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Ann Thorac Surg 2002;73:156-162
© 2002 The Society of Thoracic Surgeons

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Original article: cardiovascular

Myocardial immediate early gene activation after cardiopulmonary bypass with cardiac ischemia-reperfusion

^a Department of Cardiology, Children’s Hospital, Harvard Medical School, Boston, MA, USA
^b Department of Cardiac Surgery, Children’s Hospital, Harvard Medical School, Boston, MA, USA
^c Division of Hematology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

Accepted for publication August 2, 2001.

* Address reprint requests to Dr Nelson, Division of Pediatric Cardiology, Children’s Hospital Medical Center, OSB-4, 3333 Burnet Ave, Cincinnati, OH 45229, USA
e-mail: davenelson{at}chmcc.org

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. The inflammatory process after cardiopulmonary bypass is accompanied by alterations in gene expression for various inflammatory mediators.

Methods. To analyze differential gene expression after myocardial ischemia-reperfusion, subtraction hybridization was used to discover induction of TIS7/PC4, an immediate early gene heretofore not observed in the heart. This prompted characterization of the related immediate early genes c-fos and c-jun, by Northern analysis and in situ hybridization in human and lamb myocardium subjected to cardiopulmonary bypass with myocardial ischemia. For comparison, we analyzed expression of inducible nitric oxide synthase (iNOS), which requires cytokine-activation, resulting in a "delayed" response.

Results. In ischemic-reperfused myocardium at end-cardiopulmonary bypass, c-fos, c-jun, and TIS7/PC4 were induced, whereas iNOS transcripts were undetectable. Expression patterns of c-fos and c-jun by in situ hybridization were markedly different; myocardial c-fos expression was diffuse and homogeneous, whereas c-jun expression was patchy with areas of intense focal localization.

Conclusions. Cardiopulmonary bypass with myocardial ischemia rapidly induces the immediate early genes TIS7/PC4 (discovered by subtraction hybridization), and c-fos and c-jun (precursors to the transcriptional regulator AP-1). Immediate early genes presumably contribute to activation of inflammatory mediators after cardiopulmonary bypass and differences in their tissue expression patterns, as observed for c-fos and c-jun, presumably modulate their effect upon downstream gene activation.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) circulatory support techniques used for cardiac operations, are known to contribute to postoperative inflammatory tissue injury and organ system dysfunction [1, 2]. Initiators of inflammation include activation of blood components by the bypass apparatus and ischemia-reperfusion injury, resulting from the obligate period of ischemia (and pursuant reperfusion) that occurs with cardiopulmonary bypass. Inflammatory tissue injury after cardiac operation contributes to morbidity and mortality because the organs most significantly affected include the heart, lungs, and central nervous system [3].

Investigations from our group and others have demonstrated that the inflammatory process after CPB is accompanied by alterations in gene expression for various inflammatory mediators and adhesion molecules [4–6]. Because the molecular pathways linking CPB and ischemia-reperfusion to changes in gene expression remain obscure, we used subtraction hybridization to identify other induced genes in neonatal lamb hearts subjected to CPB with associated myocardial ischemia-reperfusion. One of the clones isolated by subtraction hybridization was the early response gene TIS7/PC4 [7–9], an immediate early gene cloned in neurally-derived tissue but not previously observed in the heart. The subtraction hybridization results prompted us to characterize expression of the related immediate early gene precursors of the transcription factor AP-1, c-fos and c-jun [10], in human and ovine myocardium subjected to CPB and associated ischemia-reperfusion. For comparison, we assessed myocardial mRNA expression of inducible nitric oxide synthase (iNOS), a "delayed response gene" requiring cytokine-activation [11].

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Neonatal lamb CPB/DHCA model
Myocardial tissues for subtraction-hybridization, Northern analysis and in-situ hybridization were harvested from neonatal lambs subjected to CPB/DHCA immediately upon termination of bypass (n = 3) or control animals undergoing sternotomy with 3 hours anesthesia and no myocardial ischemia (n = 3). Tissue samples were immediately frozen in liquid nitrogen upon harvest.

The neonatal lamb CPB/DHCA model has been previously characterized and described [12]. Briefly, neonatal lambs were anesthetized, paralyzed, and ventilated. The heart was exposed by sternotomy and catheters placed for hemodynamic monitoring. After systemic heparinization, a 5-French sheath was inserted into the apex of the left ventricle for venting during bypass. The CPB circuit was primed with homologous donor blood to achieve a hematocrit of 20%. Cardiopulmonary bypass was initiated through the femoral artery and right atrial cannulas. After 5 minutes normothermic bypass (37°C), animals were cooled to 20°C for more than 25 minutes using pH stat strategy, and DHCA was initiated. The heart was topically cooled with cold saline during DHCA. After 2 hours DHCA, CPB was reinitiated with warm perfusate. The animals were warmed to a rectal temperature of 35°C for more than 30 minutes and weaned from bypass. Animals were immediately sacrificed and tissues were harvested.

