INTRODUCTION

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CARDIOVASCULAR COMPLICATIONS are a major cause of morbidity and mortality after surgery in patients with coronary artery disease. Identification of factors associated with increased perioperative risk is the subject of intense investigation. Diabetes mellitus and hyperglycemia are important independent risk factors for the development of perioperative cardiovascular complications in patients undergoing anesthesia and surgery (20, 22, 24, 25). The mechanisms responsible for this increased risk have not been completely elucidated. Ischemic preconditioning is impaired by diabetes (8, 10, 18); however, whether reductions of myocardial infarct size produced by volatile anesthetics are similarly attenuated is unknown. Volatile anesthetic agents, such as isoflurane, have previously been shown to precondition myocardium against infarction through similar signal transduction pathways as ischemic preconditioning, including activation of ATP-regulated potassium (K_ATP) channels (17). Our previous work suggests that an important interaction exists between blood glucose concentration and activation of K_ATP channels (13). Therefore, we tested the hypothesis that isoflurane-induced preconditioning is impaired by chemically induced diabetes in a canine model of experimental myocardial infarction.

	METHODS

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All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional 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" of the American Physiological Society and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996).

General preparation. Implantation of instruments has been previously described in detail (15). Briefly, mongrel dogs of either sex were anesthetized with barbital sodium (200 mg/kg) and pentobarbital sodium (15 mg/kg) and were ventilated with positive pressure with an air and oxygen mixture after tracheal intubation. Arterial blood pH was maintained within a physiological range by adjustment of tidal volume and respiratory rate. End-tidal concentrations of isoflurane were measured at the tip of the endotracheal tube by an infrared anesthetic analyzer that was calibrated with known standards before and during experimentation. Temperature was maintained with a heating blanket. A 7-Fr, dual micromanometer-tipped catheter was inserted into the aorta and left ventricle (LV) for measurement of aortic and LV pressures and the maximum rate of increase of LV pressure (+dP/dt_max). After thoracotomy, heparin-filled catheters were inserted into the left atrial appendage and the right femoral artery for administration of radioactive microspheres and withdrawal of reference blood flow samples, respectively. A catheter was also inserted in the right femoral vein for fluid or drug administration. A 1-cm segment of the left anterior descending coronary artery (LAD) was isolated immediately distal to the first diagonal branch, and a silk ligature was placed around the vessel for production of coronary artery occlusion and reperfusion. Hemodynamics were continuously monitored on a polygraph during experimentation and digitized with the use a computer interfaced with an analog-to-digital converter.

Experimental protocol. Ninety minutes after instrumentation was completed and calibrated, baseline systemic hemodynamics were recorded. Dogs were randomly assigned to receive isoflurane (0, 0.32, or 0.64% end-tidal concentration corresponding to 0, 0.25, or 0.5 minimum alveolar concentration) in the absence or presence of diabetes in six experimental groups. Diabetic dogs received intravenous alloxan (40 mg/kg) and streptozotocin (25 mg/kg) and were studied 3 wk after induction of diabetes as previously described (18). Only dogs with sustained hyperglycemia (blood glucose > 200 mg/dl) without ketosis or renal insufficiency (blood urea nitrogen < 40 mg/dl) were studied. Isoflurane was administered for 30 min and then discontinued for 30 min (washout) before LAD occlusion, a procedure that has been previously shown to precondition myocardium against infarction (17). All dogs were subjected to a 60-min LAD occlusion followed by 3 h of reperfusion. Regional myocardial blood flow was measured at baseline, after 30 min of LAD occlusion, and 1 h after reperfusion. Dogs that developed intractable ventricular fibrillation and those with a subendocardial coronary collateral blood flow >0.15 ml · min¹ · g¹ were excluded from the analysis (9).

Measurement of myocardial infarct size. At the end of each experiment, myocardial infarct size was measured as previously described (31). Briefly, the LV area at risk for infarction (AAR) was separated from the normal area, and the two regions were incubated at 37°C for 20-30 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 overnight storage in 10% formaldehyde. Infarct size was expressed as a percentage of the AAR.

