Isoflurane Preconditions Myocardium Against Infarction via  Activation of Inhibitory Guanine Nucleotide Binding Proteins

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. All procedures were in conformity with the Guiding Principles in the Care and Use of Animals  of the American Physiologic Society 11and were performed in accordance with the Guide for the Care and Use of Laboratory Animals . 12 

The experimental methods have been previously described in detail. 13Briefly, mongrel dogs (weight = 23 ± 1 kg; mean ± SEM) were anesthetized with sodium barbital (200 mg/kg) and sodium pentobarbital (15 mg/kg) and ventilated with an air/oxygen mixture (fraction of inspired oxygen = 0.25) after tracheal intubation. Tidal volume and respiratory rate were adjusted to maintain arterial blood gas tensions within a physiologic range. A double pressure transducer–tipped catheter was inserted into the aorta and left ventricle (LV) via  the left carotid artery to measure aortic and LV pressures, respectively. The maximum rate of increase of LV pressure (+dP/dt^max) was obtained by electronic differentiation of the LV pressure waveform. The femoral artery and vein were cannulated for the withdrawal of reference blood flow samples and fluid administration, respectively. A thoracotomy was performed at the left fifth intercostal space. A heparin-filled catheter was inserted into the left atrial appendage for administration of radioactive microspheres. A 1.0-cm segment of the left anterior descending (LAD) coronary artery was dissected immediately distal to the first diagonal branch, and a silk ligature was placed around this vessel for production of coronary artery occlusion and reperfusion. Regional myocardial perfusion was measured in the ischemic (LAD) and normal (left circumflex coronary artery) zones using radioactive microspheres. Myocardial infarct size was determined with triphenyltetrazolium chloride staining at the completion of each experiment as previously described. 4End-tidal concentrations of isoflurane were measured at the tip of the endotracheal tube by an infrared anesthetic gas analyzer. The canine minimum alveolar concentration value of isoflurane used in the present investigation was 1.28%. 14Hemodynamic data were continuously monitored throughout the experiment, recorded on a polygraph, and digitized using a computer interfaced with an analog-to-digital converter.

The experimental design is illustrated in figure 1. Forty-eight hours before each dog was subjected to a 60-min LAD occlusion followed by 3 h of reperfusion, they were randomly assigned to receive an intravenous bolus of vehicle (0.9% saline) or PTX (10 μg/kg; Sigma Chemical, St. Louis, MO). In four separate experimental groups, dogs pretreated with vehicle or PTX were studied in the presence or absence of administration of 1.0 minimum alveolar concentration isoflurane (end-tidal concentration) that was discontinued immediately before the 60-min LAD occlusion. These experiments tested the hypothesis that isoflurane-mediated myocardial protection involves activation of Gⁱproteins. In two additional groups of experiments, vehicle- or PTX-pretreated dogs received intravenous nicorandil (100 μg/kg bolus and 10 μg · kg⁻¹· min⁻¹infusion) initiated 15 min before LAD occlusion and discontinued at the onset of reperfusion. This dose of nicorandil has been previously shown to reduce myocardial infarct size in the absence of systemic hemodynamic effects in dogs. 15These experiments tested the hypothesis that direct activation of K^ATPchannels protects ischemic myocardium independent of Gⁱproteins. At the completion of each experiment, dogs received intravenous injections of acetylcholine (4 and 10 μg/kg), and mean arterial and LV pressure responses were recorded. The latter experiments verified the efficacy of PTX-induced Gⁱ-protein inhibition, as previously described. 9,16 

Statistical analysis of data within and between groups was performed using multiple analysis of variance for repeated measures with post hoc  analysis by the Student t  test with Bonferroni’s correction for multiplicity. Changes within and between groups were considered statistically significant at P < 0.05. All data are expressed as mean ± SEM.

Forty-three dogs were instrumented to obtain 38 successful experiments. Two dogs were excluded because of intractable ventricular fibrillation during LAD occlusion or reperfusion (one control; one PTX plus nicorandil). Two dogs were excluded because transmural coronary collateral blood flow exceeded 0.2 ml · min⁻¹· g⁻¹(one control; one PTX plus isoflurane). One dog (control) was excluded because of the presence of heartworms.

