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(Circulation. 1995;92:244-252.)
© 1995 American Heart Association, Inc.

Articles

Metabolic Adaptation to a Gradual Reduction in Myocardial Blood Flow

Andrew E. Arai, MD; Susan E. Grauer, MD; Cheryl G. Anselone, BA; George A. Pantely, MD; J. David Bristow, MD

From the Division of Cardiology, Oregon Health Sciences University, Portland, Ore.

Correspondence to Andrew E. Arai, MD, Bldg 1, Rm B3-07, 1 Center Dr, MSC 0105, Laboratory of Cardiac Energetics, NHLBI/National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-0105.

	Abstract

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Background Studies during 20% to 50% reductions in regional coronary blood flow have revealed a number of metabolic and functional adaptations that suggest the heart downregulates energy requirements and contractility in response to ischemia. In contrast to prior studies of sudden changes in coronary blood flow, we tested whether the heart could reduce ATP consumption commensurate with a gradual decrease in coronary blood flow or whether transient metabolic abnormalities are a necessary trigger in this process.

Methods and Results From 0 to 35 minutes, mean left anterior descending coronary artery blood flow was reduced by 1% per minute in 10 acutely anesthetized and instrumented swine. Coronary blood flow then was held constant between 35 and 60 minutes at the resulting 35% net blood flow reduction. Although systemic hemodynamics remained stable, a significant decrease in regional left ventricular systolic wall thickening developed (from control value of 45±11% to 18±11% at 60 minutes, P<.001) without a sustained decrease in the phosphorylation potential (as assessed by a <2% decrease in either the transmural or subendocardial phosphocreatine-to-ATP ratio) and with minimal myocardial lactate production (4±44 µmol · min^-1 · 100 g^-1).

Conclusions Metabolic markers of ischemia such as ratio of phosphocreatine to ATP, ATP content, lactate content, and lactate production were blunted during this protocol of gradually worsening ischemia. Thus, contractile abnormalities of mild ischemia can develop with minimal metabolic evidence of ischemia. The downregulation of myocardial energy requirements can almost keep pace with the gradual decline in coronary blood flow.

Key Words: myocardium • adenosine triphosphate • lactate • ischemia • metabolism

	Introduction

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Myocardial hibernation has been postulated to represent reversible regional left ventricular (LV) dysfunction in response to long-term reduction in coronary blood flow.^{1 2} Because high-energy phosphate turnover occurs two to six times per minute during normal myocardial workloads,³ a 50% reduction in coronary blood flow would completely deplete ATP and phosphocreatine (PCr) in <1 minute unless myocardial ATP consumption is reduced during ischemia. Thus, if the myocardium is expected to survive in a state of hibernation for any length of time, ATP consumption cannot exceed ATP production for longer than brief periods.

Two recent reviews focus attention on the physiological observations consistent with myocardial hibernation in humans and animals.^{4 5} Transient metabolic markers of ischemia despite sustained regional contractile abnormalities during mild to moderate myocardial ischemia may provide important clues to the mechanisms leading to myocardial hibernation. A sudden 20% to 50% reduction in coronary blood flow results in metabolic and functional changes that have been well characterized.⁴ This degree of ischemia causes a graded reduction in contractile function, decreased PCr and ATP levels, and decreased lactate production. Surprisingly, the heart adapts to a prolonged steady 20% to 50% reduction in coronary blood flow over 60 minutes. This results in restoration of PCr levels, less lactate production, and decreasing tissue lactate levels despite persistently abnormal regional myocardial blood flow and contractile function.^{6 7 8 9}

These metabolic adaptations indicate that the heart downregulates ATP consumption during moderate myocardial ischemia.^{7 8} In addition, a significant degree of contractile reserve remains in the ischemic zone that can be recruited by dobutamine infusion at the expense of worsening myocardial metabolism.⁸ Furthermore, metabolism and contractile function can remain stable for 5 hours without evidence of infarction.^{10 11} This new steady state of improved myocardial metabolism with abnormal contractile function characterizes what has been called acute myocardial hibernation. The triggers or mechanisms that initiate the rapid reduction in ATP use are unknown.

