Increased Tolerance to Sustained Low-Flow Ischemia by a Brief Episode of No-Flow Ischemia Without Intermittent Reperfusion
Abstract Ischemic preconditioning (IP) and myocardial hibernation (MH) are both adaptive phenomena during acute myocardial ischemia, characterized by preserved myocardial viability and attenuated alterations of energy metabolism. Recent data from isolated buffer-perfused rabbit hearts pointed to a further link between IP and MH, in that an initial stimulus of no-flow ischemia was required to permit the development of MH during subsequent sustained low-flow ischemia. In the present study, we therefore investigated in the in situ pig heart whether a brief episode of no-flow ischemia enhances the myocardial tolerance to subsequent sustained low-flow ischemia. By blocking ATP-dependent potassium channels, we attempted to further determine whether such increased tolerance to ischemia is related to IP or MH, since blockade of ATP-dependent potassium channels abolishes the cardioprotection achieved by IP but not by MH. In 8 enflurane-anesthetized pigs serving as controls (group 1), the inflow into the cannulated left anterior descending coronary artery was reduced to achieve a 90% reduction in the anterior myocardial work index (sonomicrometry) for 90 minutes. In 15 pigs (group 2), a 10-minute no-flow ischemic episode preceded 80 minutes of sustained ischemia at a blood flow reduction identical to that in pigs of group 1. In 8 additional pigs (group 3), glibenclamide was administered before the 10-minute no-flow ischemic episode. In all pigs after 120 minutes of reperfusion, infarct size (IS, percentage of area at risk) was determined by triphenyltetrazolium chloride staining. In group 2, IS was reduced (6.8±6.0% [mean±SD], P<.05) when compared with groups 1 (13.2±9.8%) and 3 (16.7±8.3%). In group 2, subendocardial blood flow of tissue that remained viable averaged 0.06±0.02 mL · min−1 · g−1. This blood flow was lower than that in groups 1 (0.11±0.04 mL · min−1 · g−1, P<.05) and 3 (0.10±0.06 mL · min−1 · g−1, P=NS), indicating an increased ischemic tolerance of the myocardium in pigs of group 2. Conclusions are as follows: (1) A brief episode of no-flow myocardial ischemia without intermittent reperfusion increases the tolerance to sustained low-flow ischemia. (2) The cardioprotective effect is mediated by activation of ATP-dependent potassium channels and therefore relates to IP rather than to MH.
- ischemic preconditioning
- myocardial hibernation
- myocardial ischemia
- ATP-dependent potassium channels
- infarct size
Myocardial ischemia, even if it persists for a prolonged period of time, does not inevitably induce irreversible damage. Recent studies have identified two phenomena that are characterized by endogenous cardioprotective features during acute myocardial ischemia: ischemic preconditioning1 and myocardial hibernation.2 3
Ischemic preconditioning refers to the reduction of infarct size resulting from prolonged and severe myocardial ischemia by one or more preceding short episodes of ischemia and reperfusion, and this phenomenon has been confirmed in a number of animal species,1 4 5 6 7 and more recently in humans.8 Myocardial ATP and creatine phosphate (CP) levels remain somewhat higher in preconditioned hearts than in hearts subjected to prolonged ischemia without ischemic preconditioning.9 10 Also, glycolysis and lactate production are attenuated in ischemic preconditioned hearts.11 12
Myocardial hibernation2 3 refers to a chronic reduction of contractile function in ischemic myocardium that is fully reversible upon reperfusion, indicating maintained viability.13 14 Myocardial CP content, after an initial reduction, recovers toward control values,15 16 17 18 and lactate production is attenuated over time,18 19 despite a persistent reduction in myocardial blood flow and contractile function. The lower limit of myocardial blood flow compatible with the development of myocardial hibernation amounts to ≈25% of baseline in experimental20 and clinical21 settings.
