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Physiol. Genomics 25: 364-374, 2006. First published March 22, 2006; doi:10.1152/physiolgenomics.00013.2006
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Received 27 January 2006; accepted in final form 3 March 2006.
Physiological Genomics 25:364-374 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Transcriptome analysis of the ischemia-reperfused remodeling myocardium: temporal changes in inflammation and extracellular matrix

Sashwati Roy 1, Savita Khanna 1, Donald E. Kuhn 1, Cameron Rink 1, Willis T. Williams 1, Jay L. Zweier 2 and Chandan K. Sen 1

1 Laboratory of Molecular Medicine, Department of Surgery Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio
2 Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
cDNA microarray analysis was performed to screen 15,000 genes and expressed sequence tags (ESTs) to identify changes in the ischemia-reperfused (I-R) rat myocardial transcriptome in the early (day 2) and late (day 7) inflammatory phases of acute myocardial infarction. Lists of candidate genes that were affected by I-R transiently (2 or 7 days only) or on a more sustained basis (2 and 7 days) were derived. The candidate genes represented three major functional categories: extracellular matrix, apoptosis, and inflammation. To expand on the findings from microarray studies that dealt with the two above-mentioned time points, tissues collected from days 0, 0.25, 2, 3, 5, and 7 after reperfusion were examined. Acute myocardial infarction resulted in upregulation of IL-6 and IL-18. Genes encoding extracellular matrix proteins such as types I and III collagen were upregulated in day 2, and that response progressively grew stronger until day 7 after I-R. Comparable response kinetics was exhibited by the candidate genes of the apoptosis category. Caspases-2, -3, and -8 were induced in response to acute infarction. Compared with the myocardial tissue from the sham-operated rats, tissue collected from the infarct region stained heavily positive for the presence of active caspase-3. Laser microdissection and pressure catapulting technology was applied to harvest infarct and adjacent noninfarct control tissue from a microscopically defined region in the rat myocardium. Taken together, this work presents the first evidence gained from the use of DNA microarrays to understand the molecular mechanisms implicated in the early and late inflammatory phases of the I-R heart.

heart; oxygen; perceived hyperoxia; wound healing


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AN ACUTE MYOCARDIAL INFARCTION induces ventricular remodeling, a process that can influence ventricular functions and survival outcomes (22). Ventricular remodeling is directly implicated in postinfarction development of ventricular dilatation, a predictive sign of future congestive heart failure. Time-dependent changes in ventricular architecture occur in the infarcted and noninfarcted regions, resulting in hypertrophy of the viable regions of the affected site (6). Cardiac remodeling after myocardial infarction depends on cellular responses, characterized by hypertrophy of myocytes and hyperplasia of interstitial fibroblasts (18). Animals that survive with large transmural infarctions develop heart failure without another ischemic event, as is typically seen in humans. A rat model of myocardial infarction has been extensively studied to understand the functional, structural, and molecular changes associated with clinical ischemic heart disease (23, 33). Substantial alterations in gene expression are needed to afford such profound changes within cells of the remodeling myocardium (33).