Uncertainty regarding the type of ischemic insult necessary to initiate myocardial hibernation was highlighted at a recent conference. One proposal involved a series of 2-minute complete coronary occlusions.¹² A second model challenged a rat heart with zero perfusion for 10 minutes followed by a period of low-pressure perfusion.¹³ The third set of experiments were performed on chronically instrumented swine with an ameroid constrictor around the left anterior descending coronary artery (LAD). This group concluded that the regional contractile abnormalities were due to bouts of excess demand ischemia resulting in stunning.¹⁴

In contrast, results of our previous work suggest that acute myocardial hibernation can be initiated by relatively mild ischemic insults. The rapidity with which these adaptations occur¹⁵ raised the following questions. Can the myocardium adapt to a gradual reduction in myocardial blood flow without developing metabolic signs of ischemia? Alternatively, is the period of lactate production and high-energy phosphate catabolism a necessary trigger in the development of myocardial hibernation? To differentiate these possibilities, we studied 10 domestic swine in a protocol involving a gradual reduction in coronary blood flow. LAD blood flow was reduced by 1% per minute for 35 minutes and then held constant for the remainder of a 60-minute period. The myocardium rapidly adjusted during the period of gradually worsening ischemia. This adaptation resulted in blunted metabolic markers of ischemia despite ultimately similar blood flow reduction and functional consequence compared with animals previously studied during sudden-onset ischemia.^{6 7}

	Methods

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The methods for this experiment were similar to those of our previous work.^{6 7 15} Briefly, 35- to 45-kg domestic swine were surgically prepared while under general anesthesia with intravenous -chloralose (100 mg/kg initial dose followed by 50 mg/kg every 1.5 to 3 hours). The -chloralose was dissolved in distilled water and propylene glycol. Premedication consisted of a single intramuscular dose of ketamine (10 mg/kg) and xylazine (2 mg/kg). Morphine (0.5 to 1.5 mg/kg IV) was administered 30 minutes before we started the protocol. Intravascular volume status was maintained with intravenous normal saline. Acid-base status was maintained within normal limits by titration of mechanical ventilation and administration of intravenous bicarbonate.

The hearts were exposed by a midline sternotomy and supported by a pericardial cradle. Instrumentation included a polyvinyl hydraulic occluder and an electromagnetic flow probe around the LAD before the first septal perforator. Catheters were placed in the distal LAD to measure coronary perfusion pressure, in the anterior interventricular cardiac vein to measure regional oxygen and lactate consumption, in the left atrium for microsphere injections, in the aorta to obtain microsphere blood flow reference samples, and in the femoral vein to administer intravenous fluids and medications. A catheter-tip manometer was inserted through the LV apex to measure pressure and dP/dt. A pair of ultrasonic crystals was used to monitor transmural LV wall thickness in the LAD zone.

Transmural LV biopsy specimens (3 mm) were obtained with a drill biopsy gun¹⁶ and injected into liquid nitrogen within 1 to 2 seconds. While still frozen, biopsy specimens were divided into subendocardial, middle, and subepicardial thirds as previously described.^{6 15} Chemical extraction and enzyme-linked chemical analysis of lactate, ATP, and PCr were done following the methods of Lowry and Passonneau.^{6 7 17} Blood oxygen content was determined with an IL hemoximeter (Instrumentation Laboratory).

Protocol
Fig 1 summarizes the experimental protocol and timing of measurements. LAD blood flow was gradually reduced, with an aim of 65% of control flow by 30 to 35 minutes. Coronary flow was then held constant at this reduced level for the last 30 minutes of the experiment. Coronary blood flow was regulated by manually making small adjustments to the hydraulic occluder every 5 to 15 seconds while watching mean LAD flow and pressure. A custom-built screw-type mechanism allowed fine control of pressure within the syringe regulating the hydraulic occluder. As a technical note, during the first 10 minutes, LAD perfusion pressure was gradually reduced to 60 mm Hg, which is close to the break-point of coronary blood flow autoregulation under these experimental conditions. This resulted in a 10% decrease in LAD blood flow. After 10 minutes, it was easier to rely on the coronary flow measurements.