Determination of regional myocardial blood flow. Carbonized plastic microspheres [15 ± 2 (SD) µm in diameter] labeled with ¹⁴¹Ce, ¹⁰³Ru, or ⁹⁵Nb were used to measure regional myocardial perfusion as previously described (15). Transmural tissue samples were selected from the ischemic region (distal to the LAD occlusion) and were subdivided into subepicardial, midmyocardial, and subendocardial layers of approximately equal thickness. Samples were weighed and placed in scintillation vials, and the activity of each isotope was determined. Similarly, the activity of each isotope in the reference blood flow sample was assessed. Tissue blood flow (ml · min¹ · g¹⁾ was calculated as Q_r · C_m · C_r¹, where Q_r is the rate of withdrawal of the reference blood flow sample (ml/min), C_m is the activity (cpm/g) of the myocardial tissue sample, and C_r is the activity (cpm) of the reference blood flow sample. Transmural blood flow was considered as the average of subepicardial, midmyocardial, and subendocardial blood flows.

Statistical analysis. Statistical analysis of data within and between groups was performed with analysis of variance (ANOVA) for repeated measures followed by Student-Newman-Keuls test. The relationship between blood glucose concentration or transmural collateral blood flow and myocardial infarct size was evaluated with analysis of covariance. Changes within and between groups were considered statistically significant when the P value was <0.05. All data are expressed as means ± SE.

	RESULTS

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Fifty dogs were instrumented to obtain 45 successful experiments. Four dogs were excluded because subendocardial collateral blood flow exceeded 0.15 ml · min¹ · g¹ (1 in the 0.32% isoflurane group, 1 in the 0.64% isoflurane group, and 2 in the 0.32% isoflurane + diabetes group). One diabetic dog receiving 0.64% isoflurane was excluded because of intractable ventricular fibrillation during LAD occlusion. Blood glucose concentrations were significantly increased at baseline and throughout experimentation in all dogs with diabetes (Table 1).

Systemic hemodynamics. There were no differences in hemodynamics between experimental groups after instrumentation was completed (baseline data in Table 2). Isoflurane caused dose-dependent decreases in heart rate, mean arterial and LV systolic pressures, and LV +dP/dt_max in the absence or presence of diabetes. Hemodynamics returned to baseline values 30 min after isoflurane was discontinued (preocclusion data in Table 2). LAD occlusion and reperfusion produced increases in LV end-diastolic pressure and decreases in LV +dP/dt_max in each experimental group.

