INTRODUCTION

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INTERVENTIONS THAT REDUCE myocardial necrosis consequent to coronary artery occlusion (CAO), e.g., coronary artery reperfusion (CAR) and ischemic preconditioning (IPC), result in patchy (heterogeneous) rather than confluent (homogeneous) necrosis. It is also well recognized that myocardial blood flow in the absence of ischemia is heterogeneous. The spatial heterogeneity of myocardial blood flow has been observed in various species, including dogs (2, 35), rabbits (3), and baboons (6, 13). Recently we reported (6) that the spatial heterogeneity of myocardial blood flow could predict salvage or necrosis before CAO and reperfusion in conscious baboons. We found that infarcted tissues had higher pre-CAO myocardial blood flow, whereas the salvaged tissues were characterized by lower pre-CAO myocardial blood flow. We reasoned that interventions that induce cardioprotection may also induce a similar pattern of spatial redistribution of blood flow, particularly if the shift to lower blood flow might reflect reduced metabolic demands and consequently elicit cardioprotection.

To test this hypothesis, we examined the effects of two interventions known to induce cardioprotection; the first was an adenosine agonist, and the second was IPC. The phenomenon of IPC, first described by Murry et al. (18) in 1986, is perhaps the most powerful intervention for cardioprotection. Adenosine, potentially through the A₁ receptor, has been implicated in the mechanism of preconditioning in various animal species (1, 8, 17, 27, 28, 31). To test the above hypothesis, we first needed to determine whether an A₁ agonist and IPC would induce cardioprotection in conscious pigs. The second goal was to examine myocardial blood flow before and after infusion of a selective A₁-receptor agonist and before and after IPC to determine whether these interventions would alter the pre-CAO spatial distribution of myocardial blood flow. It was also necessary to demonstrate in the control group that small samples of myocardium with higher pre-CAO blood flow would indeed result in a higher incidence of infarct. Pigs were used in this study because they have few native collaterals (34) and no detectable xanthine oxidase (19, 22) in their myocardium, two important aspects similar to the human heart. In addition, experiments in conscious, chronically instrumented animals are not complicated by the acute effects of surgery and anesthesia (4, 32). Furthermore, the infarct size was measured by using pathological techniques after reperfusion for 3 days, eliminating the possibility that the infarct size might have been underestimated due to potential errors in the triphenyltetrazolium chloride technique (26).

	METHODS

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Animal preparation. Twenty-four Yorkshire farm pigs (22 ± 1 kg body wt) were pretreated with Telazol (2-3 mg/kg im) and atropine (0.05 mg/kg im). General anesthesia was induced with thiamylal sodium (10-20 mg/kg iv), the animals were intubated, and anesthesia was maintained with halothane (0.5-1.5 vol%). With the use of a sterile surgical technique, thoracotomy was performed at the left fifth intercostal space. Tygon catheters (Norton Plastics, Akron, OH) were implanted in the descending aorta and in the left atrium for pressure measurements and radioactive microsphere injection and withdrawal. A solid-state miniature pressure gauge was implanted in the left ventricular (LV) cavity to obtain measurements of LV pressure and the first derivative of pressure (dP/dt). The left circumflex coronary artery was isolated, and a hydraulic occluder, made of Tygon tubing, was implanted; in four pigs a Transonics flow transducer was also implanted on that vessel. Two pairs of 5-MHz piezoelectric crystals for wall thickness determination were implanted across the LV wall supplied by the left anterior descending coronary artery (nonischemic area) and by the left circumflex artery (potential ischemic area). The wires and catheters were externalized between the scapulae. The incision was closed in layers, and the chest was evacuated. Each animal was treated with 1 g of cephalothin sodium (Keflin, Lilly, Indianapolis, IN) before surgery and daily for 1 wk after surgery. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals [National Research Council, revised 1996].

