Volatile anesthetics produce pharmacological preconditioning in animal models of myocardial ischemia-reperfusion injury and humans with coronary artery disease (1). However, such a preconditioning strategy requires advance knowledge of when the ischemic event will occur. Volatile anesthetics may also be capable of producing protection against ischemic injury when administered solely upon reperfusion (2–5). This observation is potentially important from a clinical perspective because the precise timing of coronary artery occlusion is unknown in the majority of patients with acute myocardial infarction. Our laboratory recently demonstrated that brief administration of isoflurane immediately before and during early reperfusion after prolonged coronary artery occlusion markedly reduces myocardial infarct size in rabbits (5). This “anesthetic-induced postconditioning” was mediated by activation of the pro-survival phosphatidylinositol-3-kinase (PI3K)-Akt (protein kinase B) signaling cascade (5). The PI3K-Akt pathway has also been implicated in myocardial protection during early reperfusion produced by brief, repetitive ischemic stimuli (5–8) and other drugs including bradykinin (9), adenosine receptor agonists (9), insulin (10), statins (11), and opioids (12). Selective δ1-opioid receptor agonists (e.g., TAN-67 and BW373U86) and morphine (a μ1-opioid receptor agonist with δ1 agonist properties) augment isoflurane-induced preconditioning in vivo (13,14). In the current investigation, we tested the hypothesis that morphine enhances isoflurane-induced postconditioning against myocardial infarction via PI3K or opioid receptor activation. Previous studies demonstrated that volatile anesthetics (15) and opioids (16,17) reduce ventricular myocyte apoptosis in response to tissue injury in vitro or ischemia-reperfusion in vivo. Whether volatile anesthetics or opioids also preserve myocardial integrity by attenuating apoptosis during early reperfusion remains unknown. Thus, the current investigation also tested the hypotheses that brief exposure to isoflurane or morphine before and during early reperfusion reduces apoptotic cell death.
All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. Furthermore, all conformed to the Guiding Principles in the Care and Use of Animals (18) of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals (19).
Male New Zealand white rabbits weighing between 2.5 and 3.0 kg were anesthetized with IV sodium pentobarbital (30 mg/kg) as previously described (5,20). Briefly, a tracheostomy was performed through a midline incision, and each rabbit was ventilated with positive pressure using an air-oxygen mixture (fractional inspired oxygen concentration = 0.33). Arterial blood gas tensions and acid-base status were maintained within a normal physiological range (pH between 7.35 and 7.45, Paco2 between 25 and 40 mm Hg, and Pao2 between 90 and 150 mm Hg) by adjusting the respiratory rate or tidal volume. Body temperature was maintained with a heating blanket. Heparin-filled catheters were inserted into the right carotid artery and the left jugular vein for measurement of mean arterial blood pressure and fluid or drug administration, respectively. Maintenance fluids (0.9% saline) were administered at 15 mL·kg-1·h−1 for the duration of each experiment. A thoracotomy was performed at the left fourth intercostal space and the heart was suspended in a pericardial cradle. A prominent branch of the left anterior descending coronary artery (LAD) was identified, and a silk ligature was placed around this vessel approximately halfway between the base and the apex for the production of coronary artery occlusion and reperfusion. IV heparin (500 U) was administered immediately before LAD occlusion. Coronary artery occlusion was verified by the presence of epicardial cyanosis and regional dyskinesia in the ischemic zone, and reperfusion was confirmed by observing an epicardial hyperemic response. Hemodynamic data were continuously recorded on a polygraph throughout each experiment.
The experimental design is illustrated in Figure 1. Baseline hemodynamic data and arterial blood gas tensions were recorded 30 min after instrumentation was completed. All rabbits underwent a 30-min LAD occlusion followed by 3 h of reperfusion. In separate experimental groups (n = 6 to 8 per group), rabbits were randomly assigned to receive 0.9% saline (control), the selective PI3K inhibitor wortmannin (0.6 mg/kg), or the nonselective opioid antagonist naloxone (6 mg/kg) 30 min before prolonged LAD occlusion, 0.5 or 1.0 minimum alveolar concentration (MAC) isoflurane (1.0 MAC = 2.05% in the rabbit) administered for 5 min, beginning at 3 min before until 2 min after reperfusion, or morphine (0.05 or 0.1 mg/kg in 0.9% saline) administered as a continuous IV infusion over 5 min beginning 3 min before and ending 2 min after reperfusion. Isoflurane was administered for 3 min before reperfusion to establish a plasma concentration of the volatile anesthetic when the coronary blood flow was restored. Additional groups of rabbits were pretreated with wortmannin or naloxone and received 1.0 MAC isoflurane or 0.1 mg/kg morphine before and during early reperfusion. Three final groups of rabbits received the combination of isoflurane (0.5 MAC) and morphine (0.05 mg/kg) in the presence or absence of wortmannin or naloxone pretreatment.
