Fig. 4
Isoproterenol (ISO) injection leads to cardiac fibrosis and expansion of Tcf21-expressing cells. (a) Overview of the ISO injection schedule. (b) Cardiac fibrosis area of Masson trichrome-stained sections after ISO injection. Dunnett’s test was performed for post-hoc analysis (biologically n = 3). (c) Tcf21 expression before and after ISO injection assessed by qPCR. Dunnett’s test was performed for post-hoc analysis (biologically n = 3). (d) Tcf21-positive area calculated based on β-galactosidase staining (biologically n = 4). The detected area was normalized to the cardiac cross-sectional area. (e,f) Tcf21-positive cells accumulated in the fibrotic area after 3 or 7 days of ISO injection. Tcf21-positive cells were calculated in the Masson trichrome (MT) positive or negative area. Scale bar, 100 µm (biologically n = 3). (g) Immunostaining of a section of heart from Tcf21LacZ/+ transgenic mice. Tcf21 expression as assessed by β-galactosidase (green) and periostin (red) staining coexisted after 3 days of ISO injection. Scale bar, 100 µm. (h) Immunostaining of Ki67 (brown) and Tcf21 expression by β-galactosidase (blue). Ki67-positive cells accumulated in the Tcf21-positive area. Scale bar, 100 µm. (i) Proliferation capacity of Ki67-positive cells. Each sample was counted in a high-power microscopic field more than three times, and the average values were used for analysis. Unpaired two-tailed t-tests with Bonferroni correction were used for post-hoc analysis (biologically n = 4). (j) Fibroblast-associated gene expression increased after ISO injection. Dunnett’s test was performed for post-hoc analysis (biologically n = 3).
