Induction of Senescence by Loss of Gata4 in Cardiac Fibroblasts

Figure 1. Gata4 expression and inactivation in Tcf21 lineage traced cardiac fibroblasts. (A) Gata4 expression in Tcf21 lineage traced cardiac fibroblasts. Following tamoxifen administration, 8-weeks old Tcf21iCre:R26RtdT mouse hearts were harvested, and heart sections were processed and immunostained for Gata4. Scale bar, 100 µM. (B) Schematic illustration of generation of cardiac fibroblast specific, inducible Gata4 knockout mice. Tcf21iCre mice in which MerCreMer cDNA cassette was inserted into exon 1 (E1) were crossed with Gata4 floxed mice which contain a pair of loxP sites encompassing exons 3, 4, and 5 (E3, 4, and 5) of Gata4. Alternatively, Tcf21iCre:R26RtdT mice were crossed with Gata4 floxed mice. Activated MerCreMer by tamoxifen administration recombines two loxP sites to delete exons 3, 4, and 5 of Gata4, thereby inactivating Gata4 in Tcf21 lineage traced cardiac fibroblasts. (C) Immunofluorescent analysis to demonstrate Gata4 inactivation in Tcf21 lineage traced cardiac fibroblasts. Following tamoxifen administration, Tcf21iCre:R26RtdT (Control) and Tcf21iCre:Gata4fl/fl: R26RtdT (Gata4 KO) mouse hearts were harvested, and heart sections were processed and immunostained for…

Figure 2. Senescence induction by Gata4 loss in cardiac fibroblasts in the heart. (A) Demonstration of senescence by SA β-gal staining. SA β-gal staining was performed on heart sections 2- or 7 weeks following tamoxifen administration into Tcf21iCre:Gata4fl/fl (Gata4 KO) or Tcf21iCre (Control) mice. Scale bar, 40 µM, and 100 µM. (B) Demonstration of senescence by p16 immunostaining. The heart sections from Tcf21iCre:Gata4fl/fl (Gata4 KO) and Tcf21iCre (Control) mice were immunostained for p16 2-week (2 wks) or 7-week (7 wks) following tamoxifen administration. Scale bar, 100 µM. (C,D) Summary of quantification of senescent cells in cardiac fibroblast specific Gata4 deficient hearts. Three independent experiments are presented as mean ± s.d. n = 3, ** p < 0.01; *** p < 0.001.

Figure 3. Senescence induction in Gata4 deficient cardiac fibroblasts in vitro. (A) Inactivation of Gata4 in vitro. Ad-Cre or Ad-vector viruses were infected into adult cardiac fibroblasts isolated from Gata4fl/fl mice. The infected cells were immunostained for alpha-smooth muscle actin (SMA) and Gata4. Representative immunofluorescent images of cells infected with Ad-vector (Control) or Ad-Cre (Gata4 KO) were shown. Gata4-expressing cells were quantified using high-content imaging analysis. Five independent experiments are presented as mean ± s.d. n = 5, *** p < 0.0001. Scale bar, 200 µM. (B) The proliferation of Gata4 deficient cardiac fibroblasts. Gata4fl/fl cardiac fibroblasts infected with Ad-vector (Control) or Ad-Cre (Gata4 KO) were EdU labeled or immunostained for Ki67. Representative immunofluorescent images used for high-content imaging analysis were shown. Three independent experiments are presented as mean ± s.d. n = 3, * p < 0.05; ** p < 0.005. Scale bar, 200 µM. (C) Demonstration of senescence in Gata4 deficient cardiac fibroblasts. Gata4fl/fl cardiac fibroblasts infected with Ad-vector (Control) or Ad-Cre (Gata4 KO) were stained for SA β-gal or immunostained for γH2AX.…

Figure 4. Generation of cardiomyocyte-specific, inducible Gata4 KO mice and evaluation of senescence induction. (A) Schematic illustration of generation of cardiomyocyte-specific, inducible Gata4 knockout mice. The transgenic mice which containing αMHC promoter driven MerCreMer cDNA cassette and were cross-bred with Gata4 floxed mice which contained a pair of loxP sites encompassing exons 3, 4, and 5 (E3, 4, and 5). Activated MerCreMer by tamoxifen administration recombines two loxP sites to delete exons 3, 4, and 5 of Gata4, thereby inactivating Gata4 in αMHC lineage traced cardiomyocytes. (B) Immunofluorescent analysis to demonstrate Gata4 inactivation in αMHC lineage traced cardiomyocytes. A week after tamoxifen administration, αMHCiCre (Control) and αMHCiCre:Gata4fl/fl (Gata4 KO) mouse hearts were harvested, and heart sections were processed. The heart sections were immunostained for Gata4 and stained for wheat germ agglutinin (WGA) to demarcate the cell boundary. Gata4+ nuclei in the WGA-stained interstitial area indicate Gata4+ interstitial cells. Scale bar, 100 µM. (C) Quantification of Gata4+ cells in the heart. Gata4+DAPI+ cells were quantified as Gata4+ cells in the heart. Three…