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,1
* Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland;
Institute of Biochemistry and Clinical Biochemistry, Universita' Cattolica del S .Cuore, Rome, Italy; and
Department of Medicine, Surgery, and Dentistry, University of Milan, San Paolo Hospital, Milan, Italy
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Abstract |
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Key Words: chronic hypoxia • intermittent hypoxia • hypoxic preconditioning • isolated perfused heart • reoxygenation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At present, no universal model is available to adequately mimic chronic heart perfusion with hypoxic blood. The most commonly used approach includes animal raising in hypoxic or hypobaric chambers for extended periods of time followed by sacrifice, heart excision and perfusion with oxygenated media, and application of ischemia as required. In this approach, there are at least two events that potentially lead to heart reoxygenation before it is intended. First, the design of the hypoxic or hypobaric chambers usually allows exposure of the animal to room air whenever the chamber is opened for maintenance. Second, to obtain the baseline values necessary to assess ischemia tolerance, hearts from hypoxic animals are often perfused with oxygenated media. Both of these events may induce some degree of reoxygenation injury before baseline values are taken. The reoxygenation injury, which is more severe in hypoxic than ischemic hearts (13), is a major factor that induces preconditioning, thereby potentially leading to apparently improved ischemia tolerance. However, as far as we know, the question of whether repeated in vivo reoxygenation episodes affect ischemia tolerance has not yet been addressed.
The aim of this study was to test the hypothesis that CH represents a risk factor with respect to post-hypoxia dysfunction, and that repeated reoxygenation episodes in vivo precondition the heart. For these purposes, we used a hypoxic chamber that prevents any exposure of the animal to room air during maintenance. The experimental protocol included the removal of the heart under hypoxia, and heart perfusion with a hypoxic medium, followed by hyperoxic perfusion. With this design, we monitored the myocardial changes during either the first oxygenation after continuous 2-week exposure to hypoxia, or the reoxygenation after hypoxia with repeated reoxygenation episodes (intermittent hypoxia, IH). In addition, we measured some markers of oxidative stress. We will show that although they differed by only 1 hr of daily exposure to room air, CH and IH induced different patterns of animal homeostasis and myocardial resistance to the reoxygenation-induced injury.
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Materials and Methods |
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All animals had free access to water and conventional laboratory diet containing 90 mg/kg 
-tocopherol until 24 hr before the experiment. Water and food consumption was assessed every 2 days. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Hypoxic Chambers.
The design of the cage used in this study prevents exposure of the animals to room air during feeding, cleaning operations, and sacrifice (Fig. 1
). The assembly is composed of two types of chambers. The hypoxic chamber, a 350 x 350 x 200-mm transparent plastic box for three animals each, is equipped with a single 165-mm diameter window with a plastic sleeve (Iris Sleeve; Nufer Medical, Gumligen, Switzerland). The compensation chamber, with the same dimensions of the hypoxic chamber, has two windows in the facing walls. Both chambers are independently flushed with gas containing 0.100 ± 0.001 O2 (Carbagas, Lausanne, Switzerland). The O2 tension inside the chambers is continuously monitored by an O2 electrode (Servomex Oxygen Analyzer 570 A, Zurich, Switzerland) through a vent hole in the wall.
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Heart Perfusion.
Rats were anesthetized with an i.p. injection of sodium thiopental (10 mg/100 g body weight) and heparin (500 units). Hearts were rapidly excised and immersed in isotonic saline at ambient temperature (25°C). These operations were performed in the compensation chamber. The aorta was immediately cannulated and the heart was perfused at 37°C with hypoxic Krebs-Henseleit containing 2.0 mM/L free Ca2+ and 11 mM/L glucose, pH 7.33 ± 0.01. A roller pump (Ismatec SA; Labortechnik-Analytik, Glattbrugg-Zurich, Switzerland) delivered the medium (flow = 15 ml/min) to a 8-µm pore size, 47-mm diameter filter (MSI, Westboro, MA), a membrane oxygenator (Dideco, Mirandola, Italy), a pre-heater, and the cannula. The gas was provided from cylinders (Carbagas) containing O2, CO2, and N2 in the following proportions: 0.10/0.06/0.84 or 0.94/0.06/0.00 for the hypoxic and hyperoxic perfusions (nominal accuracy 0.001), respectively. The temperature of the heart and of the perfusion medium was maintained at 37°C by an external water bath. A latex balloon filled with saline was introduced into the left ventricle and was connected to a pressure transducer (MPC-500; Millar Instruments Inc., Houston, TX) to monitor performance. An additional transducer was inserted above the aortic cannula to monitor the coronary perfusion pressure. A cannula was inserted into the pulmonary artery to collect the venous return and to monitor venous PO2 by an O2-sensing electrode (model 5300 Oxygen Monitor; Yellow Springs Inc., Yellow Springs, OH).
