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(Circulation. 1997;95:1278-1285.)
© 1997 American Heart Association, Inc.

Articles

Increased Tolerance of the Chronically Hypoxic Immature Heart to Ischemia

Contribution of the K_ATP Channel

John E. Baker, PhD; Brian D. Curry, MS; Gordon N. Olinger, MD; Garrett J. Gross, PhD

the Departments of Pharmacology and Toxicology (J.E.B., B.D.C., G.J.G.) and Cardiothoracic Surgery (J.E.B., G.N.O.), Medical College of Wisconsin, Milwaukee.

Correspondence to John E. Baker, PhD, Department of Cardiothoracic Surgery, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail jbaker{at}post.its.mcw.edu

	Abstract

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Background Hypoxia from birth in immature rabbits increases the tolerance of isolated hearts to ischemia compared with age-matched normoxic rabbits. We determined whether this increased tolerance to ischemia was due to an alteration in the ATP-sensitive potassium (K_ATP) channel and whether increased K_ATP channel activation was associated with increases in intracellular lactate.

Methods and Results Isolated immature rabbit hearts (7 to 10 days old) were perfused with bicarbonate buffer at 39°C in the Langendorff mode at a constant pressure. Saline-filled latex balloons were placed in the left and right ventricles for measurement of developed pressure. A K_ATP channel agonist (bimakalim) or a K_ATP channel antagonist (glibenclamide) was added 15 minutes before a global ischemic period of 18 minutes, followed by 35 minutes of reperfusion. Rabbits raised from birth in hypoxic conditions (FIO₂=0.12) displayed significantly enhanced recovery of developed pressure. The right ventricle was more tolerant of ischemia than the left ventricle in normoxic and hypoxic hearts. Bimakalim (1 µmol/L) increased the recovery of left ventricular developed pressure in normoxic hearts to values not different from those of hypoxic controls (43±3% to 67±5%) and slightly increased developed pressure in hypoxic hearts (67±5% to 72±5%). Glibenclamide (3 µmol/L) abolished the cardioprotective effect of hypoxia (67±5% to 43±5%). Constant-flow studies indicated that the effects of bimakalim and glibenclamide were independent of their actions on coronary flow. Ventricular lactate and lactate dehydrogenase concentrations were elevated in hypoxic hearts compared with normoxic control hearts.

Conclusions Increased tolerance to ischemia exhibited by chronically hypoxic rabbit hearts is associated with increased activation of the K_ATP channel. This increased K_ATP activity may be the result of increased intracellular concentrations of lactate.

Key Words: cardiovascular diseases • heart defects, congenital • hypoxia • ions • ischemia

	Introduction

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We have developed a rabbit model of hypoxia from birth that simulates the essential characteristics of cyanotic congenital heart disease and have demonstrated that hypoxia from birth increases the tolerance of the heart to ischemia compared with age-matched normoxic control rabbits.¹ The mechanism for adaptation to prolonged hypoxia that subsequently confers increased tolerance to myocardial ischemia is currently unknown.

We recently demonstrated that a brief period of acute hypoxic perfusion of the dog heart before a subsequent period of sustained ischemia protects the heart against injury as defined by a reduction in the ratio of infarct size to the area at risk.² The protective effect of acute hypoxic preconditioning was blocked by the ATP-sensitive potassium channel (K_ATP) antagonist glibenclamide. Tajima and coworkers³ recently found that chronic hypoxia in a rat model protected the heart against subsequent ischemia, although no mechanisms were uncovered to explain the protective effect of chronic hypoxia.

We hypothesized that K_ATP is responsible for conferring increased protection against ischemia in hearts previously exposed to chronic hypoxia. Lactate has been shown to activate the K_ATP channel in myocytes,⁴ and hypoxia has been shown to result in the elevation of ventricular lactate.¹ The objectives of our study were to determine whether increased tolerance to ischemia in rabbit hearts that are hypoxic from birth is the result of increased activation of the K_ATP channel and whether such alterations are associated with elevations in myocardial lactate concentrations.

	Methods

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Animals
Animals used in this study received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and Guidelines for the Care and Use of Laboratory Animals (DHEW [DHHS] Publication No. NIH 85-23, revised 1985).

