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J Thorac Cardiovasc Surg 2003;126:1521-1530
© 2003 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass, USA
b Divisions of Medical Genetics, Boston, Mass, USA
c Informatics Program, Children's Hospital and Harvard Medical School, Boston, Mass, USA
d Genomics Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass, USA
Received for publication December 5, 2002; revisions received January 21, 2003; revisions received February 5, 2003; accepted for publication March 11, 2003.
* Address for reprints: Frank W. Sellke, MD, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, LMOB 2A, 110 Francis St, Boston, MA 02215, USA
fsellke{at}bidmc.harvard.edu
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Abstract |
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METHODS: Atrial myocardium and skeletal muscle were harvested from 16 patients who underwent coronary artery bypass grafting before and after cardiopulmonary bypass and cardioplegic arrest. Ten sample pairs were selected for patient similarity, and oligonucleotide microarray analyses of 12,625 genes were performed using matched precardiopulmonary bypass tissues as controls. Array results were validated with Northern blotting, real-time polymerase chain reaction, in situ hybridization, and immunoblotting. Statistical analyses were nonparametric.
RESULTS: Median durations of cardiopulmonary bypass and cardioplegic arrest were 74 and 60 minutes, respectively. Compared with precardiopulmonary bypass, postcardiopulmonary bypass myocardial tissues revealed 480 up-regulated and 626 down-regulated genes with a threshold P value of .025 or less (signal-to-noise ratio: 3.46); skeletal muscle tissues showed 560 and 348 such genes, respectively (signal-to-noise ratio: 3.04). Up-regulated genes in cardiac tissues included inflammatory and transcription activators FOS; jun B proto-oncogene; nuclear receptor subfamily 4, group A, member 3; MYC; transcription factor-8; endothelial leukocyte adhesion molecule-1; and cysteine-rich 61; apoptotic genes nuclear receptor subfamily 4, group A, member 1 and cyclin-dependent kinase inhibitor 1A; and stress genes dual-specificity phosphatase-1, dual-specificity phosphatase-5, and B-cell translocation gene 2. Up-regulated skeletal muscle genes included interleukin 6; interleukin 8; tumor necrosis factor receptor superfamily, member 11B; nuclear receptor subfamily 4, group A, member 3; transcription factor-8; interleukin 13; jun B proto-oncogene; interleukin 1B; glycoprotein Ib, platelet, alpha polypeptide; and Ras-associated protein RAB27A. Down-regulated genes included haptoglobin and numerous immunoglobulins in the heart, and factor H-related gene 2, protein phosphatase 1, regulatory subunit 3A, and growth differentiation factor-8 in skeletal muscle.
CONCLUSIONS: By establishing a profile of the gene-expression responses to cardiopulmonary bypass and cardioplegia, this study allows a better understanding of their effects and provides a framework for the evaluation of new cardiac surgical modalities directly at the genome level.
Transcriptional profiling using high-density microarrays provides unique data about disease mechanisms, drug responses, regulatory pathways, and gene function by comparing the level of mRNA transcribed in cells in a given pathologic state versus a control. This allows for the elucidation of complex pathophysiologic mechanisms directly at the gene expression level.8 We examined the cardiac and skeletal muscle gene expression profiles of patients undergoing cardiac surgery with CPB and cardioplegic arrest to improve the understanding of these modalities and provide a reference framework for the evaluation of prognostic gene markers and new intraoperative strategies.
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Methods |
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Right atrial samples consisted of nontraumatized tissue that was not involved in a purse string suture. Pre-CPB tissues were taken from the right atrial appendage before venous cannulation, and post-CPB atrial samples were harvested after the construction of a second purse string suture on the right atrial appendage and decannulation. Skeletal muscle samples were taken from the left intercostal muscle bed, lateral to the internal thoracic artery harvest site. Atrial and skeletal muscle samples (
10 x 10 x 2 mm) were snap-frozen in liquid nitrogen and immediately stored at -80°C.
To minimize baseline phenotypic variation, 5 patients aged between 55 and 75 years, who were normocholesterolemic, nonsmoking, nondiabetic, of the same ethnicity and gender, and who had similar procedural characteristics, were selected from the 16 subjects whose tissues were harvested; 10 atrial (ie, 5 pre- and 5 post-CPB and cardioplegic arrest) and 10 skeletal muscle (ie, 5 pre- and 5 post-CPB) samples obtained from these patients were therefore used for the microarrays analyses and the validation studies (Table 1).