Myocardial infarct size was measured as previously described (21). Briefly, the LAD was reoccluded at the completion of each experiment and 3 mL of patent blue dye was injected IV. The left ventricular (LV) area at risk (AAR) for infarction was separated from surrounding normal areas (stained blue), and the 2 regions were incubated at 37°C for 20 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer adjusted to pH 7.4. Infarcted and noninfarcted myocardium within the AAR were carefully separated and weighed after storage overnight in 10% formaldehyde. Myocardial infarct size was expressed as a percentage of the AAR. Rabbits that developed intractable ventricular fibrillation and those with an AAR <15% of total LV mass were excluded from subsequent analysis.
Apoptosis was evaluated using mitochondrial cytochrome c translocation and Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) staining. Immunohistochemistry was performed using previously described techniques (5,22). Briefly, LV myocardium was also obtained from rabbits randomly assigned to receive 0.9% saline, isoflurane (0.5 and 1.0 MAC), morphine (0.05 or 0.1 mg/kg), or the combination of 0.5 MAC isoflurane and 0.05 mg/kg morphine as described above. All animals underwent a 30-min LAD occlusion. Hearts were rapidly excised after 5 min reperfusion, and the LV was isolated and stored at −70°C. Transverse cryostat sections (5 μm) of the LV were mounted on positively charged Colorfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA), fixed for 20 min in 100% acetone at −20°C, and rinsed with phosphate-buffered saline. Sections were then incubated with 1:500 dilutions of primary antibodies for cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA) in phosphate-buffered saline at 37°C for 1 h. Sections were washed 3 times for 5 min with phosphate-buffered saline and incubated with 1:1000 dilutions of biotinylated secondary antibodies (Santa Cruz Biotechnology) in phosphate-buffered saline at 37°C for 30 min. Sections were washed with phosphate-buffered saline before conjugation with 10 μg/mL streptavidin-labeled fluorescein isothiocyanate (Pierce, Rockford, IL) at 37°C for 15 min. Nuclear staining was achieved with 1 μM TO-PRO-3 (Molecular Probes, Eugene, OR) at 37°C for 30 min. Images were obtained using a laser fluorescence imaging system and a confocal microscope with a 40× objective that yielded a 400× end magnification on a 292 × 195 μm2 digital image (768 × 512 pixels). A Krypton-Argon-Helium laser was used for excitation wavelengths of 490 and 642 nm and emitted fluorescence was determined after long pass filtering at corresponding wavelengths of 520 and 661 nm for fluorescein isothiocyanate and TO-PRO, respectively. For each rabbit heart, 20 to 30 images were obtained.
TUNEL staining was performed on additional transverse cryostat sections (5 μm) of the LV using the ApopTag detection kit (Chemicon). Sections were incubated with buffer at room temperature for 10 s, followed by incubation in terminal deoxynucleotidyl transferase (1:2 dilution) at 37°C for 1 h. The enzymatic reaction was stopped by incubating the sections with 3% stop/wash buffer at room temperature for 10 min. Sections were washed 3 times for 1 min with phosphate-buffered saline and subsequently incubated with fluorescein-conjugated anti-digoxigenin (1:1 dilution) at room temperature for 30 min. Sections were then washed 4 times for 2 min with phosphate-buffered saline. TO-PRO-3 (1 μM at 37°C for 30 min) was used for nuclear counterstaining and images were obtained using confocal microscopy as described above. For each rabbit heart, 20 to 30 images were obtained.
Statistical analysis of data within and among groups was performed with analysis of variance for repeated measures followed by the Student-Newman-Keuls test. Changes were considered statistically significant when P < 0.05. All data are expressed as mean ± sd.
One-hundred-eight rabbits were instrumented to obtain 102 successful experiments. Two rabbits were excluded because of technical problems during instrumentation. Four rabbits were excluded because intractable ventricular fibrillation occurred during LAD occlusion: 1 each in the morphine (0.1 mg/kg)+wortmannin, isoflurane (0.5 MAC)+morphine (0.05 mg/kg), isoflurane (0.5 MAC)+morphine (0.05 mg/kg)+wortmannin, and isoflurane (0.5 MAC)+morphine (0.05 mg/kg)+naloxone groups.