Creation of Hypoxia From Birth
Pregnant New Zealand White rabbits were obtained from a commercial breeder. Throughout the study, the mother remained in a normoxic environment (FIO₂=0.21). For the hypoxic studies, the kits were born in a normoxic environment and immediately after their first feeding were transferred to a hypoxic environmental chamber where FIO₂ had been previously reduced to 0.12. The oxygen in the chamber was maintained at this level throughout the remainder of the study. The kits were transferred in their nesting box back to their mother once a day for 20 minutes for feeding and were then returned to the environmental chamber. For the normoxic studies, the kits were raised under identical conditions except that FIO₂ in the environmental chamber remained at 0.21 for the duration of the study. Table 1 gives the body weight and temperature data.

View this table: [in this window] [in a new window]   Table 1. Differences in Blood Gases and Several Gross and Biochemical Markers in Rabbits Born in a Normoxic or Hypoxic Environment

Validation of Hypoxia From Birth
Gross and Extracellular Changes
Arterial blood samples were withdrawn from the cannulated abdominal aorta for blood gas, glycolytic metabolite, hemoglobin, and hematocrit analysis in the rabbits (n=8 per group) raised from birth under hypoxic (FIO₂=0.12) conditions within the environmental chamber. Hypoxic rabbits breathed a mixture of 12% oxygen and 88% nitrogen during blood collection. In a parallel study, arterial blood samples were withdrawn from the rabbits (n=6 per group) allowed to develop from birth under normoxic (FIO₂=0.21) conditions. Normoxic rabbits breathed a mixture of 21% oxygen and 79% nitrogen during blood collection. Blood pH, carbon dioxide tension (PCO₂), oxygen tension (PO₂), and hemoglobin concentrations were measured with an ABL-2 blood gas analyzer (Radiometer). Values for bicarbonate, total carbon dioxide, and oxygen saturation were then derived from these data. The values for arterial oxygen tension (PaO₂) were corrected for body temperature of the rabbit (39°C) by the method of Severinghaus.⁵ Hematocrit was measured as percent packed cell volume after centrifugation of whole blood in a microcapillary tube (Sherwood Medical). Body temperature was measured with a needle thermometer inserted into the abdomen. Glucose and lactate levels were determined enzymatically with an Extachem autoanalyzer (Eastman Kodak). The heart was then excised, and its mass and the extent of right ventricular hypertrophy were determined.⁶

Intracellular Changes
To determine whether tissue concentrations of lactate were elevated in hearts from hypoxic animals, we determined the myocardial concentrations of lactate, lactate dehydrogenase, and the relative distribution of the H (LDH₁) and M (LDH₅) isoforms of lactate dehydrogenase (LDH) present in the free wall of the left ventricle of normoxic and hypoxic neonates at 9 days of age. The relative distribution of the H and M subunits of LDH are regulated in part by the ambient oxygen concentration in the cell and have been used as an index of the ability of glycolysis to produce energy by aerobic and anaerobic means, respectively.¹ Hearts (n=8/group) were perfused for 30 minutes with oxygenated Krebs-Henseleit bicarbonate buffer,⁷ and the free wall of the left ventricle was excised. The methodologies used to determine the concentrations of lactate and the different isoforms of LDH have been described previously.¹ Enzyme activities were expressed as international units per gram of wet weight. Fractions of LDH isoenzymes were calculated from the scanned peaks.

Perfusion System
The isolated rabbit heart model was used for these studies and was instrumented as previously described.⁸ Heparin (150 IU/kg IP) was administered before anesthesia. Anesthesia was then induced and maintained with halothane (4% and 1% to 2%, respectively). After 1 minute, the heart was rapidly excised and placed in cold (4°C) perfusion medium. Within 30 seconds, the aorta was attached to a stainless steel cannula, the pulmonary artery was incised to permit adequate coronary drainage, and the heart was perfused at 39°C in the Langendorff mode⁹ at a constant perfusion pressure of 43 mm Hg, which is equivalent to the mean aortic pressure for the age of the rabbits at the time of study.¹ Several other groups of hearts were perfused at a constant flow. Saline-filled latex balloons (Biomedix) were placed in the left and right ventricular cavities and connected through rigid saline-filled catheters to separate pressure transducers. A three-way tap located immediately above the site of cannulation allowed the entire perfusate to be diverted away from the heart to produce global, no-flow ischemia. Reperfusion was achieved by repositioning the tap to allow perfusate to be delivered to the heart. The heart and perfusion fluids were immersed in nongassed physiological saline solution within temperature-controlled chambers to maintain the myocardium at 39°C, normothermia for the rabbit.