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Microarray processing
cDNA was prepared according to protocols provided with the Affymetrix U95 GeneChip system (Affymetrix, Santa Clara, Calif).9 Five micrograms of total RNA were used in the first-strand cDNA synthesis with T7-d(T)24 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-[dT]24) and Superscript II (Gibco-BRL, Gaithersburg, Md). The second-strand cDNA synthesis was performed at 16°C by adding Escherichia coli DNA ligase, DNA polymerase I, and RNase H to the reaction, followed by T4 DNA polymerase to blunt the ends of newly synthesized cDNA. The cDNA was purified through phenol/chloroform and ethanol precipitation. By using a BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), the purified cDNA was incubated for 5 hours at 37°C in an in vitro transcription reaction to produce cRNA-labeled with biotin.
Microarray hibridization
cRNA was fragmented by incubating in a buffer containing 200 mmol/L Tris-acetate, 500 mmol/L KOAc, and 150 mmol/L MgOAc for 35 minutes at 94°C. Fifteen micrograms of fragmented cRNA were mixed with eukaryotic hybridization controls (containing control cRNA and oligonucleotide B2) and hybridized with a pre-equilibrated human U95Av2 Affymetrix chip for 16 hours at 45°C. The chips were washed in a fluidic station with low-stringency buffer (6x standard saline phosphate with EDTA, 0.01% Tween 20, and 0.005% antifoam) for 10 cycles and in high-stringency buffer (100 mmol/L N-morpholino-ethanesulfonic acid, 0.1 mol/L NaCl, and 0.01% Tween 20) for 4 cycles and stained with streptavidin phycoerythrin. This process was followed by incubation with normal goat immunoglobulin G and biotinylated mouse anti-streptavidin antibody, and restaining with streptavidin phycoerythrin.
Statistical analysis of microarray data
The chips were scanned in an HP G2500A ChipScanner (Affymetrix) to detect hybridization signals. Image output files were visually examined for major chip defects and hybridization artifacts, and raw data were processed with GeneChip Microarray Analysis Suite 5.0 software (Affymetrix). The image from each chip was scaled such that the absolute signal intensities were adjusted to target intensity and reported as a non-negative numerical quantity. Data were imported into SAS version 7 (SAS, Cary, NC) for further analysis. Statistical techniques were separately applied to each type of tissue and involved tests that were paired for each patient.
Median post- to pre-CPB and cardioplegic arrest gene expression ratios were computed for each gene by using the relative signal intensities of the probe cells. Two statistical methods were combined to assess differential gene expression between the pre- and post-CPB and cardioplegic arrest tissues.10 To ensure a minimal number of false positives, only the probe sets commonly yielded by the 2 methods were included in the list of genes differentially expressed in post- versus pre-CPB and cardioplegic arrest samples.
Method 1 identified probe sets with significant intensity differences between pre- and post-CPB and cardioplegic arrest tissues. For each tissue type, a Wilcoxon signed-rank test was applied to the absolute signal intensities of each gene in the pre- versus post-CPB and cardioplegic arrest data set. The use of a nonparametric, paired Wilcoxon test was selected over that of a standard t test to avoid a distributional assumption;11,12 the threshold was set at a P value of .0253 or less, which corresponds to the lowest possible P value in the particular experimental setting of the study (observed when 5 of 5 patients show a similar pattern of differential expression). By using this P value, the number of genes that could have reached significance by chance alone was determined and compared with the number of observed significant genes, and a signal-to-noise ratio was computed.
Method 2 determined the differentiability of a probe set by its signal intensity fold change. Nonparametric determination of genes with significantly different fold changes between the pre- and post-CPB and cardioplegic arrest states was performed by computing and ordering by increasing size the median relative signal intensities of each probe set. A particular gene was considered to be up-regulated after CPB and cardioplegic arrest in a given tissue type if a median fold change of 4 (4:1) or more was observed in post- versus pre-CPB and cardioplegic arrest samples. Conversely, a gene was considered down-regulated if the median fold change of post- versus pre-CPB and cardioplegic arrest samples was 0.25 (1:4) or less. Only genes identified by both methods were considered to be differentially expressed and were reported.
Microarray validation
Northern analysis
cDNA probes of human DUSP1 and CYR61 (2 significantly altered genes), and GADP and ß-actin (2 genes that were not altered) were labeled with 
32P-dCTP (New England Nuclear, Boston, Mass) using a random-priming labeling kit (Boehringer, Indianapolis, Ind) and purified from unincorporated nucleotides using G-50 Quick Spin Columns (Boehringer). The blots were hybridized overnight at 48°C using an Ambion Northern Max kit (Ambion, Austin, Tex). After hybridization, the blots were washed twice in 2x standard saline citrate with 0.1% sodium dodecyl sulfate (SDS) for 15 minutes each at room temperature and then washed twice in 0.1x standard saline citrate with 0.1% SDS for 15 minutes each at 60°C. Autoradiography was performed for 20 hours at -80°C. Blots were analyzed after digitization and quantification of x-ray films with the Image-Quant software (Molecular Dynamic, Sunnyvale, Calif), quantified as a ratio of the gene mRNA to loading band density, and presented as a median (minimum, maximum) ratio of post- to pre-CPB and cardioplegic arrest mRNA expression.