Current treatment of myocardial infarction is directed to restore blood flow to the ischemic region by thrombolysis, coronary artery bypass surgery, or percutaneous transluminal coronary angioplasty. Depending on the degree of success of the therapeutic intervention, the area at risk remains either hypoxic or is fully salvaged. When the area at risk remains hypoxic, the myocardial tissue loses its contractile function and becomes necrotic, leading to the initiation of a wound-healing process (3). This situation is experimentally modeled using the permanent ligation of coronary artery approach (33). In contrast, when blood flow through the myocardium is reestablished in time, hibernating myocardial tissue may regain its function but may also experience additional damage due to the reperfusion process itself. It is well known that reoxygenation induces free radical injury. Recently, we have demonstrated that reoxygenation may also trigger cardiac remodeling by inducing the differentiation of cardiac fibroblasts to myofibroblasts (28–30, 32). A comprehensive understanding of the myocardial biology of the infarction-affected site warrants a global approach directed toward analyzing the fundamental mechanisms as a function of time. To that end, the cDNA microarray technology has been productively employed to query 4,000+ genes in response to permanent partial ligation of the coronary artery (33). The ischemia-reperfused myocardium, however, has not been subjected to such comprehensive analysis. In this study, we employed a high-density cDNA microarray analysis approach to screen 15,000 genes and expressed sequence tags (ESTs) in the rat myocardium. The goal was to examine temporal changes in the myocardial transcriptome in response to acute ischemia-reperfusion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Survival Model for Coronary Artery Occlusion and Reperfusion
Sprague-Dawley rats weighing 500–550 g were subjected to ischemia-reperfusion of the heart as described previously (30). Parallel groups of rats were used as sham-operated controls. The studies were approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University. Rats were anesthetized, intubated, and mechanically ventilated on a positive pressure respirator with room air. The body temperature was maintained at 36–37°C with a heated small-animal operating table. A left thoracotomy was performed via the fifth intercostal space to expose the heart. A 30-min occlusion of left anterior descending coronary artery (LAD) was followed by reperfusion. Laser Doppler flow measurement was used to verify ischemia and reperfusion (I-R). On successful reperfusion, the thorax was closed, and negative thoracic pressure was reestablished for survival. The rats were killed 6 h to 7 days after reperfusion. For tissue harvesting, the infarcted I-R area was visualized under a dissecting microscope. Tissue samples were collected from the infarction (I-R) site. For control, tissues were collected from a location corresponding to the I-R sites in sham-operated rats. These rats were exposed to all surgical procedures except I-R.

GeneChip Probe Array Analysis
To identify sets of I-R-induced cardiac remodeling genes, we utilized the GeneChip approach (27, 30, 31). The total RNA was extracted from heart of 2- and 7-day I-R or sham tissue samples using Trizol (Gibco BRL) (29, 30). A further cleanup of RNA was performed using the RNeasy Kit (Qiagen). Quality of RNA was checked using the Agilent 2100 Bioanalyzer (29) Targets were prepared for microarray hybridization according to previously described protocols (27, 30, 31). To assess quality of hybridization, the samples were hybridized for 16 h at 45°C to GeneChip test arrays. Satisfactory samples were hybridized to the Rat Genome arrays (U230A) for the screening of >15,000 genes and ESTs. An EST is a short subsequence of a transcribed protein-coding or non-protein-coding DNA sequence. It was originally intended as a way to identify gene transcripts but has since been instrumental in gene discovery and sequence determination. An EST is produced by one-shot sequencing of a cloned mRNA, and the resulting sequence is a relatively low-quality fragment whose length is limited by current technology to ~500–800 nucleotides. ESTs are a useful resource for designing probes for DNA microarrays used to determine gene expression. The arrays were washed, stained with streptavidin-phycoerythrin, and then scanned with the GeneArray scanner (Affymetrix) in our own facilities. To allow for statistical treatment, data were collected from three individual animals in each group (Gene Expression Omnibus accession no. GSE-4105).

Data Analysis
Raw data were collected and analyzed using Affymetrix Microarray Suite 5.0 (MAS) software. After the scanning, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches. 3'/5' Ratios for GAPDH and ß-actin were confirmed to be within acceptable limits. BioB spike controls were present on the chips, with BioC, BioD, and CreX also present in increasing intensity. When scaled to a target intensity of 1,500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background values, Q-values, and mean intensities. Additional processing of data was performed using dChip software (14) and Data Mining Tool software (DMT, Affymetrix). Initial filtering of data was performed using DMT on absolute files generated from MAS. As an initial filter, t-test was utilized to identify differentially expressed genes between the sham vs. the I-R group (n = 3 replicates in each group). Genes that significantly (P < 0.05) changed (increased or decreased) in the I-R group compared with sham were selected. Next, for data visualization and clustering, genes initially filtered using the statistical (t-test) approach by DMT were subjected to hierarchical clustering using dChip (v1.3) software. The following major clusters of genes, which were different in the I-R group compared with sham group, were selected for further data processing: 1) up- or downregulated only on day 7 post-I-R, 2) up- or downregulated only on day 2 post-I-R, and 3) up- or downregulated on both days 2 and 7 post-I-R. To minimize the number of false positives, the selected genes, using clustering approach, were further subjected to comparison analysis using the dChip software with the use of the following criteria: 1) t-test, P < 0.05; 2) present call in all experimental (I-R) samples for upregulated genes; and 3) present call in all baseline (sham) samples for downregulated genes. To handle the multiple-comparison issue in the context of microarray data, the empirical false discovery rate (FDR) was estimated by permutations (38). Gene lists with a median FDR <5% have been presented. The significant genes were functionally categorized, and the following three major categories of genes were identified and have been presented in tabular form: 1) extracellular matrix, 2) apoptosis, and 3) inflammation. Functional categorization was performed using the following software/web resources: Gene Ontology Data Mining Tool (Affymetrix); Kyoto Encyclopedia of Genes and Genomes (KEGG); Database for Annotation, Visualization, and Integrated Discovery Verification (DAVID) (5), and LocusLink (Swiss-Prot) as described previously (30). Microarray data were verified using real-time PCR assay.