Recently, Ferrari et al22 demonstrated in isolated buffer-perfused rabbit hearts that an initial stimulus of very severe ischemia was required to permit the development of myocardial hibernation during subsequent sustained low-flow ischemia, raising the possibility of a mechanistic link between ischemic preconditioning and myocardial hibernation. In this model, they observed full recovery of myocardial ATP content and reversal of lactate production toward lactate consumption after 4 hours of low-flow ischemia (10% of baseline flow) when hearts underwent a preceding 10-minute no-flow ischemic period. During reperfusion following the sustained ischemia, only a transient creatine kinase release occurred, and contractile function rapidly recovered to 86% of the control value. Thus, Ferrari et al proposed that the development of myocardial hibernation requires an initial period of no-flow ischemia, in which the rapid decrease in pH initiating the decrease in contractile function allowed for a renewed balance between energy supply and energy demand.
Alternatively, the brief episode of no-flow ischemia might have increased the resistance to sustained low-flow ischemia by enhanced production and/or release of cardioprotective substances. Such a mechanism in which a brief episode of ischemia, but without intermittent reperfusion, increases the tolerance to sustained ischemia would then relate to the phenomenon of ischemic preconditioning. However, ischemic preconditioning without intermittent reperfusion is a controversial issue. In a study by Koning et al23 that involved anesthetized pigs, infarct size after a 60-minute coronary artery occlusion was reduced in pigs subjected to a preceding 30-minute partial coronary stenosis (which was sufficient to decrease coronary blood flow by 70%) when compared with infarct size in pigs subjected only to 60 minutes of coronary artery occlusion. In contrast, Ovize et al,24 who used a 15-minute partial coronary stenosis in dogs (which was sufficient to reduce coronary blood flow by 50%), did not observe reduced infarct size after a subsequent 60-minute coronary artery occlusion when intermittent reperfusion was avoided.
Therefore, in the present study we tested whether a prolonged period of severe myocardial ischemia can be tolerated without irreversible tissue damage when preceded by a brief episode of no-flow ischemia without intermittent reperfusion in the in situ pig heart. Pigs were chosen for study for the following reasons: (1) In this species, infarct development most closely resembles that observed in humans because of the sparsity of the collateral circulation.25 (2) Both ischemic preconditioning4 23 26 27 and myocardial hibernation16 17 18 19 20 have been characterized in detail in this species. By blocking ATP-dependent potassium channels, we attempted to further determine whether such increased tolerance to ischemia is related to ischemic preconditioning or to myocardial hibernation, since we have previously shown that blockade of ATP-dependent potassium channels abolishes the cardioprotection achieved by ischemic preconditioning27 but not by myocardial hibernation.28
Materials and Methods
The experimental protocols used in the present study were approved by the Bioethical Committee of the district of Düsseldorf, and they adhere to the guiding principles of the American Physiological Society.
Thirty-one Göttinger minipigs (20 to 40 kg) of either sex were initially sedated with ketamine hydrochloride (1 g IM) and then anesthetized with thiopental (Trapanal, 500 mg IV). Through a midline cervical incision, the trachea was intubated for connection to a respirator (Dräger). Anesthesia was then maintained with enflurane (1% to 1.5%) with an oxygen/nitrous oxide mixture (40%:60%). Arterial blood gases were monitored frequently in the initial stages of the preparation until stable and then periodically throughout the study (Radiometer). By use of a heated surgical table and drapes as well as by infusion of warmed saline solution, the rectal temperature of each individual pig was maintained between 37°C and 38°C.
Through the cervical incision, both common carotid arteries and internal jugular veins were isolated. The arteries were cannulated with polyethylene catheters, one for measurement of arterial pressure and the other to supply blood to the extracorporeal circuit. The jugular veins were cannulated for volume replacement by using warmed 0.9% NaCl and for the return of blood to the animal from the coronary venous line (see below).
A left lateral thoracotomy was performed in the fourth intercostal space, and the pericardium was opened. A micromanometer (model P7, Konigsberg Instruments) was placed in the left ventricle through the apex, together with a saline-filled polyethylene catheter (used to calibrate the micromanometer in situ). Ultrasonic dimension gauges were implanted in the left ventricular (LV) myocardium to measure the thickness of the anterior and posterior (control) walls (System 6, Triton Technologies, Inc).
