Eur J Cardiothorac Surg 2001;20:992-995
© 2001 Elsevier Science NL
Stimulated expression of cyclooxygenase-2 in porcine heart after bypass circulation and cardioplegic arrest
Pekka Uotilaa,b,
Antti Sarastec,
Tommi Vähäsiltad,
Erkki Kentalae,
Timo Savunend,
a Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland
b Department of Clinical Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland
c Department of Anatomy, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland
d Department of Surgery, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland
e Department of Anaesthesiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland
Received 25 January 2001;
received in revised form 20 June 2001;
accepted 18 July 2001.
Corresponding author. Tel.: +358-2-313-1611; fax: +358-2-313-2284
 |
Abstract
|
|---|
Objectives: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes catalyze the initial step in the formation of prostaglandins, which have a role in the regulation of circulation and in inflammatory reactions. As hypoxia is reported to stimulate the expression of COX-2, we have investigated the effects of bypass circulation and cardioplegic arrest on the expression COX-1 and COX-2 in the myocardium of porcine hearts. Methods: Anaesthetized pigs were connected to cardiopulmonary bypass and the hearts were arrested by cold crystalloid cardioplegia for 30 min and reperfused thereafter for 90 min. Then the mRNA and protein levels of COX-1 and COX-2 were measured from the transmural specimens of the left ventricular myocardium by Northern and Western blot analyses. Reference specimens were from the hearts of unoperated control pigs and from sham-operated pigs, which were connected to cardiopulmonary bypass for 120 min without any aortic clamping. Results: COX-1 mRNA was expressed in unoperated control porcine hearts, whereas the expression of COX-2 mRNA was weak in control hearts. The expression of COX-2 mRNA increased to 170% of the control level in the hearts of sham-operated pigs and to 180% in arrested hearts, while the level of COX-1 mRNA was not changed. Both COX-1 and COX-2 proteins were detected by Western blot analysis in the myocardial specimens of control hearts. After cardioplegic arrest, the level of COX-2 protein increased to 280% of the control level in arrested hearts, whereas the level of COX-1 protein remained unchanged. Conclusions: These results indicate that the expression of the COX-2 gene is stimulated in the ventricular myocardium of the porcine heart after bypass circulation and cardioplegic arrest.
Key Words: Cyclooxygenase-1 • Cyclooxygenase-2 • Cardiopulmonary bypass • Cardiac arrest • Cardioplegia • Porcine
|
1. Introduction
|
|---|
The initial step in the formation of prostaglandins from arachidonic acid is catalyzed by the constitutive cyclooxygenase-1 (COX-1) and the inducible cyclooxygenase-2 (COX-2) enzymes [1,2]. COX-1 is expressed in vascular endothelial cells and the low basal activity of COX-2 in endothelial cells can be stimulated by cytokines and hypoxia [3,4]. The main prostaglandin formed in vascular endothelium is prostacyclin, which has both vasodilatory and antiaggregatory effects [5]. COX-2 is upregulated, for example in inflammation, when the formation of proinflammatory prostaglandins is thus stimulated [2].
During open heart operations, the myocardium is subjected to global ischemia, when the aorta is clamped and the heart is arrested by cardioplegic solution. As hypoxia is reported to stimulate the expression of COX-2, we have investigated the expression COX-1 and COX-2 in the myocardium of porcine hearts arrested with cold crystalloid cardioplegia during cardiopulmonary bypass circulation.
|
2. Materials and methods
|
|---|
2.1. Animals and the surgical procedure
The study was approved by the Ethical Committee for Animal Research at the University of Turku. All animals received humane care in compliance with the European Convention on Animal Care. Sixteen Finnish domestic pigs of both genders weighing 27–30 kg (mean, 28 kg) and aged 12 weeks were used. The animals were divided into three groups: unoperated controls; sham-operated controls; and in the third group, porcine hearts were arrested and reperfused. Six animals served as control animals, which were not operated on. These control animals were killed and transmyocardial samples of the left ventricular free wall were frozen. Five sham-operated pigs were connected to cardiopulmonary bypass circulation for 120 min without aortic clamping. Thus, the hearts of the sham-operated pigs were not arrested. Five pigs in the arrested heart group were subjected to cardiac arrest for 30 min during cardiopulmonary bypass circulation and reperfused thereafter for 90 min.
