Anesthetic and ischemic preconditioning share similar signal transduction pathways. The authors tested the hypothesis that the beta1-adrenergic signal transduction pathway mediates anesthetic and ischemic preconditioning in vivo.
Pentobarbital-anesthetized (30 mg/kg) rabbits (n = 96) were instrumented for measurement of systemic hemodynamics and subjected to 30 min of coronary artery occlusion and 3 h of reperfusion. Sixty minutes before occlusion, vehicle (control), 1.0 minimum alveolar concentration desflurane, or sevoflurane, and esmolol (30.0 mg x kg(-1) x h(-1)) were administered for 30 min, respectively. Administration of a single 5-min cycle of ischemic preconditioning was instituted 35 min before coronary artery occlusion. In separate groups, the selective blocker esmolol or the protein kinase A inhibitor H-89 (250 microg/kg) was given alone and in combination with desflurane, sevoflurane, and ischemic preconditioning.
Baseline hemodynamics and area at risk were not significantly different between groups. Myocardial infarct size (triphenyltetrazolium staining) as a percentage of area at risk was 61 +/- 4% in control. Desflurane, sevoflurane, and ischemic preconditioning reduced infarct size to 34 +/- 2, 36 +/- 5, and 23 +/- 3%, respectively. Esmolol did not alter myocardial infarct size (65 +/- 5%) but abolished the protective effects of desflurane and sevoflurane (57 +/- 4 and 52 +/- 4%, respectively) and attenuated ischemic preconditioning (40 +/- 4%). H-89 did not alter infarct size (60 +/- 4%) but abolished preconditioning by desflurane (57 +/- 5%) and sevoflurane (61 +/- 1%). Ischemic preconditioning (24 +/- 7%) was not affected by H-89.
The results demonstrate that anesthetic preconditioning is mediated by the beta1-adrenergic pathway, whereas this pathway is not essential for ischemic preconditioning. These results indicate important differences in the mechanisms of anesthetic and ischemic preconditioning.
ISCHEMIC and anesthetic preconditioning against myocardial infarction share similar signal transduction pathways, including guanine regulatory protein (G protein)–coupled receptors, protein kinase C,1inhibitory G proteins (Gi),2and mitochondrial and sarcolemmal adenosine triphosphate–regulated potassium (KATP) channels.3–5Stimulation of G protein–coupled receptors, such as adenosine type 1,6opioid Δ1,7or α1-adrenergic receptors,8plays a major role in the preconditioning process. The role of the G protein–coupled β1-adrenergic receptor (β1-AR) in the signaling cascade of preconditioning is unclear. The β1-AR subtype is coupled to a Gsprotein, responding to catecholamine stimulation in the myocardium with an increase of intracellular levels of cyclic adenosine monophosphate. The subsequent activation of protein kinase A (PKA) results in phosphorylation of L-type calcium channels, phospholamban, and myofilaments, thereby evoking contractile and relaxant responses in the heart.9Persistent stimulation of β1-AR results in dilatative cardiomyopathy, heart failure, and increased apoptosis.10However, transient stimulation of β1-AR induces cardioprotective effects that are similar to ischemic preconditioning.11,12Moreover, the blockade of β-adrenergic receptors attenuates ischemic preconditioning,13,14thus indicating a prominent role for β1-AR in preconditioning. The goal of the current study was to elucidate the role of the β1-adrenergic pathway in anesthetic and ischemic preconditioning. Therefore, the hypothesis was tested that the inhibition of the β1-adrenergic signal transduction pathway blocks desflurane preconditioning, sevoflurane preconditioning, and ischemic preconditioning against myocardial infarction in an in vivo model of myocardial infarction in rabbits.