The proximal left anterior descending coronary artery was dissected over a distance of 1.5 cm, ligated, and cannulated, and the distal left anterior descending coronary artery was perfused from an extracorporeal circuit. Before coronary cannulation, the pigs were anticoagulated with 20 000 IU sodium heparin; additional doses of 10 000 IU were given at hourly intervals. The system included a roller pump, windkessel, and a side port for the injection of radiolabeled microspheres. Coronary perfusion pressure was measured from the side arm of a polyethylene T connector (Cole-Parmer) used as catheter tip with an external transducer (Bell and Howell type 4-327I). The large epicardial vein parallel to the left anterior descending coronary artery was dissected and cannulated. Coronary venous blood was drained to an unpressurized reservoir and then returned to a jugular vein through use of a second roller pump. Heart rate was controlled throughout the study by left atrial pacing (Hugo Sachs Elektronik type 215/T).
Regional Myocardial Function
End diastole was defined as the point when LV dP/dt started its rapid upstroke after crossing the zero line. Regional end systole was defined as the point of maximal wall thickness within 20 milliseconds before peak negative LV dP/dt.29 Regional myocardial function was assessed as systolic wall thickening, which was calculated as end-systolic wall thickness minus end-diastolic wall thickness divided by the end-diastolic wall thickness, and a regional myocardial work index described previously.30
Regional Myocardial Blood Flow
Radiolabeled microspheres (15-μm diameter, 141Ce, 114In, 51Cr, 113Sn, 103Ru, 95Nb, or 46Sc; NEN, Du Pont Co) were injected into the coronary perfusion circuit (1 to 2×105 suspended in 1 mL saline) to determine the regional myocardial blood flow and its distribution throughout the left anterior descending coronary artery perfusion bed. This procedure for the determination of blood flow has been validated extensively.30 Blood flow to the tissue at the site of the ultrasonic crystals was reported, and this piece of tissue was divided into transmural thirds of approximately equal thickness.
In addition, total subendocardial blood flow to the left anterior descending coronary artery–perfused territory was also measured and related to myocardial infarct size. To determine the threshold for infarct development, the averaged blood flow of all viable (triphenyltetrazolium chloride [TTC]–positive; see below) subendocardial tissue pieces of the left anterior descending coronary artery–perfused territory was determined.
Regional Myocardial Metabolism
Oxygen content was measured by using anaerobically sampled blood drawn simultaneously from the coronary vein and an artery (Cavitron/LexO2-Con-k, Dr B.G. Schlag). Oxygen consumption of the anterior myocardial wall (MV̇o2) was calculated by multiplying the arterial−coronary venous oxygen difference by the mean transmural blood flow at the crystal site. Lactate was measured in simultaneously drawn coronary venous and arterial blood samples by using enzymatic dehydrogenation and subsequent photometry of NADH,31 and lactate consumption was calculated by multiplying the arterial−coronary venous difference by the mean transmural blood flow at the crystal site. In addition, the arterial and coronary venous blood pH was measured (Radiometer).
Transmural myocardial biopsies (≈10 mg each) were taken with a modified dental drill from the left anterior descending coronary artery perfusion bed for analysis of ATP and CP contents. Care was taken to ensure that the biopsies originated from within the left anterior descending coronary artery perfusion territory (using epicardial arteries as landmarks) but distal to the site of the ultrasonic dimension gauges and blood flow measurements. Samples requiring >1 to 2 seconds for acquisition were not used for this analysis. The tissue samples were stored at −70°C. For homogenization, tissue samples were cooled in liquid nitrogen and ground together with 100 μL frozen 0.3 mol/L perchloric acid (Micro Dismembrator, B. Braun Melsungen). After thawing and rinsing with 400 μL water, the precipitated protein was removed by centrifugation, and the supernatant was neutralized with a solution of 0.89 mol/L KOH, 0.17 mol/L K2SO4, and 57 mmol/L Tris.