Pertussis toxin pretreatment significantly (P < 0.05) reduced mean arterial and LV systolic pressures at baseline (table 1). No other differences in baseline systemic hemodynamics were observed between experimental groups. Isoflurane decreased heart rate, mean arterial and LV systolic pressures, rate–pressure product, and LV +dP/dt^max. Isoflurane produced similar hemodynamic effects in the presence and absence of PTX pretreatment. Nicorandil caused minimal cardiovascular effects. LAD occlusion increased LV end-diastolic pressure in all experimental groups. Hemodynamics were similar between groups during LAD occlusion and reperfusion.

Transmural myocardial blood flow in the ischemic (LAD) and normal (left circumflex coronary artery) regions is summarized in table 2. There were no intergroup differences in myocardial blood flow before, during, or after LAD occlusion.

The area at risk was similar between groups (control, 40 ± 3%; isoflurane alone, 38 ± 2%; PTX alone, 42 ± 2%; PTX and isoflurane, 42 ± 3%; nicorandil, 45 ± 1%; PTX and nicorandil, 40 ± 2% of the LV). Isoflurane significantly (P < 0.05) reduced myocardial infarct size to 7 ± 2% of the area at risk (fig. 2) compared with control experiments (26 ± 2%). PTX pretreatment abolished the protective effects of isoflurane (21 ± 3%) but had no effect on infarct size when administered alone (31 ± 4%). Nicorandil decreased infarct size independent of PTX (11 ± 1% and 11 ± 2% in the presence and absence of PTX pretreatment, respectively;fig. 2).

Mean arterial pressure responses to acetylcholine are depicted in figures 3 and 4. Acetylcholine (4 and 10 μg/kg) decreased mean arterial pressure in control experiments (65 ± 5% and 62 ± 6% of baseline values, respectively). Pretreatment with PTX significantly attenuated these effects.

Experimental evidence indicates that volatile anesthetics exert protective actions during ischemia and reperfusion by activating K^ATPchannels. 4–7Opening of K^ATPchannels has also been shown to play a pivotal role in mediating the protective effects of ischemic preconditioning (IPC). 17These findings suggest that the intracellular signal transduction pathways responsible for both APC and IPC may be similar. The mechanism by which APC or IPC activates the K^ATPchannel is incompletely understood. It has been proposed that IPC causes activation of adenosine A¹receptors, which are coupled to Gⁱproteins. Activation of PKC 18,19may subsequently phosphorylate and enhance K^ATPchannel opening by decreasing its sensitivity to inhibition by ATP. 20The beneficial effects of IPC are abolished by pharmacologic blockade of A¹receptors, 17Gⁱproteins, 9,21,22and PKC. 23Blockade of both A¹receptors and PKC also attenuates the protective effects of isoflurane to enhance recovery of stunned myocardium 1,2and blocks the protective effects of isoflurane 3,5and halothane 3during experimental myocardial infarction. These findings suggest that Gⁱproteins may also be involved in signal transduction during APC.

The present results indicate that Gⁱproteins are an essential element of isoflurane-induced K^ATPchannel activation. Gⁱ-protein blockade with PTX alone did not alter myocardial infarct size but completely abolished the protective effects of isoflurane independent of the alterations of systemic hemodynamics produced by this volatile agent. In contrast, PTX pretreatment did not block the protective effects of the direct K^ATP-channel agonist nicorandil. These findings indicate that K^ATPchannels remain functionally intact during inhibition of Gⁱproteins, and direct stimulation of these channels by nicorandil, at a site presumably independent of the ATP inhibition site, is capable of producing a cardioprotective effect. Thus, the present findings support the contention that volatile anesthetics may activate K^ATPchannels through second messengers.

