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1Division of Cardiothoracic Surgery, Institutes of 2Pathophysiology and 3Physiology, University of Essen, 45122 Essen, Germany
Submitted 13 January 2003 ; accepted in final form 22 September 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and the protein expression of endothelial nitric oxide synthase (eNOS) were measured. Postischemic left ventricular function recovered to 66 ± 6% and 67 ± 5% of baseline in Norm and Hypo, respectively. In contrast, postischemic right ventricular function was 93 ± 2% of baseline in Hypo vs. 67 ± 3% in Norm (P < 0.05). Improved postischemic right ventricular function in Hypo (93 ± 2% and 96 ± 2% of baseline) was observed with 95% O2 or 21% O2 in the perfusate, and it was not attenuated by glibenclamide (5 and 10 µmol/l) (86 ± 4% and 106 ± 6% recovery). HIF-1 mRNA and eNOS protein expression were increased in both left and right hypoxic ventricles. In conclusion, postischemic right, but not left, ventricular function was improved by preceding chronic hypoxia. ATP-sensitive K+ channels are not responsible for the increased right ventricular tolerance to ischemia after chronic hypoxia in adult rat hearts.
chronic hypoxia; glibenclamide; hypoxia-inducible factor-1; endothelial nitric oxide synthase
Chronic hypoxic preconditioning also increases the tolerance of the myocardium to ischemia. In an isolated adult rat heart model, chronic hypoxia improved postischemic systolic function and protected against prolonged periods of ischemia (27). Although similarities of the phenomena of hypoxic and ischemic preconditioning have been found in protecting the myocardium, the additive cardioprotective effects of hypoxic and ischemic preconditioning suggest different mechanisms of protection (27).
Baker et al. (3, 4) reported that the increased tolerance to ischemia in isolated crystalloid-perfused immature rabbit hearts exposed to chronic hypoxia is associated with an activation of the KATP channels. The beneficial effects of chronic hypoxic preconditioning on left and right ventricular (RV) developed pressure were abolished by the KATP channel antagonist glibenclamide. The KATP channel agonist bimakalim enhanced postischemic left ventricular (LV) function in normoxic hearts similar to that of hypoxic hearts (3). As in ischemic preconditioning, these results suggested a role for the KATP channel in hypoxic preconditioning.
Because the protection by ischemic preconditioning and by chronic hypoxia is discussed to be age dependent (2), we now tested the protection afforded by chronic hypoxia in blood-perfused working adult rat hearts. The role of KATP channels was investigated using the KATP channel antagonist glibenclamide.
Hypoxia-inducible factor (HIF)-1 mediates a compensatory response to decreases in oxygen tension (24) leading to a characteristic pathophysiological phenotype with pulmonary polycythemia, hypertension, and RV hypertrophy (14). LV function of isolated working hearts from HIF-1 wild-type mice was preserved after ischemia-reperfusion after chronic hypoxia, whereas no cardioprotection after chronic hypoxia and ischemia-reperfusion was seen in HIF-1+/– heterozygous mice. These results suggested a role for HIF-1 in hypoxic preconditioning (6). We expected the expression of HIF-1 to relate to the different responses of right and left ventricles to chronic hypoxia and therefore measured HIF-1 mRNA.
Cardioprotective effects after ischemia-reperfusion have also been described for nitric oxide (NO), with postischemic application of a NO donor in isolated guinea pig hearts (16). In chronic hypoxic isolated immature rabbit hearts, increased production of NO and limited superoxide anion generation have been found and correlated with a better LV tolerance to ischemia (25). We therefore also assessed the endothelial NO synthase (eNOS) expression in normoxic and hypoxic left and right ventricles.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Perfusion solutions. A red blood cell perfusate was prepared, consisting of human red blood cells at a final hematocrit of 30%. The perfusion buffer used was a modified Krebs-Henseleit bicarbonate solution (in mmol/l: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.2 CaCl2, 10 glucose, and 5.0 pyruvate). Human serum albumin was added at a final concentration of 50 mg/ml. White cell-depleted human donor blood was centrifuged at 4°C with 3,000 revolutions/min for 10 min. The supernatant was removed and the resulting packed cells were mixed 1:1 with Krebs-Henseleit buffer. The centrifugation and resuspension steps were repeated three times. After the red blood cells were prepared, the perfusate was immediately used for the experiment.