High-Performance Liquid Chromatographic Analysis
Aliquots of the neutralized supernatant were chromatographed at a flow of 1 mL/min on a Protein Pak DEAE 5PW column (Waters Millipore Corp). A linear gradient starting with 100% buffer A (3 mmol/L Tris/H2SO4, pH 9.0) reached 100% buffer B (3 mmol/L Tris/H2SO4, 290 mmol/L K2SO4, pH 9.0) at 7 minutes. CP and ATP were eluted at 6.8 and 11.5 minutes, repectively. The UV detector (wavelength, 214 nm; M 440) and the peak integration software (maxima 820) were obtained from Waters Millipore. To compare myocardial CP and ATP contents obtained in the present study by high-performance liquid chromatography (HPLC) with results previously obtained by bioluminescence,18 27 simultaneous measurements were performed in 28 tissue samples. The regression lines for myocardial CP content (HPLC=1.015*bioluminescence−0.051, r=.97) and myocardial ATP content (HPLC=0.973*bioluminescence+0.074, r=.98) obtained from simultaneous measurements with HPLC and bioluminescence were not different from the respective lines of identity determined by ANCOVA.
At the end of each study, the heart was removed and sectioned from base to apex into five transverse slices in a plane parallel to the atrioventricular groove. The tissue slices were immersed in a 0.09 mol/L sodium phosphate buffer (pH 7.4) containing 1.0% TTC (Sigma) and 8% dextran (molecular weight, 77 800) for 20 minutes at 37°C to facilitate the identification of infarcted tissue.
In group 1 (n=8), after control measurements, coronary inflow was reduced to achieve an ≈90% reduction of the anterior myocardial work index, and this level of hypoperfusion was maintained for 90 minutes. Coronary inflow then averaged 30% of the control baseline flow. At 5 and 85 minutes of the ischemic period, pairs of arterial and coronary venous blood samples were simultaneously withdrawn. During the blood sampling, microspheres were injected into the left anterior descending coronary artery perfusion system for the measurement of regional myocardial blood flow, and hemodynamic and regional dimension data were recorded. Coronary perfusion pressure was continously monitored during the microsphere injection to ensure that it was unaffected by the injection. Immediately after the microsphere injection, myocardial biopsies were taken. A set of measurements was obtained within 2 to 3 minutes. After 90 minutes of ischemia, the myocardium was reperfused for 120 minutes to allow the identification of infarcted tissue. Data for group 1 have been published previously.27
In group 2 (n=15), a 10-minute no-flow ischemic period was followed immediately by 80 minutes of low-flow ischemia and 120 minutes of reperfusion. The coronary inflow during the low-flow ischemia was set to 30% of the control baseline flow, analogous to that in group 1. Under control conditions and at 5 and 80 minutes of the low-flow ischemic period, measurements of systemic hemodynamics, regional myocardial blood flow, function, and metabolism were performed.
In group 3 (n=8), after control measurements, glibenclamide was given at a dosage of 0.5 mg/kg IV, followed by a continous infusion of 50 μg/min. This dose of glibenclamide has previously been shown to effectively block ATP-dependent potassium channels.27 Ten minutes after the start of the glibenclamide infusion, measurements were repeated. Except for the glibenclamide infusion, the protocol of group 3 was identical to that of group 2.
At the end of each study, the digital reading of the roller pump was calibrated by collecting arterial blood in a graduated cylinder.
Data Analysis and Statistics
Hemodynamic data were recorded on an eight-channel recorder (Gould MK 200A) and stored directly on the hard disk of an AT-type computer. Hemodynamic and functional parameters were digitized and recorded over a 20-second period during each microsphere injection (≈33 consecutive beats over at least two complete respiratory cycles) by using cordat ii software (Triton Technologies, Inc).32 Hemodynamic parameters reported are heart rate, LV end-diastolic and peak pressure, LV dP/dtmax, and mean left anterior descending coronary artery pressure. Regional wall function was assessed as anterior systolic wall thickening and the anterior myocardial work index. Metabolic parameters include the myocardial contents of ATP and CP, the consumption of oxygen and lactate (positive value indicates myocardial uptake), and the arterial and coronary venous pH. Calculation of all hemodynamic parameters was performed on a beat-to-beat basis, and data were then averaged.
