Ann Thorac Surg 1997;63:1669-1675
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Expression of Immediate Early Genes After Cardioplegic Arrest and Reperfusion
Hermann Aebert, MD,
Torsten Cornelius, BS,
Tobias Ehr, BS,
Stephan R. Holmer, MD,
Dietrich E. Birnbaum, MD,
Günter A. J. Riegger, MD,
Heribert Schunkert, MD
Departments of Thoracic and Cardiovascular Surgery and Internal Medicine II, University Hospital, Regensburg, Germany
Accepted for publication December 16, 1996.
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Abstract
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Background. Induction of protooncogenes such as c-fos, c-jun, and EGR-1 has been implicated in cellular growth and differentiation. Heat-shock proteins (HSPs) such as hsp 70 may mediate resistance to ischemia after heat shock and ischemic preconditioning. The effects of cardioplegia on the regulation of these immediate early genes are unclear.
Methods. Isolated rat hearts were subjected to different cold (5°C) or normothermic (35°C) cardioplegic solutions and reperfused with normothermic Krebs-Henseleit buffer. Right atrial biopsy specimens from patients undergoing coronary artery bypass grafting with cold cardioplegic arrest were taken before and after cardiopulmonary bypass. Analysis of immediate early gene messenger RNAs was performed using Northern blots. Related proteins were localized by immunohistochemistry.
Results. In rat hearts, cold cardioplegia for 40 minutes with Bretschneider or St. Thomas' II solution followed by 40 minutes' reperfusion resulted in a significant increase in left ventricular c-fos, EGR-1, and c-jun messenger RNA levels (4.0-, 3.1-, and 3.0-fold, respectively, with Bretschneider solution and 3.7-, 2.8-, and 2.1-fold, respectively, with St. Thomas' II solution) compared with control hearts perfused at 35°C with Krebs-Henseleit buffer. Normothermic cardioplegia with St. Thomas' II solution was without effect, whereas sequential perfusion with Krebs-Henseleit buffer at 5°C and 35°C resulted in a similar increase in protooncogene messenger RNA levels. Only cold Bretschneider solution was related to a 5.2-fold induction of hsp 70 messenger RNA levels. Likewise, rat atrial tissues and samples from patients after cardiopulmonary bypass displayed a significant induction of these immediate early genes. Monoclonal antibodies against c-FOS and HSP 70 proteins stained nuclei and perinuclear spaces of endothelial cells and cardiac myocytes.
Conclusions. Cold cardioplegic arrest and normothermic reperfusion are potent triggers for immediate early gene induction. Hypothermia emerged as the prime stimulus for the examined protooncogenes. In contrast, hsp 70 induction was dependent on the cardioplegic solution.
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Introduction
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Heat shock proteins (HSPs) may modulate protein folding, assembly, and translocation between different cell compartments; they may also serve as transcription factors [1]. Experimentally, HSPs can be induced by different forms of cellular stress [2–4]. In animal hearts, induction of HSP 70 by hyperthermia has been shown to parallel improved recovery after ischemia or prolonged cardiac arrest [5–7].
Like HSP induction, induction of protooncogenes can be observed to occur rapidly after changes in the cellular milieu. In particular, stimulation of c-fos, c-jun, and EGR-1 has been found in response to hypoxia, ischemia, neurohormonal stimulation, or wall stress in animal cardiac tissue [3, 8–11]. These protooncogenes may act as transcription factors and thereby regulate other genes [12–16]. In addition, they have been linked to complex cellular responses like growth, differentiation, and programmed cell death [8, 16, 17]. The aim of this study was to evaluate the effects of cardioplegic arrest and reperfusion on the expression of these immediate early genes (IEGs).