View larger version (48K): [in this window] [in a new window]   Figure 1. Plot of the gradual-ischemia protocol and comparison with sudden-onset ischemia. Rectangles indicate 100% of control blood flow. Circles delineate coronary blood flow as a percentage of control during the gradual-onset–ischemia protocol. Blood flow in the left anterior descending coronary artery (LAD) decreased by 1% per minute. Thus, after 35 minutes, LAD blood flow was only 65% of control. Beyond 35 minutes, LAD blood flow remained relatively constant by design. The timing of specific measurements obtained during the gradual-ischemia protocol is outlined at the bottom. Bx indicates transmural myocardial biopsy; AV, arterial and coronary vein blood sampling; and MS, radioactive microsphere blood flow determination. Triangles represent coronary blood flow after a sudden 35% reduction in flow. To compare findings among these types of experiments, a time-flow deficit is calculated as the cumulative integral of flow reduction (percent below control) multiplied by time in minutes. Thus, the entire shaded area represents the time-flow deficit for the sudden-ischemia protocol. Cross-hatched area indicates the time-flow deficit during the gradual-ischemia protocol. The time-flow deficit by 60 minutes into the gradual-ischemia protocol is almost equal to the time-flow deficit at 45 minutes into the sudden-ischemia protocol shown here. (See text and Table 4 for more details.)

Hemodynamics were monitored continuously. Ultrasonic LV wall thickness, heart rate, and LV end-diastolic pressure (EDP) were determined every 5 minutes. Arterial and anterior interventricular coronary venous blood samples (listed as AV in the figure) were taken at control and every 5 minutes during the protocol except at the time of biopsies. These blood samples provided determinations of myocardial oxygen consumption and lactate consumption across the ischemic zone. Transmural biopsies (Bx in the figure) were obtained at control and at 30, 45, and 60 minutes. Blue dye painted on the epicardial surface before biopsy helped differentiate subendocardium from subepicardium. Radioactive microspheres (MS in the figure) were injected into the left atrium at control and at 25, 40, and 55 minutes to help validate the electromagnetic flow probe measurements and to evaluate regional myocardial blood flow (13-µm spheres; ⁵¹Cr, ⁹⁵Nb, ¹⁰³Ru, and ¹⁴¹Ce).

Subendocardial, midmyocardial, and subepicardial blood flows were determined for the ischemic and nonischemic zones by dividing the formalin-fixed heart as previously described. The ischemic zone was differentiated from the nonischemic zone by the injection of colored dyes into each coronary artery simultaneously at constant rate. The border zones between ischemic and nonischemic regions and the area surrounding biopsies were discarded. Five 1-g aliquots of tissue per region were counted with a Micra multichannel analyzer with a germanium detector.

Statistical Analysis
All primary results are reported as mean±SD. One-way ANOVA with repeated measures was used to detect significant treatment effects over time. When appropriate, Tukey's test was performed to detect differences between time periods.¹⁸ Use of repeated-measures ANOVA requires an equal number of measurements in each block over the course of the experiment. Technical problems resulted in irrevocable data loss from some animals. If more than one data point was missing for a given animal, that particular set of data were not analyzed. This resulted in a smaller group size for some parameters (n8 for all measurements). Remaining individual holes were estimated, and statistics were corrected with a bias factor.¹⁸ Comparison of different linear relationships was performed with ANCOVA. If a significant difference was detected, Tukey's test was used to reveal variations in slope and, if appropriate, elevation.¹⁸

In the analysis contrasting the gradual-ischemia protocol with similar degrees of ischemia produced by sudden coronary constrictions, a t test was used to compare groups at similar time-flow deficits. To facilitate the visual analysis comparing mean values between groups, the corresponding figures plot the mean and SEM.

	Results

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Twelve swine were studied, but two animals were excluded due to ventricular fibrillation after the second biopsy. Arterial pH was 7.40±0.04, PCO₂ was 37.9±3.0 mm Hg, PO₂ was 124±17 mm Hg, and rectal temperature was 37.4±0.8°C at the beginning of the protocol.

Validation of Gradual Reduction in Blood Flow
With the hydraulic coronary occluder, LAD perfusion pressure and LAD blood flow were gradually reduced, as illustrated in Fig 2. Overall, LAD blood flow was reduced to 65% of control levels during the first 35 minutes of the experiment. Mean LAD pressure decreased from 99±17 to 55±6 mm Hg, and LAD flow decreased by 10% during the first 10 minutes. On average, we reduced LAD blood flow by 1% per minute during the first 35 minutes of the experiment. LAD blood flow remained constant between 35 and 60 minutes. By ANOVA, LAD pressure and flow were significantly below control levels at 5 and 10 minutes, respectively. Both remained significantly below control levels throughout the remainder of the experiment (P<.001 before the first biopsy).