Perfusion Media
The standard perfusate used was Krebs-Henseleit bicarbonate buffer⁷ (mmol/L): NaCl 118.5, NaHCO₃ 25.0, KCl 4.8, MgSO₄.6H₂O 1.2, KH₂PO₄ 1.2, pH 7.4 (when gassed with 95% O₂/5% CO₂), in which the calcium content is reduced to 1.8. Glucose (11.1 mmol/L) was also added to the perfusate. Before use, all perfusion fluids were filtered through cellulose acetate membranes with a 5.0-µm pore size. To this perfusate, we added the K_ATP antagonist or K_ATP agonist as needed.

Assessment of Ventricular Function
Left and right ventricular function was monitored continuously throughout each experiment. A latex balloon filled with boiled and degassed saline was inserted into the left ventricle through an incision in the left atrium and secured in place with two 4-0 silk sutures, one in the mitral annulus and the other in the interatrial septum. This reliably held the balloon in place and prevented herniation through the mitral annulus. A second balloon was then inserted across the pulmonic valve into the right ventricle and secured in place with a ligature. Each balloon was slightly larger than the ventricular cavity. The balloons were then connected with rigid fluid-filled catheters to separate pressure transducers (model 8148, Deseret Medical) for measurement of ventricular pressures and heart rate.

The transducer outputs were amplified with a universal signal conditioner (model 20-4615-58, Gould) and recorded on an analog chart recorder (Astromed). End-diastolic pressure was initially set to 3 mm Hg for 2 minutes, and developed pressure (systolic minus diastolic pressure) was measured during steady-state levels of function. The balloons were progressively inflated with a microsyringe to set end-diastolic pressures to 8 mm Hg for the left ventricle and 4 mm Hg for the right ventricle, and developed pressure was recorded during steady-state conditions. Coronary flow rate was measured throughout the experiment by timed collections of the coronary effluent from the right side of the heart into a graduated cylinder. Coronary flow rate was expressed as milliliters per minute.

Perfusion Sequence
Constant-Pressure Studies
The following experiments were performed in a random manner in six groups to test the null hypothesis that tolerance to ischemia of developing rabbit hearts, either normoxic or hypoxic from birth, is not affected by changes in activation of the K_ATP channel. The six experimental groups were as follows: group 1, normoxic; group 2, normoxic plus glibenclamide (3 µmol/L); group 3, normoxic plus bimakalim (1 µmol/L); group 4, hypoxic; group 5, hypoxic plus glibenclamide (3 µmol/L); and group 6, hypoxic plus bimakalim (1 µmol/L). Fig 1 illustrates the experimental protocol. Immediately after aortic cannulation, hearts were perfused at a constant pressure of 43 mm Hg in the Langendorff mode for 30 minutes, during which time balloons were placed in both the left and right ventricles. Biventricular function and coronary flow rate were then recorded under steady-state conditions. Hearts were then perfused with either a K_ATP agonist (bimakalim 1 µmol/L) or a K_ATP antagonist (glibenclamide 3 µmol/L) for another 15 minutes before an 18-minute period of global, no-flow ischemia at 39°C. After the ischemic period, hearts were reperfused for 35 minutes, during which time the various indexes of cardiac function were again measured under steady-state conditions. Thus, each heart served as its own control. Dose-response studies (4 hearts per group) performed before these experiments indicated that these doses of bimakalim and glibenclamide were optimal for increasing the recovery of left ventricular developed pressure in normoxic hearts and antagonizing the protective effect of chronic hypoxia in hypoxic hearts, respectively.

View larger version (14K): [in this window] [in a new window]   Figure 1. Illustration of the experimental protocol used to study chronic hypoxia and KATP channels in the isolated immature heart. RV indicates right ventricular; LV, left ventricular.