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Material and Methods
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Isolated Rat Heart Preparation
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). Male Wistar rats weighing 370 to 430 g were injected intraperitoneally with 15 mg of sodium pentobarbital. The thorax was opened, and the heart was rapidly removed. Hearts were placed in a constant-temperature chamber, and perfusion was resumed through the aortic root with 35°C modified Krebs-Henseleit buffer (KH) (118 mmol/L NaCl, 4.7 mmol/L KCl, 2.0 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 25 mmol/L NaHCO3, 5.5 mmol/L glucose, 1 mmol/L lactate, and 0.5% albumin). The perfusate was equilibrated with 95% oxygen and 5% carbon dioxide for a pH between 7.36 and 7.44. Perfusion pressure was 80 mm Hg. For rapid changes between solutions and temperatures, two perfusion tanks were employed sequentially. Heart temperatures were checked with a thermistor probe in the right ventricle. Before the perfusion protocols were performed, all hearts were allowed to stabilize for 10 minutes.
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Perfusion Protocols
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Groups of 8 hearts were subjected to cold (5°C) cardioplegia with St. Thomas' II solution (ST) (91.6 mmol/L NaCl, 14.8 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 15 mmol/L MgCl2, 1.2 mmol/L CaCl2, 25 mmol/L NaHCO3, and 1 mmol/L procaine hydrochloride; Köhler Chemie, Alsbach, Germany) or Bretschneider solution (BS) (15 mmol/L NaCl, 9 mmol/L KCl, 4 mmol/L MgCl2, 1 mmol/L KH-2-ketoglutarate, 18 mmol/L histidine hydrochloride, 180 mmol/L histidine, 2 mmol/L tryptophan, and 30 mmol/L mannitol; Köhler Chemie) by perfusion at a pressure of 80 mm Hg for 5 minutes and placement in cold isotonic NaCl solution of the same temperature. After 40 minutes of cardiac arrest, hearts were transferred to a normothermic (35°C) temperature chamber, and perfusion was resumed with 35°C KH for 40 minutes. Additional groups of 5 hearts were perfused with cold KH, normothermic ST, or normothermic BS for 40 minutes followed by 40 minutes of normothermic KH reperfusion. A group of 5 hearts, continuously perfused at 35°C with KH for 40 minutes, served as control for potential induction of IEGs by the experimental setup.
To evaluate the time course of IEG induction by cold BS cardioplegia, groups of 3 hearts were arrested by the same procedure for 20, 40, 60, and 90 minutes followed by 40 minutes of normothermic KH reperfusion. To determine the influence of reperfusion time, groups of 3 hearts were subjected to cold BS cardioplegia for 40 minutes followed by 0 (ie, no reperfusion), 20, 40, 60, and 90 minutes of normothermic KH reperfusion. Levels of messenger RNAs (mRNAs) were compared with baseline (ie, unperfused hearts) and with hearts not subjected to cardioplegia (ie, 35°C KH perfusion for 40 minutes).
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Patient Studies
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For human studies, approval was given by the institutional committee on medical ethics. Informed consent was obtained from 18 patients scheduled for elective coronary artery bypass grafting with multiple distal anastomoses.
Cardiopulmonary bypass (CPB) was started with a prime of 1,400 mL of Ringer's solution and 250 mL of 20% mannitol. Moderate systemic hypothermia (30° to 32°C) was applied, and flow rates were kept at 2.4 to 2.6 L•min-1•m-2 of calculated body surface area. Cold cardioplegia was administered according to the regimen routinely used in our institution [18]: After cross-clamping of the aorta, 100 mL of cold (4°C) Kirsch solution (80 mmol/L magnesium aspartate, 11 mmol/L procaine hydrochloride, and 247 mmol/L xylitol; Köhler Chemie) was injected into the aortic root for induction of cardiac arrest. Immediately afterward, cold (4°C) cardioplegic solution (5 mmol/L KCl, 0.5 mmol/L CaCl2, 2 mmol/L magnesium aspartate, 25 mmol/L NaCl, 25 mmol/L NaHCO3, 4 mmol/L procaine hydrochloride, 10 mmol/L glucose, 200 mmol/L mannitol, and 60 g hydroxyethyl starch with a molecular weight of 450,000; Fresenius AG, Bad Homburg, Germany) was applied through the aortic root at a pressure of 60 to 70 mm Hg. Initial volume was 1 L. Application of cardioplegia was repeated after approximately 40 minutes in the case of longer cross-clamp times. In addition, hearts were cooled externally with cold (4°C) Ringer's solution. After application of cardioplegia, temperatures between 5° and 9°C were measured in the right atrial appendage.