There were no statistically significant differences in baseline hemodynamics among groups (Table 1). Coronary artery occlusion significantly (P < 0.05) decreased mean arterial blood pressure and rate-pressure product in many experimental groups. Decreases in heart rate and rate-pressure product were observed during reperfusion in all experimental groups. There were no differences in hemodynamics among groups before, during, and after LAD occlusion.
Body weight, LV mass, AAR weight, and the ratio of AAR to LV mass were similar among groups (Table 2). Brief exposure to 1.0 but not 0.5 MAC isoflurane during early reperfusion reduced infarct size (21% ± 4% and 44% ± 6% of the LV AAR, respectively) as compared with control (41% ± 4%; Fig. 2). Administration of 0.1 but not 0.05 mg/kg morphine during early reperfusion also reduced infarct size (19% ± 4% and 41% ± 6%, respectively). Wortmannin and naloxone alone did not affect infarct size (38% ± 6% and 40% ± 8%, respectively) but blocked the protection produced by 1.0 MAC isoflurane and 0.1 mg/kg morphine (44% ± 6% and 42% ± 6%, respectively, for naloxone). The combination of 0.5 MAC isoflurane and 0.05 mg/kg morphine reduced infarct size (18% ± 9%). This protective effect was also abolished by pretreatment with wortmannin (44% ± 5%) or naloxone (45% ± 4%).
Brief exposure to isoflurane during early reperfusion produced a dose-related decrease in the number of cytochrome c translocated cells (1.3% ± 1.4% and 0.7% ± 0.7% for 0.5 and 1.0 MAC, respectively; Fig. 3) as compared with control (2.3 ± 2.0%). Morphine also reduced mitochondrial cytochrome c translocation (0.6% ± 0.7% and 1.3% ± 1.1% for 0.05 and 0.1 mg/kg, respectively). Administration of isoflurane or morphine during early reperfusion decreased TUNEL staining, consistent with an antiapoptotic effect (11.9% ± 5.7% and 11.1% ± 5.6% for 0.5 and 1.0 MAC isoflurane, respectively, as compared with 17.7% ± 6.7% during control; Fig. 4). Combined administration of 0.5 MAC isoflurane and 0.05 mg/kg morphine did not further reduce cytochrome c translocation or the number of TUNEL-positive ventricular myocytes as compared with either drug alone (Figs. 3 and 4).
The current results confirm our previous findings (5) demonstrating that brief exposure to 1.0, but not 0.5, MAC isoflurane immediately before and during early reperfusion produces myocardial protection against irreversible ischemic injury by activating PI3K-mediated signal transduction. Our results from experiments conducted in rabbits also support previous data obtained in rats (12), indicating that administration of morphine on reperfusion reduces infarct size through a PI3K-dependent mechanism. The current findings further demonstrate that morphine, when administered in a dose that does not alter infarct size alone, enhances isoflurane-induced preconditioning against infarction. The protective effects of the combination of subthreshold doses of morphine and isoflurane during early reperfusion were abolished by pretreatment with wortmannin, indicating that the observed decrease in the extent of infarction is mediated by PI3K activation. We have previously demonstrated that selective δ1-opioid receptor agonists or morphine administered before prolonged coronary occlusion enhanced preconditioning by isoflurane in rats by activating adenosine triphosphate-regulated potassium (KATP) channels and opioid receptors (13,14). The current results indicate that the nonselective opioid antagonist naloxone inhibited the protective effects of morphine, isoflurane, and their combination during early reperfusion. These data suggest that opioid receptor activation mediates isoflurane-induced postconditioning and its augmentation by morphine. Gross et al. (12,23,24) have previously shown that myocardial protection by opioids before ischemia and during reperfusion occurs as a result of δ1-opioid receptor activation. These data collectively imply that postconditioning against infarction by isoflurane and its enhancement by the clinically relevant opioid morphine may also be mediated by the δ1-opioid receptor. Nevertheless, the δ1-opioid receptor was not specifically examined in the current investigation, and additional study will be required to examine this latter hypothesis.