Constant-Flow Studies
To separate the effects of K_ATP agonists and K_ATP antagonists on coronary flow from those on contractile function, we repeated the previous experimental protocols under conditions of constant flow in an additional six experimental groups. The six experimental groups were as follows: group 7, normoxic; group 8, normoxic plus glibenclamide (3 µmol/L); group 9, normoxic plus bimakalim (1 µmol/L); group 10, hypoxic; group 11, hypoxic plus glibenclamide (3 µmol/L); and group 12, hypoxic plus bimakalim (1 µmol/L). For these studies, hearts were perfused initially at constant pressure for 20 minutes until steady-state levels of developed pressure and coronary flow rate were achieved. Coronary perfusion was then continued at a constant flow with a peristaltic pump (Masterflex model 7523-30, Cole Palmer) at the identical flow rate present during the preceding period of constant pressure perfusion. During the reperfusion period, hearts were perfused at constant flow throughout reperfusion and recovery of cardiac function measured.

Statistical Analysis
Recovery of developed pressure was expressed as a percentage of its predrug value. A minimum of six hearts was used for each of the 12 conditions studied, and the results are expressed as mean±SD. Statistical analysis was performed by use of repeated-measures ANOVA, with the Greenhouse-Geisser adjustment used to correct for the inflated risk of a type I error.¹⁰ After ANOVA, the data were corrected for multiple comparisons. Significance was accepted at a level of P<.05.

	Results

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Validation of Hypoxia From Birth
Gross and Extracellular Changes
Table 1 shows the effect that hypoxia from birth in the developing rabbit exerted on body weight, heart weight, body temperature, components of the arterial blood gas system, and circulating lactate and glucose levels compared with age-matched normoxic control rabbits. Body weight was not different in the hypoxic neonates at 7 to 10 days of age compared with that of age-matched normoxic control rabbits. Body temperature in the hypoxic rabbits was consistently lower than that observed in normoxic rabbits. However, the differences between the two groups were not significantly different. In all chronically hypoxic neonates, arterial PO₂ and oxygen saturation were significantly lower compared with age-matched normoxic controls. There were no hypoxia-induced changes in arterial bicarbonate, total carbon dioxide, base excess, or pH for 7- to 10-day-old rabbits. Hematocrit, hemoglobin, and lactate levels were elevated in hypoxic rabbits compared with normoxic control rabbits. There were no differences in arterial glucose levels between hypoxic and normoxic rabbits. Mortality rates for hypoxic rabbits at 7 to 10 days of age were comparable to those of normoxic control rabbits (10% to 15%).

Gross and microscopic analyses demonstrated that rabbits raised from birth in our hypoxic chamber develop anatomic cardiovascular systems that are similar to their human neonatal counterparts with cyanotic congenital heart disease. In 7 of 9 hypoxic rabbits examined, the ductus arteriosus was anatomically patent. None of 10 normoxic rabbits examined had a patent ductus arteriosus. Furthermore, microscopic analysis of the right ventricle revealed both hypertrophy and hyperplasia of the myocardial cells consistent with that observed in children with cyanotic congenital heart disease.

The ratio of the right ventricular free wall weight to the combined weights of the septum and left ventricular free wall was 0.60±0.11 (mean±SD) immediately after birth. Table 1 shows that this value remained unchanged in hypoxic hearts at 7 to 10 days of age but decreased in normoxic age-matched control hearts (P<.05).

Intracellular Changes
Table 1 shows that hypoxia resulted in a 200% increase in ventricular lactate and a 135% increase in ventricular LDH compared with normoxic controls. Fig 2 shows that the predominant LDH isoform in both hypoxic and normoxic ventricles was the H (LDH₁) subunit. The imposition of hypoxia from birth caused a shift in isoenzyme distribution, resulting in an increase in the M-subunit fraction and a decrease in the H-subunit fraction for all five isoforms compared with normoxic hearts.