Materials and Methods
All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the local authorities (Government of Unterfranken, Würzburg, Germany). Furthermore, all procedures conformed to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals .15
General Preparation of Animals
Male New Zealand White rabbits were anesthetized with sodium pentobarbital (30 mg/kg intravenous bolus, followed by an infusion of 20–30 mg · kg−1· h−1) via the left marginal auricular vein. Depth of anesthesia was verified by recurrent testing of palpebral reflexes and hind paw withdrawal throughout the experiment. After tracheotomy and tracheal cannulation, animals were artificially ventilated (Cicero®; Dräger, Lübeck, Germany) using positive pressure with an air–oxygen mixture (70% air–30% O2). Arterial blood drawn from the auricular artery was analyzed using an ABL 505 blood gas analyzer (Radiometer, Copenhagen, Denmark), and blood gases were maintained within a normal physiologic range by adjusting the respiratory rate or tidal volume. End-tidal concentrations of desflurane and sevoflurane were measured at the tip of the endotracheal tube by an infrared anesthetic analyzer that was calibrated with known standards before and during experimentation. The rabbit minimum alveolar concentrations (MACs) of desflurane and sevoflurane used in the current investigation were 8.9%16and 3.7%,17respectively. Left ventricular (LV) pressure and the maximum increase of LV pressure (+dP/dtmax) were measured with a saline-filled PE-50 polyethylene catheter inserted into the left ventricle via the right carotid artery. Mean arterial pressure was monitored by insertion of a 2.5-French microtipped catheter (Millar Instruments Inc., Houston, TX) via the right femoral artery into the descending aorta. Rectal body temperature was maintained at 38.5°± 0.5°C by a servo-controlled heating pad (Föhr Instruments, Seeheim, Germany). After a left fourth thoracotomy and pericardiotomy, the left heart was exposed and suspended in a pericardial cradle. A 4-mm ultrasound probe (Transonic®; Ithaca, NY) was placed around the pulmonary artery for measurement of cardiac output. A silk ligature (2-0) was placed halfway between the base and the apex of the heart around a prominent branch of the left anterior descending coronary artery to form a snare. By tightening of the snare, a coronary artery occlusion was produced. Reperfusion was instituted by loosening the snare. Each rabbit received 300 U/kg heparin 5 min before coronary artery occlusion for anticoagulation. Coronary artery occlusion was verified by epicardial cyanosis, regional dyskinesia in the ischemic zone, and electrocardiographic changes. Adequate reperfusion was confirmed by epicardial hyperemic response and reversion of electrocardiographic changes. Hemodynamic parameters, body temperature, and electrocardiogram were continuously recorded and analyzed using a personal computer (Hewlett Packard, Palo Alto, CA; 4 gigabyte) and hemodynamic data acquisition and analysis software (Notocord® hem 3.5; Croissy sur Seine, France). Data were digitized at a sampling rate of 1,000 Hz.
The experimental protocol used in this investigation is illustrated in figure 1. Baseline systemic hemodynamics were recorded following a 30-min equilibration period after instrumentation and calibration were completed. All rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. Rabbits were randomly assigned to receive vehicle (0.9% saline [control]), 1.0 MAC desflurane, 1.0 MAC sevoflurane, or the β1-selective blocker esmolol (30.0 mg · kg−1· h−1) for 30 min, or a single 5-min cycle of ischemic preconditioning. All interventions were completed 30 min before coronary artery occlusion; thus, a memory period of 30 min was allowed in all groups. Esmolol was coadministered in three separate groups with desflurane, sevoflurane, or a single 5-min cycle of ischemic preconditioning. In a second set of experiments, the selective PKA inhibitor H-89 was given alone and in combination with desflurane, sevoflurane, and single-cycle ischemic preconditioning. H-89 (250 μg/kg) was administered directly into the left ventricle 5 min before the onset of desflurane, sevoflurane, and ischemic preconditioning.
Measurement of Myocardial Infarct Size
Infarct size and area at risk (AAR) were gravimetrically determined according to standard procedures.18Briefly, at the end of each experiment, the coronary artery was reoccluded, and the AAR was determined by infusion of 2 ml patent blue (0.1 g/ml; Sigma-Aldrich, Taufkirchen, Germany). The rabbits were then killed with a lethal dose of pentobarbital, and the heart was rapidly excised. The heart was cut into five slices from apex to base, and the nonstained red myocardium (AAR) was separated from the nonischemic blue-stained LV normal areas. The samples of ischemic and nonischemic regions were incubated at 37°C for 20 min in 1% 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich, Steinheim, Germany) in 0.1 m phosphate buffer adjusted to pH 7.4. After overnight storage in 10% formaldehyde, infarcted (pale) and noninfarcted (brick-red) myocardium within the AAR were carefully separated and weighed. Infarct size was expressed as a percentage of the AAR. Rabbits with an AAR less than 10% of LV mass and those with intractable ventricular fibrillation or LV pump failure were excluded from the study.