mRNA Quantitation
Tissue mRNA was quantified by real-time PCR assay using double-stranded DNA-binding dye SYBR Green-I as described previously (28). The primer sets used for the individual genes are listed in Supplemental Table S1 (available at the Physiological Genomics web site).1

Histology
Histochemistry.
Formalin-fixed tissues were embedded in paraffin and sectioned (4 µm), followed by hematoxylin and eosin staining.

Immunostaining.
The sections were stained with the following primary antibodies: mouse monoclonal antibodies to p21 (1:50; Pharmingen), anti-ED-1 (1:100; Serotec), anti-smooth muscle actin (1:1,000; Sigma), anti-desmin (1:100; Dako), and anti-active caspase-3 (1:200, Cell Signaling).

Laser Microdissection and Pressure Catapulting
Laser microdissection and pressure catapulting (LMPC) was performed using the Microlaser System from P.A.L.M. Microlaser Technologies (Bernreid, Germany). Rat hearts with experimental occlusions were isolated, frozen in optimum cutting temperature (OCT) compound, and then cut into 10-µm sections with the use of a cryomicrotome. The sections were placed on polyethylene napthalate (PEN) membrane glass slides (P.A.L.M. Microlaser Technologies) that had been RNasin (Ambion, Austin, TX) and UV treated, for cutting and catapulting as described by our group recently (11). Sections were stored at –80°C and then thawed at room temperature for 5–10 min before use. Sections were stained using hematoxylin QS and the infarct site identified as reported (11). Infarcted areas of the section stained blue, whereas noninfarcted areas stained purple. Matched area (0.8–1.0 x 106 µm2) of noninfarct and infarct area was captured in chaotropic RNA lysis solution as described (11).

RNA Extraction and Reverse Transcription
RNA was extracted from catapulted elements using the RNAqueous-Micro Kit from Ambion according to their laser capture microdissection (LCM) protocol. Elements from three sections of same animal were pooled and incubated at 42°C for 30 min for RNA isolation. Extracted RNA was DNase treated at 37°C for 30 min, and the resulting RNA solution was used in reverse transcription reactions.

Reverse transcription was carried out using the Full Spectrum RNA Amplification Kit from System Biosciences (SBI, Mountain View, CA) according to the manufacturer's instructions. Reactions were performed in 10-ml volumes containing RT buffer, primers, dNTPs, DTT, and RT. Reaction conditions included 4-min incubation at 70°C with primer and RNA alone followed by 5-min incubation at 25°C. Transcription buffer, dNTPs, DTT, and RT were then added, and the resulting solution was incubated at 42°C for 50 min followed by 4 min at 70°C.

Total cDNA Amplification and mRNA Quantification
cDNA from reverse transcription reactions was amplified using the Full Spectrum RNA Amplification Kit from SBI. Each reaction solution contained PCR buffer, dNTPs, proprietary primers, and DNA polymerase. PCR cycling was done according to the manufacturer's instructions, except that the number of cycles was increased to 25. Cycling conditions included 95°C for 4 min and 68°C for 5 min followed by 95°C for 25 s, 58°C for 1 min, and 68°C for 1.5 min. Next, 25 cycles at 95°C for 25 s and 68°C for 1.5 min were carried out followed by a 2-min extension step at 68°C. Typically, 1 µl of this solution was used for real-time PCR. Real-time PCR assay was performed using double-stranded DNA-binding dye SYBR Green-I as described previously (28, 30).