Measurements of Myocardial and Metabolic Function.
Myocardial performance was recorded by a LabView system (National Instruments, Austin, TX) running on a PC. The measured parameters included the end-diastolic pressure (EDP), the heart rate (HR), the left-ventricle developed pressure (LVDP), the coronary perfusion pressure (CPP), and the venous PO2. As the membrane oxygenator used in this study allows complete equilibration of the liquid phase with the gas (14), the arterial PO2 was inferred from the nominal O2 content in the gas cylinder. The O2 consumption was calculated from this value, the measured venous PO2, the coronary flow, and the O2 solubility coefficient in water (15). The rate • pressure product (RPP, i.e., LVDP•HR) is an integrated index of myocardial performance. We did not pace hearts because pacing induces biochemical changes that interfere in this study (16). The vascular resistance was calculated as (CPP - EDP)/flow/(ventricle weight) (17). Samples of the venous effluent were taken at the end of the hypoxic and hyperoxic perfusions and were frozen at -80°C for lactate assay by enzymatic methods (COBAS FARA II; Hoffman-La Roche, Basel, Switzerland). When glucose is the only oxidizable substrate, the total turnover of ATP is calculated as (lactate release) + (6·VO2) (14).
Experimental Protocol.
Hearts were subjected to a 30-min hypoxic perfusion (coronary flow = 15 ml/min, PO2 = 67 mmHg). During this period, we measured the intraventricular balloon volume needed to increase EDP from 0 to 10 mmHg (V0
10), an index of ventricular volume. Afterward, the balloon volume was kept constant. After the hypoxic perfusion, hearts were oxygenated for 30 min (hyperoxic perfusion and coronary flow = 15 ml/min, PO2 = 670 mmHg).
Blood.
Immediately after heart mounting on the perfusion apparatus, a blood sample was withdrawn into a 5-ml heparinized tube. After mixing, the sample was divided into two aliquots. One was centrifuged (2500 rpm x 10 min) and plasma was stored at -80°C until use. In the other, hemoglobin concentration, hematocrit, and red blood cell count were measured (Abbott Cell-dyn 3500 R System, Baar, Switzerland).
In separate experiments, blood gases (ABL 700 analyzer; Radiometer, Copenhagen, Denmark) from femoral artery blood were measured in anesthetized rats.
Plasma Levels of Coenzyme Q and -Tocopherol.
The thawed plasma sample (0.5 ml) was mixed with 0.1 M Na-dodecilsulphate (0.5 ml), added with ethanol-isopropanol (2 ml, 95/5, v/v), and was extracted with n-hexane (4 ml) for 5 min while being continuously shaken in a vortex mixer. The mixture was centrifuged (1000 rpm x 2 min), and the upper phase was recovered and evaporated to dryness at 40°C under N2. The residue was dissolved in ethanol (200 µl), and 40 µl of this solution was injected into a HPLC apparatus (Beckmann Gold, Fullerton, CA) equipped with Chrompack column (ODS, 4.6 x 10 mm; Varian, Appleton, WI). The eluent (ethanol-methanol, 70/30, v/v) was flushed at 0.5 ml/min under isocratic conditions. Coenzyme Q9 and -tocopherol were detected photometrically at 
= 275 nm. -Tocopherylacetate (Merck, Darmstadt, Germany) was the internal standard. The plasma levels of coenzyme Q9 and -tocopherol were calculated from the areas of the respective peaks by a four-point calibration curve obtained by injecting the standard mixture at known concentrations. The within-run and between-run coefficients of variation for this determination are 0.8% and 3.8%, respectively.
Morphological Measurements.
At the end of the perfusion, hearts were removed from the perfusion apparatus, excess water was absorbed on tissue paper, and the heart mass was weighed. Atria were excised, and the free walls of the right and left ventricles as well as the septum were dissected free, dried at 90°C for 48 hr, and weighed separately. Right ventricular hypertrophy was assessed from the weight ratio (right ventricle)/(left ventricle + septum).
Statistics.
Data are expressed as mean ± SE. The significance level was P = 0.05 (two-tailed). To detect significant differences among the three groups, we performed one-way analysis of variance (ANOVA). If significant, the differences between selected pairs of data were tested using the Fischer comparison procedure (StatView; Abacus Concepts, Berkeley, CA).