Isolated rat heart preparation. The rats were anesthetized with enflurane inhalation. Heparin sodium (1,000 U, Braun; Melsungen, Germany) was administered intraperitoneally 1 h before anesthesia. The chest was opened, the ascending aorta was cannulated, and retrograde Langendorff perfusion (15) of the nonworking heart was initiated with a red blood cell solution maintained at 37°C and at a constant pressure of 80 mmHg. Thereafter, the hearts were rapidly excised and perfused in a closed recirculating system at 37°C with a membrane oxygenator (Lilliput 1 D901, Dideco; Mirandola, Italy) in contact with 95% O2-5% CO2 or 21% O2-5% CO2-74% N2 gas mixtures to achieve a PO2 of the blood cell perfusate between 500 and 600 mmHg (for 95% O2) or 100 and 130 mmHg (for 21% O2). Two different O2 concentrations were used to investigate a potential influence of normoxic as opposed to hyperoxic reoxygenation.
During the initial retrograde Langendorff perfusion, hearts were trimmed of excess tissue, and superior and inferior caval veins were ligated. In left heart preparations, the left atrium was cannulated via the pulmonary venous orifices. A cannula was placed into the main pulmonary artery to permit drainage of the coronary sinus effluent. The hearts were switched to the working mode (20) after 15 min of aerobic, nonworking, and retrograde aortic perfusion and were perfused at a left atrial preload of 10 cmH2O and an aortic afterload of 75 mmHg. During the working mode, heart rate and peak systolic pressure were recorded with the use of a physiological recording system with CORDAT II software (26) and a pressure transducer (model P23Db, Statham; Oxnard, CA) in the aortic line. Aortic output was measured using a blood flowmeter (model SP 2202, Statham) around the aortic cannula. The same type of flowmeter was placed around the pulmonary artery to determine coronary flow throughout the experiment.
For right heart preparations, openings of the superior vena cava and the thoracic veins were ligated. The inferior vena cava was cannulated. This catheter was connected to a fluid-filled column and the preload was adjusted to 10 cmH2O. A cannula was placed into the main pulmonary artery and tied securely. The pulmonary cannula was connected to a fluid-filled column, where pulmonary afterload was adjusted to 15 mmHg. RV cardiac output and coronary flow were measured using flowmeters (model SP 2202, Statham) placed around the main pulmonary and aortic cannulas. After 15 min of Langendorff perfusion, the RV working mode (28) was initiated.
Experimental protocols. Right and left heart preparations underwent the same perfusion protocol. Two periods of perfusion in working mode, W1 (15 min) and W2 (15 min), were separated by 15 min of global warm ischemia, with hearts bathed in 37°C Krebs-Henseleit buffer, and 15 min of reperfusion in a nonworking Langendorff mode.
The rat hearts were assigned to the following 11 groups: group 1, normoxic hearts performing LV work, gassed with 95% O2 (n = 7); group 2, hypoxic hearts performing LV work, gassed with 95% O2 (n = 8); group 3, normoxic hearts performing RV work, gassed with 95% O2 (n = 8); group 4, hypoxic hearts performing RV work, gassed with 95% O2 (n = 6); group 5, normoxic hearts performing LV work, gassed with 21% O2 (n = 6); group 6, hypoxic hearts performing LV work, gassed with 21% O2 (n = 4); group 7, normoxic hearts performing RV work, gassed with 21% O2 (n = 6); group 8, hypoxic hearts performing RV work, gassed with 21% O2 (n = 6); group 9, normoxic hearts performing RV work + 5 µmol/l glibenclamide, gassed with 21% O2 (n = 6); group 10, hypoxic hearts performing RV work + 5 µmol/l glibenclamide, gassed with 21% O2 (n = 12); and group 11, hypoxic hearts performing RV work + 10 µmol/l glibenclamide, gassed with 21% O2 (n = 4).