Statistics
Data shown as bar graphs are means ± SD. Student's t-test or ANOVA was used to test significance of difference between means. P < 0.05 was interpreted as significant difference between means. For Fig. 8D, the simple {chi}2-test was used. Statistics related to DNA microarray data processing are described above in Data Analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The compensation for myocardial infarction by myocardial gene expression in the peri-infarct site is known to occur at an early phase after myocardial infarction, and myocardial dysfunction is thought to begin from adjacent to remote peri-infarcted myocardium during progressive cardiac remodeling (18). A survival model of left anterior descending coronary artery occlusion for 30 min followed by reperfusion in rats was employed to examine changes in the transcriptome of the I-R heart tissue. The loss of blood flow during occlusion was verified by laser Doppler flowmetry, and the infarct tissue was subjected to histological characterization. Masson trichrome staining of the infarct region revealed progressive receding of the myocyte line indicating the time course of infarction-induced loss of myocyte viability (Fig. 1A). Distinct changes were observed in day 2, which we refer to in this work as the early inflammatory phase. The histological changes in the peri-infarct site were progressive and continued until day 7 (Fig. 1A) beyond which the changes were not as marked as a function of time (not shown). Thus we chose to compare findings from day 2 with those from day 7, which we refer to in this work as the late inflammatory phase. Histological characterization of the late inflammatory phase revealed the receding myocyte line, which was cohabited by infiltrating macrophages (Fig. 1B, a and b). The peri-infarct site was also habited by the characteristic myofibroblasts, which are produced by the differentiation of resident cardiac fibroblasts. The myofibroblasts were identified as {alpha}-smooth muscle actin-positive (Fig. 1Bc) and desmin-negative cells (Fig. 1Bd). Consistent with our previous reports demonstrating that p21 plays a central role in triggering the differentiation of cardiac fibroblasts to myofibroblasts at the peri-infarct site of myocardial infarction, this cell cycle regulatory protein was detected at the peri-infarct site (Fig. 1Be).


Figure 1
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  		Fig. 1. Histological characterization of remodeling cardiac tissue used for genomic studies. Occlusion (30 min) of left anterior descending coronary artery was followed by reperfusion. Ischemia was documented by laser Doppler flow measure. On successful reperfusion, the thorax was closed, and negative thoracic pressure was reestablished for survival. Formalin-fixed paraffin sections from day 7 postreperfusion heart were stained using the following. A: Masson trichrome procedure (days 2, 5, and 7 postreperfusion). This procedure results in blue-black nuclei and blue collagen and cytoplasm; cardiac muscle fibers are stained red. Ba: Masson trichrome-stained day 7 postreperfusion section showing the remodeling cardiac tissue. Next, immunostainings were performed to characterize the tissue illustrated in Ba. Bb: monocyte/macrophage (ED-1, brown): recognizes a single-chain glycoprotein of 110 kDa that is expressed predominantly on the lysosomal membrane of rat monocyte/macrophages. The antigen recognized by ED-1 (brown) in the rat is a homolog of human CD68. Blue (nuclei) is counterstaining with hematoxylin. Bc: {alpha}-smooth muscle antibody that recognizes {alpha}-isotype of smooth muscle actin (brown) present on myofibroblasts and smooth muscle and myoepithelial cells. Bd: desmin (brown): antibody recognizes skeletal, cardiac, and smooth muscle cells. Be: p21 (brown, nuclear stain): recognizes the p21/WAF1 protein, which is an important cell cycle regulatory protein. p21 inhibits cell cycle progression at both the G1 and G2 checkpoints. Scale bar = 100 µm.