All animals in these studies received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (National Institutes of Health, Publication No. 86–23).

Collection of paired human atrial tissues
Pairs of human atrial samples were collected from patients undergoing cardiac surgery for repair or palliation of congenital heart defects, as previously described [4, 6]. The study protocol was approved by the Committee for Clinical Investigation at Children’s Hospital (Boston, MA). Ten pediatric patients (ages, 1 month to 32 months) were enrolled in the study after informed consent was obtained from their parents. The cardiac lesions of these patients included double outlet right ventricle [3], complete atrioventricular canal [4], truncus arteriosus [2], and heterotaxy/common atrium [1]. Median cross-clamp and total CPB pump times were 85 and 131 minutes, respectively (ranges, 67–103 minutes and 109–159 minutes). Circulatory arrest times were minimal ( 11 minutes). Surgical technique and tissue collection has been previously described [4, 6]. Briefly, paired samples of human atrium were obtained just before CPB initiation and again at CPB termination. Specimens for RNA isolation were snap frozen in liquid nitrogen and stored at -80°C. Specimens for in-situ hybridization were soaked in 4% paraformaldehyde for 6 hours, in 30% sucrose overnight, then drained and stored at -80°C.

RNA preparation
Total RNA was extracted from human or lamb tissues by guanidinium-thiocyanate method with RNAzol B (Cinna/Biotecx, Friendswood, TX). Oligo-dT selected RNA was isolated by Oligotex-dT (Quiagen, Inc, Chatsworth, CA). The RNA was suspended in 0.5% SDS and quantitated spectrophotometrically.

Subtraction-hybridization and isolation of subtracted cDNA clones
Ischemic ovine ventricle for subtraction hybridization and cDNA library construction was obtained from neonatal lamb hearts subjected to CPB with 2 hours hypothermic arrest. Subtraction-hybridization was performed using the Subtractor kit (Invitrogen Corp, San Diego, CA) according to the manufacturer’s instructions. The induced pool of first-strand cDNA was prepared from 1 µg of ischemic-reperfused ovine oligo-dT-selected ventricular RNA by reverse transcription with oligo-dT primer (200 ng) at 42°C for 60 minutes. Template mRNA was removed by alkali treatment and induced first-strand cDNA was purified by phenol-chloroform extraction and ethanol precipitation. The control (noninduced) pool consisted of 10 µg oligo-dT-selected RNA from control (nonischemic) ovine ventricle. Control mRNA was photobiotinylated with photobiotin-acetate irradiated with a bright light. The photobiotinylated mRNA was then purified by multiple phenol-chloroform extractions. The large excess of photobiotinylated control mRNA driver was hybridized with induced first-strand cDNA for 48 hours at 68°C. Photobiotinylated mRNA-cDNA duplexes were removed by treatment with streptavidin followed by phenol-chloroform extraction, thus leaving the subtracted (induced - control) first-strand cDNA.

Subtraction probe was generated from subtracted cDNA by random-primed Klenow DNA polymerase reaction. The ³²P-labeled probe was used to screen approximately 10,000 plaques of an ischemic sheep ventricle cDNA library [5], plated sparsely (500 to 1000 plaques per 150 mm plate) to allow isolation of single phage colonies. Fifty "subtracted" clones were obtained. Actin clones, presumed to be major contaminants of the subtraction screening, were screened and discarded by screening replica filters with human -actin probe. Phage clones isolated after the subtraction screening were excised by ExAssist helper phage (Stratagene), and resultant pBluescript plasmids were purified for further analysis. Restriction mapping and DNA sequencing were used to assess the identity of subtracted cDNA clones, and Northern analysis was used to confirm induction of subtracted clones by CPB.

Northern analysis
Samples of total RNA (15 µg/lane) or oligo-dT selected RNA (1 µg/lane) were denatured, separated by electrophoresis, and transferred to Magnagraph membranes (MicronTechnology, Westborough, MA). Hybridization probes included cDNA templates isolated by subtraction-hybridization, human c-fos, murine c-jun and human -actin labeled with ³²P by random primed Klenow DNA polymerase reactions. Blots were hybridized overnight at 42°C, washed, and exposed. Hybridization of blots with -actin probe served as an internal control for RNA loading. Signals were quantified using ImageQuant software after overnight exposures on PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA).