Hemodynamic and myocardial infarction size measurements. Hemodynamics were recorded continuously on a 14-channel magnetic tape recorder (Honeywell, Denver, CO) and played back on a multiple-channel ink-writing oscillograph (Gould-Brush, Cleveland, OH). Aortic and left atrial pressures were measured by strain gauge manometers (Statham Instruments, Oxnard, CA) connected to the respective fluid-filled catheters. The solid-state LV pressure gauge was cross-calibrated against measurements of systolic aortic and left atrial pressures. LV dP/dt was calculated with an operational amplifier connected as a differentiator, which has a frequency response of 700 Hz. Mean arterial pressure was determined using a resistance-capacitance filter with a 2-s time constant. A cardiotachometer triggered by the LV pressure pulse provided instantaneous and continuous records of heart rate. Percent wall thickening was calculated as [(end-systolic wall thickness  end-diastolic wall thickness)/end-diastolic wall thickness] × 100.

Protocol. The experiments were conducted 10-14 days after surgery. During this postoperative period, the pigs were introduced to a sling for training. Valium was administered at 0.5-1.0 mg/kg for tranquilization before initiation of the experimental protocol and additionally as required, i.e., if the pig became transiently agitated. An intravenous catheter was introduced into an ear vein for delivery of drugs. Twenty-four pigs were divided into three groups. 1) In the control group, nine pigs had saline vehicle infused into the ear vein for 10 min, and after an additional 10 min, the left circumflex coronary artery was occluded for 60 min. 2) In the A₁ agonist group (GR-79236X dissolved in normal saline) (9, 12), eight pigs received 10-min infusion of the A₁ agonist (3.5 µg/kg iv). After a 10-min washout, the animals underwent CAO for 60 min. 3) In the IPC group, seven pigs received IPC as two episodes of 10-min CAO with 10-min CAR before 60-min CAO. Hemodynamic data were recorded before CAO and throughout the first 3 h after CAR. Hemodynamic data were recorded again 24, 48, and 72 h after CAR. The first injection of microspheres (baseline 1) was injected as a baseline. The second injection of microspheres (baseline 2) was injected after infusion of vehicle (control), A₁ agonist, or IPC, i.e., before 60-min CAO. At 5 min after CAO, the third injection of microspheres was given. During CAO, multiple ventricular premature contractions were treated with bolus injection of lidocaine through the left atrium. Lidocaine was also injected intravenously immediately before CAR. All animals received a similar total dose of lidocaine (10-15 mg/kg). The microspheres were given again at 5 min before and at 3 and 24 h after CAR.

Data analysis. Data among the groups were compared using analysis of variance with a repeated-measures design. Individual groups at various time intervals were compared using grouped t-tests with Bonferroni correction. Spatial distribution of myocardial blood flow in the AAR for all groups was analyzed with histogram distribution analysis. Blood flow distributions in the ischemic and nonischemic zones were evaluated by calculating the coefficient of variation, defined as the ratio of the standard deviation of the distribution of measurements to its mean. The normality of the distribution was tested with a Kolmogorov-Smirnov test and by plotting the normal probabilities. Because the distributions were not all characterized by normal distribution, myocardial blood flows measured at baselines 1 and 2 were compared with a Wilcoxon test. The hemodynamics and pathology data were compared using a one-way analysis of variance with individual group comparisons using Fisher's least significant differences test. Significance was taken at P < 0.05. Values are means ± SE.

	RESULTS

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Hemodynamics. Hemodynamic variables at baseline 1 (before interventions) are summarized in Table 1. The changes from baseline 1 to baseline 2 are also noted in Table 1. Figure 1 summarizes mean values for systemic hemodynamics (mean aortic pressure, left atrial pressure, LV dP/dt, and heart rate) during both baseline and at 5 and 55 min after CAO and at 3, 24, 48, and 72 h after CAR. Control animals showed no significant hemodynamic changes during infusion of vehicle (saline). However, pigs receiving the A₁ agonist showed significant (P < 0.05) reductions in mean arterial pressure (31 ± 3 mmHg), LV dP/dt (1,013 ± 75 mmHg/s), and heart rate (35 ± 3 beats/min) and increases in mean left atrial pressure (+4 ± 1 mmHg). However, regional wall thickening was not decreased (Table 1). IPC reduced LV dP/dt compared with baseline 1 (482 ± 141 mmHg/s; P < 0.05) but less than observed with the A₁ agonist, and IPC also reduced posterior wall thickening significantly (13 ± 3%; P < 0.05), indicating myocardial stunning.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Hemodynamic responses during coronary artery occlusion (CAO) and coronary artery reperfusion (CAR). Baseline 1 represents data before any intervention. Baseline 2 represents hemodynamic data after infusion of vehicle in control group, infusion of A1 agonist, and ischemic preconditioning (IPC) by 2 episodes of 10-min CAO. A1 agonist reduced arterial pressure, heart rate (HR), and first derivative of left ventricular pressure (LV dP/dt) during CAO. LV dP/dt tended to recover more fully in IPC group. MAP, mean aortic pressure; LAP, left atrial pressure. CAO5' and CAO55', values at 5 and 55 min after CAO, respectively; CAR3H, CAR24H, CAR48H, and CAR72H, values at 3, 24, 48, and 72 h after CAR, respectively. * P < 0.05 vs. control.