In groups 9–11, glibenclamide (5 and 10 µmol/l) was put into the perfusate 15 min before ischemia. Hemodynamic variables were determined at the end of W1 and at the end of W2 (coronary flow, aortic and pulmonary flow, and heart rate). Preischemic cardiac output of left hearts ranged between 31 to 85 ml/min (average 51 ± 3 ml/min). Preischemic cardiac output of right hearts ranged between 22 and 99 ml/min (average 57 ± 3 ml/min). Pre- and postischemic external heart work was computed as the product of mean aortic or pulmonary pressure, total output, and a conversion factor (1.333222 x 10–4) for expression in Joules per minute (9). Postischemic recovery was defined as postischemic external heart work over preischemic external heart work and expressed in percent.
Extraction and quantification of HIF-1 mRNA. Hearts from control (n = 10) and chronically hypoxic animals (n = 10) were removed, and the RVs and LVs were separated and immediately frozen in liquid nitrogen. Frozen myocardial tissue was homogenized in 4 M guanidinium thiocyanate containing 0.1% 
-mercaptoethanol. Total RNA was isolated by acid phenol-chloroform extraction (7) and redissolved in water. RNA concentration was determined by measurement of optical density at 260 nm. Total RNA (1 µg) was reversely transcribed into cDNA using oligodT15 as a primer for RT (AMV RT, Promega; Heidelberg, Germany). Quantification of HIF-1 cDNA was carried out by real time PCR (Gene Amp 5700, PE Applied Biosystems; Weiterstadt, Germany) using the SYBRGreen PCR Master Mix (PE Applied Biosystems). Sense and antisense primers for HIF-1 were 5'-gctggccccagccgctggag-3' and 5'-gagtgcagggtcagcactac-3' (GenBank Accession No. AF208487
[GenBank]
) resulting in a cDNA fragment of 214 bp length. Serial dilutions of the HIF-1-specific cDNA fragment were used as standard. Real-time PCR was run for 35 cycles (cycle profile: denaturation 94°C, 15 s; annealing and elongation 60°C, 1 min) after an initial denaturation at 95°C for 10 min. Each sample was quantified in triplicate.
eNOS protein content. Hearts from control and chronically hypoxic animals (n = 6 each) were removed, and RVs and LVs were separated and immediately frozen in liquid nitrogen. Briefly, tissue was homogenized in 20 vol of cell lysis buffer. The homogenates were centrifuged twice for 10 min at 20,000 g, and the combined supernatants were considered as cell extract. Samples were boiled in SDS buffer at 95°C and applied to 10% polyacrylamide gels. The proteins were separated by electrophoresis (25 µA, for 2 h at 4°C) and transferred to nitrocellulose membranes for electroblotting (40 V, for 1 h, at 20°C). After overnight incubation at 4°C with the primary antibodies for eNOS (eNOS/NOS Type III, BD Biosciences, Heidelberg, Germany) and extensive washing, the blots were transferred to a blocking buffer containing secondary antibodies linked to horseradish peroxidase (phototope horseradish peroxidase detection kit, New England Biolabs; Frankfurt, Germany), and incubated for 1 h at room temperature. Finally, blots were incubated in LumiGlo substrate protein (Amersham Pharmacia Biotech; London, UK) for 1 min, wrapped in plastic, and opposed to imaging films for 1 to 2 min to detect the chemiluminescent signal. Films were processed by routine methods and analyzed by quantitative two-dimensional densitometry using commercially available software (Herolab; Wiesloch, Germany).