Myocardial infarct size. The LV AAR was similar among groups (control, 43 ± 2%; diabetes alone, 43 ± 2%; 0.32% isoflurane, 37 ± 3%; 0.64% isoflurane, 44 ± 2%; 0.32% isoflurane + diabetes, 38 ± 3%; 0.64% isoflurane + diabetes, 37 ± 3%). Myocardial infarct size expressed as a percentage of the AAR was 29 ± 3% (n = 8 dogs) in control dogs. Isoflurane (0.32 and 0.64% end-tidal concentration) reduced infarct size to 15 ± 2 (n = 8) and 13 ± 1% (n = 7) of the AAR, respectively (Fig. 1). Diabetes alone (n = 8) did not alter myocardial infarct size (30 ± 3%) but blocked the protective effect of 0.32% isoflurane (27 ± 2%; n = 7). In contrast, diabetes did not affect the reduction of infarct size produced by 0.64% isoflurane (18 ± 3%; n = 7).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Histograms illustrating myocardial infarct size expressed as a percentage of the left ventricular area at risk in dogs receiving 0, 0.32, or 0.64% end-tidal concentrations of isoflurane (Iso) in the absence (Con) or presence of diabetes (Diab). * Significantly (P < 0.05) different from control experiments in the absence of Iso.  Significantly (P < 0.05) different from experiments during diabetes in the absence of Iso. § Significantly (P < 0.05) different from experiments during diabetes and 0.32% Iso.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Relationship between myocardial infarct size expressed as a percentage of the left ventricular area at risk and transmural collateral blood flow in control experiments and during 0.32 and 0.64% Iso, respectively. Myocardial infarct size was inversely related (y = 140.5x + 37.9; r = 0.82; P < 0.05) to collateral blood flow in control experiments. Iso produced similar protection against infarction at 0.32 and 0.64% end-tidal concentrations, and these data were pooled for regression analysis. Iso significantly shifted the regression relationship downward (y = 45.1x + 17.5; r = 0.44; P < 0.001) compared with control experiments, indicating myocardial protection. There were no differences in the regression relationships between 0.32 and 0.64% Iso concentrations (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Relationship between myocardial infarct size expressed as a percentage of the left ventricular area at risk and transmural collateral blood flow in diabetic dogs (Diab) in the absence and presence of Iso. There was no relationship (P > 0.05) between infarct size and collateral blood flow in diabetic animals.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Relationship between myocardial infarct size expressed as a percentage of the left ventricular area at risk and blood glucose concentration in Diab dogs in the absence or presence of Iso. Iso (0.32%) did not protect against infarction in Diab animals, and therefore, data from unprotected experiments (Diab and Diab + 0.32% Iso) were pooled. Blood glucose concentration was directly related to myocardial infarct size (y = 0.05x + 15.1; r = 0.67; P < 0.01) in Diab animals in the absence or presence of 0.32% Iso. In contrast, higher concentrations of Iso (0.64%) abolished this relationship (y = 0.0002x + 17.6; r = 0.0015; P > 0.05) and shifted the regression relationship downward compared with the unprotected groups (P < 0.01).

	DISCUSSION

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Volatile anesthetics were first demonstrated to enhance recovery of stunned myocardium (30) over a decade ago, but the mechanisms responsible for this protection against ischemic injury were unknown. Observations that halothane (21) and isoflurane (2) produced coronary vasodilation through activation of K_ATP channels suggested that volatile anesthetics may produce protective effects by activating similar signal transduction pathways as those implicated in ischemic preconditioning. Subsequently, we demonstrated that isoflurane-induced improvements in the functional recovery of stunned myocardium were blocked by the nonselective K_ATP channel antagonist glyburide (12, 15). Isoflurane was also shown to precondition myocardium against infarction with an acute memory period. Reductions of myocardial infarct size produced by isoflurane exposure were comparable in size to that of ischemic preconditioning (17). The myocardial protective pathways activated by halothane, isoflurane, desflurane, and sevoflurane are also remarkably similar to ischemic preconditioning (3) and include signaling through A₁ adenosine receptors (4, 11, 14, 27), inhibitory guanine regulatory (G_i) proteins (29), protein kinase C (4, 29), and sarcolemmal and mitochondrial K_ATP channels (11, 17, 26-28).

The current results demonstrate that isoflurane-induced preconditioning is impaired by diabetes and, furthermore, that anesthetic and blood glucose concentrations are interactive determinants of myocardial infarct size in a canine model of diabetes. The findings extend our previous work indicating that both chemically induced diabetes (18) and acute hyperglycemia produced by intravenous administration of dextrose (16) abolish the protection produced by ischemic preconditioning or direct activation of mitochondrial K_ATP channels with diazoxide (13). Diazoxide (2.5 and 5.0 mg/kg) caused similar reductions of infarct size (10 ± 1 and 11 ± 2%, respectively) in our previous study compared with isoflurane (15 ± 2 and 13 ± 1% at 0.32 and 0.64% concentrations, respectively) in the current investigation. Moderate hyperglycemia (blood glucose concentration of ~250-300 mg/dl) abolished the protection afforded by the low doses of diazoxide, but severe hyperglycemia (>500 mg/dl) was required to attenuate the beneficial actions of the higher dose of the mitochondrial K_ATP channel opener. A direct relationship between blood glucose concentration and myocardial infarct size was observed in the present and previous investigations during diabetes and acute hyperglycemia, and during administration of diazoxide (13) with or without ischemic preconditioning (18). In contrast, blood glucose concentration and myocardial infarct size were unrelated during experiments in which higher concentrations of isoflurane (0.64%) were used. Blood glucose concentrations were only moderately increased by diabetes (300 mg/dl) and did not exceed 450 mg/dl in any dog receiving isoflurane. Thus it remains possible that a higher blood glucose concentration may be required to attenuate the protective effects of isoflurane during administration of end-tidal concentrations >0.5%. Nonetheless, the findings suggest that volatile anesthetics and glucose may exert opposing actions on K_ATP channel-mediated modulation of myocardial injury in vivo.