Arrhythmias. The total number of arrhythmic beats was similar among the three groups during CAO, but IPC caused increased frequency of arrhythmias during the early reperfusion period (Fig. 2). For example, the number of premature ventricular contractions at 15 min of reperfusion after 60 min of CAO was 32 ± 13 beats/min (P < 0.05) compared with 11 ± 3 beats/min in the control group. Ventricular fibrillation in the control, A₁ agonist, and IPC groups occurred in four of nine, one of eight, and three of seven pigs, respectively (not significant by -square). All pigs were resuscitated rapidly and completed the protocol. However, there was no ventricular fibrillation during CAR in any animals.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Frequency of arrhythmias during CAO and CAR. Values during CAO represent total no. of arrhythmias during 60-min CAO, whereas values during CAR represent no. of arrhythmic beats per minute. IPC increased arrhythmias during early reperfusion. * P < 0.05 vs. control.

Pathology. The size of AAR expressed as a percentage of the left ventricle (AAR/LV) was not significantly different among the three groups of pigs (Fig. 3). The control pigs had an average infarct size of 55.1 ± 2.9% of the AAR (Fig. 3). Infarct size in the A₁ agonist pigs was reduced significantly to 27.1 ± 6.6% (P < 0.05 vs. control). Myocardial infarct size was reduced to a greater extent by IPC (11.6 ± 5.1%, P < 0.05 vs. control and A₁ agonist).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   A: myocardial infarct size expressed as a percentage of area at risk (infarct/AAR). B: size of AAR, expressed as a percentage of LV (AAR/LV). A1 agonist reduced infarct size, but IPC decreased infarct size even more. * P < 0.05 vs. control; # P < 0.05 vs. A1 agonist.

Regional myocardial blood flow. The initial baseline myocardial blood flow was not significantly different among the three groups of animals (Table 2). Baseline 2 transmural myocardial blood flow in the ischemic zone was significantly reduced after infusion of the A₁ agonist (1.27 ± 0.19 vs. 0.74 ± 0.10 ml · min¹ · g¹) and after IPC (1.27 ± 0.11 vs. 0.96 ± 0.09 ml · min¹ · g¹). No change in myocardial blood flow was observed after infusion of the vehicle (1.20 ± 0.10 vs. 1.23 ± 0.09 ml · min¹ · g¹). Myocardial blood flow was decreased significantly during CAO by 99 ± 1, 97 ± 1, and 93 ± 4% in control, A₁ agonist, and IPC groups, respectively.