Statistical analysis. Data are reported as means ± SE. Differences between only two groups and one time point were compared by unpaired Student's t-test. Data of more than two groups were tested by one-way or two-way ANOVA (depending on whether one or more time points were present). When this analysis indicated a significant difference between groups, values were further examined with a Student-Newman-Keuls test. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preischemic hemodynamic data. Cardiac output was similar in all groups, except for group 9, where cardiac output was lower than that of group 8. The heart rate in group 1 was lower than in groups 3, 7, 8, 10, and 11. LV external heart work in groups 1, 2, 5, and 6 and RV external heart work in the other groups were not different from each other (Table 4). Baseline hemodynamic values before administration of the KATP channel antagonist glibenclamide were similar in groups 9–11 (Table 5). Glibenclamide (5 µmol/l) decreased preischemic cardiac output and external heart work in normoxic right ventricles, whereas coronary flow and heart rate remained unchanged. There were no hemodynamic changes when hypoxic right ventricles were treated with glibenclamide (5 and 10 µmol/l). Coronary flows expressed as percentage of total cardiac output were between 10% and 17% in untreated normoxic and hypoxic left and right ventricles [LV norm 14 ± 1%, LV hypo 17 ± 2% (P = 0.5), RV norm 10 ± 1, and RV hypo 15 ± 3 (P = 0.08)].
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Postischemic hemodynamic data. Postischemic coronary flow did not differ significantly between any of the groups and there was no difference compared with preischemic coronary flow within groups (Table 4). Postischemic recovery of coronary flow is shown in Fig. 1. There was no significant difference between groups, although postischemic recovery of coronary flow in hypoxic LV tended to be improved compared with normoxic LV. Postischemic cardiac output of untreated and glibenclamide-treated normoxic right ventricles (groups 7 and 9) was significantly lower than preischemic values of the respective groups. Hypoxic right ventricles (gassed with 21% O2) had higher postischemic cardiac outputs than the respective normoxic controls both with and without the addition of glibenclamide. Except for group 1, postischemic heart rate was similar in all groups. Postischemic external heart work in groups 1 and 2 was significantly lower than the respective values before ischemia.
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Recovery of function. Postischemic recovery of external heart work did not differ between hypoxic and normoxic LVs gassed with 95% O2. However, hypoxic right ventricles recovered significantly better than normoxic right ventricles both when gassed with 95% or 21% O2 (Fig. 2). The lowest postischemic functional recovery was observed when normoxic right ventricles were treated with 5 µmol/l glibenclamide. On the other hand, glibenclamide (5 and 10 µmol/l) did not influence postischemic recovery of hypoxic right ventricles (Fig. 2).
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Expression of HIF-1 mRNA and eNOS. HIF-1 mRNA expression in hypoxic left and right ventricles was significantly higher than in normoxic controls. HIF-1 mRNA in hypoxic LVs was higher than in all other groups (P < 0.05) (Fig. 3). eNOS protein levels were higher in hypoxic left and right ventricles than in normoxic controls (Fig. 4).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA and eNOS expression were increased in hypoxic LV and RV myocardium over that in normoxic controls. Postischemic recovery of coronary flow did not significantly differ between the groups. A prior study (3) in immature rabbit hearts demonstrated postischemic recoveries of hypoxic RVs and LVs that were similarly improved over those of the respective normoxic controls. In contrast, in the present study using adult rat hearts, chronic hypoxia improved postischemic RV but not LV function. Indeed, the effect of chronic hypoxia on ischemic tolerance has been shown to be age dependent in the LV. In a Langendorff model, LVs of immature rabbit hearts (hypoxic from birth) were more tolerant to ischemia than LVs of adult rabbits subjected to chronic hypoxia (2). Differences concerning postischemic LV functional recovery between our and earlier investigations (3) could thus be explained by different responses of immature and adult LVs to chronic hypoxia. Also, the respective models differed in that we used blood perfusion and a normoxic PO2, whereas the cited study used crystalloid perfusion and a PO2 of 500–600 mmHg (3).
To the best of our knowledge, no prior study has looked at the effects of chronic hypoxia on RV recovery in adult hearts. The role of RV hypertrophy in the observed improved tolerance to ischemia remains uncertain. In principle, similar kinase pathways are activated during hypertrophy and preconditioning (18). Other mechanisms to induce RV hypertrophy, such as pulmonary artery banding, could help to find out in the future whether the development of RV hypertrophy is an independent mediator of ischemic tolerance in the right ventricle.