Small tissue samples were removed before CPB (n = 9; 7 men and 2 women; mean age, 58.9 years, and range, 37 to 74 years) and after cardiac arrest and reperfusion (n = 9; 7 men: mean age, 60.9 years, and range, 53 to 70 years). After placement of a pursestring suture around the base of the right atrial appendage, the tip was removed as the pre–CPB sample. A two-stage venous cannula was inserted, and CPB was started. For the post–cardioplegia/reperfusion sample, a pursestring suture was placed initially at the tip of the right atrial appendage, and the venous cannula was inserted. After termination of CPB, a second pursestring suture was placed around the base of the atrial appendage, and the tissue between the two sutures was taken for analysis.
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Messenger RNA Measurement
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Rat left ventricles and atria were divided from other cardiac tissue and separately snap frozen in liquid nitrogen for further analysis. Tissue samples obtained from patients were snap frozen in the operating room immediately after removal. Isolation of RNA and Northern blot analyses were performed as previously described in detail [8, 19]. Samples were blotted on nylon filters (Genescreen; New England Nuclear, Boston, MA). After a prehybridization period of 4 hours, blots were hybridized overnight with alpha 32P-labeled complementary DNA probes [8]. After washing, blots were exposed to X-ray film for 24 hours. To control for possible sample variability, identical Northern blots were hybridized with a control complementary DNA probe for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was employed as a recovery marker. In addition, slot-blot analyses were carried out using three different concentrations of each sample. Autoradiograms generated by Northern blots or slot blots were scanned with a microdensitometer (LKB Instruments, Paramus, NJ) with background set at zero for each autoradiograph. Regression lines were calculated from the integral values obtained by scanning serial concentrations of each sample. The relative signals of specific mRNAs were estimated from the slope of the regression line, and only r values greater than 0.9 were accepted. The signal for IEG mRNA was divided by the signal for GAPDH mRNA for each sample. Results are expressed as IEG/GAPDH mRNA ratios.
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Immunohistochemistry
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Small tissue samples were either directly immersed in OCT 4583 (Miles Inc, Elkhart, IN) and frozen in liquid nitrogen or previously fixed in Bouin's solution. Cryostat sections (unfixed samples were postfixed in acetone at -20°C) were stained with monoclonal antibodies (anti- c-FOS; Medac, Hamburg, Germany; and anti-HSP 72; Amersham Buchler, Braunschweig, Germany) according to the peroxidase-antiperoxidase method [20]. Adjacent sections and a portion of those already processed immunohistochemically were stained with hematoxylin and eosin for better identification of cell types. Negative controls were performed by replacing antibodies with unspecific immunoglobulins.
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Statistical Analysis
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All data are presented as the mean ± the standard error. Messenger RNA ratios from rat hearts were compared using unpaired t tests with Bonferroni's correction with ratios of negative control (KH perfusion at 35°C) designated as 1. Relationships between rat left ventricular and atrial measurements of IEG mRNA levels were determined using linear regression analysis. In patient samples, a Kolmogorov-Smirnov normality test and unpaired t tests were performed for IEG/GAPDH mRNA ratios before CPB and after cardiac arrest and reperfusion. A probability value of less than 0.05 was considered indicative of a significant difference.