Spatial distribution of myocardial blood flow. The myocardial blood flow distribution in the AAR before and after infusion of vehicle in the control group is shown in Fig. 4. Baseline 1 myocardial blood flow was characterized by a heterogeneous distribution, with flows ranging from 0.1 to 3.0 ml · min¹ · g¹, averaging 1.20 ± 0.10 ml · min¹ · g¹, with a coefficient of variation at 26.5%. There was no significant change in the distribution of myocardial blood flow at baseline 2 (1.23 ± 0.09 ml · min¹ · g¹, with a coefficient of variation at 25.7%). However, there was a shift in myocardial blood flow distribution in the AAR toward lower flow in the A₁ agonist group after infusion of the A₁ agonist (1.27 ± 0.19 ml · min¹ · g¹, with a coefficient of variation at 29.1%, vs. 0.74 ± 0.10 ml · min¹ · g¹, with a coefficient of variation at 30.3%) (Fig. 5). A similar shift was observed in the nonischemic zone (1.13 ± 0.15 ml · min¹ · g¹, with a coefficient of variation at 25.4%, vs. 0.61 ± 0.09 ml · min¹ · g¹). Myocardial blood flow distribution in the AAR of the IPC group also shifted toward lower blood flows (1.27 ± 0.11 ml · min¹ · g¹, with a coefficient of variation at 23.7%, vs. 0.96 ± 0.09 ml · min¹ · g¹, with a coefficient of variation at 24.4%) (Fig. 6). However, in contrast to the A₁ agonist group, there was no significant shift to lower blood flows (1.32 ± 0.11 ml · min¹ · g¹, with a coefficient of variation at 25.4%, vs. 1.20 ± 0.13 ml · min¹ · g¹, with a coefficient of variation at 27.4%) in the nonischemic zone (data not shown). As shown in Fig. 7, the shift in myocardial blood flow in both the IPC and A₁ agonist groups demonstrated a similar pattern; tissue samples with high baseline flow elicited greater shifts after intervention compared with tissue samples with lower baseline myocardial blood flow.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Spatial distribution of myocardial blood flow in AAR (baseline 1) and after infusion of vehicle (baseline 2) in control animals. Frequency (n) is no. of samples at each myocardial blood flow level. There was no significant shift of myocardial blood flow.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Spatial distribution of myocardial blood flow in AAR before (baseline 1) and after infusion of A1 agonist (baseline 2). There was a significant (P < 0.05) shift of myocardial blood flow toward lower blood flows after infusion of A1 agonist.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Spatial distribution of myocardial blood flow in AAR before (baseline 1) and after IPC (baseline 2). There was a significant (P < 0.05) shift of myocardial blood flow toward lower blood flows after IPC.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Relationship between baseline 1 myocardial blood flow (abscissa) and change in blood flow between baseline 1 and baseline 2 (ordinate) in control, A1 agonist, and IPC groups. Tissue samples characterized by high baseline myocardial blood flow demonstrated greater reduction in flow from baseline 1 to baseline 2 compared with samples with low baseline myocardial blood flow in both A1 agonist and IPC groups. * Slopes of lines indicated were different (P < 0.05).

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Spatial distribution of pre-CAO myocardial blood flows in AAR in 9 animals in control group compared for salvaged samples and infarcted samples. Distribution of salvaged samples is shifted to left (P < 0.05), whereas distribution of infarct samples is skewed to right, i.e., to higher pre-CAO myocardial blood flows.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Relationship between infarct size (infarct/AAR) and change in blood flow between baseline 1 and baseline 2 in control and IPC groups. Animals characterized by lower infarct size demonstrated greater reductions in flow from baseline 1 to baseline 2 (r = 0.79, P < 0.01).

	DISCUSSION

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The major finding of the current investigation was that both the adenosine A₁ agonist and IPC significantly shifted myocardial blood flow distribution in the AAR toward lower flows before prolonged CAO. This suggests the possibility that pre-CAO segments of myocardium with high flow, potentially due to higher metabolism, are at greater risk. In support of this concept, we have previously reported (6) that the spatial heterogeneity of myocardial blood flow could predict necrosis or salvage with CAO and CAR in conscious baboons and that regions in the left ventricle characterized by high levels of resting myocardial blood flows are more susceptible to infarction than are regions with low resting myocardial blood flows. This was clearly observed in the samples for the AAR of the pigs in the control group in this study (Fig. 8). Importantly, an additional analysis in this study demonstrated that the higher flows shifted to a greater extent than did lower flows during IPC or A₁ agonist (Fig. 7). These data not only offer insight into the mechanism of IPC but also help explain the patchy nature of myocardial necrosis in the AAR.