Determination of β Receptor Selectivity of Esmolol
The β1selectivity of esmolol was determined in radioligand binding experiments as described before.19Briefly, Chinese hamster ovary cells (CHO-K1 cells; CCL61; American Type Culture Collection, Rockville, MD), stably transfected with plasmid DNA coding for human β-ARs, were grown adherently and maintained in Dulbecco's Modified Eagle's Medium with nutrient mixture F12 at 37°C in 5% CO2–95% air. For the competition binding experiments with esmolol, crude membrane fractions were prepared and incubated in 50 mm Tris–HCl buffer, pH 7.4, with approximately 50 pm 125I-CYP in the case of β1and β2receptors, or approximately 80 pm 125I-CYP for β3receptors. The incubation buffer contained routinely 100 μm GTP. Membranes were incubated for 90 min at 30°C, filtered through Whatman GF/C filters, and washed three times with ice-cold assay buffer. Samples were counted in a γ-counter (Wallac 1480 Wizard 3”). The Kivalues of esmolol were calculated by nonlinear curve fitting with the program SCTFIT20and are expressed as geometric means with 95% confidence intervals.
Statistical analysis of data within and between groups was performed with one-way and two-way analysis of variance followed by post hoc Duncan test. Changes were considered statistically significant when the P value was less than 0.05. All data are expressed as mean ± SEM.
Ninety-six rabbits were instrumented to obtain 82 successful experiments. Six rabbits were excluded because of intractable ventricular fibrillation during the experimental protocol (2 control, 1 desflurane, 1 sevoflurane plus esmolol, and 2 desflurane plus H-89), 6 were excluded because of left ventricular pump failure (1 desflurane, 1 desflurane plus esmolol, 1 sevoflurane plus esmolol, 1 H-89, and 2 desflurane plus H-89), and 2 were excluded because LV AAR was less than 10% of the LV mass (1 desflurane plus esmolol and 1 desflurane plus H-89).
There were no differences in hemodynamics under baseline conditions between the experimental groups receiving vehicle, desflurane, sevoflurane, esmolol alone or in combination (table 1). Ischemic preconditioning had no effect on systemic hemodynamic variables, whereas desflurane and sevoflurane significantly reduced mean arterial pressure, rate–pressure product, and +dP/dtmax during administration. Esmolol alone or in combination with ischemic preconditioning, desflurane, or sevoflurane significantly reduced heart rate and rate–pressure product. Mean arterial pressure and +dP/dtmax were decreased by coadministration of esmolol and desflurane or sevoflurane. Cardiac output and left ventricular end-diastolic pressure were not significantly different throughout the study protocol among all experimental groups. All hemodynamic parameters returned to baseline values before coronary artery occlusion in all experimental groups. During reperfusion, mean arterial pressure and rate–pressure product were reduced similarly in all groups. In the experimental groups receiving H-89, there were no significant differences from the corresponding groups not receiving H-89 (data not shown).
Myocardial Infarct Size
The LV AAR was similar between groups (table 2). Myocardial infarct size expressed as a percentage of the AAR was 61 ± 4% (n = 8) in control. Desflurane, sevoflurane, and ischemic preconditioning significantly reduced infarct size to 34 ± 2% (n = 8), 36 ± 5% (n = 8), and 23 ± 3% (n = 8) of the AAR, respectively (fig. 2). Esmolol (n = 8) alone did not alter myocardial infarct size (65 ± 5%) but abolished the protective effects of desflurane and sevoflurane (57 ± 4%, n = 8 and 52 ± 4%, n = 7, respectively). Esmolol significantly attenuated ischemic preconditioning to 40 ± 4% (n = 8). In this group, myocardial infarct size was still significantly smaller than control but significantly larger than ischemic preconditioning alone. The specific PKA inhibitor H-89 alone did not alter infarct size (60 ± 4%, n = 4) but completely abolished desflurane-induced (57 ± 5%, n = 5) and sevoflurane-induced preconditioning (61 ± 1%, n = 5), whereas single-cycle ischemic preconditioning (24 ± 7%, n = 5) was not affected (fig. 3).