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Results
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Rat Heart Experiments
Cardioplegic arrest with cold BS or ST for 40 minutes followed by 40 minutes of normothermic reperfusion was related to a significant induction of left ventricular protooncogene mRNA levels (Figs 1, 2
). Levels of c-fos, EGR-1, and c-jun were increased 4.0 ± 0.8, 3.1 ± 0.6, and 3.0 ± 0.3 fold, respectively, after cold BS and 3.7 ± 0.6, 2.8 ± 0.4, and 2.1 ± 0.3 times, respectively, after cold ST compared with those perfused with normothermic KH. Perfusion with cold KH resulted in similar induction of protooncogene mRNA levels as the cardioplegic solutions (see Figs 1, 2). In contrast, when cardioplegia was carried out with ST under normothermic conditions, no protooncogene induction was detectable. Hearts perfused with BS at 35°C were not used for analysis, as normothermic perfusion with this solution resulted in a rapid decrease in coronary flow at the preselected perfusion pressure of 80 mm Hg and contracture. In contrast to all other groups, none of the hearts in the normothermic BS group resumed contractility.
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Fig 1. . Representative Northern blots of protooncogene messenger RNAs in isolated, perfused rat hearts. Twenty micrograms of total RNA have been loaded per lane. Type and temperature of solutions are indicated across the top. (GAPDH = glyceraldehyde 3-phosphate dehydrogenase; KH = Krebs-Henseleit buffer; Neg. Control = negative controls, ie, KH perfusion at 35°C.)
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Fig 2. . Rat left ventricular (A) c-fos and (B) c-jun messenger RNA (mRNA) levels at different temperatures (5°C and 35°C) with different solutions (BS = Bretschneider solution; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; KH = Krebs-Henseleit buffer; ST = St. Thomas' II solution; * = p < 0.05 versus KH 35°C.)
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Left ventricular hsp 70 mRNA induction was detected only after cardioplegic arrest with cold BS and reperfusion with normothermic KH. The increase was 5.2 ± 1.1 times compared with normothermic KH (Fig 3). No change in hsp 70 mRNA level was found for any other perfusion protocol.
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Fig 3. . (A) Northern blots and (B) bar graphs of rat left ventricular heat shock protein 70 (hsp 70) messenger RNA (mRNA) levels. (BS = Bretschneider solution; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; KH = Krebs-Henseleit buffer; Neg. Controls = negative controls, ie, KH perfusion at 35°C; ST = St. Thomas' II solution; * = p < 0.05 versus KH 35°C.)
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Time course experiments with cold BS revealed a significant increase in IEG mRNA levels after 20 minutes of cardiac arrest and 40 minutes of reperfusion (Fig 4A). Vice versa, a reperfusion period of 20 minutes preceded by 40 minutes of cardioplegic arrest was sufficient for a significant induction of IEGs (Fig 4B). The time course of EGR-1 and hsp 70 mRNA induction was similar to the causes shown for other protooncogenes, with a significant increase detectable after 20 minutes of cardioplegia and 20 minutes of reperfusion, respectively.
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Fig 4. . Time courses of c-fos and c-jun messenger RNA (mRNA) levels in rat hearts. (A) Effects of various lengths of cold Bretschneider cardioplegia followed by 40 minutes of reperfusion. (B) Time course of normothermic reperfusion after 40 minutes of cold Bretschneider cardioplegia. (GAPDH = glyceraldehyde 3-phosphate dehydrogenase.)
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Induction of IEGs after cardioplegia was similar in atria and left ventricles of isolated, perfused rat hearts. Linear regression analyses for atrial and left ventricular mRNA levels of hsp 70 (r = 0.92, p < 0.0005), c-jun (r = 0.88, p < 0.001), and c-fos (r = 0.62, p < 0.05) revealed comparable levels at the two cardiac sites.
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Patient Studies
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For post-cardioplegia/reperfusion samples, mean aortic cross-clamp time was 69 ± 22 minutes (± standard deviation) and reperfusion time, 38 ± 12 minutes (± standard deviation). Expression of IEG mRNA levels in right atrial tissue was increased significantly after cardioplegic arrest and reperfusion (Figs 5, 6). Levels of c-fos, c-jun, and EGR-1 mRNA were 5.4 ± 0.7 (p < 0.001), 5.0 ± 0.6 (p < 0.001), and 3.5 ± 0.7 (p < 0.05) times higher than prebypass levels. Induction of hsp 70 mRNA was 2.1-fold (± 0.2) (p < 0.001) higher than baseline (before CPB).