View larger version (15K): [in this window] [in a new window]   Figure 2. Hypoxia-induced changes in expression of lactate dehydrogenase isoforms. Open bars represent data from normoxic hearts; hatched bars, data from hypoxic hearts. Data are mean±SD (n=8 hearts per group). *P<.05, normoxic vs hypoxic isoforms.

Preischemic Function
Table 2 gives baseline functional data for immature rabbit hearts. Coronary flow rate in hypoxic hearts was unchanged compared with normoxic control hearts. Right ventricular developed pressure was higher in hypoxic ventricles compared with normoxic control hearts. The K_ATP channel antagonist glibenclamide (3 µmol/L) depressed heart rate, coronary flow rate, and developed pressures in both the left and right ventricles in normoxic and hypoxic hearts under nonischemic conditions. The K_ATP channel antagonist bimakalim (1 µmol/L) increased preischemic heart rate and coronary flow rate in normoxic and hypoxic hearts. Bimakalim (1 µmol/L) depressed preischemic left ventricular developed pressure in hypoxic but not normoxic hearts.

View this table: [in this window] [in a new window]   Table 2. Hemodynamic Values for Each Group From Constant-Pressure Studies

The hemodynamic stability of the model was assessed by perfusing six nontreated normoxic and hypoxic hearts in the Langendorff mode for 120 minutes with nonrecirculating perfusate. No statistically significant changes were found with the use of ANOVA in developed pressure or heart rate from the values reported in Table 2 until after 85 minutes of perfusion. In the protocol that tested our hypothesis, hearts were perfused in the Langendorff mode for a maximum of 80 minutes, well within the stability limits of the preparation.

Postischemic Function
Constant-Pressure Studies
Table 2 and Fig 3 show that hearts from hypoxic rabbits perfused at constant pressure were more tolerant of ischemia than normoxic control hearts. The right ventricle was also more tolerant of ischemia than the left ventricle in both normoxic and hypoxic hearts. Recovery of postischemic left ventricular developed pressure in normoxic and hypoxic hearts was 43±3% and 67±5%, respectively. The K_ATP antagonist glibenclamide (3 µmol/L) did not affect recovery of developed pressure in normoxic hearts (42±3%) but decreased recovery in hypoxic hearts from 67±5% to 43±5% (Fig 4). Thus, glibenclamide was able to decrease the extent of recovery in hypoxic hearts to that of drug-free normoxic controls. In contrast, the K_ATP agonist bimakalim (1 µmol/L) increased recovery in normoxic hearts from 43±3% to 67±5% (Fig 5). Bimakalim did not significantly increase recovery of function in hypoxic hearts (72±5%). Thus, bimakalim increased the extent of recovery in normoxic hearts to that observed in hypoxic hearts. Recovery of function in the right ventricle for all drug-treated groups paralleled the changes observed in the left ventricle.

View larger version (14K): [in this window] [in a new window]   Figure 3. Postischemic recovery of function in left and right ventricles after 35 minutes of constant-pressure reperfusion in groups 1 and 4. Open bars represent data from the right ventricle; solid bars, data from the left ventricle. Data are mean±SD (n=6 hearts per group). *P<.05, left ventricle vs right ventricle; +P<.05, normoxic vs hypoxic.

View larger version (16K): [in this window] [in a new window]   Figure 4. Results after a 15-minute pretreatment period with glibenclamide before 18 minutes of global ischemia and 35 minutes of reperfusion in groups 2 and 5. Open bars represent data from control hearts; hatched bars, data from glibenclamide-treated (3 µmol/L) hearts. Data are mean±SD (n=6 hearts per group). LV indicates left ventricular. *P<.05, control vs glibenclamide; +P<.05, normoxic vs hypoxic.

View larger version (19K): [in this window] [in a new window]   Figure 5. Results after a 15-minute pretreatment period with bimakalim before 18 minutes of global ischemia and 35 minutes of reperfusion in groups 3 and 6. Open bars represent data from control hearts; hatched bars, data from bimakalim-treated hearts (1 µmol/L). Data are mean±SD (n=6 hearts per group). LV indicates left ventricular. *P<.05, control vs bimakalim; +P<.05, normoxic vs hypoxic.

