Activation of CCL21-GPR174/CCR7 on cardiac fibroblasts underlies myocardial ischemia/reperfusion injury

Figures

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  FIGURE 1. Identification of DEGs. (A) Volcano plots and pie charts showing DEGs for end-ischemia vs. cardiac arrest, reperfusion vs. end-ischemia, and reperfusion vs. cardiac arrest. (B) Heatmaps of DEGs. (C) Venn’s diagram showing common DEGs among comparisons.

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  FIGURE 2. Functional enrichment analyses of DEGs. (A) Circos plot showing gene overlaps and genes of the same enriched ontology term. Purple curves link common genes, and blue curves link genes belonging to the same enriched term. Dark orange indicates genes that hit multiple lists, and light orange indicates genes unique to a list. (B) Heatmap showing top 20 enriched terms, colored by p-values. (C) Characteristics of top 20 enriched terms. Count (%) indicates the number (percentage) of genes in the given ontology term. (D) Networks colored by cluster IDs, where nodes with the same ID are close to each other. (E) Networks colored by p-values. (F) Networks with pie charts, color-coded by identities of the genes.

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  FIGURE 3. Key candidate genes and PPI network analysis. (A) Interactions among hub proteins in MCODE cluster 1. (B) Interactions among hub proteins, colored by identities of the proteins. (C) Top 10 enriched BP, CC, and MF terms, and enriched KEGG pathway. (D) Protein-protein interaction networks and functions.

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  FIGURE 4. Gene set enrichment analysis of the ischemia and reperfusion samples. (A) Enriched pathways in ischemia samples. (B) Enriched pathways in reperfusion samples.

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  FIGURE 5. Myocardial I/R injury in mice. (A) Evans blue/TTC staining showing myocardial infract size in the sham and I/R groups. (B) HE staining showing myocardial histological damage. Scale bar = 100 μm (left) and 50 μm (right). (C) TUNEL staining showing the apoptosis rate in the myocardium. Scale bar = 100 μm n = 3. **p < 0.01 vs. the sham group. I/R, ischemia reperfusion; AAR, area at risk; IA, infarct area; LV, left ventricle.

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  FIGURE 6. mRNA and protein expression of key genes and related receptors. (A) mRNA expression of CCL21, XCR1, CXCL13, CASR, and EDN1. (B) Protein expression of CCL21, CXCL13, and XCR1. (C) Protein expression of GPR174/CCR7 (the receptors of CCL21) and CXCR5 (the receptors of CXCL13). n = 3–6. *p < 0.05, **p < 0.01 vs. the sham group. I/R, ischemia reperfusion.

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  FIGURE 7. Expression of GPR174 and CCR7 on cardiac fibroblasts during myocardial I/R injury. (A) Immunofluorescence staining showing that GPR174 did not present on cardiomyocytes in the sham or I/R groups. (B) Immunofluorescence staining showing the expression of GPR174 on cardiac fibroblasts. (C) Immunofluorescence staining showing the expression of CCR7 on cardiac fibroblasts in the sham and I/R groups. Scale bar = 50 μm n = 3. **p < 0.01 vs. the sham group. I/R, ischemia reperfusion.