In contrast to the generalized cellular destruction and profound inflammatory response that typify necrosis, the highly regulated, ATP-dependent phenomenon of apoptosis is characterized by selective DNA lysis associated with the creation of apoptotic bodies, the appearance of condensed chromatin, and the absence of inflammation concomitant with maintenance of cell membrane architecture (25). The majority of evidence collected supports the contention that reperfusion after a prolonged ischemic insult either initiates or hastens the apoptotic process (26,27). The current results indicate that brief exposure to isoflurane before and during early reperfusion reduces cytochrome c translocation from mitochondria (an important early marker of apoptosis) (28) and decreases the number of TUNEL-positive ventricular myocytes in situ. These data suggest, for the first time, that brief administration of isoflurane before and during early reperfusion preserves myocardial integrity after prolonged coronary occlusion in part by attenuating apoptotic cell death. Volatile anesthetics have been shown to abolish norepinephrine-induced apoptosis in rat ventricular myocytes, as indicated by reductions in TUNEL-positive cell staining, attenuation of increases in annexin V staining (an index of DNA laddering), and inhibition of increased caspase-9 activity (15). The current findings also suggest that morphine inhibits apoptosis when administered before and during early reperfusion. Both doses of morphine reduced cytochrome c release, whereas only the 0.05 mg/kg dose of morphine attenuated TUNEL-positive cell staining in situ. The explanation for this observation is unclear, but a longer duration of reperfusion may have been required for TUNEL analysis in morphine-pretreated rabbits. A recent study demonstrated that morphine mimics the antiapoptotic effect of preconditioning by activating inositol (1,4,5) triphosphate [I(1,4,5)P3]-mediated signal transduction in rat ventricular myocytes (17). Administration of morphine before prolonged coronary artery occlusion attenuated myocardial apoptosis after ischemia-reperfusion injury through a δ-opioid receptor-dependent process in rabbits (16). Thus, the possibility that morphine-induced reductions in apoptosis may have occurred by a PI3K-independent mechanism cannot be completely excluded from the analysis. Whether the reduction in apoptosis produced by isoflurane or morphine during early reperfusion is also dependent on I(1,4,5)P3 signaling, δ-opioid receptors, or the PI3K cascade will require additional investigation to ascertain.
The current results must be interpreted within the constraints of several potential limitations. Wortmannin has been shown to be a selective inhibitor of PI3K at the dose used in the current investigation. Our previous immunoblot results confirmed the PI3K selectivity of wortmannin (5). Nevertheless, the possibility that wortmannin may have inhibited other protein kinases involved in myocardial protection cannot be completely excluded from the analysis. Regulation of ion channels implicated in myocardial protection (e.g., KATP channels) by phosphatidylinositol phosphates has been also suggested (29). Myocardial infarct size is determined primarily by the size of the AAR and extent of coronary collateral perfusion. The AAR expressed as a percentage of total LV mass was similar among groups in the current investigation. Rabbits have also been shown to have little if any coronary collateral blood flow (30). Thus, it appears unlikely that differences in collateral perfusion among groups account for the observed results. However, coronary collateral blood flow was not specifically quantified in the current investigation. The reductions in myocardial infarct size and apoptosis produced by brief administration of isoflurane during early reperfusion in the presence and absence of morphine occurred independent of changes in major determinants of myocardial oxygen consumption. Nevertheless, the current results require qualification because coronary venous oxygen tension was not directly measured and myocardial oxygen consumption was not calculated in the current investigation. Reductions in cytochrome c translocation from mitochondria and the number of TUNEL positive stained ventricular myocytes produced by isoflurane suggest that this volatile anesthetic may preserve myocardial viability by inhibiting apoptotic cell death during early reperfusion. Nevertheless, other indices of the apoptotic process, including phosphorylation of pro-apoptotic proteins (e.g., Bad, Bax), inhibition of caspase activity, and cleavage of poly(ADP)ribose polymerase, were not specifically assessed in the current investigation.
In summary, the current results demonstrate that administration of morphine immediately before and during early reperfusion enhances reductions in myocardial infarct size produced by isoflurane after prolonged coronary artery occlusion in rabbits. This enhancement of the protective effects of isoflurane by morphine during early reperfusion is mediated by activation of PI3K and opioid receptors. The findings also suggest that isoflurane-induced reductions in apoptotic cell death contribute to the preservation of myocardial viability observed with the administration of this anesthetic during early reperfusion in vivo.
The authors thank David A. Schwabe BSEE (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin) for technical assistance, Garrett J. Gross PhD (Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin) for his insights and suggestions, and Mary Lorence-Hanke AA (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin) for assistance in preparation of the manuscript.
1. Tanaka K, Ludwig LM, Kersten JR, et al. Mechanisms of cardioprotection by volatile anesthetics. Anesthesiology 2004;100:707–21.
2. Schlack W, Preckel B, Stunneck D, Thamer V. Effects of halothane, enflurane, isoflurane, sevoflurane and desflurane on myocardial reperfusion injury in the isolated rat heart. Br J Anaesth 1998;81:913–9.