Experiments performed in six hearts with PEG (0.03% vol/vol in NaOH/95% ethanol) as the vehicle for glibenclamide and in six hearts with dimethyl sulfoxide (0.02% vol/vol in Krebs buffer) as the vehicle for bimakalim demonstrated that these agents did not exert any effect on developed pressure, heart rate, or coronary flow rate (with ANOVA) from the preischemic predrug values reported in Table 2. Experiments performed in six hearts demonstrated that the cardiodepressive effect of glibenclamide (3 µmol/L) on developed pressure as shown in Table 2 was reversed within 10 minutes from the onset of perfusion with drug-free buffer.

Constant-Flow Studies
There was greater within-group variation in the results observed for the constant-flow studies in groups 7 through 12 compared with the results of the constant-pressure studies observed in groups 1 through 6. Table 3 and Fig 6 show that hearts from hypoxic rabbits perfused at constant flow were more tolerant of ischemia than normoxic controls. Glibenclamide (3 µmol/L) did not affect recovery of developed pressure in normoxic hearts (51±15% versus 42±17%) but decreased recovery in hypoxic hearts from 68±11% to 51±11%. Table 3 and Fig 7 show that bimakalim (1 µmol/L) increased recovery in normoxic hearts from 51±15% to 80±15% and increased recovery in hypoxic hearts from 68±11% to 83±11%. Thus, the overall direction of change effected by glibenclamide and bimakalim on recovery of postischemic function under conditions of constant-flow perfusion were the same as for those observed under conditions of constant-pressure perfusion.

View this table: [in this window] [in a new window]   Table 3. Hemodynamic Values for Each Group From Constant-Flow Studies

View larger version (18K): [in this window] [in a new window]   Figure 6. Results after a 15-minute pretreatment period with glibenclamide at constant-flow perfusion before 18 minutes of global ischemia and 35 minutes of reperfusion in groups 8 and 11. Open bars represent data from control hearts; hatched bars, data from glibenclamide-treated (3 µmol/L) hearts. Data are mean±SD from a minimum of eight hearts in each group. LV indicates left ventricular. *P<.05, control vs glibenclamide; +P<.05, normoxic vs hypoxic.

View larger version (22K): [in this window] [in a new window]   Figure 7. Results after a 15-minute pretreatment period with bimakalim at constant-flow perfusion before 18 minutes of global ischemia and 35 minutes of reperfusion in groups 9 and 12. Open bars represent data from control hearts; hatched bars, data from bimakalim-treated hearts (1 µmol/L). Data are mean±SD from a minimum of eight hearts in each group. LV indicates left ventricular. *P<.05, control vs bimakalim; +P<.05, normoxic vs hypoxic.

	Discussion

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We have demonstrated that in rabbits, chronic exposure to hypoxia from birth increases the tolerance of the heart to subsequent ischemia compared with age-matched normoxic control hearts. Bimakalim, an ATP-sensitive potassium channel opener, markedly enhanced the recovery of left ventricular developed pressure in normoxic hearts to that observed in hypoxic hearts. Bimakalim only slightly increased functional recovery in hypoxic hearts. Glibenclamide, an ATP-sensitive potassium channel antagonist, abolished the cardioprotective effect observed in rabbits chronically exposed to hypoxia from birth. Glibenclamide did not affect the recovery of function in normoxic hearts. The observed effects of bimakalim and glibenclamide on recovery of postischemic function were independent of their actions on coronary flow. Ventricular lactate and LDH concentrations were elevated in hypoxic hearts compared with those observed in normoxic controls. These results suggest that enhanced activation of the K_ATP channel observed in hearts obtained from rabbits exposed to a hypoxic environment for 7 to 10 days after birth results in an enhanced tolerance to an ischemic insult. The mechanism responsible for the increased K_ATP activity may involve increased cellular concentrations of lactate, a substance previously shown to activate K_ATP channels in cardiac myocytes.⁴

Creation of Hypoxia From Birth
A reproducible, nonsurgical model of cyanosis from birth has been developed by this laboratory in which the myocardium is chronically perfused with hypoxic blood. Our model has proved to simulate the essential characteristics of cyanotic congenital heart disease. The model is characterized by decreased arterial oxygen levels, polycythemia, right ventricular hypertrophy, decreased weight gain, and overall failure to thrive,¹ characteristics similar to those seen in children with cyanotic congenital heart disease.¹¹ The resulting changes in the physiology and biochemistry of the heart are achieved without surgical manipulation. With this model, we have shown that hypoxia from birth in the immature rabbit increases the tolerance of the isolated heart to subsequent ischemia compared with age-matched normoxic control hearts during early postnatal development.