The fact that postischemic recovery of coronary flow did not correlate with postischemic recovery of function is in accordance with our earlier studies in isolated hearts (16). However, the conclusions drawn from coronary flow measurements in this model are limited because in the working right heart preparation a large portion of flow running through the aortic cannula perfuses the left anterior descending and the circumflex artery of the nonworking LV. We cannot determine the right coronary artery flow, which certainly is a limitation of this working heart model. The same limitations apply for the working left heart preparation.
KATP channel inhibition using glibenclamide was only used in right heart preparations because no cardioprotective effect of hypoxic preconditioning was observed in left hearts. Hypoxic preconditioning was not inhibited by glibenclamide in our model, but glibenclamide also depressed preischemic function in normoxic, but not in hypoxic hearts. This is in contrast to earlier studies, in which glibenclamide was reported to abolish the protective effects of hypoxic preconditioning (3, 4). As mentioned earlier, the models used in the respective studies were different. Our results may suggest a different sensitivity to KATP channel modulation in preischemic and postischemic normoxic and hypoxic hearts. This notion is supported by the triggering of arrhythmias by the KATP channel agonist diazoxide in normoxic, but not in chronically hypoxic adult rat hearts (1).
The increase in HIF-1 mRNA expression in hypoxic left and right ventricles obviously does not support an involvement of HIF-1 in the observed hypoxic preconditioning of right ventricles. However, the sensitivity for a preconditioning effect mediated by HIF-1 may be different in hypoxic RV and LV, and this has not yet been investigated. A recent report (11) describes HIF-3 to represent a rapidly reacting component of the HIF system. The heart, however, contained the lowest amounts. Nevertheless, the fact that only one HIF-subunit was measured may be a limitation of the present study. A further limitation is that HIF-1 mRNA levels do not indicate function or activity of the protein, which could not be measured because it is degraded very rapidly.
The cardioprotective effects of NO have been reported for different species (5, 16, 17). Again, the elevation of eNOS in both right and left hypoxic ventricles in our model suggests that the observed protection is probably not mediated by NO. However, again, the NO effect on a hypertrophied chronically hypoxic ventricle may well be different from the effect on a nonhypertrophied normoxic ventricle. A recent review (21) suggests a role for both the constitutive and the inducible forms of NOS in cardioprotection. The effect of cell cycle regulators, such as G1 cyclins and associated cyclin-dependent kinases, activated during cardiac hypertrophy and thought to mediate apoptosis in hypoxic cells, are, on the other hand, attenuated by NO (21), possibly relating to the effects observed in the present study. Future experiments with direct inhibition of NOS may help to identify the functional sequences of endogenous NO blockade in hypoxic right ventricles.
In conclusion, chronic hypoxia improved RV functional recovery in adult rat hearts, whereas postischemic LV function remained unchanged. KATP channel blockade with glibenclamide did not abolish the cardioprotection afforded by chronic hypoxia. The relation of our experimental data to the clinical results in children with cyanotic heart disease undergoing surgery is not clear in detail. Cyanosis did not influence the surgical outcome in infants (0–12 mo), but in children >12 mo old, cyanosis was associated with greater troponin I release and worse outcome (12).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA expression. FASEB J 17: 1541–1543, 2003.This article has been cited by other articles:
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  M.-G. Ryou, J. Sun, K. N. Oguayo, E. B. Manukhina, H. F. Downey, and R. T. Mallet Hypoxic Conditioning Suppresses Nitric Oxide Production upon Myocardial Reperfusion Experimental Biology and Medicine, June 1, 2008; 233(6): 766 - 774. [Abstract] [Full Text] [PDF]
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E. B. Manukhina, H. F. Downey, and R. T. Mallet Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia. Experimental Biology and Medicine, April 1, 2006; 231(4): 343 - 365. [Abstract] [Full Text] [PDF]
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 L. P. Thompson and Y. Dong Chronic Hypoxia Decreases Endothelial Nitric Oxide Synthease Protein Expression in Fetal Guinea Pig Hearts Reproductive Sciences, September 1, 2005; 12(6): 388 - 395. [Abstract] [PDF]
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 P. La Padula and L. E. Costa Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. I. Mechanical activity J Appl Physiol, June 1, 2005; 98(6): 2363 - 2369. [Abstract] [Full Text] [PDF]
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