Analysis of region specific gene expression patterns in the heart and systemic responses after experimental myocardial ischemia

Figures

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  Figure 1. A. Biopsy Donor Site. Overview photography of a porcine heart after LAD occlusion shows the extension of affected myocardium and exact locations of three different myocardial regions respectively of the biopsy donor sites. B. Study design. DEG = differentially expressed genes; TF = transcription factor.

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  Figure 2. A. Venn diagram showing up-regulated genes with significant changes in all three areas of myocardium 24h after induction of acute myocardial infarction compared to control myocardium (CZ). Infarct core zone (IZ, n = 2483); border zone (BZ, n = 285); remote myocardium zone (RZ, n = 135). B. Venn diagram showing downregulated genes with significant changes in all three areas of myocardium 24h after induction of acute myocardial infarction compared to control myocardium (CZ). Infarct core zone (IZ, n = 6420); border zone (BZ, n = 71); remote myocardium zone (RZ, n = 78). C. Principal Component Analysis displaying mRNA expression data of the different areas of infarcted hearts and untreated healthy hearts. Infarct core zone (IZ, red dots); border zone (BZ, orange dots); remote myocardium (RZ, grey dots); control zone (CZ, blue dots). D. Validation of microarray results by RT-PCR. mRNA levels of nine selected genes were quantified by RT-PCR. Shown are mean ± SD of log FC values. RT-PCR data were normalized to beta-actin.

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  Figure 3. A.-C: Heat map showing the expression values (blue…upregulation; red…downregulation) of transcripts significantly altered (adjusted P value ≤ 0.05) in the infarct core zone A., border zone B. and remote zone C. compared to control myocardium. Functional annotation clustering of the regulated genes in the infarcted heart (A…up-/downregulated; B+C…all differentially regulated genes) identified pathways and biological processes affected by myocardial infarction. Pathways are sorted by decreasing p-values.

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  Figure 4. A-C. Identification of over-represented transcription factor binding sites (TFBS) in sets of differentially expressed genes in myocardium by oPOSSUM. A. In the infarct core zone members of the ETS transcription factor family (blue diamonds), key regulators of the hypertrophic transcriptional program (orange diamonds), signaling transducer and activator of transcription 3 (STAT3, green diamond) and kruppel-like factor 4 (Klf4, red diamond) binding sites were significantly enriched within the promoter regions. B. and C. In the border and remote zone AT-Rich Interaction Domain 3 (ARID3A, green diamond) and Nkx2-5 (red diamond) binding sites were significantly enriched within the promoter regions. D. Rank test of selected transcription factors based on the Fisher exact test score for different myocardial regions (IZ…infarct core zone, BZ … border zone, RZ … remote zone). A rank% value of 100 indicates that the TF mentioned are most probably functionally relevant. A rank% value of < 70 indicates that these TF may not be involved in gene regulation. Klf4 shows high values in the IZ and low values in the BZ and RZ. E. Five different down-stream targets of Klf4 show a significant up-…

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  Figure 5. Representative IHC images of porcine A. and human B. left ventricles after myocardial infarction. Positive immunoreactive signals for Klf4 (red dots), nucleic acid (blue dots) and the merging of both stainings are shown for the infarct core zone (IZ), border zone (BZ) and remote zone (RZ). For human samples only tissue specimens from the site of infarction (IZ, BZ) were available. The mean percentage of Klf4 positive cells (±SD) is given in the merged picture.

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  Figure 6A-6B. Venn diagram showing A. up- and B. downregulated genes in hepatic (red circle) and splenic (green circle) probe sets and the intersection of both tissue types. Among upregulated transcripts, ten genes could be identified, among downregulated transcripts five genes could be found in both probe sets. C-D: Heat map showing the expression values (blue = upregulation; red = downregulation) of transcripts significantly altered (adjusted P value ≤ 0.05) in hepatic C. and splenic D. probe sets. Functional annotation clustering of the up-/downregulated genes identified pathways and biological processes affected by myocardial infarction. Pathways are sorted by decreasing p-values.

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  Figure 7. A-B: Identification of over-represented transcription factor binding sites (TFBS) in sets of differentially expressed genes in hepatic E and splenic F tissue by oPOSSUM. Members of the ETS transcription factor family (blue diamonds), key regulators of the hypertrophic transcriptional program (orange diamonds), signaling transducer and activator of transcription 3, (STAT3, green diamond), AT-Rich Interaction Domain 3 (ARID3A, green diamond), Nkx2-5 (red diamond) and kruppel-like factor 4 (Klf4, red diamond) binding sites are highlighted within the promoter regions. Binding sites for SPIB, Nkx2-5 and Klf4 are specifically enriched in the promoter regions of differentially regulated genes in both organs (liver and spleen) after MI. C. Rank test of selected transcription factors based on the fisher exact test score for liver and spenic tissue. A rank% value of 100 indicates that the mentioned TF are most probably functionally relevant. A rank% value of < 70 indicates that these TF may not be involved in gene regulation. Klf4 shows high values in both tissue types, comparable to those in the myocardial infarct core zone.

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  Figure 8. A variety of organ systems are involved in the coordination of the organism´s response to myocardial infarction by the production and release of paracrine factors For initiation of this paracrine feedback loops a variety of transcription factors are regulated in different organs.