Contribution of the K_ATP Channel
Acute hypoxia in dogs² or chronic hypoxia in rats³ before subsequent ischemia has been shown to be cardioprotective. The present study demonstrates that chronic hypoxia before an acute ischemic event is also cardioprotective in immature rabbit hearts. The cardioprotective effect of hypoxic adaptation most likely results from oxygen deprivation because normoxic perfusion before the study did not increase tolerance to subsequent ischemia. Our study is the first to suggest a role for the K_ATP channel in long-term adaptation to hypoxic stress.

The evidence supporting a role for the K_ATP channel is based on the observation that the cardioprotective effects resulting from adaptation to chronic hypoxia are blocked by the K_ATP channel antagonist glibenclamide at a concentration that has no effect on the recovery of function in normoxic hearts. Furthermore, the K_ATP channel opener bimakalim produced a marked cardioprotective effect in normoxic hearts but did not significantly add to the cardioprotection already present in hearts chronically hypoxic from birth. These results suggest that K_ATP channels are nearly maximally activated in these hypoxic hearts, so that no further protection would be expected in the presence of a K_ATP channel opener such as bimakalim.

Mechanism of K_ATP Channel Activation
These results suggest that enhanced activation of the K_ATP channel is an important endogenous cardioprotective mechanism capable of adapting to chronically stressful situations. The mechanism by which these channels become more active is uncertain, but one possible mechanism relates to observed changes in tissue lactate concentrations and alterations in the isoforms of LDH.

Elevation of myocardial lactate and LDH was present in hearts from hypoxic rabbits compared with normoxic control rabbits. The relative expression of the isoforms of LDH is regulated in part by the ambient oxygen concentration in the myocardial cell.¹ Myocardium from hypoxic hearts exhibited a relative decrease in the expression of the H (LD₁) isoform and a relative increase in the expression of the M (LD₅) isoform of LDH compared with normoxic ventricles. This shift in isoenzyme distribution suggests an increased dependency on anaerobic glycolysis for energy production in hypoxic hearts.¹

One of the consequences in the myocardium of increased glycolysis is the production of lactate and protons. Accumulation of these metabolites would normally be toxic to the myocardial cell. In contrast, for example, to the state of ischemia produced by coronary occlusion, in rabbits subjected to chronic hypoxia from birth, coronary flow rate is maintained and can wash out lactate and protons from the myocardium. The result is that lactate is transported outside the myocardial cell into the circulation during hypoxic conditions. Lactate is known to activate the K_ATP channel in ventricular myocytes.⁴ Lactate may reduce the sensitivity of the K_ATP channel to ATP,^{12 13} or it may act like cromokalin¹⁴ and pinacidil¹⁵ and open the K_ATP channel. In our study, tissue lactate levels in hypoxic hearts are elevated by 1 mmol/L over normoxic control hearts. This relatively small increase may be sufficient for lactate to modulate K_ATP activation. Intracellular acidification also activates the K_ATP channel.¹⁶ Hypoxia-induced K_ATP channel activation in this chronic model may be the result of increased lactate and proton production. Thus, an adaptation to chronic hypoxia designed to increase energy production by glycolysis may confer increased tolerance of the myocardium to a subsequent period of ischemia by activation of the K_ATP channel.

There are other potential mechanisms by which increased activation of the K_ATP channel may occur. Increased activation of the K_ATP channel may be caused not only by a decrease in ATP itself but also by a change in the ATP/ADP ratio.¹⁷ Increased levels of ADP would lead to a decrease in the ATP/ADP ratio, and this may act as the physiological regulator of activation of the K_ATP channel.^{18 19} In support of this hypothesis, patch clamp studies in rat cardiac tissue have shown that increases in ADP result in increased K_ATP channel activation.¹⁹ Metabolic products produced during the initial stages of ischemia may also modulate activation of the K_ATP channel. Adenosine, the breakdown product of ATP, increases in the interstitial fluid after myocardial stunning.²⁰ Adenosine has also been shown to improve postischemic myocardial function through the A₁ receptor.²¹ Thus, in addition to changes in lactate and protons, other factors such as the ATP/ADP ratio and increases in adenosine may be responsible for increased K_ATP channel activity.