Previous studies (1, 5, 7, 17, 23, 27) support the hypothesis that IPC is mediated by the adenosine A₁ receptor and that pretreatment with an A₁ agonist will offer similar cardioprotective effects. However, these studies were conducted in anesthetized, open-chest animals. Anesthesia and acute surgery affect hemodynamics and autonomic control (32), free radical production (15), calcium metabolism (21), and, consequently, the development of infarction. Also, different anesthetics affect myocardial preconditioning (10). The present study demonstrated that both intravenous pretreatment with an adenosine A₁-receptor agonist and IPC provide cardioprotection against myocardial infarction in chronically instrumented pigs. The extent of protection was significantly greater with IPC, suggesting the possibility that the mechanisms involved may be more complex than simple A₁ agonist stimulation. The administration of the A₁ agonist in the current study resulted in significant decreases in heart rate and mean arterial pressure that may have contributed to its cardioprotective effects. However, other studies include indirect evidence that A₁ agonist administration induces salvage independent from bradycardia and hypotension (12, 27, 29, 30).

With comparison of the effects of IPC and the A₁ agonist, insight might be gained regarding the mechanisms that underlie the cardioprotective effects of IPC. As noted above, administration of the A₁ agonist elicited significant negative inotropic and chronotropic effects and decreased mean arterial pressure. This may explain the shift of myocardial blood flow distribution seen in both the ischemic and nonischemic zones. However, the entire shift may not solely be due to hemodynamic effects. Indeed, IPC induced a significant shift toward lower myocardial blood flows in the AAR, despite no major change in systemic hemodynamics. Whereas this was not associated with major changes in global hemodynamics, there was a decrease in regional contractility in the previously ischemic zone due to myocardial stunning, which could result in a decrease in oxygen consumption and might explain the shift in myocardial blood flow. It should be mentioned that postischemic myocardial dysfunction is not thought to be associated with decreased myocardial oxygen consumption and an impairment of the normal relation between coronary blood flow and myocardial oxygen utilization (14). Nonetheless, the extent of contraction and wall thickening during systole in the AAR was reduced significantly with IPC, which was associated with reduced coronary blood flow. Thus the severity of the stunning elicited by IPC appears to be associated with reduced myocardial oxygen demand as opposed to less severe stunning. With the A₁ agonist, wall thickening was preserved, potentially due to the decrease in afterload and heart rate. Thus both interactions tended to reduce oxygen requirements, but in different ways, suggesting that the mechanism of A₁ and IPC protection links changes in metabolism to blood flow distribution and cellular alterations, i.e., changes in PKC- or ATP-dependent K⁺-channel regulation (1, 5, 7, 31).

To determine whether the A₁ agonist reduced coronary blood flow secondary to its effects on afterload and heart rate, the A₁ agonist was administered to four conscious pigs with heart rate maintained constant with electrical pacing and afterload held constant with a simultaneous infusion of phenylephrine. Under these conditions, coronary blood flow fell by 13 ± 5% without a major change in hemodynamics. These data suggest that the major mechanism by which the A₁ agonist decreases coronary blood flow involves its effects on heart rate and afterload.

Although it is well accepted that IPC renders the heart more tolerant to subsequent prolonged ischemia, its protective influence on ventricular arrhythmias during reperfusion is still debated. Several studies (11, 16, 25, 33) have demonstrated that preconditioning limits reperfusion-induced arrhythmias after ischemia in the rat heart. In our study, the frequency of arrhythmias during early reperfusion was significantly higher after IPC compared with that in the control group. Indeed, Ovize et al. (20) have reported that preconditioning accelerates the time to ventricular fibrillation in a pig model. However, this apparent contradiction may be species dependent.

In conclusion, we demonstrated that the reduction of infarct size after CAO in conscious pigs by intravenous pretreatment with a selective adenosine A₁ agonist and by IPC was accompanied by a shift in the spatial distribution of blood flow to a pattern with lower blood flows. These interventions, despite markedly different hemodynamic profiles, both elicited a heterogeneous pattern of salvaged myocardium. This shift in spatial distribution of myocardial blood flow may reflect an alteration in metabolic state of the myocardium and consequently may play a role in mediating IPC, and this shift can also explain the patchy pattern of necrosis observed after interventions that salvage ischemic myocardium.