View larger version (23K): [in this window] [in a new window]   Figure 2. Plots of pressure and blood flow in the left anterior descending coronary artery (LAD). A, Mean coronary pressure distal to the occluder plotted every 5 minutes for each animal. Group average is indicated by squares. B, Time course of LAD blood flow in each animal as determined by the electromagnetic flow probe. Group average is indicated by circles.

Although the flow probe was used to monitor the timing of changes in coronary flow continuously during the experiment, statistical comparisons were made only every 5 minutes. Microsphere blood flow determinations were used to define magnitude of blood flow reduction at 25, 40, and 55 minutes in the protocol. Control transmural blood flow was 1.10±0.29 mL · min^-1 · g^-1 with a normal subendocardial-to-subepicardial distribution (Table 1). By 25 minutes, transmural LAD microsphere flow was reduced by 32% (P<.01), which closely corresponds to the degree of blood flow reduction determined with the electromagnetic flow probe. By the end of the experiment, microsphere blood flow was 36% below the control-period level (P<.01). Subendocardial microsphere blood flow was reduced 48% and 54% below control levels at 25 and 55 minutes of ischemia, respectively (P<.001).

View this table: [in this window] [in a new window]   Table 1. Microsphere Blood Flow During Gradual Onset of Ischemia

The relative uniformity of the rate of change in myocardial perfusion is important in terms of combining data for the group averages in other analyses. Thus, LAD pressure and LAD blood flow reduction for each individual animal are shown in Fig 2A and 2B as dotted lines. The group average values are represented by the symbols superimposed on Fig 2A and 2B. Defining the ultimate blood flow reduction as an average of the last four electromagnetic flow probe determinations (45 to 60 minutes of ischemia), each animal reached the plateau by 37.5±3.5 minutes (range, 25 to 45 minutes). These points were selected because all animals appeared at a final plateau by 45 minutes of ischemia (Fig 2B).

Hemodynamics and Regional Function During Ischemia
The degree of ischemia produced had only mild effects on systemic hemodynamics (Table 2). LV systolic pressure was 119±14 mm Hg at control and decreased 14% by the end of the experiment. All LV systolic pressures after 35 minutes were statistically lower than during the control period (P<.001). Heart rate started at 83±10 beats per minute and increased to 92±16 beats per minute by the end of the experiment. When compared with the control heart rate, only the 40-, 50-, 55-, and 60-minute values were statistically different. These trends resulted in no significant change in rate-pressure product over the course of the experiment. LVEDP started at 10±3 mm Hg and was statistically different from control only at the 25-minute time period (13±2 mm Hg).

View this table: [in this window] [in a new window]   Table 2. Hemodynamics During Gradual Onset of Ischemia

As a reflection of external determinants of energy requirements, microsphere blood flow to the nonischemic zone did not change over time in any layer. During the control period, the transmural blood flows to the ischemic and nonischemic zones were not significantly different (1.10±0.29 versus 1.14±0.33 mL · min^-1 · g^-1, respectively). During the ischemic period, the subendocardial LAD blood flow was 50% below the non-LAD subendocardial blood flow level at the three times it was measured (Table 1; P<.001). Thus, by comparing ischemic zone blood flow with either its own control flow or the corresponding flow in the nonischemic zone, significant subendocardial blood flow reductions were found to have occurred.

LV systolic wall thickening in the LAD zone (Fig 3) decreased with a time course similar to the decrease in LAD blood flow. During the control period, wall thickening averaged 45±11%. Wall thickening measurements were significantly different from control by 20 minutes of ischemia and throughout the remainder of the experiment. Wall thickening before myocardial biopsies averaged 24±12% at 30 minutes, 22±10% at 45 minutes, and 18±11% at 60 minutes of the protocol. These measurements were all significantly lower than control levels (P<.001). The rate of change in contractile function was quite similar in each animal (Fig 3). Thus, most of the standard deviation is due to interanimal differences at baseline. Wall thickening was not assessed in the non-LAD zone.