 
The specific objective of this study was to identify changes in the I-R rat myocardial transcriptome in the early (day 2) and late (day 7) inflammatory phases of acute myocardial infarction. Identical tissue harvested from sham-operated rats subjected to the same surgical procedures, where the ligature was inserted in the heart but not tied, was used as control for comparison purposes. Three independent experiments were performed to enable appropriate statistical analyses. Figure 2 depicts the experiment design and data analysis approach, which are consistent with our previous reports (27, 30). Data processing was initially performed using MAS and DMT (v2.0) softwares. Additional data filtration was performed using dChip, employing the following specific criteria: 1) t-test, P < 0.05 (median FDR <5%); 2) present call in all I-R test samples for upregulated genes; and 3) present call in all sham control samples for downregulated genes. This approach for data analysis identified 119 genes that were significantly and uniquely upregulated in day 2, 193 genes that were upregulated commonly in days 2 and 7, and 590 genes that were uniquely upregulated in day 7 after I-R. Overall, fewer genes were downregulated by I-R. It was identified that 38 genes were downregulated significantly and uniquely in day 2, 216 genes were downregulated commonly in days 2 and 7, and 286 genes were uniquely downregulated in day 7 (Fig. 2). Hierarchical cluster images of these candidate genes are illustrated in Fig. 3. A list of candidate genes of each category is provided as Supplement Materials (Supplemental Tables S2–S7). The candidate genes represented three major functional categories: extracellular matrix, apoptosis, and inflammation. The pattern of expression of genes representing these specific functional groups in the post-I-R remodeling heart is presented in Fig. 4. A complete list of all candidate genes belonging to the specific functional groups illustrated in Fig. 4 is presented in Tables 1Go–3.


Figure 2
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  		Fig. 2. Experimental design and data analysis scheme. GeneChip data analysis scheme used to identify differentially expressed genes from cardiac tissue harvested from rats subjected to ischemia (30 min) and reperfusion (I-R) for 2 days (2d) or 7 days (7d) compared with control tissue, i.e., rats that underwent sham surgery (sham). Data processing was initially performed using Microarray Suite 5.0 (MAS) and Data Mining Tool 2.0 (DMT) softwares. Additional data filtration was performed using dChip, employing the following specific criteria: 1) t-test, P < 0.05 [median false discovery rate (FDR) <5%]; 2) present call in all I-R test samples for upregulated genes; and 3) present call in all sham control samples for downregulated genes. Details of software and other resources for data analysis are provided in MATERIALS AND METHODS.

 

Figure 3
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  		Fig. 3. Hierarchical cluster images illustrating remodeling-sensitive genes in cardiac tissue induced or downregulated after I-R. Specific clusters of genes showing an increase (A) or decrease (B) in expression after ischemia (30 min) and reperfusion (2d or 7d). Genes that were found significantly up- or downregulated in all replicates after I-R compared with sham-operated samples were selected. These select candidate genes were subjected to hierarchical clustering to identify clusters of genes that are induced/downregulated by I-R after 2d or 7d postreperfusion. Cluster i: cluster of genes upregulated specifically after 7d of reperfusion. Cluster ii: cluster of genes upregulated after both 2d and 7d of reperfusion. Cluster iii: cluster of genes upregulated specifically after 2d of reperfusion. Cluster iv: cluster of genes downregulated specifically after 2d of reperfusion. Cluster v: cluster of genes downregulated specifically after 7d of reperfusion. Cluster vi: cluster of genes downregulated after both 2d and 7d of reperfusion. Data shown are from 3 individual animals (animals 1–3). Graphs next to each image (at right) illustrate the average pattern of gene expression in the corresponding cluster. Samples 1–3 in each group represent the replicates. Red-to-green gradation in color represents higher-to-lower expression signal.

 

Figure 4
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  		Fig. 4. Visualization of the expression pattern of candidate genes representing the specific functional groups in the remodeling myocardium after I-R. Genes from the following major functional groups were subjected to hierarchical clustering to visualize expression patterns of these genes after I-R. A: extracellular matrix. B and C: apoptosis. D: immune/inflammation. Graphs next to each image (at right) illustrate the average pattern of gene expression in the corresponding cluster. Samples 1–3 in each group represent the replicates. Red-to-green gradation in color represents higher-to-lower expression signal.

