Here, we demonstrate for the first time that a CM senescence‐like phenotype is a feature of normal physiological human and murine ageing and provide a novel mechanistic model by which senescence occurs in rarely dividing/post‐mitotic tissues. Persistent DNA damage foci, which co‐localise with telomere regions, increase in cardiomyocytes with age independently of telomere length, telomerase activity or DNA replication and can be induced by mitochondrial dysfunction in vitro and in vivo . Global gene expression analysis of purified CMs, isolated from young and old mice, indicates activation of the classical senescence growth arrest pathways and of a distinct non‐canonical SASP with functional effects including the potential to promote myofibroblast differentiation in fibroblasts and CM hypertrophy. Furthermore, specific induction of length‐independent telomere dysfunction in CMs induces several senescence markers, including hypertrophy, which importantly is also associated with CM senescence in vivo . Finally, we demonstrate that suicide gene‐mediated ablation of p16 Ink4a ‐expressing senescent cells and treatment with the senolytic drug, navitoclax, reduces CMs containing dysfunctional telomeres and attenuates cardiac hypertrophy and fibrosis in aged mice.

Critically short telomeres, induced by breeding of multiple generations of mice lacking the catalytic subunit of telomerase Terc , lead to cardiac dysfunction and myocardial remodelling (Wong et al , 2008 ). However, replicative senescence‐induced telomere shortening is unlikely to reflect normal physiological myocardial ageing, considering that the majority of CMs are post‐mitotic, withdrawing from the cell cycle shortly after birth. While the heart has a limited potential for regeneration, CM turnover rates in both humans and mice are extremely low (Senyo et al , 2013 ; Bergmann et al , 2015 ; Richardson et al , 2015 ). We and others have reported that stress‐induced telomere damage can also lead to telomere dysfunction and induce senescence (Fumagalli et al , 2012 ; Hewitt et al , 2012 ). Therefore, we investigated whether this phenomenon occurs in CMs and whether CM senescence contributes to myocardial remodelling during ageing.

Clinical heart failure (HF) represents a worldwide problem, and over 50% of all patients exhibit HF with preserved ejection fraction (HFpEF), which is a disease of the older population (Borlaug, 2014 ). As the population continues to age, the prevalence of HFpEF is increasing, currently at a rate of over 10% per decade (Borlaug & Paulus, 2011 ). During ageing, the heart undergoes distinct physiological and molecular changes, including cardiomyocyte (CM) hypertrophy and increased fibrosis, which contribute to increased ventricular stiffness causing a reduction in diastolic but not systolic cardiac function (Strait & Lakatta, 2012 ). Despite the growing need for interventions, effective treatments for HFpEF have yet to be identified (Borlaug & Paulus, 2011 ).

Telomere shortening has been proposed as a major inducer of senescence (Bodnar et al , 1998 ). Telomeres are repetitive sequences of DNA, associated with a protein complex known as shelterin (de Lange, 2005 ), which facilitates the formation of a lariat‐like structure to shield the exposed end of DNA (Griffith et al , 1999 ), thus protecting the ends of chromosomes from being recognised as DNA damage (d'Adda di Fagagna et al , 2003 ). The current dogma of telomere biology suggests that telomere shortening with each successive cell division eventually disrupts the protective cap, leading to a sustained DNA damage response (DDR) and activation of the senescence programme (Griffith et al , 1999 ). This hypothesis may explain age‐related degeneration of tissues maintained by constant contribution of stem cell pools, such as the skin and hematopoietic systems; however, it is insufficient to explain how senescence contributes to ageing in primarily post‐mitotic tissues such as the heart. As such, the mechanisms that drive senescence in post‐mitotic cells and the contribution of post‐mitotic cell senescence (PoMiCS) to tissue degeneration during ageing are an emerging area of interest (Anderson et al , 2018 ; Sapieha & Mallette, 2018 ).

The role of cellular senescence in tissue maintenance, repair and ageing is currently a rapidly evolving area of research under intense focus. Cellular senescence is classically defined as an irreversible loss of division potential of mitotic cells, often accompanied by a senescence‐associated secretory phenotype (SASP). While senescence can be detrimental during the ageing process, it has also been implicated in fundamental biological processes such as tumour suppression, embryonic development and tissue repair (Muñoz‐Espín & Serrano, 2014 ).