View larger version (25K): [in this window] [in a new window]   Figure 3. Plot of percent systolic wall thickening in the ischemic zone. Data for individual animals are indicated by dotted lines; group average, by circles.

Metabolic Changes During Ischemia
All animals showed net arteriovenous lactate consumption across the LAD zone (53±36 µmol· min^-1 · 100 g^-1) before the protocol was started. Lactate consumption declined during the protocol (Fig 4). On average as a group, these animals never developed significant lactate production (4 µmol · min^-1 · 100 g^-1). However, as suggested by the standard deviations, 7 of 10 animals developed net lactate production at some time during the protocol. Lactate production occurred on average for 2.5 samples per animal of 9 samples taken during ischemia. Whether lactate production happened during the middle or end of the protocol was inconsistent. Five of 7 animals reverted to net lactate consumption after some lactate production earlier during the protocol despite similar or more severe blood flow reduction.

View larger version (17K): [in this window] [in a new window]   Figure 4. Plot of lactate consumption and production across the ischemic zone. Units of measure are micromoles per minute per 100 g of myocardium.

Because lactate production was quite variable from animal to animal, we analyzed regional wall thickening 5 minutes before any detected lactate production in each animal (Fig 5). By this analysis, wall thickening was significantly decreased compared with control before net arteriovenous lactate production was detected (P<.01). Thus, net arteriovenous lactate production need not be present for regional hypokinesis to develop. Net arteriovenous lactate production also was not present at the end of the experiment despite the greatest decrease in regional wall thickening; 7 of 10 animals showed net lactate consumption at that time.

View larger version (45K): [in this window] [in a new window]   Figure 5. Bar graph of systolic wall thickening before any detected lactate production. Rectangles and lines indicate percent systolic wall thickening for each animal at control and 5 minutes before any detected lactate production in that animal. Bars indicate the average and SD values.

Subendocardial ATP content decreased significantly below control levels but appeared stable between 30 and 60 minutes of ischemia (Table 3). Subendocardial PCr levels decreased to 65% of control levels during the first 30 minutes of the protocol (P<.01 versus control). By 60 minutes, subendocardial PCr levels returned to 87% of control levels (P=NS versus control).

View this table: [in this window] [in a new window]   Table 3. Metabolic Measurements During Gradual Onset of Ischemia

The phosphorylation potential represents an index of cellular energetic state. The phosphorylation potential at control, as assessed by the PCr-to-ATP ratio, was 1.61±0.09 in the subendocardium and 1.68±0.06 transmurally. Neither changed significantly over time (Table 3), but there was a trend toward increasing PCr-to-ATP ratios toward the end of ischemia, particularly in the subendocardium. Because subendocardial ATP content decreased slightly (Table 3; P<.001), the phosphorylation potential must have fallen at some points during the protocol. Taken together, these data indicate that the phosphorylation potential decreased briefly during the procedure but must have rapidly normalized.

	Discussion

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The following conclusions can be drawn from the present study. The downregulation of myocardial energy requirements can almost keep pace with the progressive but gradual decline in coronary blood flow. Contractile abnormalities occur at a rate that parallels the reduction in myocardial perfusion. Abnormal regional function develops without a sustained decrease in the phosphorylation potential, as reflected by the PCr-to-ATP ratio. These events occur without significant lactate production. Thus, contractile abnormalities associated with a gradual reduction in coronary blood flow can exist with no or minimal measurable metabolic signs of ischemia. This indicates that there is a tightly regulated feedback or feedforward mechanism maintaining the balance between myocardial oxygen consumption and supply.

To put the results of the current protocol of gradually worsening ischemia in perspective, we first compared these results with data obtained in our laboratory after sudden reductions in coronary blood flow. The new results are also compared with experimental models of hibernating myocardium from other laboratories.