 

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  		Table 1. Extracellular matrix protein-encoding genes upregulated after ischemia (30 min) and 7d reperfusion compared with 7d sham-operated animals

 

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  		Table 2. Apoptosis genes up- and downregulated after ischemia (30 min) and 7d reperfusion compared with 7d sham-operated animals

 

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  		Table 3. Immune/inflammatory genes upregulated after ischemia (30 min) and 2d reperfusion compared with 2d sham-operated animals

 
To verify accuracy of the microarray analyses, four candidate genes were randomly selected from Supplemental Table S3 for real-time PCR analyses. The four genes were thrombospondin-4 (TSBP4), endothelial type gp91-phox gene (Cybb), follistatin-like (Fstl), and fibroblast activation protein (FAP). Quantitative real-time PCR analyses of the individual genes provided results that were consistent with microarray findings (Fig. 5). Next, we identified candidate genes from the three functional groups to verify whether the microarray analyses could be reproduced by quantitative real-time PCR. To expand on our microarray findings dealing with two time points, days 2 and 7, we examined tissues collected from days 0, 0.25, 2, 3, 5, and 7. In the category of inflammation, the expression of interleukin-6 (IL-6) and IL-18 was investigated. Acute myocardial infarction resulted in upregulation of both of these cytokines. Significant induction of both IL-6 as well as IL-18 was noted in day 2, and the effect was more prominent in day 3. After day 3, the inducible response for both cytokines subsided (Fig. 6A). Myocardial healing response is characterized by the deposition of type I and III collagen (9, 18). The progression of response to acute myocardial infarction in the functional category of extracellular matrix was not akin to the inflammatory mediators. In contrast, genes encoding extracellular matrix proteins such as types I and III collagen were appreciably upregulated in day 2, and that response progressively grew stronger until day 7 after I-R (Fig. 6B). Comparable response kinetics was exhibited by the candidate genes of the apoptosis category. Caspases-2, -3, and -8 were induced in response to acute infarction. For all three genes, the change was significant in day 2 after I-R (Fig. 6C). The effect was more prominent in day 7 after infarction, consistent with the observation for genes in the extracellular matrix category (Fig. 6B). There is mounting evidence that apoptosis is important in the pathogenesis of myocardial infarction. One of the key events in the process of apoptosis is activation of caspase-3 (43). Thus we chose to histologically evaluate the presence of caspase-3 protein in the myocardial tissue that was harvested 7 days after infarction. Compared with the myocardial tissue from the sham-operated rats, tissue collected from the infarct region stained heavily positive for the presence of active caspase-3 (Fig. 7). \. The LMPC technology has the unique ability to harvest spatially defined tissue that is typically not possible to collect while tissue in the infarct region is harvested surgically. Our laboratory (11) has recently standardized the application of this technology to examine the spatially resolvedbiology of myocardial infarction. In this study, we applied LMPC to harvest infarct and adjacent noninfarct control tissues from microscopically defined regions in the rat myocardium. Our results show a very tight response demonstrating that type I collagen and type III collagen are potently induced in the infarct region but not in the tissue harvested beyond 500 µm away from the infarct site (Fig. 8). \. When compared, the LMPC approach was observed to generate tighter data with lower standard deviation than results derived from samples harvested by conventional surgical excision (Fig. 8).


Figure 5
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  		Fig. 5. Real-time PCR validation of GeneChip microarray expression analysis. Expression levels of selected genes identified using GeneChip analysis were independently determined using real-time PCR. For comparison, the real-time PCR data (normalized to GAPDH, a housekeeping gene) were proportionately adjusted to fit to the scale with GeneChip expression values (normalized using global scaling approach). Thbs4, thrombospondin-4; Cybb, endothelial type gp91-phox gene; Fstl, follistatin-like; FAP, fibroblast activation protein. Data represent means ± SD. *P < 0.05: significantly higher compared with the corresponding sham samples.

 

Figure 6
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  		Fig. 6. Expression kinetics of representative genes from major functional groups as identified using microarray in a reperfused myocardium. Expression kinetics of selected genes in reperfused (6 h to 7d) myocardium after 30 min of ischemia. These genes belonged to 3 functional groups A: inflammation. B: extracellular matrix. C: apoptosis. Functional groups and representative genes were identified using the microarray approach. mRNA expression analysis was performed using real-time PCR. Real-time PCR data were normalized to GAPDH, a housekeeping gene. Casp, caspase; Col, collagen; IL, interleukin. Data are mean ± SD. *, P < 0.05 compared with day 0.