Results

Length‐independent telomere damage in aged cardiomyocytes in vivo Previously, we defined a novel mechanism of cellular senescence via the induction of irreparable telomere damage that occurs in the absence of telomere shortening (Hewitt et al, 2012). These data led us to hypothesise that this form of telomere damage could be the process by which senescence occurs in post‐mitotic, or rarely dividing cells, which are not subject to proliferation‐associated telomere shortening. We therefore investigated telomere dysfunction in adult mice throughout ageing by using dual Immuno‐FISH to quantify co‐localisation between DDR proteins γH2A.X, 53BP1 and telomeres, hereafter referred to as telomere‐associated foci (TAF) in CMs (Fig 1A). CMs were identified by CM‐specific markers α‐actinin, troponin‐C and the perinuclear protein PCM1 (Richardson, 2016; Richardson et al, 2015; Fig EV1A–C). Figure 1.Telomere dysfunction increases with age in mouse cardiomyocytes A. Representative images of γH2AX immuno‐FISH in troponin‐C‐positive/α‐actinin‐positive (top left and bottom left panels, respectively) mouse CMs (white—troponin‐C/α‐actinin; red—telo‐FISH; green—γH2AX). Images are Z‐projections of 0.1 μm stacks taken with a 100× oil objective. Scale bar: 20 μm. Right panels represent a single z‐planes where co‐localisation between a γH2AX foci and telomere was observed. Scale bars 1 μm. (Below) 3D reconstruction of Immuno‐FISH using STED microscopy for γH2A.X and telomeres in a CM from 30‐month‐old mice. Scale bars as indicated and scale the same for both individual TAF examples using STED.

B, C. Mean number of TAF (B) and % of TAF‐positive (C) α‐actinin‐positive CMs from C57BL/6 mice. Data are mean ± SEM of n = 4–7 mice per age group. 100 α‐actinin‐positive CMs were quantified per mouse.

D, E. Mean number of total γH2A.X foci (D) and % of γH2A.X foci‐positive nuclei (E) in α‐actinin‐positive CMs from C57BL/6 mice. Data are mean ± SEM of n = 4–7 mice per age group. 100 α‐actinin‐positive CMs were quantified per mouse.

F. Fold enrichment of γH2AX at telomere repeats by real‐time PCR. Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control, 3‐ and 30‐month‐old whole mouse hearts. Data are mean ± SEM of n = 3 mice per age group.

G. Quantitative PCR‐ELISA TRAP assay comparing telomerase activity of 3‐ and 30‐month‐old C57BL/6 mice whole heart lysates. Data are mean ± SEM of n = 4 mice per age group.

H. Histograms displaying distributions of telomere intensity in CMs analysed by 3D Q‐FISH in young (4 months) and old (30 months) wild‐type mice. Data are from n = 3 mice. > 100 CMs were analysed per mouse.

