Ischemic preconditioning provides a powerful means to reduce myocardial infarct size in vivo and has been proposed to limit the extent of myocardial infarction in patients. In contrast, hyperglycemia correlates with increases in mortality after acute myocardial infarction. Thus we hypothesized that acute hyperglycemia alters the protection afforded by ischemic preconditioning, and this hypothesis was tested in acutely instrumented dogs subjected to a prolonged (60 min) coronary artery occlusion and 3 h of reperfusion. Ischemic preconditioning was elicited by four 5-min occlusion-reperfusion periods in the presence or absence of an intravenous infusion of 15% dextrose in water to produce acute hyperglycemia (plasma glucose concentration of 300 mg/dl). The dose-dependent effects of hyperglycemia on myocardial infarct size independent of preconditioning stimuli were further evaluated in dogs subjected to increases in plasma glucose concentrations to either 300 or 600 mg/dl. Infarct size (triphenyltetrazolium staining) was 24 ± 2% of the area at risk in control dogs and was significantly (P < 0.05) decreased by ischemic preconditioning (8 ± 1%). Modest degrees of hyperglycemia (300 mg/dl) had no effect on infarct size (34 ± 4%) but abolished the protective effect of ischemic preconditioning (30 ± 5%). In contrast, profound hyperglycemia (600 mg/dl) increased infarct size (44 ± 6%). Hemodynamics and coronary collateral blood flow (radioactive microspheres) were similar between groups. Thus acute hyperglycemia adversely modulates myocardial injury in response to ischemia in vivo.
- myocardial infarction
- infarct size
- prolonged coronary occlusion
ischemic preconditioning powerfully limits myocardial infarct size in vivo (18) and may play an important role to reduce the extent of myocardial infarction in patients with a history of angina (14, 19). In contrast, hyperglycemia has been correlated with increases in mortality after acute myocardial infarction in patients with diabetes (10, 15, 16) and those with “asymptomatic” hyperglycemia (2, 10). Recent evidence indicates that diabetes impairs ATP-sensitive potassium (KATP) channel function of human arterioles (17), and KATPchannels are an important mediator of ischemic preconditioning (24). However, it is unknown if hyperglycemia contributes to increases in mortality during myocardial infarction through effects to adversely modify the protection afforded by ischemic preconditioning. We tested the hypotheses that acute hyperglycemia abolishes ischemic preconditioning-induced myocardial protection and increases myocardial infarct size in a dose-related fashion in acutely instrumented, barbiturate-anesthetized dogs.
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 conformed to the “Guiding Principles in the Care and Use of Animals” of the American Physiological Society and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Revised, 1996).
General Preparation. Implantation of instruments has been previously described (12). Briefly, mongrel dogs were anesthetized with barbital sodium (200 mg/kg) and pentobarbital sodium (15 mg/kg) and ventilated with an air and oxygen mixture after tracheal intubation. Arterial blood gases were maintained within a physiological range. Dogs were instrumented for measurement of arterial and left ventricular pressures and the maximum rate of increase of left ventricular pressure (dP/dt max). A silk ligature was placed around the left anterior descending coronary artery (LAD) immediately distal to the first diagonal branch for production of occlusion and reperfusion. Hemodynamics were continuously monitored on a polygraph and digitized using a computer interfaced with an analog-to-digital converter.
Experimental protocol. The experimental design is illustrated in Fig.1. Baseline hemodynamics were recorded 90 min after instrumentation was completed. Dogs were randomly assigned to one of five groups. All dogs were subjected to a 60-min LAD occlusion period followed by 3 h of reperfusion. Control experiments (n = 13) had no intervention before prolonged LAD occlusion and reperfusion. A second group of dogs (n = 13) underwent ischemic preconditioning (PC) with four 5-min LAD occlusions interspersed with 5-min reperfusion periods, followed by prolonged (60 min) LAD occlusion and reperfusion. This preconditioning protocol has previously been shown to cause marked reductions in myocardial infarct size (18). The effects of hyperglycemia to abolish the protection afforded by preconditioning were evaluated in dogs receiving intravenous infusion of 15% dextrose in water to produce acute hyperglycemia (plasma glucose = 300 mg/dl) for a period of 30 min before and during preconditioning coronary occlusions (H300 + PC,n = 15). The plasma glucose concentration was allowed to return to baseline values after the onset of prolonged LAD occlusion. The dose-dependent effects of hyperglycemia on myocardial infarct size were further evaluated in dogs receiving intravenous dextrose to produce acute hyperglycemia [plasma glucose concentration = 300 (H300,n = 14) or 600 (H600,n = 11) mg/dl] before prolonged LAD occlusion. Normoglycemic dogs received an equivalent volume of normal saline. Plasma glucose (Tracer II glucometer), potassium, and insulin concentrations (Marshfield Laboratories) were measured at selected intervals. Regional myocardial blood flow was measured with radioactive microspheres at baseline, 30 min after the onset of prolonged LAD occlusion, and 1 h after final reperfusion. Dogs that developed intractable ventricular fibrillation and those with a subendocardial coronary collateral blood flow ≥0.15 ml ⋅ min−1 ⋅ g−1were excluded from data analysis (8, 18).