 

Figure 7
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  		Fig. 7. Histological evaluation of expression of active caspase-3 in reperfused myocardium. Active caspase-3 expression in sham (A) vs. I-R (B) heart of rats harvested 7d after infarction. By use of the microarray approach, caspase-3 mRNA was identified to be upregulated after 7d of I-R. To verify the results at the protein expression level, immunostaining was performed using anti-active caspase-3 antibody (brown). Counterstaining was performed using hematoxylin (blue, nuclei). Scale bar = 100 µm.

 

Figure 8
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  		Fig. 8. mRNA expression analysis of histologically defined areas of I-R rat hearts using laser microdissection and pressure catapulting (LMPC). A and B: frozen sections (10 µm) from heart of rats subjected to ischemia (30 min) and reperfusion (7d) were stained with hematoxylin to histologically define infarct and control areas in the same microscopic field. A: representative stained frozen section showing distinct I-R and control regions. B: same section as shown in A after the elements from the I-R and control areas have been cut and captured. Scale bar = 100 µm. C: mRNA expression analysis of collagen I and collagen III from laser-captured I-R and control areas. mRNA analysis was performed using real-time PCR. Real-time PCR data were normalized to GAPDH, a housekeeping gene. Data are means ± SD (n = 3). **P < 0.005 compared with control. D: comparison of collagen I mRNA expression analysis using conventional surgical tissue excision or LMPC techniques. mRNA analysis was performed on frozen section samples using real-time PCR. Data are presented as %change compared with corresponding control area. Data are shown as individual %change data points (as scatter plot) as well as means ± SD (as bar graph overlay). *P < 0.05: simple {chi}2-test demonstrated lower SD in LMPC compared with conventional surgical procedure.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Myocardial infarction is associated with an inflammatory response. The inflammatory phase contributes to cardiac remodeling and eventual host outcome (8, 17, 26). Cytokine release is a hallmark of the early inflammatory phase. Our observation that IL-6 is induced in the early inflammatory phase is consistent with the prior observation that reperfusion rapidly induces IL-6 in the heart (12). It has been demonstrated recently that, following a preconditioning stimulus, IL-6 is obligatorily required for the activation of the JAK-STAT pathway, the ensuing upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the development of a cardioprotective phenotype (4). We noted that IL-18, a potent proinflammatory cytokine, was induced in the early inflammatory phase of reperfusion. Endogenous IL-18 has been reported to play a significant role in ischemia-reperfusion-induced human myocardial injury (24). Neutralization of IL-18 attenuates lipopolysaccharide-induced myocardial dysfunction (25). Furthermore, serum levels of IL-18 have been observed to be strong predictors of cardiac mortality in patients with coronary artery disease (37). The CCR2 chemokine receptor mediates leukocyte chemoattraction, which is involved in the pathogenesis of coronary heart disease. Our results show that CCR2 gene expression was induced in the early inflammatory phase. The CCR2 genotype is known to predispose patients for myocardial infarction before the age of 65 yr (19). Cybb (34) codes for gp91-phox, a component of NADPH oxidase. The expression of Cybb was induced in the early inflammatory phase, which is consistent with the previous finding that angiotensin AT(1) receptor activation results in NADPH oxidase gene expression in the ischemia-reperfused heart (20).