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Results |
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shows the changes induced by 2-week CH or IH. During normal maintenance, the O2 level in the hypoxic chamber never increased by >1%. During sacrifice, the O2 level in the compensation chamber never increased by >2%. The mortality rate was 3/12 and 2/9 for CH and IH, respectively. Arterial PO2 ranged between 34 and 38 mmHg in both CH and IH rats vs. 60 and 70 mmHg in N rats.
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Hearts weighed less in CH as compared with N and IH. The heart weight/body weight ratio, and thus hypertrophy, was higher in hypoxic animals (both CH and IH). The weight ratio (right ventricle)/(left ventricle + septum) increased significantly in all hypoxic animals, indicating right ventricular hypertrophy. The balloon volume needed to increase EDP from 0 to 10 mmHg (V010, an index of the left ventricular volume) was less in IH than CH and N hearts.
Myocardial Performance.
We previously tested the apparatus by perfusing hearts (n = 6) under aerobic conditions (PO2 = 670 mmHg, flow = 15 ml/min) for 110 min: the changes of LVDP, HR, and EDP accounted for less than 8.3 ± 4.3 mmHg, 22 ± 10 min-1, and 1.2 ± 0.9 mmHg, respectively (18). Absolute values for those parameters were 113 ± 1 mmHg, 270 ± 4 min-1, and 10.2 ± 0.1 mmHg, respectively.
Because the balloon volume was fixed at the start of the perfusion to yield EDP = 10 mmHg, EDP was the same in the three groups during the hypoxic perfusion (Fig. 2). The oxygenation of CH hearts did not immediately alter EDP; however, the changes became evident >10 min after the onset of hyperoxia. The reoxygenation of IH and N hearts decreased EDP more markedly in IH than N. At the end of the hyperoxic perfusion, the difference in EDP between N and IH hearts vanished (P = 0.11). Thus, EDP was higher in CH than IH and N hearts.
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). HR and LVDP were 157 ± 8 min-1 and 52 ± 3 mmHg, respectively. During the hyperoxic perfusion, HR increased by 31.9% ± 2.2% in all groups. In contrast, LVDP increased by 22.2 ± 3.8 mmHg in CH hearts vs. 32.6 ± 5.7 and 39.7 ± 4.9 in N and IH, respectively. As a result of these changes, CH hearts appeared less protected than IH and N hearts with respect to the systolic performance.
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). Resistance remained steady throughout the hypoxic perfusion in the order of N>CH>IH. The hyperoxic perfusion increased resistance to a larger extent in N and CH hearts than in IH hearts (inset of Fig. 4). Thus, at the end of the hyperoxic perfusion, the resistance ranked N>CH>IH as during the hypoxic perfusion, but with more marked differences.
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). The venous PO2 at the end of the hypoxic perfusion ranged between 0 and 3 mmHg in all groups. After (re)oxygenation, venous PO2 ranked CH>IH>N. Venous lactate concentration at the end of the hypoxic perfusion was the highest in N hearts, intermediate in IH, and lowest in CH hearts. After (re)oxygenation, lactate was undetectable in all groups.
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, which summarizes myocardial bioenergetics, shows the anaerobic, e.g., lactate release, and aerobic, e.g., O2 consumption, contributions to total ATP production. During the hypoxic perfusion, the O2 consumption was the same in all the groups. Thus, the higher ATP production in N hearts reflects their higher lactate release. After the (re)oxygenation, the lactate release was virtually zero for all the hearts. Although both the O2 consumption and ATP turnover were the lowest for IH, in CH hearts, these parameters were ranked between N and IH.Figure 7 summarizes the findings relative to coenzyme Q (Q9 is the predominant form in rodents) and -tocopherol. Coenzyme Q level in plasma was nearly doubled in CH than IH and N rats, whereas -tocopherol remained constant in all the groups.
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Discussion |
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In IH rats, hypoxia-induced deterioration could offset the normally expected weight gain. In contrast, CH rats, despite unlimited access to food and water, underwent weight loss, as in humans exposed to high altitude (21). Hypoxia exposure induces right ventricular hypertrophy as a consequence of pulmonary hypertension (22). Table I
shows that despite different body weights, right ventricular hypertrophy was the same in CH and IH animals.
