INTRODUCTION

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CORONARY ARTERY DISEASE accounts for nearly 50% of all deaths in patients with diabetes mellitus (13). This striking cardiovascular mortality in diabetic patients cannot be explained by conventional risk factors (23). Recently, the relative role of hyperglycemia in the macrovascular complications of diabetes has been intensely scrutinized. Some previous data suggested that hyperglycemia alone does not play a substantial role in the development of cardiovascular disease in diabetic patients, but more recent evidence disputes this contention and indicates that hyperglycemia and poor glycemic control increase cardiovascular risk without an apparent threshold of blood glucose concentration (3, 20).

Acute hyperglycemia and diabetes both impair coronary microcirculatory responses to ischemia in vivo (14) and attenuate endothelium-dependent vascular responses in vitro (19). We have recently shown that acute hyperglycemia also abolishes the protection afforded by ischemic preconditioning in a canine model (16). However, the nature of the relationship between myocardial infarct size and blood glucose concentration during acute hyperglycemia or diabetes is controversial (21, 25) and has been incompletely evaluated. Thus we tested the hypothesis that the extent of myocardial infarction is directly related to blood glucose concentration in diabetic and hyperglycemic dogs, independent of serum osmolality, insulin concentration, coronary collateral blood flow, or hemodynamics.

	METHODS

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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 (7th ed. Washington, DC: Nat. Acad. Press, 1996).

Chemical induction of diabetes. Dogs were fasted for 24 h before induction of diabetes. Alloxan (40 mg/kg) and streptozotocin (25 mg/kg) (1, 6) were dissolved in citrate buffer and injected intravenously over 1 min. Dogs were subsequently studied 3 wk after induction of diabetes, which is beyond a period of acute renal toxicity that may be associated with administration of alloxan (4). Only dogs with sustained hyperglycemia (blood glucose > 200 mg/dl), without ketosis or renal insufficiency (blood urea nitrogen < 40 mg/dl), were studied.

General preparation. Mongrel dogs (18-22 kg) were anesthetized with barbital sodium (200 mg/kg) and pentobarbital sodium (15 mg/kg), intubated, and ventilated with room air (supplemented with oxygen), using positive pressure ventilation. Tidal volume and respiratory rate were adjusted to maintain arterial carbon dioxide tension and acid-base status in the normal range. The oxygen flow rate was adjusted in each experiment to maintain arterial blood oxygen tension near values demonstrated in conscious dogs. A double-pressure transducer-tipped catheter (PC771, Millar) was inserted into the aorta and left ventricle (LV) via the left carotid artery to measure arterial and LV pressures, respectively, and the maximum rate of increase of LV pressure (dP/dt_max). The right femoral vein and artery were catheterized for administration of fluid or supplemental anesthetics and for withdrawal of reference arterial blood samples, respectively. A thoracotomy was performed in the left fifth intercostal space, the lung gently retracted, and the heart suspended in a pericardial cradle. A 1.5- to 2-cm segment of the proximal left anterior descending coronary artery (LAD), distal to the first diagonal branch, was isolated and a silk ligature placed around the vessel to produce coronary artery occlusion and reperfusion. A catheter was placed in the left atrium for injection of radioactive microspheres. All hemodynamic data were monitored continuously on a polygraph and digitized with a computer interfaced with an analog-to-digital converter.

Measurement of myocardial infarct size. At the conclusion of each experiment, the LAD was reoccluded and cannulated at the occlusion site (17). Ten milliliters each of saline and patent blue dye were injected at equal pressure into the LAD and the left atrium to delineate the anatomic area at risk (AAR) and the normal zone, respectively. The heart was fibrillated, removed, and sliced into serial 6- to 7-mm-wide transverse sections. The unstained AAR was separated from the normal area, 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 (IF) was expressed as a percentage of the AAR.

Measurement of regional myocardial perfusion. Carbonized plastic microspheres [15 ± 2 µm (SD) diameter] labeled with ⁹⁵Nb, ¹⁴¹Ce, and ¹⁰³Ru were used to measure myocardial perfusion as previously described (15). Briefly, microspheres were administered into the left atrium as a bolus and flushed in with 10 ml of warm (37°C) saline. A few seconds before injection, a timed collection of reference arterial flow was started from the femoral arterial catheter at a rate of 7 ml/min for 3 min. Transmural tissue samples were selected from the ischemic region and 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 _r · C_m · C_r¹, where _r is rate of withdrawal of the reference blood flow sample (ml/min), C_m is activity (cpm/g) of the myocardial tissue sample, and C_r is activity (cpm) of the reference blood flow sample. Transmural blood flow was considered to be the average of the subepicardial, midmyocardial, and subendocardial blood flows.