The extracellular matrix (ECM) is a key component in the remodeling process, and increases in collagen occur in the infarct area to replace necrotic myocytes and form a scar. The ECM is coupled to the cell through cell surface receptors, primary of which are the integrins. In addition, the matrix metalloproteinases coordinate ECM turnover through degradation of ECM components. These processes ultimately, when favorable, pave the way for angiogenesis and cellular regeneration. Collagen is the major ECM protein in the heart and represents a crucial target for anti-remodeling and cardioprotective therapy (41). In the myocardium, collagen represents the most abundant structural protein of the connective tissue network. Its structural organization consists of a complex weave of collagen fibers that surrounds and interconnects myocytes, groups of myocytes, muscle fibers, and muscle bundles. In the current study, the largest functional group of genes observed to be induced in the late inflammatory phase was represented by those that encode proteins of the ECM. When findings from the microarray studies were expanded, including additional time points, it was observed that both collagen I and III were induced in the early phase, but the induction got progressively stronger from day 2 to day 7 after reperfusion. The expression of these two specific types of collagen is known to be induced by reperfusion of the heart (18). Wound healing begins with the proliferation of young fibroblasts positive for type I, III, and V collagens at the wound margin. Next, vascular granulation tissue grows into the injured myocardium followed by deposition of fibrous components immunoreactive with types I and III (9). Fibroblast activation protein-{alpha} (FAP) is a cell surface serine protease expressed at sites of tissue remodeling in embryonic development. FAP is not expressed by mature somatic tissues except activated melanocytes, tumor stroma, and fibroblasts in wound healing (13, 15). Our study suggests that FAP is induced in the remodeling phase of ischemia-reperfused heart. Thrombospondins are a family of extracellular calcium-binding proteins that are involved in cell proliferation, adhesion, and migration. Thrombospondin-4 binds specifically to both collagenous and noncollagenous ECM proteins via its COOH-terminal domains (16). Thrombospondin-4 is expressed by vascular cells and influences the vessel wall by modulating the proliferation of endothelial cells and smooth muscle cells (35). Recently, an elevated level of expression of thrombospondin-4 has been reported in scar tissue (21). Our results provide the first evidence demonstrating elevated expression of thrombospondin-4 in the late inflammatory phase. Polymorphic thrombospondin-4 gene has been identified to perturb calcium homeostasis (36) and contribute to the development of myocardial infarction in humans at any age (39).

Seven years ago it was observed that the pharmacological inhibitor of caspase ZVAD-fmk effectively reduces myocardial reperfusion injury (40). Ever since, interest in the significance of caspases in the infarcted myocardium has soared. The postinfarct treatment with caspase inhibitors improved left ventricular remodeling and dysfunction through inhibition of granulation tissue cell apoptosis (7). Caspases, which are the executioners of apoptosis, comprise two distinct classes, initiators and effectors. Although general structural features are shared between the initiator and the effector caspases, their activation, inhibition, and release of inhibition are differentially regulated. Long-term caspase inhibition ameliorated apoptosis, reduced myocardial troponin I cleavage, protected left ventricular function, and attenuated remodeling in rats with myocardial infarction (2). Recent studies show that treatment with a caspase-3 inhibitor improves survival and prevents ventricular dilation and dysfunction after permanent coronary artery occlusion (1). In this study, we noted concurrent induction in the expression of caspases-2 (initiator), -3 (effector), and -8 (initiator). The induction for all three genes was more intense in the late inflammatory phase than in the early inflammatory phase. Our observation that active caspase-3 protein expression in the reperfused myocardium is high in the late inflammatory phase is consistent with the recent report demonstrating an elevated presence of activated caspase-3 in the infarcted human myocardium (43). Caspase-2 appears to be necessary for the onset of apoptosis triggered by several insults, including DNA damage and inflammation (42). Caspase-8 is required for killing induced by the death receptors Fas, tumor necrosis factor receptor-1, and death receptor-3. Moreover, caspase-8-deficient mice die in utero as a result of defective development of heart muscle and display fewer hematopoietic progenitor cells, suggesting that the FADD/caspase-8 pathway is absolutely required for the growth and development of myocardial cells (10). It is yet unknown whether such cytotrophic function of caspase-8 is implicated in postinfarction myocardial remodeling.

Taken together, this work presents the first evidence gained from the use of DNA microarrays to understand the molecular mechanisms implicated in the early and late inflammatory phases of the ischemia-reperfused heart. With the use of the high-density oligonucleotide microarray approach, an excess of 15,000 well-annotated full-length genes and ESTs were surveyed in the early and later inflammatory phases of acute myocardial infarction. Definite patterns of candidate genes emerged and are indicative of the functional responses underlying myocardial infarction. The reported findings represent a resource that may be utilized to develop testable hypotheses examining the fundamental mechanisms implicated in the reperfused myocardium.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-073087 to C. K. Sen.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. K. Sen, Davis Heart & Lung Research Institute, 473 W. 12th Ave., Columbus, OH 43210 (e-mail: Chandan.Sen{at}osumc.edu).

1 The Supplemental Material for this article (Supplemental Tables S1–S7) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00013.2006/DC1. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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