The oxidative stress was greater in IH than CH rats (Fig. 7). The total (oxidized and reduced) plasma level of coenzyme Q is under consideration as a potential biomarker of oxidative stress (23). Coenzyme Q is, among lipophylic antioxidants, the only one produced by endogenous biosynthesis. Its plasma level is the result of a balance between biosynthesis and destruction by peroxidative phenomena or elimination (24). Increased coenzyme Q level in CH rats is consistent with high values observed in hypoxemic fetuses (25) and the faster biosynthesis early during altitude adaptation (26). Coenzyme Q is known to reduce tocopheryl radical and semidehydroascorbate back to tocopherol and ascorbate, thereby acting as antioxidant (27). Its low level in IH rats may reflect either its utilization to maintain the plasma level of -tocopherol or its internalization in the cells to contrast the reoxygenation (24). Being available only from dietary sources, -tocopherol did not change appreciably among the groups in our study, despite reduced food intake in CH and IH rats, in agreement with the observed decrease in coenzyme Q, as opposed to maintained level of -tocopherol, in CCl4-treated rats (23).
Isolated Heart Perfusion.
Hearts were isolated and Langendorff-perfused with a blood-free medium according to an established technique. The described set-up rules out all the potentially confusing blood-related responses, including the presence of hormones and coagulation factors, lymphocyte-mediated inflammatory processes, uneven spacing of red cells within capillaries, hematocrit heterogeneity, and hypoxia-induced changes in the hemoglobin-O2 affinity. Absence of hemoglobin reduces the blood O2 capacity, but high PO2 and flow partially compensated for that reduction. The accurate determination of O2 delivery to heart under the various conditions used in this study requires knowledge of variables that were not measured, i.e., hemoglobin-O2 saturation in arterial and coronary sinus blood, or myocardial O2 extraction. However, the selected experimental conditions represent a first approach to unravel the response of hypoxic hearts to abrupt increase in O2 supply.
The inter-group differences in cardiac weight, hypertrophy, ventricular volume, and body weight might mismatch the initial perfusion conditions. However, the employed perfusion protocol could reduce the impact of those variables. First, the balloon volume was set for each heart to a value that yielded the same EDP, irrespective of the group. Thus, myocardial load and performance were the same in all the groups during the initial phase, and the performance changes in response to abrupt (re)oxygenation could therefore be monitored. Second, other critical parameters such as vascular resistance, lactate release, O2 consumption, and ATP turnover are expressed in relation to heart weight.
The lesser V010 in IH hearts might be explained as a result of increased diastolic stiffness or decreased left ventricular volume. As the ventricle volume/heart weight ratio was 65.9 ± 7.7 and 41.0 ± 4.5 µl/mg (P = 0.007) in CH and IH hearts, respectively, it is likely that CH hearts develop hypertrophy accompanied by dilatation, whereas IH hearts show signs of hypertrophy only.
Myocardial Performance.
Although hypoxic perfusion did not reveal striking differences among the groups, the (re)oxygenation induced different responses in CH, IH, and N hearts. In the Langendorff heart with fixed-volume intraventricular balloon, higher EDP indicates impaired diastolic compliance. Thus, diastolic performance was more protected in IH than N and CH hearts. The EDP change during (re)oxygenation represents a compromise between two contrasting effects. On one hand, reoxygenation of hypoxic hearts reverts the hypoxia-induced Ca2+ load (28), thereby leading to EDP decrease. On the other hand, the oxidative stress associated with hypoxia reoxygenation leads to diastolic contracture in this model (14). It is difficult to assess the relative contribution of these effects, but in IH and N hearts, the deleterious effect of the oxidative stress was apparently less than in CH hearts. Perhaps the repeated reoxygenation episodes induced a greater antioxidant protection in the IH hearts.
In analogy to diastolic performance, myocardial contractility too was more protected upon (re)oxygenation in IH and N than CH hearts. Again, performance improvement due to restoration of aerobic mechanisms with highly efficient energy-yielding paths is challenged by the oxidative stress derived from hypoxia reoxygenation. Apparently, this compromise was more favorable in IH and N than CH hearts.
Low resistance in IH and CH hearts shows that the effect of hypoxia-induced vasodilation, probably led by increased NO release (29), persisted in isolated hearts. Rapid switch from in vivo high-hematocrit blood perfusion to in vitro blood-free perfusion might have induced reactive hyperemia that potentially disturbs vascular resistance data. However, hematocrit and blood viscosity as well as hypertrophy were the same in CH and IH rats. Yet, the response of IH and CH hearts was different. Therefore, such differences cannot be ascribed to abrupt changes in perfusion conditions, but must be due to inherent differences between CH and IH hearts. As above, we believe that oxidative stress following repeated reoxygenation episodes in IH hearts may induce protection.