Statistical analysis was performed by using systat software. Hemodynamic and metabolic data were subjected to a two-way ANOVA for repeated measures, accounting for the three groups of pigs and the time course of the experiment. No data for regional myocardial blood flow, myocardial oxygen, and lactate consumption were obtained during no-flow ischemia in groups 2 and 3. In this heparinized preparation, only a limited number of biopsies can be taken. Therefore, in group 3, the biopsy before the glibenclamide infusion was avoided. For the comparison of myocardial ATP and CP contents before ischemia, data following glibenclamide infusion were determined for group 3. Area at risk, infarct size, and the averaged blood flow of viable subendocardial tissue were compared by one-way ANOVA. When significant differences were detected in either the one-way or two-way ANOVA, individual mean values were compared by using Tukey’s post hoc test. All data are reported as mean±SD, and a value of P<.05 was accepted as indicating a significant difference in mean values.
Linear regression analyses between subendocardial blood flow at 5 minutes of low-flow ischemia in the LV area at risk and infarct size (expressed as percentage of the area at risk) were performed for pigs of groups 1 through 3. This calculation was performed only for those pigs that developed myocardial infarction, since otherwise any such relation would be diluted rightward.20 Regression lines between infarct size and subendocardial blood flow in the three groups of pigs were compared by ANCOVA.
There were no significant differences in any measured parameter under control conditions among the three groups of pigs. In all pigs, heart rate was held constant throughout the experimental protocol by left atrial pacing, and regional systolic wall thickening of the posterior control wall remained stable throughout the experimental protocol.
Group 1: 90-Minute Low-Flow Ischemia
Hemodynamics and Blood Flow
Five minutes of low-flow ischemia reduced mean coronary arterial pressure and regional myocardial blood flow, particularly in the subendocardium. LV end-diastolic pressure tended to increase, while LV peak pressure and LV dP/dtmax tended to decrease. Prolonging ischemia to 90 minutes did not result in further significant changes in LV pressure, LV dP/dtmax, or regional myocardial blood flow.
Regional Myocardial Function
Anterior systolic wall thickening (Fig 1⇑) and the myocardial work index decreased by 85% to 90% after 5 minutes of low-flow ischemia. With prolongation of ischemia to 90 minutes, anterior systolic wall thickening tended to decrease further while the anterior myocardial work index remained unchanged.
Regional Myocardial Metabolism
At 5 minutes of low-flow ischemia, the myocardial CP and ATP contents were significantly reduced. Coronary venous pH (Fig 2⇑) and MV̇o2 were decreased, and myocardial lactate consumption was reversed to net lactate production. When ischemia was prolonged to 90 minutes, myocardial CP content had slightly recovered, while myocardial ATP content was decreased further. Coronary venous pH was increased to a value no longer significantly different from the control value. MV̇o2 remained unchanged, while lactate production tended to be attenuated.
Group 2: 10-Minute No-Flow Ischemia and 80-Minute Low-Flow Ischemia
Hemodynamics and Blood Flow
The cessation of coronary inflow increased LV end-diastolic pressure while LV peak pressure and LV dP/dtmax were reduced. During the first 5 minutes of low-flow ischemia, LV peak pressure and LV dP/dtmax increased once more but tended to remain below control values. LV end-diastolic pressure remained increased. Mean transmural myocardial and subendocardial blood flows at 5 minutes of the low-flow ischemia were decreased to values comparable to those in group 1. Prolonging the low-flow ischemia to 80 minutes (total ischemic time, 90 minutes) did not result in further significant changes in LV pressures, LV dP/dtmax, or regional myocardial blood flow.