At the conclusion of each experiment, the LAD was reoccluded and cannulated just distal to the occlusion site (12). Ten milliliters each of saline and Patent blue dye were injected at equal pressure into the LAD and left atrium to delineate the anatomic area at risk (AAR) and the nonischemic zone, respectively. The heart was fibrillated, removed, and sliced into serial traverse sections 6–7 mm in width. The unstained AAR was separated from the normal region, and the two regions were incubated for 20 min at 37°C 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 separated and weighed after being stored overnight in 10% formaldehyde. Infarct size was expressed as a percentage of the AAR.
Statistical analysis. Statistical analysis of data within and between groups was performed with analysis of variance (ANOVA) for repeated measures followed by application of Student’s t-test with Duncan’s correction. Linear regression analysis was also performed to determine the relationship between transmural collateral blood flow and infarct size expressed as a percentage of AAR. Changes within and between groups were considered statistically significant whenP < 0.05. Data are expressed as means ± SE.
Sixty-six dogs were instrumented to obtain 45 successful experiments. Eighteen dogs were eliminated from the analysis because subendocardial collateral blood flow was >0.15 ml ⋅ min−1 ⋅ g−1(2 control, 4 PC, 4 H300, 3 H600, and 5 H300 + PC). Three dogs were excluded because of intractable ventricular fibrillation (2 control and 1 H300 + PC).
Systemic hemodynamics. There were no differences in baseline hemodynamics between groups (Table1). Acute hyperglycemia produced a transient increase in heart rate, mean arterial pressure, and dP/dt max. Increases in heart rate and decreases in dP/dt max occurred during reperfusion in all groups. There were no hemodynamic differences between groups during prolonged LAD occlusion or reperfusion.
Glucose, insulin, and potassium.Intravenous administration of dextrose caused transient hyperglycemia when the plasma glucose concentration was increased to the targeted value of 300 mg/dl, and the plasma glucose concentration remained elevated during LAD occlusion when the target value of 600 mg/dl was achieved (Table 2). Acute hyperglycemia was also accompanied by increases in plasma insulin concentration. Potassium concentration decreased after 3 h of reperfusion in preconditioned, hyperglycemic dogs.
Infarct size and coronary collateral blood flow. Myocardial infarct size was 24 ± 2% of the AAR in control dogs. Ischemic preconditioning significantly (P < 0.05) decreased infarct size to 8 ± 1% AAR (Figs. 2 and3). Moderate hyperglycemia (H300) alone had no effect on infarct size (34 ± 4%); however, it abolished the protective effect of ischemic preconditioning (30 ± 5%). Marked hyperglycemia (H600) increased infarct size (44 ± 6%) compared with control experiments. These findings were observed in the absence of differences in AAR (control: 34 ± 1; PC: 36 ± 2; H300: 35 ± 1; H600: 34 ± 1; and H300 + PC: 34 ± 2% of left ventricular mass) or transmural coronary collateral blood flow (Table3) among groups.
It has been suggested that glucose is a potentially toxic molecule that may have deleterious effects if its concentration is not closely regulated (21). Hyperglycemia impairs coronary microcirculatory responses to ischemia (11), reduces availability of nitric oxide (6), attenuates endothelium-dependent vascular responses (3, 20), and enhances oxygen-derived free radical production (3). Despite evidence of hyperglycemia-induced vascular dysfunction, whether hyperglycemia alters the extent of myocardial injury during ischemia is controversial, and interactions with ischemic preconditioning are unknown. Ischemic preconditioning has been proposed to limit the extent of myocardial infarction in patients with new onset prodromal angina (19) or a previous history of angina (14). In contrast, hyperglycemia has been shown to correlate with an increase in mortality after acute myocardial infarction in both diabetic (15, 16) and nondiabetic (2) patients. An adverse interaction between hyperglycemia and ischemic preconditioning has not been demonstrated and may represent a clinically important phenomenon.
