in the 2 windows of APC. The present results confirm and extend the data obtained in rats and mice. In mice, iNOS mediates the second window of desflurane-induced preconditioning, whereas iNOS is not important for the first window of APC. The authors’ observation that eNOS, but not iNOS, is crucial for the first window of desflurane-induced preconditioning seems plausible because eNOS, but not iNOS, is expressed constitutively in the myocardium, and the interval of 30 minutes from the onset of preconditioning to the onset of myocardial ischemia in the first window of APC is too short for a translational increase of iNOS. An increased bioavailability of NO during ischemia-reperfusion injury is one of the pivotal mechanisms in transforming cells into a protected phenotype. NO activates the “classic” soluble guanylate cyclase signaling pathway36 that increases cyclic guanosine monophosphate levels and thus upregulates a multitude of proteins mediating myocardial protection.36 In addition, emerging evidence has indicated that NO can act directly on mitochondria, which is considered the major end effector of cardioprotective strategies, including APC.37 During ischemia-reperfusion injury, increased reactive oxygen species production and Ca2⫹ overload cause a loss of mitochondrial membrane potential and an opening of the mitochondrial permeability transmission pore. The loss of mitochondrial integrity leads to intracellular adenosine triphosphate depletion and to myocyte apoptosis and necrosis.37,38 By a direct inhibition of the electron transport chain, NO preserves mitochondrial integrity during ischemia-reperfusion injury by the mitigation of mitochondrial Ca2⫹ uptake, reactive oxygen species generation, and opening of the mitochondrial permeability transmission pore.37 A growing body of literature has provided compelling evidence for the strong preconditioning effects of volatile anesthetics in the experimental setup. However, according to recent meta-analyses, the preconditioning effects of volatile anesthetics in the clinical setup seem to be comparatively weak.39-41 Several issues might explain this discrepancy. Patients undergoing cardiovascular surgery often take a multitude of drugs that possibly interfere with intracellular pathways mediating APC (eg, ␤-blockers).42 Moreover, specific comorbidities impair preconditioning. Hyperglycemia abolishes isoflurane-induced preconditioning.43,44 The preconditioning effects of volatile anesthetics are impaired in senescent individuals.45 A common feature of diabetes and advanced age is the impairment of endothelial function.46 The present data provide evi-

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dence that proper endothelial function regarding NOS activity is crucial for APC, and the restoration of NOS activity in patients with impaired endothelial function may be important to allow APC in patients with cardiovascular risk undergoing surgery. The present study should be interpreted within the constraints of several potential limitations. In organisms with a constitutive knockout of a specific gene, the expression of other enzymes may be increased to compensate for the missing protein. However, in the eNOS⫺/⫺ mouse strain used in the present study, basal iNOS expression has been shown to be unaffected.47 In the myocardium, eNOS and iNOS are not the only sites of NO production. Neuronal NOS is expressed constitutively at a low level in the myocardium.48 Previous studies have implicated that nNOS is unlikely to play an important role in APC.12,30 However, the role of nNOS in desflurane-induced preconditioning was not investigated in the present study. Moreover, the role of NO produced by nonenzymatic pathways49 and the NOx levels in the myocardium were not quantified in the present study. Oxygen delivery to the animals during desflurane application has the potential to induce myocardial protection by the formation of reactive oxygen species. However, it has been shown previously that a 2-hour exposure to oxygen 24, 48, 72, and 96 hours before myocardial ischemia-reperfusion does not decrease myocardial IS, and thus the inhalation of oxygen-enriched air does not induce a second window of preconditioning.28 In the present study, a pilot group of animals was exposed to a 50/50 mixture of air and oxygen and desflurane 0.0 MAC 48 hours before CAO. The oxygen treatment did not decrease myocardial IS (data not shown). The size of myocardial AAR and the amount of coronary collateral blood flow were shown to be crucial determinants of myocardial IS. However, the AAR was not different among the groups. Coronary collateral blood flow was not measured in this study. Small rodents have been reported to have little, if any, coronary collateral blood flow,50 and coronary blood flow is not different in preconditioned and nonpreconditioned mouse hearts.51 Thus, it is highly improbable that AAR and coronary collateral blood flow contributed to differences in myocardial IS. Myocardial oxygen consumption was not quantified directly in this study. Therefore, the authors cannot exclude completely that changes in the myocardial oxygen supply/demand ratio contributed to the present results. In summary, desflurane induces a second window of preconditioning 48 hours after its administration in mice. Endothelial NOS, but not iNOS, is crucial for the first window of desflurane-induced preconditioning. The second window of desflurane-induced preconditioning is mediated by iNOS, but not by eNOS. ACKNOWLEDGMENTS The authors thank Claudia Sommer, MD, PhD (Department of Neurology, University of Würzburg) for providing the eNOS⫺/⫺ and iNOS⫺/⫺ mice.

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