Regional Myocardial Function
The anterior myocardial work index decreased to zero, and systolic wall thickening was reversed to systolic wall thinning (Fig 1⇑) during the 10 minutes of no-flow ischemia. At 5 minutes of low-flow ischemia, anterior systolic wall thickening and the anterior myocardial work index remained severely depressed. At the end of the low-flow ischemic period, both the anterior systolic wall thickening and the anterior myocardial work index had increased to values comparable to those in group 1.
Regional Myocardial Metabolism
Myocardial CP and ATP contents as well as coronary venous pH (Fig 2⇑) were decreased after the 10 minutes of no-flow ischemia. Five minutes after partial restoration of coronary inflow to values comparable to those in group 1, myocardial CP content was slightly increased, while the myocardial ATP content and coronary venous pH remained decreased. At 5 minutes of low-flow ischemia, MV̇o2 was decreased, and myocardial lactate consumption was reversed to net lactate production. With prolongation of the low-flow ischemia to 80 minutes, myocardial CP content remained unchanged, while the myocardial ATP content was further decreased. Coronary venous pH was increased once more to a value no longer different from the control value. MV̇o2 did not change further, and lactate production was attenuated.
Group 3: Glibenclamide and 10-Minute No-Flow Ischemia and 80-Minute Low-Flow Ischemia
Data are summarized in Table 3⇓ and Figs 1⇑ and 2⇑. Infusion of glibenclamide tended to increase LV peak pressure, mean coronary arterial pressure, and the anterior myocardial work index, while anterior systolic wall thickening remained unchanged. Also, MV̇o2 tended to increase after glibenclamide infusion. During ischemia, the changes in systemic hemodynamics, regional myocardial function, blood flow, and metabolism were comparable to those of group 2.
Time Course of Contractile Function
Data are summarized in Fig 3⇓. With the reduction in coronary inflow, the anterior myocardial work index decreased. This decrease in the anterior myocardial work index was more pronounced and occurred significantly faster in pigs with no-flow ischemia (groups 2 and 3) than in pigs with reduced, but persistent, coronary inflow (group 1). At 30 minutes of reperfusion, systolic wall thickening (group 1, 6.8±5.4%; group 2, 6.0±7.3%; and group 3, 5.4±4.2%) and the work index (group 1, 47±43 mm Hg · mm; group 2, 66±57 mm Hg · mm; and group 3, 58±48 mm Hg · mm) remained severely depressed but were not different between groups.
Myocardial Infarction: Regional Myocardial Blood Flow Versus Infarct Size
Data are summarized in Figs 4⇓ and 5⇓. The area at risk was comparable among the three groups of pigs (Fig 4⇓). After 90 minutes of severe low-flow ischemia (group 1), 13.2±8.9% of the area at risk was infarcted. Ten minutes of no-flow ischemia preceding the 80 minutes of low-flow ischemia reduced infarct size to 6.8±6.0% (P<.05). Blockade of ATP-dependent potassium channels with glibenclamide abolished this reduction in infarct size (16.7±8.3%).
In pigs of group 2, the blood flow of the viable subendocardial portions of the left anterior descending coronary artery–perfused territory averaged 0.06±0.02 mL · min−1 · g−1. This blood flow was lower than that of group 1 (0.11±0.04 mL · min−1 · g−1, P<.05) and group 3 (0.10±0.06 mL · min−1 · g−1, P=NS).
The relation between infarct size and subendocardial blood flow was different in group 2 pigs compared with control pigs (Fig 5⇑); glibenclamide abolished this difference.
The results of the present study demonstrate that in the in situ pig heart a brief episode of no-flow ischemia increases the tolerance to subsequent sustained severe low-flow ischemia. This effect is abolished by glibenclamide, indicating that activation of ATP-dependent potassium channels is involved in this cardioprotective mechanism.
Critique of Methods
The present experiments were performed in pigs, since both ischemic preconditioning4 23 26 27 and myocardial hibernation16 17 18 19 20 have been characterized in detail in this species. Although because of the virtual absence of collateral flow in pigs,25 30 infarct development closely resembles that observed in humans,25 occlusion of the proximal left anterior descending coronary artery may result in ventricular fibrillation and extensive infarction of the LV with subsequent pump failure. Indeed, in the present study, two pigs that received glibenclamide experienced ventricular fibrillation during the 10 minutes of no-flow ischemia and were therefore excluded from the data analysis.