Comparison of Gradual Versus Rapid Reductions in Coronary Blood Flow
We previously studied 10 swine during a sudden 22% reduction in LAD flow,⁶ 9 swine during a sudden 30% reduction,⁷ and 7 swine during a sudden 43% reduction.⁶ All of these groups had instrumentation, anesthesia, hemodynamics, and metabolic assays similar to those of the present study. To allow a comparison between the protocols involving gradual or rapid changes in coronary blood flow, a "time-flow deficit" integral was calculated. This is calculated as the cumulative integral of time multiplied by the percent transmural LAD flow reduction based on microsphere measurements for each animal. Thus, the time-flow deficit increases with time and more severe blood flow reductions. Fig 1 illustrates the time-flow deficit integral for a sudden 35% reduction in coronary blood flow (entire shaded area) and a gradual 35% reduction (cross-hatched area). After 35 minutes, coronary blood flow in both animals would be 35% below control levels. However, the time-flow deficit at 35 minutes would be 12.3 units (35 minutes multiplied by 35% flow reduction) in the sudden flow reduction group but only half as large in the gradual flow reduction group. Table 4 lists the calculated time-flow deficits at the times of major metabolic determinations for the three sudden-ischemia groups and the gradual-ischemia group.

View this table: [in this window] [in a new window]   Table 4. Calculated Time-Flow Deficits for Each Protocol

When compared with sudden ischemia of similar severity, the gradual reduction in coronary blood flow results in less flux through anaerobic glycolysis (Fig 6) and less-severe abnormalities in high-energy phosphates (Fig 7). Each sudden-ischemia group showed significantly greater subendocardial lactate accumulation than the gradual-ischemia group (Fig 6A, both P<.001). Maximal lactate production in the sudden-ischemia group was 136±81 µmol · min^-1 · 100 g^-1. This was much greater than maximal lactate production measured in the gradual-ischemia group at 4±44 µmol · min^-1 · 100 g^-1 (Fig 6B; P<.01). Subendocardial ATP content in the three sudden-ischemia groups followed nearly superimposable declines as shown in Fig 7A. In contrast, the subendocardial ATP content for the gradual-ischemia group ultimately appeared to stabilize at a higher level (P<.05 versus sudden 22% and sudden 30%; P<.01 versus sudden 43%). Subendocardial PCr levels tended to decrease less early during the gradual-ischemia protocol and rebound less quickly during the last 30 minutes of the protocol than in the sudden-ischemia groups (data not shown). This resulted in markedly different PCr-to-ATP ratios toward higher time-flow deficits (Fig 7B; P<.01 versus sudden 22% and sudden 30%; P=.06 versus sudden 43%). Thus, our prior data suggest that the downregulation of energy requirements lags behind the sudden blood flow–limited rate of ATP production. Once energy requirements have been downregulated, PCr levels increase and lactate levels decrease, but ATP levels remain depressed, probably due to the loss of adenine nucleotide precursors.^{19 20}

View larger version (21K): [in this window] [in a new window]   Figure 6. Plots of comparison between gradual-onset ischemia and sudden-onset ischemia versus lactate data. A, Subendocardial lactate levels were available for all groups except the sudden-43%-ischemia group. By this analysis, the degree to which subendocardial lactate accumulation is blunted during the gradual protocol is readily appreciated. Each group starts with a similar lactate content. Lactate accumulated to 300% of control levels in the two sudden-ischemia groups within 10 minutes. Each sudden-ischemia group showed significantly greater subendocardial lactate accumulation than the gradual-ischemia group (both P<.001). Units of measure for lactate content are micromoles per gram of wet weight. B, Arteriovenous lactate production was measured in the sudden-30%-group and the gradual-ischemia group. Maximal lactate production in the sudden-ischemia group was 136±81 µmol · min-1 · 100 g-1. This was much greater than maximal lactate production for the gradual-ischemia group at 4±44 µmol · min-1 · 100 g-1 (P<.01). Units for lactate production and consumption are micromoles per minute per 100 g; error bars indicate SEM values.

View larger version (22K): [in this window] [in a new window]   Figure 7. Plots of comparison between gradual- and sudden-onset ischemia versus ATP content and ratio of phosphocreatine (PCr)-to-ATP data. A, Subendocardial ATP content for each sudden-ischemia protocol follows nearly superimposable patterns in this analysis. Gradual-onset–ischemia protocol results in less ATP depletion. B, PCr-to-ATP ratio changed less in the gradual-onset–ischemia protocol than the sudden-onset–ischemia protocol experiments.

