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, Data are mean ± SEM of n=3 independent experiments. >100 cells were quantified per condition. f) Expression of p21 mRNA (as a function of ?-actin and Gapdh) by Real-time PCR in TRF1-FokI-D450A and TRF1-FokI expressing CMs. Data are mean ± SEM of n=6 independent experiments; g) Mean cell surface area (?m 2 ) of FLAG-labelled CMs expressingTRF1-FokI-D450A and TRF1-FokI, Mean % of FLAG-labelled CMs positive for SA-?-Gal activity

*. P<0,

. **p<0,

, Figure 4 Aged cardiomyocytes activate senescent pathways but not a typical SASP. a) Schematic illustrating CM isolation procedure. b) Real-time PCR gene expression analysis in performed by One-Way ANOVA

*. P<0, 05. c) Mean % of p21-positive CM nuclei from 3, 15, and 30 month old C57BL/6 mice by

. Immunohistochemistry, Data are mean ± SEM of n=4 per age group. >100 CMs were quantified per age group. Statistical analysis performed using One-Way ANOVA

*. P<0, 05. d) Mean % of 3 and 24 month old mouse CMs staining positive for SA-?-Gal in vivo with representative images above (blue -SA-?-Gal

. Red--wga), Statistical analysis performed using two-tailed t-test * P<0.05. Data obtained 8 quantified

. Sasp-heatmap, Asterisks denote a statistical significance at P < 0.05 using twotailed t-test. e) Mean number of TAF (left graph) and mean % of TAF-positive nuclei (right graph) in CMs. Data are mean ± S.E.M. of n=6 per age group. 100 CMs were analysed per mouse. f) Histograms showing distribution of individual telomere intensities measured by Q-FISH in INK-ATTAC mice (28-29m old) treated with vehicle or AP20187. >150 CMs were analysed per mouse. g) Mean CM area ?m 2 . Data are mean ±S.E.M. of n=6 per age group, >150 CMs analysed per mouse. h) % of fibrotic area evaluated by Sirius Red staining, Pearson correlation clustered heatmap showing a curated list of known SASP genes (top panel) or a selection of secreted SASP proteins (bottom panel) in young (3 months) and old (20 months) mouse CMs (n=5 per age group

. **p<0, , p.1

*. P<0, , p.5