In the present study, the proximal left anterior descending coronary artery was cannulated, resulting in a large area at risk (42% of the LV mass, on average). During the sustained ischemic period, the left anterior descending coronary artery was hypoperfused at low but maintained flow, resulting in a small infarct size when expressed as a percentage of the area at risk (13.2±8.9% in group 1). However, infarct size expressed as a percentage of the total LV mass in the present study averaged 6% in the control group and was thus comparable to that in a previous study using swine, with a total occlusion of only one distal left anterior descending coronary artery branch.4
A reduction in anterior myocardial work by 70% over 90 minutes can be tolerated without the development of irreversible tissue damage (myocardial hibernation).18 Further reductions in the anterior myocardial work index will induce myocardial infarction. In the present study, we attempted to investigate whether a brief episode of no-flow ischemia enhances myocardial tolerance to subsequent sustained low-flow ischemia and permits the development of myocardial hibernation. Therefore, blood flow during the sustained low-flow ischemia was reduced to a level that would normally result in some myocardial infarction.
Time Course of Contractile Function: Changes in Coronary Venous pH
In isolated buffer-perfused rabbit hearts, the initial decrease in contractile function was significantly faster in hearts undergoing no-flow ischemia than in hearts undergoing a 90% reduction of coronary inflow. This more rapid decrease in contractile function during no-flow ischemia was accompanied by a greater decrease in interstitial pH and therefore attributed to the faster development of myocardial acidosis. This faster decrease in contractile function, by better preserving myocardial energy stores, was thought to then allow the development of myocardial hibernation.22
In the present study, the decrease in the anterior myocardial work index was also more pronounced and occurred significantly faster (Fig 3⇑) in pigs undergoing an initial no-flow ischemia (groups 2 and 3) than in pigs with reduced, but persistent, coronary inflow (group 1). The changes in coronary venous pH, however, were comparable among the three groups of pigs (Fig 2⇑). Although we realize that analysis of coronary venous pH is only a crude measure of interstitial and, even more so, of cytosolic pH, our findings do not support the idea that changes in pH are primarily responsible for the changes in contractile function. The time course of changes in contractile function was also comparable in hearts with no-flow ischemia in the presence or absence of glibenclamide. Glibenclamide, however, abolished the infarct size reduction achieved by no-flow ischemia, supporting the idea that the beneficial effect of no-flow ischemia in mediating such infarct size reduction was not secondary to a faster or more pronounced decrease in contractile function.
Myocardial Hibernation Versus Ischemic Preconditioning: Myocardial Viability
In hibernating myocardium, necrosis is absent by definition. In ischemic preconditioned hearts, infarct size for a given regional myocardial blood flow is reduced compared with nonpreconditioned hearts when the duration of the sustained ischemic episode does not exceed 90 to 180 minutes.1 33 In the present study, infarct size was significantly reduced in hearts undergoing 10 minutes of no-flow ischemia followed by 80 minutes of low-flow ischemia when compared with control hearts undergoing only 90 minutes of low-flow ischemia. Using the same animal model, we have previously demonstrated that blockade of ATP-dependent potassium channels abolishes the cardioprotection achieved by ischemic preconditioning27 but not that achieved by myocardial hibernation.28 In the present study, blockade of ATP-dependent potassium channels with glibenclamide abolished the infarct size reduction achieved by the preceding 10 minutes of no-flow ischemia, therefore pointing toward ischemic preconditioning rather than myocardial hibernation as the underlying mechanism of the observed cardioprotective effect.
Myocardial Hibernation Versus Ischemic Preconditioning: Metabolism
Both hibernating and ischemic preconditioned myocardium are characterized not only by preserved myocardial viability but also by attenuation of metabolic alterations during prolonged ischemia. In the present study, myocardial lactate production tended to be attenuated in all groups of pigs when ischemia was prolonged to 90 minutes, indicating recovery of glycolytic metabolism over time. In contrast, myocardial CP content remained severely depressed.