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Fig 5. . (A, B) Northern blots of immediate early gene messenger RNAs after cardioplegic arrest and reperfusion in human hearts. (GAPDH = glyceraldehyde 3-phosphate dehydrogenase; hsp70 = heat shock protein 70; neg = tissue sample obtained before cardioplegic arrest; rat = experimental rat heart subjected to cold Bretschneider cardioplegia for 40 minutes followed by 40 minutes reperfusion; 53, 66, 83, 111 = minutes of cardioplegic arrest.)
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Fig 6. . Immediate early gene messenger RNA (mRNA) levels in human atrial biopsy specimens before and after cardiopulmonary bypass (CPB). (GAPDH = glyceraldehyde 3-phosphate dehydrogenase; hsp70 = heat shock protein 70; * = p < 0.05 versus pre-CPB samples.)
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Immunohistochemistry
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After cardioplegic arrest and reperfusion, monoclonal antibodies against c-FOS protein revealed intense staining of endothelial cells. Cardiac myocytes were also clearly marked but less uniformly (Fig 7a). Staining for c-FOS was confined to nuclei and perinuclear spaces and was not observed in other cell types (see Fig 7a, inset). Immunoreactivity for inducible HSP 72 was also strongest in the nuclei of endothelial cells and cardiac myocytes (Fig 7b). In addition, some staining was detected in the cytoplasm of these cell types and in some nuclei of connective tissue cells. No staining was observed when specific antibodies were replaced with nonspecific immunoglobulins.
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Fig 7. . Localization of c-FOS and HSP 72 immunoreactivity in human hearts. (a) Transmission microscopy displays staining for c-FOS protein in nuclei of cardiac myocytes and the vasculature where immunoreactivity is mainly expressed in endothelial cells. (Inset: Interference contrast.) (b) HSP 72 immunoreactivity in cardiac myocytes and blood vessels in a tissue section counterstained with hematoxylin and examined by transmission microscopy. (Inset) Without counterstaining, there is a concentration of immunoreactivity in nuclei of cardiac myocytes. (a, x700 and inset, x1,250 before 40% reduction; b and inset, x500 before 40% reduction.)
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Comment
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The present study indicates that cold cardioplegic arrest of the heart followed by normothermic reperfusion is related to a marked induction of several IEGs. This was evident in both atria and left ventricles of isolated rat hearts as well as in right atrial biopsy specimens from patients undergoing coronary artery bypass grafting.
Remarkable differences in the pattern of IEG expression were observed with the various cardioplegic strategies. In rat hearts, increased levels of hsp 70 were detected only after cold BS cardioplegia. This increase was five-fold and highly significant. Neither cold or normothermic ST nor KH of different temperatures had this result in our experiments. Hearts perfused with calcium-free normothermic BS sustained irreversible damage [21] and could not be used for analysis. A potential explanation for hsp 70 induction after cold BS cardioplegia may be a deviation in calcium levels, as IEG expression is known to be sensitive to intracellular calcium levels by activation or suppression of calcium response elements [8, 22]. Alternatively, other components of BS such as histidine, tryptophan, or mannitol may mediate this effect. Irrespective of what the molecular mechanisms may be, experimental cardioplegia with cold BS seems to be in line with hyperthermia, other forms of stress, or substances such as amphetamine that have been identified to stimulate hsp 70 gene expression [2–4, 23].
The increase in hsp 70 levels in the right atrial biopsy specimens from patients undergoing coronary artery bypass grafting was significant but smaller than in the experimental protocol using cold BS. Species differences, mechanical stress during the surgical procedure, collateral flow and blood reperfusion, or other factors may play a role in respect to the differences between our experimental and clinical results. The cardioplegic solution routinely used in our institution does contain a subphysiologic concentration of calcium. If calcium levels or additives such as mannitol are implicated in induction of HSPs, our findings are not necessarily in contradiction to those of McGrath and associates [24], who did not find HSP induction in right atria of patients after blood cardioplegia.