In-situ hybridization
Hybridization was performed on cryopreserved sections of human and ovine myocardium using ³⁵S-labeled riboprobes specific for human c-jun and c-fos and ovine c-jun as previously described [6]. The cDNA for human c-jun (IMAGE clone 41019; nt 933–2221) and c-fos (EST 74940; full-length 180 nt) for production of sense and antisense riboprobes were obtained from ATCC (Rockville, MD) and cloned into pBluescript. Ovine c-jun and TIS7 were cloned as phagemids from a -ZAP-II ischemic-reperfused lamb lung library [5], and were used directly as templates for riboprobe synthesis. To evaluate expression patterns for human c-fos and c-jun, a blinded observer (EJN) assessed paired atrial sections from 3 human subjects for each gene, with sections from 2 of the subjects analyzed for both c-fos and c-jun. Ovine c-jun expression was examined in a blinded manner by the same observer in myocardial sections from 3 control lambs and 3 lambs subjected to CPB/DHCA.

Reverse transcriptase-polymerase chain reaction
The RNA from paired human atrial samples (n = 5 subjects) pre-CPB and post-CPB were subjected to Superscript reverse transcriptase (RT) (Stratagene, LaJolla, CA) and polymerase chain reaction (PCR) with primers for human iNOS and ß-actin iNOS primers were 5'ATTCAGGTACGCTGTGTTTGG 3' (sense) and 5'CATGGTGAACACGTTCTTGG 3' 351 bp predicted product - nt 2060–2411 in iNOS cDNA genbank accession number L09210). A water blank served as a negative control. The COS cells transfected with full-length iNOS cDNA served as positive controls.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Using myocardial RNA isolated from control (nonischemic) and induced (CPB/DHCA) myocardium, 50 cDNA clones were isolated by subtraction library screening. Subtraction cloning results are summarized in Table 1. After restriction mapping and sequence analysis, a total of 9 subtracted clones were analyzed by Northern analysis using ³²P labeled ECOR1 fragments of subtracted cDNA clones. Five subtraction clones were found to represent genes induced by CPB/DHCA. Among the induced genes, subtraction clone 13 was identified to be the ovine homolog of murine TIS7/rat PC4, an interferon-related immediate early gene first cloned in cultured rat astrocytes [7, 8]. Figure 1 shows a poly-A Northern blot of pre-CPB and post-CPB ovine myocardium demonstrating induction of subtraction clone 13 at termination of bypass. Homology search of clone 13 revealed homology to murine TIS7/rat PC4 (predicted ovine amino acid sequence from Blast search: KGDEQRAAAALASVLCIQLGPGIESEEVLKTLGPILKKIICDGTASIQARQTCATCFGVC). Attempted in situ hybridization of TIS7 transcripts in ovine tissue was unsuccessful, presumably due to the low prevalence of these transcripts (results not shown).

View larger version (48K): [in this window] [in a new window]   Fig 1. Immediate early gene identified in subtraction screening induced at termination of bypass and circulatory arrest in ovine ventricle. Representative Northern analysis of oligo-dT-selected RNA from control neonatal lamb ventricle and ventricle collected immediately after bypass and circulatory arrest, probed with cDNA fragment of subtraction clone 13. Blast search with clone 13 sequence identified close homology with murine TIS7/rat PC4.

View larger version (48K): [in this window] [in a new window]   Fig 2. Marked induction of c-fos and c-jun mRNA was observed in neonatal lamb ventricle at termination of bypass and circulatory arrest (n = 3 lambs). Representative total RNA Northern blot of control neonatal lamb ventricle (Pre) and ventricle collected immediately after bypass and 2 hours circulatory arrest (Post). Blots were probed with 32P labeled cDNA fragments of c-fos and c-jun, respectively. The internal control for each lane was human -actin cDNA. (DHCA = deep hypothermic circulatory arrest.)

View larger version (75K): [in this window] [in a new window]   Fig 3. Representative in-situ hybridization of lamb ventricular myocardium showing patchy, but intense focal induction of c-jun mRNA at termination of bypass and circulatory arrest (mag = 40 x objective). (A) Control (nonischemic) ventricular myocardium. (B) Ventricular myocardium at termination of bypass demonstrating mild diffuse induction and a focus of strong perivascular induction that may represent an inflammatory cell (arrow). (C) Sense riboprobe (negative control) on postbypass myocardium demonstrates minimal background staining.