The pigs for both sham-operated and arrested heart groups were premedicated with ketamine (1000 mg i.m., Ketalar®, Parke-Davis, Barcelona, Spain) and diazepam (20 mg i.v., Stesolid Novum®, A/S Dumex, Denmark). The animals were intubated via a tracheostomy after pankuronium (8 mg i.v., Pavulon®, Organon, Oss, Netherlands) was given. The animals were connected to a respirator and were ventilated with room air (tidal volume, 450 ml) with a frequency of 16–18/min. The anaesthesia was maintained with continuous intravenous infusion of ketamine (7 mg/kg per min) and pankuronium (0.3 mg/kg per min). In addition, the animals received Ringer's solution (7 ml/kg per min, Ringer Acetat®, Pharmacia & Upjohn, Sweden) and a solution containing hydroxyethylstarch (6%, 4 ml/kg per min, Plasmafusin®, Pharmacia & Upjohn) throughout the experiment. The ECG, heart rate, rectal temperature, and systemic and pulmonary artery pressures were monitored.
After median sternotomy, the pericardium was incised and tented. The animals received 100 mg of heparin intravenously and were placed on a total vented cardiopulmonary bypass. The venous return from the caval veins was directed into a membrane oxygenator. The system was primed with 1.5 l of fresh porcine blood containing 570 mg of sodium citrate and 50 mg of heparin to prevent coagulation. The oxygenated blood was returned into the ascending aorta. During the cardiopulmonary bypass, the mean perfusion pressure was maintained at 40–80 mmHg by adjusting the flow rate in the aortic line. Systemic normothermia was used. The aortas of five pigs in the arrested heart group were clamped for 30 min, and these hearts were arrested and protected by infusing 500 ml of cold crystalloid cardioplegic solution (+4°C, modified St. Thomas II) into the proximal ascending aorta. In addition, topical cooling of the heart with ice slush was used. The aorta was declamped after 30 min and the beating heart was reperfused for 90 min. The animals were weaned from cardiopulmonary bypass and the effect of heparin was antagonized by 100 mg of protamine (Protamini Sulfas®, Leiras, Turku, Finland). The animals were sacrificed with potassium chloride injection into the left ventricle. The hearts were excised and transmyocardial samples from the mid-left ventricular free wall were frozen in liquid nitrogen and stored at -70°C.
2.2. Northern blot analysis
About 0.1 g of the frozen ventricular transmyocardial specimen was sliced and homogenized in denaturating solution, and the total RNA was extracted by the guanidinium isothiocyanate method [6]. Total RNA (20 µg) was separated in agarose gel containing formaldehyde and transferred to a nylon membrane. The RNA membrane was hybridized with antisense cRNA probes for ovine COX-1 and human COX-2, which were generated by in vitro transcription as described earlier [7,8]. To detect the mRNA of glyceraldehyde phosphate dehydrogenase (GAPDH), a 1.3 kb rat cDNA probe was used [9].
For cRNA probes, the RNA membrane was prehybridized for at least 6 h at 65°C in a standard solution. Hybridization was carried out at 66°C overnight in the same solution after adding the 32P-labelled cRNA probe. After hybridization, the membranes were washed first at room temperature and then at 65°C [8]. With the cDNA probe for GAPDH, the membranes were hybridized at 42°C and washed at 60°C. Hybridized membranes were exposed to phosphor imaging plates and the amounts of COX-1, COX-2 and GAPDH mRNAs were determined by a phosphor imaging plate scanner. The results were corrected for the loading of GAPDH.
2.3. Western blot analysis
The level of COX-1 and COX-2 protein in porcine left ventricular wall was analyzed by Western blot analysis. Frozen transmyocardial ventricular specimens were crushed and homogenized in extraction buffer and centrifuged to collect the supernatant containing proteins. The proteins (20 µg) were separated by 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred electrophoretically to a nitrocellulose membrane. After blocking the non-specific binding sites with bovine serum albumin and goat normal serum, the protein membrane was incubated with the monoclonal antibody for ovine COX-1 or human COX-2 (Cayman, Ann Arbor, MI) at a 1:1000 dilution for 1 h. Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin was used as a secondary antibody, which was visualized by the enhanced chemiluminescence system (ECL, Amersham, Buckinghamshire, UK) to detect the immunoreactive proteins. The blots were detected by X-ray films. The amount of actin protein was analyzed by a mouse monoclonal antibody for actin (ICN, Costa Mesa, CA) to determine the loading of the proteins.