Evidence for Adaptation of K_ATP Channels to Chronic Stress
The most common naturally occurring long-term stress in humans is hypertension, resulting in myocardial hypertrophy. In hypertrophic cardiomyopathic human patients²² and rats,²³ ATP concentrations are decreased. Although ATP has not been shown to decline enough to open K_ATP channels in humans except during cardiac failure, cells from hypertrophied ventricles have been shown to display a decreased sensitivity of the K_ATP channel to inhibition by ATP.²⁴ Activation of the K_ATP channel has also been shown to increase in spontaneously hypertensive rats.²⁵ Although the function of the K_ATP channel in myocardial hypertrophy is not understood, it is apparent that the chronic stress of hypertension does cause K_ATP channel activation to be altered.

Adaptation to Hypoxia, a Unique Form of Preconditioning
There appear to be similarities between adaptation to chronic hypoxia and brief periods of ischemia or hypoxia to "precondition" the myocardium against a subsequent period of ischemia. Ischemic preconditioning involves the initial release of endogenous myocardial substances after receptor activation such as lactate⁴ and adenosine^{20 21} to activate K_ATP channels. Once activated, the K_ATP channel may inhibit the inward slow calcium current directly or indirectly by stimulating a cGMP-dependent phosphodiesterase to decrease levels of cAMP.²⁶ The protection afforded by acute hypoxic preconditioning can be blocked by the K_ATP channel antagonist glibenclamide.² We have shown that prolonged exposure to hypoxia from birth results in a number of adaptive processes that may confer increased tolerance to subsequent ischemia. These include (1) increased expression of existing proteins (LDH activity is elevated 135% of normoxic values in hypoxic hearts), (2) a twofold increase in myocardial lactate levels in hypoxic myocardium, (3) increased release of cGMP from hypoxic hearts (unpublished data, 1995), and (4) maximal activation of the K_ATP channel in hypoxic hearts. Thus, the operative mechanisms by which adaptation to chronic hypoxia and by which brief ischemia or hypoxia preconditions to protect the heart during subsequent ischemia may be similar.

Increased Tolerance of the Right Ventricle to Ischemia
Decompensating children with congenital cyanotic heart disease generally display right-sided heart failure. Most studies to date have investigated failure of the left side of the heart. Our model is unique in its ability to simultaneously measure the recovery of both the right and left ventricles in neonatal rabbit hearts and make comparisons between them. Our study indicates that the rate and extent of postischemic recovery in normal right ventricles from normoxic and hypertrophied right ventricles from hypoxic hearts is greater than for the corresponding left ventricle. Recovery of function in the right ventricle for hearts treated with a K_ATP antagonist and agonist paralleled responses in the left ventricle.

We conclude that increased tolerance to ischemia in rabbit hearts hypoxic from birth during early postnatal development is associated with increased activity of the K_ATP channel. Further studies are needed to determine age-dependent differences between normoxic and hypoxic heart responses to ischemia during postnatal development and the role of the K_ATP channel at the cellular, membrane, and molecular levels in the mechanism of adaptation to hypoxia and tolerance to subsequent ischemia. Hypoxia from birth may represent a new, unique model of preconditioning in the face of chronic sublethal ischemia.

View this table: [in this window] [in a new window]   Table 2C. Continued

View this table: [in this window] [in a new window]   Table 3C. Continued

	Acknowledgments

 
This work was supported in part by grants HL-08311 and HL-45048 from the National Institutes of Health, Ronald McDonald's Children's Charities, and the Children's Hospital of Wisconsin Research Foundation. We are grateful to Patricia Holman for technical support and Mary Lynne Koenig for secretarial assistance.

	Footnotes

 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995.

Received August 28, 1996; revision received October 15, 1996; accepted October 28, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

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