Our immunohistochemical studies showed staining for HSP 72 protein in cardiac myocytes and endothelial cells, which are supposed to be prime target cells for injury after cardioplegic arrest and reperfusion. Further studies are needed to identify whether HSP induction by cardioplegia relates to functional improvement, which is suggested by studies [5–7, 25] demonstrating that HSP 70 may ameliorate myocardial damage after prolonged cardiac arrest or ischemia and reperfusion.
Induction of the protooncogenes c-fos, c-jun, and EGR-1 was found to depend on temperature rather than type of cardioplegic solution. In rat hearts, even continuous perfusion with KH at 5°C resulted in a significant increase in protooncogene levels. In contrast, normothermic arrest with ST had no effect. In the right atrial biopsy samples from patients undergoing coronary artery bypass grafting, cold cardioplegic arrest and reperfusion was associated with a marked induction of protooncogenes. Increased protooncogene mRNA levels have been found in human saphenous vein grafts stored at 4°C [26]. In this context, recent findings in rat brain showing a modulation of ischemia-induced IEG expression by hypothermia may be of interest [27]. Ischemia seems unlikely to be a stimulus for protooncogene induction in our study, as continuous perfusion with cold oxygenated KH also resulted in an increase in respective mRNA levels. Further, warm cardioplegic arrest by ST perfusion did not result in protooncogene induction. Neurohormonal factors and mechanical stress are known to be mediators of increased protooncogene transcription under experimental conditions [3, 8, 10]. In our patient studies, we cannot exclude a possible influence of these stimulants. In isolated unloaded rat hearts, however, these mechanisms appear unlikely.
Like other protooncogenes, c-FOS protein acts as a transcription factor and exerts its effects in the nucleus. Cytoplasmic regulatory mechanisms may modulate c-FOS levels after translation [28]. Therefore, our immunohistochemical findings with localization of c-FOS protein in nuclei of endothelial cells and cardiac myocytes appear important for discussion of possible consequences of increased mRNA levels. FOS and JUN proteins may form a heterodimer (AP-1) that binds to defined sequences of the 5` flanking regions of several genes [12, 13]. Possible target genes of the AP-1 complex include transforming growth factor beta, atrial natriuretic peptide, and alpha actin [3, 14, 29]; the alpha-myosin heavy-chain gene has been identified as a target of EGR-1 [15]. However, recent evidence suggests a role for c-fos and c-jun protooncogenes also in complex cellular responses such as growth and programmed cell death [8, 16, 17].
The functional consequences of IEG induction in the context of cardioplegic arrest have yet to be defined. Therefore, it is difficult to assess the clinical impact of our studies. However, growing evidence supports an important role for IEGs in cellular responses to stress. We found different patterns of IEG mRNA induction with different cardioplegic solutions and different temperatures in left ventricles and atria of rat hearts. In humans, IEG induction was observed after cold cardioplegic arrest and reperfusion. In addition, we collected evidence of localization of respective proteins in endothelial cells and cardiac myocytes. Further studies are necessary to define the molecular and pathophysiologic implications of IEG induction in the setting of cardioplegia. Eventually, this may lead to better measures for organ protection.
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Footnotes
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Address reprint requests to Dr Aebert, Department of Thoracic and Cardiovascular Surgery, Regensburg University Hospital, Franz-Josef-Strauss-Allee, D-93042 Regensburg, Germany.
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J. P. Schmitt, J. Schroder, H. Schunkert, D. E. Birnbaum, and H. Aebert
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H. Aebert, S. Kirchner, A. Keyser, D. E. Birnbaum, E. Holler, R. Andreesen, and G. Eissner
Endothelial apoptosis is induced by serum of patients after cardiopulmonary bypass
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W. Zhang, S. D. Yan, A. Zhu, Y. S. Zou, M. Williams, G. C. Godman, B. M. Thomashow, M. E. Ginsburg, D. M. Stern, and S.-F. Yan
Expression of Egr-1 in Late Stage Emphysema
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Abstract
Introduction