2.4. Statistical analysis
Statistical analyses were performed by analyses of variance (ANOVA) using Fisher's protected least significant differences test. P values less than 0.05 were considered statistically significant.
|
3. Results
|
|---|
The expression of COX-1 mRNA was detected in specimens of the left ventricular wall of control porcine hearts, whereas the expression of COX-2 mRNA was rather weak in control hearts. The size of the COX-1 mRNA transcript was about 3 kb and that of COX-2 was about 4.6 kb. When porcine hearts were arrested for 30 min by cold crystalloid cardioplegia and reperfused thereafter for 90 min, the level of COX-2 mRNA was increased to 180±10% (mean, SEM, n=5) of the control level, while the expression of COX-1 mRNA was not significantly changed (Fig. 1). The level of COX-2 mRNA was similarly increased to 170±30% (n=5) of the control value in the hearts of sham-operated pigs, which were connected to cardiopulmonary bypass circulation, although the hearts of these sham-operated pigs were not arrested with cardioplegia (Fig. 1).
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 1. The mRNA level of COX-1 and COX-2 in the left ventricular wall of unoperated control and sham-operated pigs as well as in porcine hearts, which were arrested for 30 min with cold crystalloid cardioplegia during cardiopulmonary bypass circulation and reperfused thereafter for 90 min. The hearts of sham-operated pigs (sham) were not arrested, although these pigs were also connected to cardiopulmonary bypass circulation. The means and standard errors of the means from five or six experiments are indicated. The mean value of the control experiments is expressed as 100%. Compared with the corresponding controls: *P<0.05 and ***P<0.001.
|
|
In Western blot analysis, the level of COX-1 protein in the hearts arrested by cold cardioplegia was 110±20% (n=5) of the unoperated control level and was thus not significantly changed (Fig. 2). COX-2 protein was detected in left ventricular specimens of both control and arrested hearts (Fig. 2). The level of COX-2 protein was increased to 280±70% (n=5) of the control value in the myocardium of arrested hearts, which had been arrested for 30 min and reperfused thereafter for 90 min (Fig. 2).
View larger version (54K):
[in this window]
[in a new window]
|
Fig. 2. Western blot analysis of COX-1, COX-2 and actin proteins in the left ventricular wall of control (C) and arrested (A) porcine hearts, which were arrested and protected for 30 min with cold crystalloid cardioplegia and reperfused thereafter for 90 min. Actin protein is shown to indicate the loading of the total protein.
|
|
|
4. Discussion
|
|---|
The present study indicates that the expression of the COX-2 gene is increased after cardioplegic arrest in the porcine heart, while the expression of COX-1 is not changed. The increase was detected at both the COX-2 mRNA and protein level.
The expression of COX-2 mRNA was, however, also stimulated in the ventricular myocardium of sham-operated pigs, which were connected to cardiopulmonary bypass for 120 min without aortic clamping. During bypass circulation, the hearts of sham-operated pigs were beating and their coronary circulation was normal.
Therefore, the increased expression of COX-2 in arrested hearts is obviously not solely due to myocardial ischemia reperfusion injury. Increased expression of COX-2 in arrested hearts could be related to ischemia during cardioplegic arrest, because the expression COX-2 is stimulated in vascular endothelial cells by hypoxia [4] and is enhanced in infarcted areas of human myocardium [10], and because the expression of COX-2 and the formation of prostaglandins was stimulated by ischemia in rabbit hearts [11]. In ischemic rabbit hearts, COX-2 was detected to mediate the cardioprotective effects, which were abolished by specific inhibitors of COX-2 [11]. Also, the reperfusion could contribute to the induction of COX-2 expression, since the expression of COX-2 was upregulated in rat brain during the reperfusion after transient cerebral ischemia [12,13].
It seems, however, that the connection of pigs into the cardiopulmonary bypass circulation as such contributed to the increased myocardial expression of COX-2, since the level of COX-2 mRNA was also stimulated in the hearts of sham-operated pigs. Increased expression of COX-2 may thus be related to surgery, extracorporeal circulation, additional porcine blood, or anaesthesia.
Increased expression of COX-2 may have a protective function in the heart, since it may increase the formation of prostacyclin and other prostaglandins. In rabbit hearts, the induction of COX-2 after myocardial ischemia resulted in salutary effects, which were obviously related to the increased formation of prostacyclin and/or prostaglandin E2 [11]. It is, however, possible that there are also some deleterious effects, because some of the formed prostaglandins may have inflammatory effects. Further studies are thus needed to assess the benefits and adverse effects of COX-2 induction in the heart after bypass circulation and cardioplegic arrest.