View larger version (138K): [in this window] [in a new window]   Fig 4. Representative in-situ hybridization of human atrial myocardium demonstrating patchy, focal induction of c-jun mRNA at termination of bypass with associated myocardial ischemia (mag = 40x objective). (A) and (B) Two separate examples of "hot spots" (arrows) for c-jun mRNA in briefly reperfused human atrial myocardium at end-bypass. Transcripts for c-jun were not detected in prebypass atrial tissue (data not shown).

View larger version (102K): [in this window] [in a new window]   Fig 5. Representative in situ hybridization of human atrial myocardium demonstrating diffuse induction of c-fos mRNA immediately upon termination of bypass with associated myocardial ischemia. Sections are shown at low magnification (10 x objective) to demonstrate diffuse nature of c-fos expression. Dark-field (A, C, and E) and corresponding bright-field (B, D, and F) photomicrographs. (A), (B) Prebypass. (C), (D) Briefly reperfused atrial tissue at end-bypass demonstrates predominantly diffuse myocardial induction, with rare high-intensity foci. (E), (F) Sense riboprobe (negative control) shows minimal staining.

View larger version (40K): [in this window] [in a new window]   Fig 6. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrates absence of iNOS mRNA expression in briefly reperfused human atrium at termination of bypass (n = 5 subjects). (A) Representative gel demonstrating iNOS fragment amplified by RT-PCR; Lane 1 = water only (negative control); Lane 2 = prebypass atrial tissue; Lane 3 = Briefly reperfused atrial tissue at end-bypass; Lane 4 = monkey kidney COS cells transfected with human iNOS cDNA. (B) Control human -actin amplified fragment, observed as expected in human atrial tissue only (lanes 2 and 3).

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Our data demonstrate rapid activation of immediate early genes (c-jun, c-fos, TIS7) in briefly reperfused myocardial tissue at end-CPB, whereas iNOS transcripts were yet undetectable even by RT-PCR. In-situ hybridization demonstrated that tissue expression patterns of c-fos and c-jun were markedly different; myocardial c-fos expression was diffusely homogeneous, whereas c-jun expression was patchy with areas of intense focal localization. Although attempts to develop an ovine c-fos riboprobe were unsuccessful, the expression pattern differences are well demonstrated in ischemic-reperfused human atrial tissue. Because both c-jun and c-fos proteins are required for functional AP-1 transcriptional regulation, differences in their tissue expression patterns presumably modulate the activation of downstream genes. These data suggest that activation of genes associated with postbypass inflammation may be modulated by spatial and temporal factors in addition to transcriptional control (ie, activation of genes induced by AP-1 will be pinpointed in areas with high expression of both c-jun and c-fos).

One of the early response genes found to be induced by CPB, the ovine homolog of murine TIS7/rat PC4 [7, 8], was identified by subtraction cloning. Subtraction hybridization was used to isolate gene transcripts induced by CPB and myocardial ischemia. Interferon-related TIS7 (TPA inducible sequence 7) is induced rapidly in cultured neural cell lines by growth factors and phorbol esters [7–9]. Its proposed actions include tumor suppressor activity [9] and transcriptional regulation of myoblast differentiation [16]. Our observation that TIS7/PC4 is induced by CPB is the first report that it may play a role in ischemia-reperfusion injury. Enthusiasm for differential gene expression analyses has been fueled by the emergence of powerful new high-throughput techniques; subtraction hybridization can complement such analyses [17]. Current microarray technologies are biased toward known genes and those genes thought to be involved in a certain process, as determined by the genes applied to commercially available chips or filters. In contrast, subtraction methods allow detection of novel or unexpected genes. Genes identified by subtraction screening are good candidates for inclusion on subsequent arrays.

Although there is evidence that iNOS activity [2, 18] is altered by CPB, the pattern and time course of expression is not well characterized. Activation of iNOS requires cytokine-activation resulting in a "delayed" response [2, 11]. Our findings would suggest that myocardial iNOS is not yet upregulated at termination of CPB. Studies in rats subjected to endotoxemia and ischemia-reperfusion injury demonstrate delayed expression of iNOS mRNA, with only slight increases in iNOS expression detectable by 2 hours reperfusion and peak levels not detected until 6 to 8 hours after the inflammatory insult [11]. The finding that iNOS transcripts were undetectable by RT-PCR in human atrial tissue at termination of bypass (with minimal reperfusion) is thus consistent with previous work.

In summary, the data suggest that immediate early gene activation occurs promptly after CPB and likely participates in signal transduction leading to the myocardial inflammatory process. Differences in tissue expression patterns of these early-response genes, as observed for c-jun and c-fos, presumably modulate their effect upon downstream gene activation.

	Acknowledgments

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Supported by NIH grant SPO1 HL48675 (JEM, JWN) and K24HL04184 (EJN).

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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