3. Siegmund B, Schlack W, Ladilov YV, et al. Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture. Circulation 1997;96:4372–9.
4. Varadarajan SG, An J, Novalija E, Stowe DF. Sevoflurane before or after ischemia improves contractile and metabolic function while reducing myoplasmic Ca2+ loading in intact hearts. Anesthesiology 2002;96:125–133.
5. Chiari PC, Bienengraeber MW, Pagel PS, et al. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits. Anesthesiology 2005;102:102–9.
6. Kin H, Zhao ZQ, Sun HY, et al. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 2004;62:74–85.
7. Zhao ZQ, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:H579–88.
8. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of “modified reperfusion” protects myocardium by activating the PI3K-Akt pathway. Circ Res 2004;95:230–2.
9. Yang XM, Krieg T, Cui L, et al. NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO. J Mol Cell Cardiol 2004;36:411–21.
10. Jonassen AK, Sack MN, Mjos OD, Yellon DM. Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res 2001;89:1191–8.
11. Bell RM, Yellon DM. Atorvastatin, administered at the onset of reperfusion, and independent of lipid lowering, protects the myocardium by up-regulating a pro-survival pathway. J Am Coll Cardiol 2003;41:508–15.
12. Gross ER, Hsu AK, Gross GJ. Opioid-induced cardioprotection occurs via glycogen synthase kinase β inhibition during reperfusion in intact rat hearts. Circ Res 2004;94:960–6.
13. Ludwig LM, Patel HH, Gross GJ, et al. Morphine enhances pharmacological preconditioning by isoflurane: role of mitochondrial K(ATP) channels and opioid receptors. Anesthesiology 2003;98:705–11.
14. Patel HH, Ludwig LM, Fryer RM, et al. Delta opioid agonists and volatile anesthetics facilitate cardioprotection via potentiation of K(ATP) channel opening. FASEB J 2002;16:1468–70.
15. Zaugg M, Jamali NZ, Lucchinetti E, et al. Norepinephrine-induced apoptosis is inhibited in adult rat ventricular myocytes exposed to volatile anesthetics. Anesthesiology 2000;93:209–18.
16. Okubo S, Tanabe Y, Takeda K, et al. Ischemic preconditioning and morphine attenuate myocardial apoptosis and infarction after ischemia-reperfusion in rabbits: role of δ-opioid receptor. Am J Physiol Heart Circ Physiol 2004;287:H1786–91.
17. Barrere-Lemaire S, Combes N, Sportouch-Dukhan C, et al. Morphine mimics the antiapoptotic effect of preconditioning via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 2005;288:H83–8.
18. World Medical Association; American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 2002;283:R281–3.
19. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Guide for the care and use of laboratory animals. 7th ed. Washington, DC: National Academy Press, 1996.
20. Tanaka K, Weihrauch D, Kehl F, et al. Mechanism of preconditioning by isoflurane in rabbits: a direct role for reactive oxygen species. Anesthesiology 2002;97:1485–90.
21. Warltier DC, Zyvoloski MG, Gross GJ, et al. Determination of experimental myocardial infarct size. J Pharmacol Methods 1981;6:199–210.
22. Ludwig LM, Weihrauch D, Kersten JR, et al. 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. Anesthesiology 2004;100:532–9.
23. Schultz Je-J, Hsu AK, Nagase H, Gross GJ. TAN-67, a delta 1-opioid receptor agonist, reduces infarct size via activation of Gi/o proteins and KATP channels. Am J Physiol Heart Circ Physiol 1998;274:H909–14.
24. Fryer RM, Wang Y, Hsu AK, Gross GJ. Essential activation of PKC-delta in opioid-initiated cardioprotection. Am J Physiol Heart Circ Physiol 2001;280:H1346–53.
25. Gottlieb RA, Engler RL. Apoptosis in myocardial ischemia-reperfusion. Ann N Y Acad Sci 1999;874:412–26.
26. Eefting F, Rensing B, Wigman J, et al. Role of apoptosis in reperfusion injury. Cardiovasc Res 2004;61:414–26.
27. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res 2004;61:448–60.
28. Kennedy SG, Kandel ES, Cross TK, Hay N. Akt/Protein kinase B inhibits cell death by preventing release of cytochrome c from mitochondria. Mol Cell Biol 1999;19:5800–10.
29. Shyng SL, Nichols CG. Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science 1998;282:1138–41.
© 2005 International Anesthesia Research Society
30. Maxwell MP, Hearse DJ, Yellon DM. Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res 1987;21:737–46.