β1Selectivity of Esmolol
The binding affinities for esmolol at β-AR subtypes were determined in competition binding experiments using membranes from Chinese hamster ovary cells stably transfected with the human subtypes. The respective Kivalues for β1-, β2-, and β3-ARs are 100 (79–127), 2,700 (2,050–3,560), and 4,830 (1,150–20,300).
In the current study, the volatile anesthetics desflurane and sevoflurane or a single cycle of ischemic preconditioning all markedly reduced infarct size in the rabbit heart in vivo . These results confirm previous studies demonstrating preconditioning against myocardial infarction induced by enflurane,21halothane,21,22and isoflurane,21–24and the now widely used volatile anesthetics desflurane22and sevoflurane.25In the current study, the degree of infarct size reduction did not differ between desflurane and sevoflurane, whereas the study by Piriou et al. 22using the same model did not demonstrate significant infarct size reduction by sevoflurane-induced preconditioning. These conflicting results might be explained by important differences in study protocols. In the study by Piriou et al. , rabbits were anesthetized with ketamine, whereas in our study, sodium pentobarbital was used. Sodium pentobarbital is known to have a negligible effect on preconditioning,26whereas ketamine inhibits sarcolemmal and mitochondrial KATPchannels, thereby attenuating anesthetic and ischemic preconditioning.27–29Furthermore, a memory period of only 15 min was instituted, in contrast to 30 min in the current study. The degree of infarct size reduction by ischemic and anesthetic preconditioning in our study is in accord with previous findings.30
To demonstrate the effect of β1-AR blockade on preconditioning, the duration of action of the corresponding β blocker must be extremely short. Otherwise, the β blocker will act throughout the ischemia and reperfusion period and thereby might confound ischemia–reperfusion injury. In this study, esmolol was chosen over other β1-selective drugs because of its extremely short duration of action. Thereby, any effects of β1-AR blockade on the ischemia and reperfusion injury could be precluded, and β1-AR blockade was maintained only during the preconditioning period. Desflurane- and sevoflurane-induced preconditioning was abolished by concomitant administration of esmolol. In contrast, ischemic preconditioning was significantly attenuated but not completely blocked by the administration of esmolol. Furthermore, the selective PKA inhibitor H-89 completely abolished anesthetic preconditioning by desflurane and by sevoflurane but had no effect on ischemic preconditioning. Both blockers given alone had no effect on myocardial infarct size. Therefore, our findings indicate important differences in the mechanisms of anesthetic and ischemic preconditioning against myocardial infarction in an in vivo model. The involvement of the β adrenergic signal transduction pathway in preconditioning is evidenced by the finding that pharmacologic stimulation of β-AR induces preconditioning. In isolated rat hearts, a short 2-min perfusion with noradrenaline or isoproterenol, followed by a 10-min washout period, increased functional recovery of the myocardium.31In addition, creatine kinase release was reduced in the groups pretreated with noradrenaline or isoproterenol. These beneficial effects were abolished by the coadministration of the nonselective β blocker timolol. By using selective β blockers, β-adrenergic preconditioning was shown to be mainly mediated by the β1-AR subtype.11In addition, xamoterol, a selective agonist at β1-AR, induces protection against ischemia–reperfusion injury in the isolated catecholamine-depleted rat heart.32Participation of β-AR in ischemic preconditioning has been demonstrated in studies in isolated rabbit13and rat hearts.14Infarct size reduction, induced by two 5-min cycles of ischemic preconditioning, is attenuated by pretreatment with the β blocker esmolol.13Likewise, coadministration of the nonselective β blockers propranolol and nipradilol abolishes cardioprotection induced by a single 3-min cycle of ischemic preconditioning in isolated rat hearts.14In the current study, esmolol attenuated cardioprotection induced by a 5-min single cycle of ischemic preconditioning. Therefore, our results confirm and extend these findings into the in vivo animal model. Downstream of the β1-AR, the PKA inhibitor H-89 had no effect on ischemic preconditioning.