The results demonstrate that myocardial infarct size was unchanged when the plasma glucose concentration was increased to 296 ± 9 mg/dl, but this clinically relevant degree of hyperglycemia completely prevented reductions of myocardial infarct size produced by ischemic preconditioning. Activation of KATP channels has been shown to mediate the effects of ischemic preconditioning in vivo (8), and recent evidence indicates that diabetes mellitus impairs KATP channel function of human arterioles in vitro (17). Impairment of myocardial KATP channel function may be a potential mechanism that accounts for the findings that hyperglycemia abolishes reductions of myocardial infarct size afforded by ischemic preconditioning. However, we previously reported that epicardial microvessels demonstrated enhanced dilation in response to the KATP channel agonist, aprikalim, in the presence of hyperglycemia (11). In contrast, the protection afforded by ischemic preconditioning was abolished by hyperglycemia. Whereas the findings of our previous study using aprikalim appear to conflict with those of the current investigation, an important difference between these two investigations exists. In our previous work, KATP channels were directly activated by aprikalim, whereas in the current investigation endogenous opening of KATP channels during ischemic preconditioning was evaluated. Ischemic preconditioning occurs via activation of a cardioprotective signal transduction mechanism, which includes activation of adenosine receptors that are coupled to KATP channels by inhibitory G (Gi) proteins (1, 13). Thus, whereas KATP channels may be directly activated by pharmacological agonists in the absence of ischemia, endogenous activation of these channels during preconditioning stimuli may be impaired by hyperglycemia. This contention is supported by evidence that coupling of adenosine receptors to Gi proteins is impaired in hyperglycemic (streptozocin-induced diabetic) rats (7). Hyperglycemia may also have differential effects on KATP channels in cardiac compared with vascular myocytes. Different sulfonylurea receptor subunits are present in KATP channels in heart and vascular smooth muscle, and the presence of different sulfonylurea receptor types appears to confer differential pharmacological properties on KATP channels in different tissues (4, 9). Activation of KATP channels is also potentiated by nitric oxide (23), and acute hyperglycemia has been shown to impair nitric oxide availability (6). Shortening of action potential duration may contribute to the cardioprotective effects of ischemic preconditioning (25), and recent evidence in ventricular myocytes suggests that acute hyperglycemia prolongs action potential duration and slows intracellular calcium clearing (22). The role of hyperglycemia to modulate interactions among adenosine receptors, Gi proteins, nitric oxide, and KATP channels and/or to alter action potential duration in the presence or absence of ischemic preconditioning requires further investigation.
The present findings must be interpreted within the constraints of several possible limitations. Hyperglycemia increased the extent of myocardial infarction when plasma glucose was increased to 585 ± 6 mg/dl. Because this degree of hyperglycemia increased infarct size alone, the effects of hyperglycemia to block preconditioning were not performed during higher (≈600 mg/dl) plasma glucose concentrations. Coronary collateral blood flow and area of the left ventricle at risk for infarction were similar among groups. Thus differences in myocardial infarct size were observed independently of changes in collateral blood flow or area at risk. Hyperglycemia caused transient hemodynamic effects that may, in part, account for the results. However, modest degrees of hyperglycemia caused no hemodynamic effects during coronary artery occlusion, and hemodynamics were not different in preconditioned dogs in the presence versus the absence of hyperglycemia. Finally, a deleterious effect of hyperinsulinemia on infarct size cannot be excluded; however, experimental diabetes in dogs increases myocardial infarct size in the absence of hyperinsulinemia (5).
In conclusion, the results demonstrate the effects of hyperglycemia to abolish the protective effects of ischemic preconditioning. The mechanism(s) responsible for these adverse effects of hyperglycemia remains to be investigated further.
The authors thank David Schwabe for technical assistance and Angela Barnes for assistance in preparation of this manuscript.
Address for reprint requests: J. R. Kersten, Dept. of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-03690 (to J. R. Kersten) and HL-54280 (to D. C. Warltier), by an American Diabetes Association Research Award (to J. R. Kersten), and by an Anesthesia Research Training Grant GM-08377 (to D. C. Warltier).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 1998 the American Physiological Society