Real-time polymerase chain reaction
Reverse transcription was performed on 2 µg of total RNA isolated from atrial and skeletal muscle tissues, using an oligo dT primer (Invitrogen) for 90 minutes at 37°C. DUSP1 primers were forward-TGGAGGAAGGGTGTTTGTCC, reverse-CAAGGCAGATGGTGGCTGA, and TaqMan-FAM-CTGCCAGGCAGGCATTTCCCG-TAMRA (Applied Biosystems, Foster City, Calif). CYR61 primers were forward-ATCAAGGACCCCATGGAGG, reverse-AGGCATCGAATCCCAGCTC, and TaqMan-FAM-CCAGGACGGCCTCCTTGGCAA-TAMRA (Applied Biosystems). Amplification conditions were as follows: stage 1, 1 cycle for 2 minutes at 50°C; stage 2, 1 cycle for 10 minutes at 95°C; stage 3, 40 cycles for 15 seconds at 95°C and 1 minute at 60°C. Real-time detection was accomplished using SYBR Green I dye and fluorescence quantified with a GeneAmp Sequence Detection System (Applied Biosystems). All experiments were performed in duplicate, and post- to pre-CPB and cardioplegic arrest ratios of polymerase chain reaction (PCR) products are presented as median (minimum, maximum) ratios.
In situ hybridization
Probes for human DUSP1 and CYR61 were generated from PCR fragments containing T3 (forward) and T7 (reverse) promoter sequences incorporated in the amplicon. Frozen sections of 10 µm were cut in a cryostat and captured onto microscope slides. Sections were fixed and acetylated, and hybridized at 68°C to the probe for 3 nights. Hybridized probes were visualized using alkaline phosphatase-conjugated anti-DIG Fab fragments and 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate (Kierkegard and Perry, Gaithersburg, Md). Sections were rinsed several times in 100 mmol/L Tris, 150 mmol/L NaCl, and 20 mmol/L EDTA pH 9.5, and coverslipped with glycerol gelatin. Control sections were incubated in an identical concentration of the sense probe transcript. In situ hybridization autoradiographs were evaluated qualitatively in random high-power fields.
Immunoblotting
Total lysate from atrial and skeletal muscle tissues was obtained by 30-second homogenization on ice in lysis buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor, and centrifuged at 12,000g for 10 minutes at 4°C to separate soluble from insoluble proteins. The supernatant protein concentration was measured spectrophotometrically at 595 nm wavelength using a BCA protein assay kit (Pierce, Rockford, Ill). Total protein was fractionated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass) using a semi-dry transfer apparatus. Membranes were stained with Ponceau S and incubated with 5% nonfat dry milk in 50 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, and 0.1% Tween 20 (TBST) buffer for 1 hour at room temperature. Membranes were incubated with antibodies (in 2.5% nonfat dry milk in TBST) (anti-DUSP1 1:1000 [v/v] dilution, rabbit polyclonal 1:1000 [v/v], or anti-CYR61 1/1000 [v/v] dilution), and rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, Calif) for 2 hours. After washing with TBST, the membranes were incubated for 1 hour in 2.5% nonfat dry milk in TBST diluted with anti-rabbit immunoglobulin G (Jackson Immunolabs, West Grove, Pa), both at 1:3000 (v/v) dilution conjugated to horseradish peroxidase. Peroxidase activity was visualized using enhanced chemiluminescence and exposed to x-ray films (Amersham, Arlington Heights, Ill). Post-CPB to pre-CPB and cardioplegic arrest ratios of protein density are presented as median (minimum, maximum) ratios.
Reporting and functional classification
Genes identified to be significant by microarray analysis with both statistical methods were the object of a literature search and reported, along with their GenBank accession number, according to the current nomenclature of Online Mendelian Inheritance in Man.13 Functional inferences
outlined in the right column of Tables 2 to 5 are based on the authors' interpretation
of literature referenced in Online Mendelian
Inheritance in Man pertaining to each gene.