The present study was carried out using pigs as experimental animals. It is, however, possible that a similar increase in COX-2 expression would occur also in the myocardium of human hearts, which are arrested by cardioplegia during bypass circulation in open heart operations, since increased blood levels of prostaglandins have been detected in humans during and after cardiopulmonary bypass [14,15], and since increased expression of COX-2 was detected in human atrial appendages exposed to cold cardioplegia and a short reperfusion during coronary artery surgery [16].
|
References
|
|---|
-
Smith W.L., Garavito R.M., DeWitt D.L. Prostaglandin endoperoxide H synthases (cyclooxygenase)-1 and -2. J Biol Chem 1996;271:33157-33160.[Free Full Text]
-
Vane J.R., Bakhle Y.S., Botting R.M. Cyclooxygenases 1 and 2. Ann Rev Pharmacol Toxicol 1998;38:97-120.[Medline]
-
Ristimäki A., Garfinkel S., Wessendorf J., Maciag T., Hla T. Induction of cyclooxygenase-2 by interleukin-1
. J Biol Chem 1994;269:11769-11775.[Abstract/Free Full Text]
-
Schmedtje J.F., Jr, Ji Y.-S., Liu W.-L., DuBois R.N., Runge M.S. Hypoxia induces cyclooxygenase-2 via NF-
B p65 transcription factor in human endothelial cells. J Biol Chem 1997;272:601-608.[Abstract/Free Full Text]
-
Vane J.R., Botting R.M. Pharmacodynamic profile of prostacyclin. Am J Cardiol 1995;75:3A-10A.[Medline]
-
Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 1987;162:156-159.[Medline]
-
Uotila P.J., Kääpä P.O. Cyclooxygenase-2 expression in human monocytes stimulated by meconium. Lancet 1998;351:878.[Medline]
-
Isoherranen K., Punnonen K., Jansen C., Uotila P. Ultraviolet irradiation induces cyclooxygenase-2 expression in keratinocytes. Br J Dermatol 1999;140:1017-1022.[Medline]
-
Fort P., Marty L., Piechaczyk M., El Sabrouty S., Dani C., Jeanteur P., Blanchard J.M. Various rat adult tissues express only one major mRNA species from the glycelaldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res 1985;13:1431-1442.[Abstract/Free Full Text]
-
Wong S.C.Y., Fukuchi M., Melnyk P., Rodger I., Giaid A.G. Induction of cyclooxygenase-2 and activation of nuclear factor-B in myocardium of patients with congestive heart failure. Circulation 1998;98:100-103.[Abstract/Free Full Text]
-
Shinmura K., Tang X.-L., Wang Y., Xuan Y.-T., Liu S.-Q., Takano H., Bhatnagar A., Bolli R. Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sci USA 2000;97:10197-10202.[Abstract/Free Full Text]
-
Miettinen S., Fusco F.R., Yrjänheikki J., Keinänen R., Hirvonen T., Roivainen R., Närhi M., Hökfelt T., Koistinaho J. Spreading depression and focal brain ischemia induce cyclooxygenase-2 in cortical neurons through N-methyl-D-aspartic acid-receptors and phospholipase A2. Proc Natl Acad Sci USA 1997;94:6500-6505.[Abstract/Free Full Text]
-
Nogawa S., Forster C., Zhang F., Nagayama M., Roos M.E., Iadecola C. Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia. Proc Natl Acad Sci USA 1998;95:10966-10971.[Abstract/Free Full Text]
-
Uotila P., Suves M., Heikkilä H., Jalonen J. Prostanoids and hemodynamics in man before and during cardiopulmonary bypass. Prostaglandins 1984;28:497-508.[Medline]
-
Faymonville M-E., Deby-Dupont G., Larbuisson R., Deby C., Bodson L., Limet R., Lamy M. Prostaglandin E2, prostacyclin, and thromboxane changes during nonpulsatile cardiopulmonary bypass in humans. J Thorac Cardiovasc Surg 1986(91):858-866.
-
Métais C., Li J., Li J., Simons M., Sellke F.W. Serotonin-induced coronary contraction increases after blood cardioplegia-reperfusion: role of COX-2 expression. Circulation 1999;100:II328-II334.
Top
Abstract
1. Introduction