Experimental protocol. The experimental design is illustrated in Fig. 1. Ninety minutes after instrumentation was completed and calibrated, baseline systemic hemodynamics were recorded. All dogs were subjected to a prolonged 60-min LAD occlusion followed by 3 h of reperfusion. Control experiments had no previous intervention before prolonged (60 min) LAD occlusion and reperfusion. Ischemic preconditioning (PC) consisted of four 5-min LAD occlusions interspersed with 5-min periods of reperfusion, followed by prolonged LAD occlusion and reperfusion (24). The actions of chemically induced diabetes to alter the extent of myocardial infarction in the absence or presence of ischemic preconditioning stimuli were investigated in two additional groups. The effects of acute hyperglycemia on infarct size were evaluated in a fifth group of dogs by infusing 15% dextrose in water to increase blood glucose to between 100 and 600 mg/dl for a period of 70 min before prolonged coronary artery occlusion. Blood glucose was then allowed to return to baseline values throughout the remainder of the experimental protocol. Finally, the effect of hyperosmolality on infarct size with and without ischemic preconditioning stimuli was investigated in dogs receiving intravenous raffinose (300 g), a nonmetabolizable sugar infused over 70 min before prolonged coronary artery occlusion, in two final groups of experiments. Dogs that developed intractable ventricular fibrillation during LAD occlusion or reperfusion and those with subendocardial coronary collateral blood flow 0.15 ml · min¹ · g¹ were excluded from data analysis (10, 24).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Schematic diagram illustrating experimental protocol.

Statistical analysis. Statistical analysis of hemodynamic and perfusion data was performed with a two-way ANOVA for repeated measures followed by application of Student-Newman-Keuls test. Myocardial infarct size was evaluated with one-way ANOVA. The relationship between blood glucose or transmural collateral blood flow and infarct size was evaluated with multiple regression analysis. Changes within and between groups were considered statistically significant when P < 0.05. Data are expressed as means ± SE.

	RESULTS

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Sixty-three dogs were instrumented to obtain forty-eight successful experiments. Two dogs were excluded because of intractable ventricular fibrillation (1 each in the diabetic and raffinose + PC groups). Eleven dogs were excluded because subendocardial blood flow exceeded 0.15 ml · min¹ · g¹ (3 each in the diabetic and diabetic + PC groups; 2 each in the control and PC alone groups; 1 in the hyperglycemic group). Two additional dogs were excluded because of heartworms (1 each in the control and hyperglycemic groups). Chemically induced diabetes caused chronic sustained hyperglycemia (blood glucose concentration: 274 ± 30 mg/dl) and hypoinsulinemia (serum insulin concentration: 2.2 ± 0.4 µU/ml).

Hemodynamics during LAD occlusion and reperfusion. There were no statistically significant differences in hemodynamics between groups under baseline conditions, before LAD occlusion, and during occlusion or reperfusion (Table 1). Heart rate and mean arterial pressure were unchanged during the experimental protocol in all experimental groups. LV dP/dt_max transiently increased in hyperglycemic dogs and those receiving raffinose but decreased after brief, repetitive occlusions in dogs subjected to PC stimuli. LV dP/dt_max was significantly reduced during reperfusion in all experimental groups.

Myocardial infarct size. The LV AAR for infarction was similar between groups (control, 42 ± 3; PC, 40 ± 3; diabetic, 42 ± 1; diabetic + PC, 39 ± 3; hyperglycemic, 40 ± 3; raffinose, 40 ± 2; raffinose + PC, 37 ± 2%). Myocardial infarct size (Fig. 2) was 24 ± 2% of the AAR in control experiments (n = 8) and was significantly (P < 0.05) reduced by PC (n = 7) to 8 ± 2%. Infarct size was similar in diabetic (n = 8; 28 ± 3%) compared with normal dogs; however, PC did not protect against infarction in diabetic dogs (n = 8; 22 ± 4%). There was an inverse relationship between infarct size and transmural collateral blood flow (P < 0.02) in normal (Fig. 3A) but not in diabetic dogs (Fig. 3B). Infarct size was linearly related to blood glucose concentration (Fig. 4) in diabetic (P < 0.002) and acutely hyperglycemic (n = 9; P < 0.001) dogs.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Histograms illustrating myocardial infarct size expressed as a percentage of the area at risk in control (CON), preconditioned (PC), and diabetic (DIAB) dogs. *Significantly (P < 0.05) different from CON; significantly (P < 0.05) different from DIAB + PC.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Relationship between myocardial infarct size and transmural collateral blood flow is depicted in control (A: without and with PC) and diabetic (B) experiments. Infarct size was inversely related to collateral blood flow in control dogs (P < 0.02), but there was no relationship between these variables in preconditioned or diabetic dogs (P = NS) with or without PC stimuli. In normoglycemic dogs (control + PC), PC significantly (P < 0.05) shifted regression line downward compared with control or diabetic dogs.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Relationship between myocardial infarct size and glucose concentration (measured immediately before coronary artery occlusion) is shown in diabetic (A) and acutely hyperglycemic (B) dogs. Infarct size was linearly related (P < 0.002) to blood glucose concentration in diabetic dogs (in presence or absence of PC stimuli) and in acutely hyperglycemic (P < 0.001) dogs. There were no differences in slope or intercept of the relation between groups.