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Results |
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Microarray signal-to-noise characteristics
Of 12,625 genes examined, 1,106 and 974 genes were identified by method 1 to have a P value of .0253 or less for their differential expression in post- versus pre-CPB and cardioplegic arrest cardiac and skeletal muscle tissues, respectively (Figure 1).
The projected number of genes that could have reached significance because of chance alone was 320 for each tissue type, corresponding to a signal-to-noise ratio of 3.46 in cardiac and 3.04 in skeletal muscle microarrays.
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Validation
DUSP1
The microarray results for DUSP1 revealed increased expression (median 12-fold; 3.2, 27.1; P = .025) in the atrium, but no significant change in the skeletal muscle after CPB and cardioplegic arrest (median 1.6-fold; 0.3, 6.5; P = .68). Validation studies revealed the median mRNA expression of DUSP1 in post- versus pre-CPB atrial samples to be increased 3-fold (1.7, 7.1; P = .025) and 6.4-fold (1.5, 16.8; P = .05) by Northern blotting and real-time PCR, respectively, with a change in protein level of 4.6-fold (1.3, 5.7; P = .05) by immunoblotting (Figure 2).
In skeletal muscle, DUSP1 was not significantly up-regulated, with a median differential mRNA expression of 1.6-fold (1.2, 4.6; P = .08) and 8.2-fold (2.5, 23.3; P = .08) by Northern blotting and PCR, respectively, and a protein level change of 2.1-fold (1.1, 3.0; P = .68) by immunoblotting. In situ hybridization revealed a striking increase in DUSP1 expression in atrial tissues, particularly localized to the endothelium and myocytes (Figure 3). No increase in expression was detected in skeletal muscle samples.
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GAPD and ß-Actin
Microarray results revealed the differential expression of these genes not to be significantly altered after CPB and cardioplegic arrest in both atrial and skeletal muscle tissues. On Northern blotting, the median differential expression of post-CPB and cardioplegic arrest to pre-CPB samples was 1.2 and 1.1 for GAPD, and 1.0 and 1.1 for ß-actin in atrial and skeletal muscle samples, respectively (P = not significant).
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Discussion |
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Several of the genes found to be differentially expressed in this study have been implicated in the pathophysiology of CPB and cardioplegia in humans. FOS and JUN were found to be up-regulated in subendocardial myocytes and endothelial cells of right atrial samples after cold crystalloid cardioplegia.14,15 Nuclear factor-kappa B activation has been proposed as an inducer of transcription in right atrial tissue exposed to ischemia-reperfusion,16 and endothelin 1 receptor blockade has been associated with minimization of left ventricular dysfunction after CPB and cardioplegic arrest in swine.17 A number of neutrophil adhesion molecules such as CD11 and CD44 have been differentially expressed,18,19 and serum levels of IL6, IL8, TNF, IL1B, SCYA2, SELE, and IL13, and CD44 levels have been shown to be increased after CPB.20-26 Elevated serum levels of S100 have been correlated with cerebral damage after CPB and circulatory arrest.27
This study also identifies multiple genes that had not been implicated in the pathophysiology of CPB and cardioplegic arrest, such as DUSP1, TCF8, CYR61, SLC2A3, MYC, TCCR, BTG2, TRIM, ALCAM, EFNB2, and many others (Tables 2-5). Although the clinical significance of these new findings is still unknown, further research may characterize the expression of these genes, quantify polymorphisms, and examine their correlation with surgical outcome.
Limitations
Microarray techniques, despite their immense potential to improve disease understanding, have inherent shortcomings related to the selection of tissues, lack of standardized methods for the statistical analysis of data, presentation of vast amounts of results, and generalizability of the findings.28 Microarray technology also does not allow for the determination of absolute expression patterns and provides a measure of transcribed mRNA rather than a functional gene product. We have attempted to limit these shortcomings by selecting phenotypically and procedurally similar subjects, combining nonparametric statistical methods to minimize false positives, validating the expression patterns, protein levels, and origin of select genes with conventional molecular techniques, and suggesting a potential functional role for genes whose expression was most strikingly altered.
For ethical reasons, we used human atrial biopsies rather than ventricular biopsies to examine the cardiac gene expression changes that result from CPB and cardioplegic arrest. Atrial sampling involves little or no intrinsic risk of morbidity, provides full-thickness samples, and is clinically reproducible. CPB and antegrade cold blood cardioplegia distribute to the right atrium.29 Although atrial myocardium differs from ventricular myocardium with respect to the relative percentage of myocytic, endothelial, connective, and neural elements, these cell types are present in each tissue type and may demonstrate ultrastructural changes in disease.30,31
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Footnotes |
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