Volatile anesthetic preconditioning was studied in vitro in the investigation by Hanouz et al. 33Human right atrial trabeculae were subjected to 30 min of simulated ischemia, and isometric contractile force was measured. Desflurane preconditioning increased functional recovery, and this beneficial effect was abrogated by propranolol.33In contrast, in a cellular model of simulated ischemia in isolated rat ventricular cardiomyocytes, propranolol did not affect protection by isoflurane and sevoflurane.34In the current in vivo study, esmolol and H-89 abolished desflurane- and sevoflurane-induced preconditioning, thus providing evidence for the role of β1-AR signaling in anesthetic preconditioning. It has been suggested that β1-ARs exclusively couple to Gsproteins.35,36However, a recent study demonstrated that activation of protein kinase C promotes functional coupling of β1-ARs to Giproteins.37Moreover, activation of PKA leads to phosphorylation of agonist-occupied β2-ARs, resulting in an altered coupling specificity of the receptor from Gsto Gi.38Activation of Giproteins results in protein kinase C activation, promoting opening of mitochondrial KATPchannels,39which play a central role in volatile anesthetic preconditioning.3–5As an intriguing possibility, anesthetic preconditioning might promote the coupling of β1-ARs toward Gicoupling. The results of the current investigation, however, demonstrate that Gs-coupled downstream signaling plays an important role in anesthetic preconditioning, because inhibition of PKA, the downstream mediator of Gssignaling, abrogated anesthetic preconditioning, whereas ischemic preconditioning was not affected. Mechanistically, this might involve altered receptor coupling of β1-AR in ischemic preconditioning, a hypothesis that needs further investigation to be proven.
There are several potential limitations to the study that must be considered in the interpretation of the results. The LV AAR for myocardial infarction and coronary collateral blood flow are important determinants of the myocardial infarct size. However, the AAR was not different among experimental groups. Coronary collateral blood flow was not determined in this study. However, coronary collateral blood flow is negligible in rabbits.40Systemic hemodynamic changes were introduced by the administration of the volatile anesthetics and esmolol. However, all hemodynamic changes that might have influenced oxygen demand returned to baseline values before coronary occlusion was established. Therefore, it is highly unlikely that hemodynamic changes may have influenced myocardial infarct size. Nevertheless, coronary venous oxygen content was not measured, and myocardial oxygen consumption was not directly quantified in this investigation; therefore, alterations in myocardial oxygen metabolism during and after administration of desflurane, sevoflurane, and esmolol cannot be completely excluded as factors involved in the reduction of infarct size. In this study, the selectivity of esmolol at β-AR subtypes was studied in vitro . Our study confirms previous results, indicating that the selectivity of esmolol to β1-AR is sufficient and equals that of other β1-AR selective agents such as atenolol.41However, effects by concomitant blockade of β2- or β3-AR subtypes cannot completely be excluded. In addition to its action on β-AR, esmolol has non–receptor-mediated effects that reduce action potential duration and plateau voltage.42It cannot be excluded that these non–receptor-mediated effects influenced infarct size. The PKA inhibitor H-89, although considered selective to PKA, also blocks several other protein kinases, including MSK1, S6K1, ROCK-II,43and protein kinase G44and directly inhibits KATPchannels in rabbit coronary artery smooth muscle cells.45However, KATPchannels play a major role in anesthetic and ischemic preconditioning, and ischemic preconditioning was not affected by H-89. Therefore, it is highly unlikely that KATPchannel inhibition is responsible for the observed effects of H-89 on anesthetic preconditioning. However, these additional effects of H-89 should be borne in mind when interpreting the current findings.
In summary, the results of the current study demonstrate that β1-adrenergic signaling plays an important role in anesthetic preconditioning. The results also indicate that activation of the β1-adrenergic pathway is not essential for ischemic preconditioning. Therefore, the results suggest important differences in signaling cascades of anesthetic and ischemic preconditioning.
The authors thank Sonja Kachler, B.A. (Research Technologist, Department of Pharmacology, Bayerische Julius Maximilians-Universität, Würzburg, Germany), for her technical assistance.