The new data indicate that gradual reduction of coronary blood flow is different from sudden-onset ischemia, at least in terms of the severity of the induced metabolic abnormalities. Overall, the downregulation of energy requirements can almost keep pace with the gradual experimental blood flow reduction. Although not perfect, these adaptations occur rapidly enough to prevent some of the metabolic signs of myocardial ischemia during the gradual reduction in coronary blood flow. That PCr levels show the smallest differences between the sudden-onset and gradual-onset ischemia groups is consistent with the greater biochemical lability of this compound. The myocardial lactate content provides a longer integration of the metabolic consequences of ischemia. Lactate production also shows some of the largest differences between the sudden- and gradual-ischemia protocols. Lower lactate levels and less arteriovenous lactate production during gradual-ischemia protocols are consistent with a smaller energy deficit during this form of ischemia and therefore less need for anaerobic glycolysis. This fits with our previous conclusion that the lack of lactate production is not secondary to substrate depletion.⁷

The strongest evidence of myocardial adaptation to ischemia is provided by considering the data on ATP and PCr-to-ATP ratio. Because ATP turnover rate is high compared with the size of cellular high-energy phosphate stores, both PCr and ATP levels decrease when ATP consumption exceeds ATP production. If the myocardial response to ischemia was completely passive, one would expect similar degrees of ATP depletion for a given time-flow deficit. This appears true when comparing the three groups of animals studied during various degrees of sudden-onset ischemia (Fig 7A). However, higher ATP levels for a given time-flow deficit during gradual ischemia suggest that the myocardium was actively adjusting energy consumption commensurate with the decrease in coronary blood flow. The difference between ATP consumption and production must have been smaller to explain these findings.

The PCr-to-ATP ratio reflects these findings in a slightly different way. The creatine kinase equilibrium will tend to shift high-energy phosphates from PCr to ADP at times of ATP depletion. Although PCr levels were not significantly different between sudden- and gradual-onset ischemia, in both protocols PCr levels were increasing during later portions of the experiment. This is consistent with relatively normal intracellular free energy of ATP hydrolysis during ischemia after adaptations have taken place. The sooner these adaptations occur, the less ATP will be degraded to adenosine and other degradation products. Thus, the trend toward a high PCr-to-ATP ratio ("PCr overshoot") seen in our sudden-ischemia protocols and in the stunned (postischemic) myocardium reflect primarily the severity of the early ATP production deficit. In the gradual-ischemia protocol, the energetic status of the myocardium quickly adapts to the relatively small changes in flow. This prevents some of the ATP depletion and results in more normal PCr-to-ATP ratios throughout the protocol.

This is an important concept should noninvasive measurement of ATP and PCr become practical in patients.²¹ Nuclear magnetic resonance techniques frequently describe the PCr-to-ATP ratio rather than absolute concentrations of either compound for technical reasons. Our data suggest that the PCr-to-ATP ratio in an ischemic segment of myocardium could be low, normal, or high depending on when the ratio is measured and how quickly the ischemia developed. It is a new concept that the PCr-to-ATP ratio is dependent on the rate of onset of ischemia.

Comparison With Other Experimental Models of Hibernating Myocardium
The experiments of Schulz et al⁸ and Zhang et al⁹ were quite comparable to our sudden-onset–ischemia protocols and led to similar conclusions, as described. Also in support of our findings, mild coronary constriction resulting in prolonged moderate regional hypokinesis in chronically instrumented swine is associated with minimal metabolic abnormality before reperfusion and minimal histological evidence of infarction.²² That ischemic dysfunction associated with severe coronary stenosis does not necessarily cause infarction has been extrapolated to humans.²³ However, there are limits to the degree of adaptation to ischemia in terms of both severity of blood flow reduction and cardiac workload.²⁴

In an isolated ferret heart preparation, Kitakaze and Marban^{25 26} defined "pure hibernation" as a change in contractile function without metabolic markers of ischemia that occurred at perfusion pressures of >60 mm Hg. However, Schulz et al²⁷ showed that myocardial function in vivo does not vary significantly within the autoregulatory range. Thus, it is difficult to extrapolate the perfused-heart data to our experiments (at least at pressures of >60 mm Hg). The isolated-heart data at coronary perfusion pressures more comparable to the severity of ischemia induced in the present study are concordant with our sudden-onset–ischemia experiments.²⁵

