Fig 1: Aged cardiomyocytes in both humans and mice are characterised by dysfunctional telomeres Representative images of ?H2A.X immuno-FISH in PCM1-positive human cardiomyocytes (blue—DAPI; yellow—PCM1; red—telo-FISH; green—?H2A.X). Images are z-projections of 4.5 µm stacks taken with 100× objective. Right panels show co-localisation between telomeres and ?H2AX, with taken from single z-planes where co-localisation was found. Graphs showing mean number of TAF (left) and mean percentage of TAF-positive nuclei (right) in PCM1-positive human cardiomyocytes from 46-year-old to 65-year-old and 74-year-old to 82-year-old human heart tissue. Data are represented as the mean for individual subjects, with the horizontal line representing group mean. *P < 0.05 statistical significance using two-tailed t-test. Scale bars as indicated.Quantification of 53BP1 Immuno-FISH in 4-month-old and 24-month-old mice. Graphs representing mean number of TAF and % of cardiomyocytes positive for TAF are in black and graphs representing mean number of 53BP1 and % of cardiomyocytes positive for 53BP1 are in orange; n = 3 mice per age group; > 100 cardiomyocytes were quantified. Asterisk denotes a statistical significance at P < 0.01 using two-tailed t-test.Above: Representative images of PCM1 and a-actinin, PCM1 and WGA and ?H2A.X, PCM1 immuno-FISH in 30-month-old mouse cardiac tissue. White arrows identify PCM1-expressing CM nuclei. Below: Graph representing mean ± SEM number of TAF in PCM1-positive versus PCM1-negative cardiac cells; n = 3 mice; > 100 cardiomyocytes were quantified. Asterisk denotes a statistical significance at P < 0.01 using two-tailed t-test. Scale bars represent 20 µm.Representative images of conventional confocal (green) versus STED microscopy (red) for detection of telomeres by Q-FISH. Blue arrows identify telomeres shown in higher magnification in right 2-panels. Note that STED microscopy is capable of discerning telomere clusters which would otherwise be detected by confocal microscopy as a single signal. (left) Main image scale bar represents 1 µm. (Right) Higher magnification images scale bars represent 500 µm.Comparison between average number of telomere FISH signals detectable per cell (left graph) and mean telomere volume (right graph) in mouse cardiomyocytes, detected by either standard confocal or STED microscopy. Data are represented as the mean ± SEM for each measurement, with the horizontal line representing the group mean.Representative image of immuno-FISH using STED microscopy for ?H2A.X and telomeres in cardiomyocytes from a 30-month-old mouse. Graph on the right side shows two telomere FISH signals of similar intensities (one showing co-localisation with ?H2A.X and the other not). Scale bar represents 1 µm.Histograms representing Q-FISH analysis by 3D STED microscopy comparing individual telomere length either co-localising (TAF) or not co-localising (non-TAF) with ?H2AX foci in mouse cardiomyocytes from n = 3 mice (aged 30 months of age). 300 cardiomyocytes (detected by troponin-C and WGA) were analysed per mouse. Mann–Whitney test reveals no statistical significance between TAF and non-TAF P = 0.13.Histograms displaying telomere intensity for telomeres co-localising (bottom) or not co-localising (top) with ?H2A.X DDR foci for PCM1-positive cardiomyocytes obtained from 46-year-old to 65-year-old (left) and 74-year-old to 82-year-old subjects. Dotted lines represent median intensity. Mann–Whitney test show no significant difference in telomere intensity between TAF and non-TAF in either 46-year-old to 65-year-old or 74-year-old to 82-year-old subjects (P > 0.05). More than 100 cardiomyocytes were quantified per subject.Graph showing values for telomere FISH intensities either co-localising (TAF) or not co-localising (non-TAF) with ?H2AX foci in 22 individual cardiomyocytes chosen randomly. Purple arrow indicates one telomere FISH signal, which is lower in intensity than the median. Green horizontal lines denote median fluorescence intensity.