The direct effects of volatile anesthetics on the K^ATPchannel in vitro  remain unclear. Using patch-clamp techniques in rabbit ventricular myocytes, Han et al.  24demonstrated that isoflurane directly inhibits K^ATP-channel activity but paradoxically increases the probability of K^ATP-channel opening. The latter action probably occurred through an effect of isoflurane to decrease channel sensitivity to inhibition by ATP. Adenosine has also been shown to enhance K^ATP-channel opening by altering channel sensitivity to ATP, and this effect is mediated by activation of A¹receptors and Gⁱproteins. 25,26Recent evidence indicates that isoflurane does not potentiate K^ATP-channel activity in the presence of adenosine in a cell-free environment 27but increases K^ATP-channel current in whole ventricular myocytes. These data suggest that cellular mechanisms underlying anesthetic-induced activation of K^ATPchannels require the presence of an intracellular second messenger system. The present results support the latter hypothesis because blockade of Gⁱproteins abolished the protective effects of isoflurane but not the actions of the direct K^ATP-channel agonist nicorandil, whose actions are thought to occur at a site distinct from the ATP regulatory site. 10,26 

Pertussis toxin was used in the present investigation to block Gⁱproteins, and the mechanism and duration of action of this toxin have been previously characterized. Endoh et al.  28demonstrated in isolated rat atria that intravenous administration of PTX blocked the Gⁱprotein–mediated negative chronotropic and inotropic effects of the muscarinic cholinergic agonist carbachol. These effects were time-dependent and most pronounced 48 h after administration. Accordingly, several in vitro  22,29,30and in vivo  9,16,21,31studies have used PTX to block Gⁱproteins, and the dose used in the present investigation has been shown to be effective in the dog. 16The efficacy of PTX to block Gⁱproteins was confirmed in the present investigation using an acetylcholine challenge, as previously described. 9,16Intravenous acetylcholine causes pronounced decreases in arterial pressure mediated through activation of Gⁱproteins, and pretreatment with PTX modulates this response. We observed marked attenuation of acetylcholine-induced decreases in arterial pressure in PTX-pretreated dogs, indicating that Gⁱproteins were effectively blocked in the present investigation. PTX pretreatment is well tolerated in a variety of animal species, but decreases in baseline arterial pressure are often observed. 9,31,32In the present investigation, PTX pretreatment also caused slight decreases in baseline mean arterial and LV systolic pressures. However, it is unlikely that these small hemodynamic changes were responsible for the failure of isoflurane to reduce myocardial infarct size in PTX-pretreated dogs.

The present findings must be interpreted within the constraints of several other potential limitations. Isoflurane-induced decreases in heart rate, mean arterial pressure, and myocardial contractility may have caused favorable alterations in myocardial oxygen supply–demand relations and contributed to a reduction in infarct size. However, blockade of Gⁱproteins with PTX completely abolished the protective effect of isoflurane without affecting the hemodynamic actions of this anesthetic agent. Nevertheless, coronary venous oxygen tension was not measured and myocardial oxygen consumption was not directly quantified in the present investigation. Interpretation of the present findings should also be qualified because only a single end-tidal concentration of isoflurane was used. Higher inspired concentrations of isoflurane may have produced reductions of myocardial infarct size via  effects on K^ATPchannels despite pretreatment with PTX. Experiments with nicorandil were completed as positive controls to demonstrate that PTX does not prevent direct K^ATP-channel activation and reductions of myocardial infarct size. Nicorandil possesses nitrate-like characteristics that could contribute to cardioprotection independent of K^ATPchannels. However, Mizumura et al.  33demonstrated that the infarct size–reducing effect of nicorandil is specifically mediated by activation of K^ATPchannels in vivo  and is not blocked by nitric oxide inhibition with methylene blue. Nicorandil has also been shown to activate mitochondrial K^ATPchannels, 34and these channels have been suggested to be critical mediators of ischemic preconditioning. 35The subcellular location (sarcolemmal vs.  mitochondrial) of K^ATPchannels modulated by isoflurane is unknown, but preliminary results with desflurane suggest that mitochondrial K^ATPchannels are also involved in anesthetic-mediated myocardial protection. 7Whether PTX-induced Gⁱ-protein inhibition differentially alters sarcolemmal versus  mitochondrial K^ATPchannel–linked cardioprotective mechanisms is unknown.

In summary, the present results indicate that Gⁱproteins play a critical role in isoflurane-mediated reductions of experimental myocardial infarct size in dogs and support the contention that ischemic preconditioning and volatile anesthetics activate similar signal transduction pathways.

The authors thank Drs. Werner List and Helfried Metzler (Department of Anesthesiology and Intensive Care Medicine, University of Graz, Austria) for their gracious support, and David Schwabe for technical assistance.