Although not specifically designed to study the time course of gradually worsening ischemia, the study of Keller et al²⁸ described the metabolic and functional consequences of sequentially decreasing perfusion pressure in isolated rat hearts. Their results compare well with our metabolic data in the gradual-ischemia protocol, which resulted in a coronary perfusion pressure of 42±4 mm Hg by 60 minutes. More severe metabolic abnormalities developed at slightly lower coronary perfusion pressures than the range we studied. Thus, the discrepancies between the findings of Keller et al²⁸ and the results of Marban and Kitikaze^{25 26} may be explained in part by differences in the ischemia rate of onset.

The chosen level of ischemia of the present study may be close to a threshold requiring greater reliance on anaerobic glycolysis for more severe perturbations. This hypothesis could explain differences between our findings and those of Downing and Chen.²⁹ After 2 hours of more severe coronary flow reduction in isolated, blood-perfused neonatal pig hearts, lactate production continued, ATP levels were 76% of control levels, and PCr was normal. As in our experiments, PCr must have decreased transiently but normalized once ATP levels stabilized.

Regarding the mechanism of contractile dysfunction during ischemia, our data indicate that the energetic state of the individual myocyte might be relatively normal once myocardial adaptations to ischemia develop despite persistent regional contractile abnormality. Observations in hypoxic perfused rat hearts support this conclusion.³⁰ Although we did not measure pH or intracellular inorganic phosphate, these cations likely were not severely influenced in light of the minor abnormalities in PCr, ATP, and lactate levels. This raises further questions as to the mechanism of ischemic contractile dysfunction that will need to be addressed in future studies.

Study Limitations
Although this experimental model is well characterized and provides great flexibility in controlling myocardial blood flow, some variations in blood flow are inevitable. As shown in Fig 2B, the variation for a given animal can be a large percentage of the net flow reduction, particularly when coronary flow is decreased by 1% per minute. Thus, minute-to-minute variation in flow may have contributed to some of the findings, such as the decrease in PCr levels. Our chemical extraction methods do not permit measurement of inorganic phosphate or intracellular pH. Inorganic phosphate and pH have been implicated as a likely factor contributing to ischemic dysfunction during ischemia of this severity.³¹ Inorganic phosphate interferes with calcium myofibrillar interactions.^{32 33} Until inorganic phosphate and intracellular pH are measured under these conditions, no firm conclusion regarding their roles can be made. We did not assess metabolism or local contractile function in the nonischemic myocardium. Thus, we would not be able to detect any interactions between the ischemic and nonischemic regions that may have contributed to the metabolic adaptations described.

Myocardial blood flow, flow reserve, and metabolic indicators of ischemia exhibit a heterogeneous distribution during myocardial ischemia.^{34 35 36 37} One could postulate that the downregulation process occurs on a patchy basis. Then, the regional metabolic determinations may appear blunted because the adaptations occur in different locations at different times during the 30 minutes of ischemia and thus are lost in the average values. Even if heterogeneity of ischemia could explain why we did not detect arteriovenous lactate production or subendocardial lactate accumulation, it would be difficult to explain why subendocardial ATP content is better preserved in the gradual-ischemia group without invoking our same conclusions of rapid downregulation of energy requirements.

From the standpoint of a patient with coronary artery disease, the present findings suggest that a regional wall motion abnormality might develop without measurable metabolic signs of ischemia. Potentially, these contractile abnormalities may develop asymptomatically. Indeed, our choice of 30 minutes for the onset of ischemia was somewhat arbitrary based on prior data for comparison and practical time constraints on the length of experiments of this nature. The metabolic difference between gradual- and sudden-onset ischemia might have been larger had we studied even slower-onset ischemia. It is not clear to what extent the mechanisms outlined explain reversible wall motion abnormalities in patients. In the clinical setting, other mechanisms, such as myocardial stunning, also may contribute.

	Acknowledgments

 
This work was funded in part by grants from the American Heart Association (86-1278, 90-793) and the American Heart Association, Oregon Affiliate.

Received June 15, 1994; revision received January 3, 1995; accepted January 9, 1995.

	References

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