Figure 6: Bmi1-mediated cardiomyocyte senescence.
From: Bmi1 limits dilated cardiomyopathy and heart failure by inhibiting cardiac senescence
(a) Cytochemical staining of SA-β-gal activity and immunostaining of Caspase 3 in paraffin sections of hearts from 15-week-old Bmi1f/f;αMHC-Cretg/+ mice and Bmi1f/ controls. Bars, 50 μm. (b) qRT–PCR analysis of Bmi1 mRNA expression in sorted populations of CMs, endothelial cells (ECs) and FBs from Bmi1f/f;αMHC-Cretg/+ and Bmi1f/ mice. Expression is standardized to β-actin and is expressed relative to the level in Bmi1f/ mice (means±s.d.; n=12, *P<0.05; Student’s t-test). (c) Proliferation rate of CM, EC and FB subpopulations measured by in vivo BrdU incorporation over 1 week. Values are means±s.d. (n=5). (d,e) Representative histograms for C12-fluorescein show the relative levels of SA-β-gal in CM, EC and FB subsets from Bmi1f;αMHCCre and control mice (d); the values above the peaks are the median fluorescence intensities of the respective populations. (e) Percentage of SA-β-gal-positive cells in CM, EC and FB subpopulations. Values are means±s.d. (n=6, *P<0.05; Student’s t-test). (f) qRT–PCR analysis of the expression of Bmi1, p16INK4a, Ezh2, Myh7, Tgfβ, p53 and p21 mRNA in total heart cells from Bmi1fl/fl;αMHC-Cretg/+;p16INK4a−/− mice and Bmi1fl/fl;αMHC-Cre+/+;p16INK4a−/− mice. Values are means±s.d.; n=10 (**P<0.001, *P<0.05; Student’s t-test). (g) The gross cardiac phenotype of 12-week-old Bmi1fl/fl;αMHC-Cre+/+;p16INK4a−/− (Bmi1fl;p16−/−), Bmi1fl/fl;αMHC-Cretg/+;p16INK4a+/+ (Bmi1fl;αMHCCre) and Bmi1fl/fl;αMHC-Cretg/+;p16INK4a−/− (Bmi1fl;αMHCCre;p16−/−) mice. Representative views are shown of H&E-stained heart cross-sections at the midventricle (bars, 1 mm), Masson’s trichrome staining of left ventricle to detect fibrosis (bars, 40 μm) and WGA staining to outline cardiomyocytes (bars, 10 μm). (h–k) HW/BW ratios (h), myocyte cross-sectional area (i), left ventricular wall thickness (j) and fractional shortening (k) in 12-week-old Bmi1fl;p16−/−, Bmi1fl;αMHCCre and Bmi1fl;αMHCCre;p16−/− mice. Data are means±s.d. n=8 mice per group (*P<0.05; Student’s t-test). (l) Cytochemical staining of SA-β-gal activity in paraffin sections of hearts from Bmi1fl;p16−/−), Bmi1fl;αMHCCre) and Bmi1fl;αMHCCre;p16−/− mice. Scale bars, 50 μm. (m,n) Representative fluorescence histograms show the relative levels of SA-β-gal in CM, EC and FB subsets from Bmi1fl;p16−/− and Bmi1fl;αMHCCre;p16−/− hearts (m); the values above the peaks are the median fluorescence intensities of the respective populations. Percentage of SA-β-gal-positive cells in CM, EC and FB subpopulations (n). Values are means±s.d. (n=6; Student’s t-test). (o) Kaplan–Meier survival curves for Bmi1fl;p16−/−, Bmi1fl;αMHCCre and Bmi1fl;αMHC-Cre;p16−/− littermates (means±s.d., **P<0.001; Student’s t-test).