Bioenergetic Metabolism.
The very low venous PO2 in all groups during hypoxic perfusion indicates that hearts extracted all available O2, in agreement with previous data obtained under acute hypoxia (14). We calculated the relative contributions of aerobic and anaerobic mechanisms to the ATP production during hypoxic perfusion from lactate release and O2 consumption, assuming ATP/lactate ratio = 1.0 (glucose as substrate without significant glycogenolysis), and ATP/O2 = 6 (no mitochondrial uncoupling). In the isolated rat heart, and in the absence of triglycerides in the perfusion medium, the contribution of intracellular triglyceride stores to ATP production is very small (30). With these assumptions, the contribution of aerobic mechanisms to total ATP turnover was 45% ± 1%, 66% ± 2%, and 55% ± 2% in N, CH, and IH hearts, respectively. Thus, IH and CH induced differential patterns of hypoxic adaptation. Yet, the performance of N, CH, and IH hearts was the same (Fig. 3). Further studies are needed to understand this feature, but the increased mitochondrial oxidative mechanisms in hypoxic hearts might result from hypoxia adaptation, which increases size and capacity of mitochondria (31). In contrast, the lower contribution of aerobic mechanisms in IH versus CH hearts might be a consequence of mitochondrial damage secondary to increased oxidative stress. As a matter of fact, during the hyperoxic perfusion, when hearts rely almost entirely on aerobic mechanisms, the ATP turnover is slightly less in IH than CH and N hearts, probably reflecting the mitochondrial damage led by repeated reoxygenation episodes.
Clinical Implications.
In this study, we show that repeated reoxygenation episodes protect hearts against hypoxia-reoxygenation, whereas continuous CH does not. This result is related to the hypoxia effect on ischemia tolerance. Most of the studies on this issue support a protective role for hypoxia. In a few of them, however, it was reported that animals were repeatedly reoxygenated during hypoxia due to chamber opening for maintenance (6, 7). In another study, it was declared that hearts were perfused with hyperoxic media, thereby undergoing reoxygenation, before baseline measurements (8). Still in other studies, both operations were performed (9–12). In the light of the present data, the improved tolerance to ischemia, a common finding in these studies, can be attributed to hypoxic preconditioning due to reoxygenation rather than to hypoxia per se. As a matter of fact, in the experimental studies supporting the deleterious effects of hypoxia, heart reoxygenation before baseline was prevented by surgically installing a cardiac bypass (3–5).
Hypoxia-induced erythropoietin was recently proposed as a local protective factor (32, 33). Although we have not measured erythropoietin in this study, Table I shows that the erythropoietic response was the same in CH and IH rats. In addition, there is no proof that myocardial tissue produces erythropoietin. Therefore, it is difficult to support a protective function of erythropoietin in hearts (34). Rather, differential response to CH and IH of mechanisms known to protect hearts, as activation of KATP channels (35) and recruitment of signaling transduction pathways (36) should be critically evaluated. It was also recently reviewed (37) that IH, although with different times and modes than those employed in this study, is more effective than CH in activating activator protein-1 and hypoxia-inducible factor-1.
Conclusion.
Despite differing by only a 1-hr daily exposure to room air, CH and IH induced different responses both at the level of whole animal homeostasis and myocardial tolerance to reoxygenation-induced injury. The tolerance to hyperoxic perfusion was impaired in CH hearts, but improved in IH hearts. This behavior is consistent with preconditioning exerted by in vivo repeated reoxygenation episodes.
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Acknowledgments |
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-tocopherol. The authors are grateful to Dideco (Mirandola, Italy) for providing the membrane micro-oxygenators. |
Footnotes |
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1 To whom requests for reprints should be addressed at Universita' di Milano, Ospedale San Paolo, Dipartimento di Medicina, Chirurgia e Odontoiatria, via di Rudini' 8, I-20142 Milano, Italy. E-mail: Michele.Samaja{at}unimi.it 
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), CH (
), and IH (
), during the hypoxic and hyperoxic perfusions. An asterisk indicates significant difference versus normal; A pound symbol indicates significant difference versus CH (P < 0.05, ANOVA and Fischer' post-test). The statistics was evaluated at the end of the hypoxic and hyperoxic perfusion steps.
) at the end of the hypoxic and hyperoxic perfusions. See legend of Figure 2