Hyperosmolality and infarction. Intravenous raffinose increased plasma osmolality from 303 ± 3 at baseline to 328 ± 3 mosmol/kg immediately before prolonged coronary artery occlusion. Raffinose-induced increases in plasma osmolality were sustained throughout the remainder of the experiment (325 ± 5 mosmol/kg at 3 h of reperfusion). Profound acute hyperglycemia (blood glucose  500-600 mg/dl) caused similar sustained increases in plasma osmolality from 302 ± 3 mosmol/kg at baseline to 318 ± 5 and 316 ± 11 mosmol/kg during hyperglycemia and after 3 h of reperfusion, respectively. Moderate (blood glucose  300 mg/dl) hyperglycemia did not increase plasma osmolality (301 ± 3 at baseline to 295 ± 7 and 296 ± 2 mosmol/kg during hyperglycemia and after 3 h of reperfusion, respectively), and plasma osmolality was similar (300 ± 7 mosmol/kg) in diabetic dogs. In contrast to results observed during diabetes and acute hyperglycemia, raffinose (Fig. 5) decreased infarct size (n = 4; 11 ± 3%) and enhanced PC-induced myocardial protection (n = 4; 2 ± 1%).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Histograms illustrating myocardial infarct size expressed as a percentage of area at risk in control (CON), PC, and those dogs receiving raffinose (RAFF). *Significantly (P < 0.05) different from CON; significantly (P < 0.05) different from RAFF; §significantly (P < 0.05) different from PC alone.

Myocardial perfusion. Transmural myocardial blood flow in the ischemic (LAD) region is summarized in Table 2. There were no significant differences in baseline tissue perfusion or coronary collateral blood flow between groups. Reperfusion blood flow was decreased in dogs receiving raffinose + PC.

	DISCUSSION

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The role of hyperglycemia in acute myocardial infarction has been unappreciated until recently. Previous data indicated that tight control of blood glucose concentration reduced the microvascular consequences of diabetes, but the relative risk of associated irreversible ischemic injury with acute or chronic increases in glucose levels was thought to be unrelated to the degree of hyperglycemia (12). New evidence convincingly demonstrates that hyperglycemia cannot be ignored as an important risk factor for the development of cardiovascular disease and the occurrence of myocardial infarction (20). In a case control study of 600 subjects, the odds ratio for developing myocardial infarction was increased sixfold when fasting glucose levels exceeded 114 mg/dl compared with findings when fasting glucose levels were less than 80 mg/dl. Remarkably, increases in fasting blood glucose also predicted an increased risk of myocardial infarction in subjects without diabetes or impaired glucose tolerance (7). Using a meta-analysis of data collected from over 95,000 subjects, Coutinho et al. (3) demonstrated that the risk of adverse cardiovascular events increased in individuals with abnormally elevated fasting or 2-h postprandial glucose concentrations. Intensive glycemic control in patients with newly diagnosed type 2 diabetes mellitus also reduced the risk of myocardial infarction by 16% (32). These data suggest that hyperglycemia may increase the risk of myocardial infarction in diabetic (3, 7, 23) and asymptomatic hyperglycemic (3, 7) patients.

These results indicate that myocardial infarct size is linearly related to blood glucose concentration in both diabetic and acutely hyperglycemic dogs. These findings confirm our previous work demonstrating that acute hyperglycemia dose dependently increased infarct size and abolished the protection afforded by ischemic preconditioning (16). The present results also demonstrate that ischemic preconditioning is abolished in dogs with chemically induced diabetes of only 3 wk duration, findings that are comparable to those previously observed during acute hyperglycemia alone. Ischemic preconditioning, a powerful endogenous cardioprotective mechanism that limits myocardial infarct size in vivo (24), occurs via a signal transduction pathway that includes adenosine receptors coupled to ATP-sensitive K⁺ channels (K_ATP) by inhibitory G (G_i) proteins (2, 18). Adenosine receptor-G_i protein coupling has previously been shown to be impaired in hyperglycemic, diabetic rats (9). Attenuation of this signal transduction pathway during diabetes might also explain the failure of ischemic preconditioning to protect myocardium against infarction in the present study. The responsiveness of coronary arterioles to a K_ATP channel agonist was also shown to be attenuated in diabetic patients (22), findings that suggest K_ATP channel dysfunction during diabetes. In addition, nitric oxide has been shown to potentiate the actions of K_ATP channel agonists on K_ATP channel activation (26). However, increases in blood glucose concentration may impair nitric oxide release or action (8, 34) via excess superoxide anion production (30). Thus diabetes may adversely modulate an interaction between nitric oxide and K_ATP channels or superoxide anion may directly attenuate K_ATP channel activation (29). Whether the present results demonstrating that ischemic preconditioning is abolished in a canine model of diabetes mellitus can be attributed to similar mechanisms remains to be investigated.