In preconditioned hearts, the accumulation of lactate is slowed down during the prolonged ischemic period compared with nonpreconditioned hearts.9 In addition, the breakdown of myocardial CP content is slowed down early during the prolonged ischemic period in preconditioned hearts1 10 ; however, the absolute CP content reached at the end of the prolonged ischemic period may not be different between preconditioned and nonpreconditioned myocardium.10 27 In hibernating myocardium, the myocardial CP content recovers to near control values during persistent ischemia,15 16 17 18 and lactate production is attenuated.18 19
Thus, from the energetic state the cardioprotection achieved by the no-flow ischemic period appears to be more closely related to ischemic preconditioning than to myocardial hibernation.
Preconditioning Without Intermittent Reperfusion
In a study by Koning et al23 in anesthetized pigs, infarct size in pigs after a 60-minute coronary artery occlusion was reduced by a preceding 30-minute partial coronary stenosis, sufficient to decrease coronary blood flow by 70% when compared with pigs only undergoing 60 minutes of coronary artery occlusion. Also, in isolated buffer-perfused rabbit hearts, 10 minutes of global hypoxia decreased infarct size after the subsequent 30 minutes of regional no-flow ischemia, demonstrating that intermittent reoxygenation is not required for ischemic preconditioning.34 In the present study, pigs subjected to 10 minutes of no-flow ischemia preceding 80 minutes of low-flow ischemia demonstrated reduced infarct size compared with pigs only undergoing 90 minutes of sustained low-flow ischemia. In contrast, Ovize et al,24 using a 15-minute partial coronary stenosis in dogs sufficient to reduce coronary blood flow by 50%, did not observe reduced infarct size after a subsequent 60 minutes of total coronary artery occlusion, when intermittent reperfusion was avoided.
The reasons for the differences among the cited and the present studies remain unclear. One explanation, although entirely speculative, relates to the severity of blood flow reduction and the associated production and/or release of cardioprotective substances during the initial ischemic period. From the classic ischemic preconditioning protocol using a single cycle of total ischemia followed by reperfusion, it is well established that a 5-minute ischemic period is required to increase the resistance to lethal myocyte injury after more prolonged periods of myocardial ischemia,5 35 suggesting that a certain threshold level of cardioprotective substances is required to reduce lethal myocyte injury. Whereas in the study by Koning et al23 as well as in the present study the blood flow reduction during the initial ischemic period was very severe (>70% reduction in coronary inflow in swine), Ovize et al24 used a more moderate ischemia (50% reduction of coronary inflow in dogs) to precondition the heart. Therefore, the production and/or release of cardioprotective substances in the study by Ovize et al might have remained below the critical threshold level necessary to reduce lethal myocyte injury.
In any event, however, it remains unclear why the first 10 minutes of ischemia at a 70% reduction in coronary inflow in the present study did not per se induce a sufficient production and/or release of cardioprotective substances and thus precondition the heart. Alternatively, the first 10 minutes of ischemia at a 70% reduction in coronary inflow did in fact precondition, and infarct size could be much larger without the preconditioning induced by the first 10 minutes of 70% flow reduction, but this hypothesis cannot be tested because there is no control group for comparison.
In conclusion, a short episode of no-flow ischemia can increase the tolerance to sustained severe myocardial low-flow ischemia. Although we thus confirm the previous observation by Ferrari et al,22 this cardioprotective effect is mediated by activation of ATP-dependent potassium channels and therefore relates to ischemic preconditioning rather than to myocardial hibernation.
Dr Wallbridge was supported by a British Heart Foundation International Research Fellowship. We thank Claus Martin, PhD, for the chemical analyses and Petra Gres, Ursula Prägler, and Anita van de Sand for their technical support.
- Received January 27, 1995.
- Accepted March 13, 1995.
- © 1995 American Heart Association, Inc.
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