The present findings may clarify recent clinical evidence that hyperglycemia is a substantial contributor and predictor of increased short- and long-term cardiovascular mortality (5-7, 23). Diabetes mellitus adversely affects the prognosis of patients with acute myocardial infarction (1), and the degree of metabolic control may determine the outcome after myocardial infarction in these patients (19). A strong correlation between blood glucose concentration at the time of hospital admission and long-term mortality was observed in a study of diabetic patients with acute myocardial infarction (23). A metaregression analysis of data published in 20 studies of more than 95,000 patients demonstrated a relation between fasting blood glucose concentration and the relative risk of sustaining a cardiovascular event (5). Hyperglycemia was also identified as an important predictor of short-term mortality after coronary artery bypass grafting (6). Taken together, these clinical and experimental findings suggest that hyperglycemia may increase cardiovascular risk in part by impairing endogenous (i.e., ischemic preconditioning) or exogenous (i.e., pharmacological) activation of K_ATP channels. Moreover, hyperglycemia-induced impairment of preconditioning associated with administration of volatile anesthetics may represent a mechanism that accounts for increased cardiovascular risk in diabetic patients undergoing anesthesia and surgery.

The results of the current investigation should be interpreted within the constraints of several potential limitations. The LV AAR for infarction and coronary collateral blood flow are important determinants of the extent of myocardial infarction. However, there were no differences in these variables among experimental groups that may account for the current findings. An inverse relationship between myocardial infarct size and coronary collateral blood flow was observed during control conditions, and this relationship was shifted downward by isoflurane indicating direct myocardial protection. In contrast, coronary collateral blood flow and infarct size were unrelated in diabetic dogs. Increases in blood glucose concentration were observed to adversely modulate ischemic injury independent of the extent of coronary collateral blood flow. It is unlikely that the hemodynamic effects of isoflurane or diabetes were responsible for the differences in infarct size observed in this investigation. Diabetes alone produced no hemodynamic effects, and isoflurane caused similar hemodynamic actions in the presence or absence of diabetes. Hemodynamics returned to baseline values 30 min after the anesthetic was discontinued, and there were no hemodynamic differences between groups before coronary artery occlusion. Nevertheless, myocardial oxygen consumption was not specifically measured, and changes in myocardial metabolism during the administration of isoflurane in the presence or absence of diabetes cannot be completely excluded from the analysis. Elevations in plasma osmolality occur concomitant with increases in blood glucose concentration during diabetes. However, we have previously demonstrated that increases in plasma osmolality produced by the administration of the nonmetabolizable sugar raffinose do not increase, but instead, decrease the size of infarction (18). Thus changes in plasma osmolality are unlikely to account for the results of the current investigation. Decreases in plasma insulin concentration produced by alloxan and streptozotocin were probably not responsible for increasing myocardial infarct size during hyperglycemia. We (18) have previously demonstrated that acute hyperglycemia with concomitant hyperinsulinemia causes similar increases in infarct size that are directly related to blood glucose concentration and are independent of plasma insulin concentrations.

In conclusion, the current results indicate that diabetes attenuates isoflurane-induced preconditioning. The results further demonstrate that the extent of infarction is dependent both on blood glucose and volatile anesthetic concentrations. The findings are consistent with the interpretation that volatile agents stimulate but glucose inhibits K_ATP channel activity in vivo. The results suggest that the perioperative management of blood glucose concentration in diabetic patients may influence cardiovascular risk during anesthesia and surgery.