Hyperinsulinemia and hyperosmolality were not responsible for increases in the extent of infarction observed during hyperglycemia in this or our previous investigation (16). Hyperinsulinemia has been shown to contribute to coronary vasospasm and sudden cardiac death (27); however, in the present investigation, the relationship between myocardial infarct size and blood glucose levels was independent of insulin concentration. Diabetic dogs demonstrated marked reduction in insulin levels (2.2 ± 0.4 µU/ml), but insulin concentration was strikingly elevated in acutely hyperglycemic dogs (from ~15 µU/ml at baseline to 100 µU/ml during hyperglycemia) (16). Hyperosmolality also did not account for increases in infarct size observed during hyperglycemia. Raffinose caused similar increases in serum osmolality (328 ± 3 mosmol/kg) as observed during acute hyperglycemia (318 ± 5 mosmol/kg) but did not increase infarct size. In contrast to findings in diabetic and acutely hyperglycemic dogs, raffinose decreased the extent of myocardial infarction during increases in serum osmolality. Ischemic preconditioning also afforded even greater protection in raffinose-treated versus untreated dogs. The mechanism of the cardioprotective effect of raffinose remains to be further explored.

Previous investigation of the extent of ischemic injury in diabetic myocardium has been controversial despite overwhelming clinical evidence that the diabetic heart is highly sensitive to such injury. Prior studies have indicated that the diabetic rat and rabbit heart may be protected against infarction (11, 21, 28). In contrast, other investigations demonstrated that diabetes exacerbates injury in response to myocardial ischemia and reperfusion (5, 6, 25, 31). It has been suggested that the disparity in these experimental findings can be explained by differences in the severity (degree of hyperglycemia) and chronicity of the diabetic state (31), as well as the experimental conditions and model used (25). The experimental model (alloxan and streptozotocin) used in the current investigation has previously been shown to reliably produce diabetes in dogs with minimal systemic toxicity (1) and is characterized by substantial increases in fasting blood glucose concentration (250-400 mg/dl) and marked insulinopenia. Other investigations demonstrating that diabetic myocardium is resistant to ischemia and reperfusion injury used small animals (typically rats) with fasting blood glucose levels in a normal range (21) or in an experimental model during which hearts were perfused with a crystalloid solution containing normoglycemic concentrations of glucose (25, 28). The duration of diabetes may also influence the extent of myocardial ischemic injury (25, 31). For example, rats were resistant to ischemia and reperfusion-induced ventricular arrhythmias 2 wk after administration of streptozotocin, but this protection was lost by 4 wk, and myocardial injury was exacerbated 8 wk after chemical induction of diabetes (31). In the present investigation, blood glucose concentration and infarct size were similarly related in diabetic (3 wk) and acutely hyperglycemic dogs, however, the hypothesis that diabetes of longer duration further exacerbates this relationship requires further clarification. Finally, models using zero-flow ischemia are more likely to demonstrate enhanced recovery from ischemia and reperfusion in diabetic myocardium (25). In contrast, clinical episodes of myocardial ischemia often occur during low flow conditions through stenotic coronary arteries or in the presence of coronary collateral blood flow.

The present findings clearly show that the risk of irreversible ischemic injury to myocardium is related to the severity of hyperglycemia, but the results may be influenced by other variables. The LV AAR for infarction and coronary collateral blood flow, important determinants of the extent of myocardial infarction, were similar between groups and do not account for the observed results. Increases in serum osmolality can augment LV contractility (33), and such an action could increase myocardial oxygen consumption and subsequent infarct size. However, rate-pressure product was similar in all experimental groups. Although coronary sinus oxygen tension was not measured and direct measurements of myocardial oxygen consumption were not made in this investigation, no intergroup differences in systemic hemodynamics were observed. Furthermore, raffinose and dextrose produced opposite effects on myocardial infarct size, despite the presence of similar hemodynamics and production of comparable increases in serum osmolality.

In summary, these results indicate that hyperglycemia produced by either intravenous dextrose or chemically induced diabetes increases myocardial infarct size. In addition, these conditions abolished the protection afforded by ischemic preconditioning in dogs. These findings confirm recent clinical evidence and implicate an adverse interaction between hyperglycemia and endogenous cardioprotective signal transduction pathways.