Figure 7.

Wnt4 upregulation in cardiac fibroblasts is crucial for cardiac function after ischemic reperfusion (I/R) injury. Col1a2-CreERT and Col1a2-CreERT: R26R^tdTomato mice subjected to sham or I/R injury after administration of pAAV-CGB-DIO-Wnt4-miR30shRNA-WPRE virus (pAAV-CGB-DIO-EGFP-Scramble-miR30shRNA-WPRE virus serve as scramble control) and tamoxifen. (A) Western blot for Wnt4 and endothelial cell marker genes in cardiac fibroblasts isolated within 24 h from whole heart at day 7 post cardiac injury and the densitometric quantification, n = 4 animals/group. (B-C) Triple immunofluorescence staining of tdTomato, Wnt4 and VECAD in injury border zone after Wnt4 knockdown in cardiac fibroblasts and the quantification, n = 3 animals/group, Scale bar: 10 µm. (D) The representative image of echocardiography M model. (E) Cardiac function assayed by echocardiography, n = 7 animals/group. (F) Masson' trichrome staining of heart tissue and quantification of fibrosis area by Image J, n = 7 animals/group. (G) Immunofluorescence staining of isolectin B4 for vascular density and quantification by Image J and AngioTool, n = 4 animals/group, Scale bar: 20 µm. (All graphs show mean ± S.E.M; *p < 0.05, **p < 0.01, ***p < 0.001, using unpaired t-test and two-way anova, Colocalization of fluorophores is indicated by arrowhead.)