I. % of telomere FISH signal loss in 30‐month‐old wild‐type mice and late generation Terc−/− mice (6 months old) in comparison with 4‐month‐old wild‐type mice. Data are from n = 3 mice. > 100 CMs were analysed per mouse. Data information: Statistical analysis was performed using one‐way ANOVA (Holm–Sidak method) for multiple comparisons and Mann–Whitney test and two‐tailed t‐test for single comparisons. ***P < 0.001; **P < 0.01. Data information: Statistical analysis was performed using one‐way ANOVA (Holm–Sidak method) for multiple comparisons and Mann–Whitney test and two‐tailed‐test for single comparisons. ***< 0.001; **< 0.01. Click here to expand this figure. Figure EV1.Aged cardiomyocytes in both humans and mice are characterised by dysfunctional telomeres Representative images of γH2A.X immuno‐FISH in PCM1‐positive human cardiomyocytes (blue—DAPI; yellow—PCM1; red—telo‐FISH; green—γH2A.X). Images are z‐projections of 4.5 μm stacks taken with 100× objective. Right panels show co‐localisation between telomeres and γH2AX, 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 α‐actinin, PCM1 and WGA and γH2A.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 γH2A.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 γH2A.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 γH2AX 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 γH2A.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 γH2AX 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. Mean TAF per CM and the total % of CMs containing TAF increased significantly with age in both mouse and human hearts (Figs 1A–C and EV1A). In contrast, the total number of γH2A.X foci did not change with age and were detected in almost all adult CMs irrespective of age (Fig 1D and E). Similar results were obtained when analysing co‐localisation between DDR protein 53BP1 and telomeres in young and old mice (Fig EV1B). Interestingly, CMs from old animals contained a significantly higher number of TAF than other cardiac cell types (Fig EV1C), suggesting that telomere dysfunction is a predominant feature of CM ageing. To confirm further the localisation of a DDR at telomeres, we performed chromatin immunoprecipitation (ChIP) on cross‐linked chromatin from heart tissue, using an antibody against γH2A.X followed by quantitative real‐time PCR for telomeric repeats. We found enrichment of γH2A.X at telomeres in 30‐month‐old mice compared to 3‐month‐old mice (Fig 1F). Although the majority of the adult CMs are considered post‐mitotic, the adult heart retains limited potential for CM proliferation (Senyo et al, 2013; Bergmann et al, 2015; Richardson et al, 2015). We next investigated whether increased TAF was the outcome of telomere shortening driven by replication and/or impaired telomerase activity. Having first observed that telomerase activity was not affected by mouse age (Fig 1G), we observed a significant reduction in telomere FISH signal when comparing 4‐ to 30‐month‐old mice (Fig 1H). However, the median telomere FISH signal detected in old wild‐type (30 months) mice was comparatively higher than that found in fourth‐generation Terc−/− (G4) where critical telomere shortening has been shown to induce cardiac dysfunction (Wong et al, 2008; Fig 1I). 3D super‐resolution stimulated emission depletion microscopy (STED) was used to more accurately detect telomere length by Q‐FISH in CMs. STED resolution is typically 50 nm in XY and 150 nm in Z and overcomes the so‐called diffraction limit of conventional confocal microscopy, which yields resolutions of ≥ 200 nm. Using STED, we could identify an increased number of telomeres detected per CM, as well as decreased individual telomere volume when compared to conventional confocal microscopy (Fig EV1D and E, and Movie EV1). STED microscopy also revealed the existence of clusters of telomeres with varying lengths, which would otherwise be identified as single telomere signals by confocal microscopy (Fig EV1D). We then proceeded to analyse telomere FISH intensities in telomeres co‐localising with γH2A.X (TAF) and those not co‐localising with γH2A.X (non‐TAF) in aged mice using STED and found no significant differences (Fig EV1F and G). Similar observations were made in CMs from aged human hearts (Fig EV1H). Analysis of telomere FISH intensities by STED in individual cardiomyocytes revealed that telomeres with low FISH intensity co‐localising with γH2A.X are relatively rare (Fig EV1I). Together, our data suggest that length‐independent telomere‐associated DNA damage is unlikely to be due to the limitations of conventional confocal microscopy and that the shortest telomeres are not preferentially signalling a DDR. While CM proliferation is negligible in adult humans and mice, it is possible that TAF may be the outcome of a small fraction of dividing CMs. To ascertain further whether age‐associated accumulation of TAF can occur in post‐mitotic cells and is not exclusively linked to CM proliferation and turnover during ageing, we analysed our data in a mathematical model of a CM population over 27 months of a mouse lifespan. Our simulations reveal that, even considering relatively high estimates of cell division rates (4–15%), CM proliferation cannot account for the rate of the age‐dependent increase in TAF. Rather, if we assume that proliferation plays no role in the TAF increase with age and that TAF are generated in non‐dividing cells, there is a high degree of correlation between the model and our experimental data (Appendix Fig S1). In the mdx4cv/mTRG2 mouse model of telomere dysfunction, reduced expression of shelterin components is suggested to underlie increased telomere erosion in CMs (Mourkioti et al, 2013; Chang et al, 2016). To test whether uncapping of telomeres contributes to induction of a DDR at telomeres during ageing, we quantified the expression of shelterin components in CMs isolated from young and old wild‐type mice and found no significant differences (Appendix Fig S2A). Similarly, we observed no significant difference in the abundance of TRF2 (an essential component of t‐loop formation) in either TAF or non‐TAF in human CMs in vivo (Appendix Fig S2B). Together, these data support the notion that TAF increase with age in CMs and this occurs as a result of a process that is independent of cell proliferation can occur independently of telomere shortening and is not a result of overt alteration of telomere regulatory factors, such as shelterin components and telomerase. Having shown the phenomenon of telomere dysfunction occurring in CMs in vivo, we also found an age‐dependent increase in TAF (but not non‐TAF) in other post‐mitotic cells, specifically in skeletal muscle myocytes and hippocampal neurons, which indicates the widespread nature of this phenomenon (Appendix Fig S3A–G).

Telomere damage is persistent in cardiomyocytes Work by us and others previously demonstrated demonstrated that stress exposure leads to activation of a persistent DDR at telomeric regions (Fumagalli et al, 2012; Hewitt et al, 2012). To investigate the kinetics and nature of the DDR in CMs, we utilised different in vitro models. We first observed that exposure to X‐ray radiation (10 Gy) resulted in both telomere‐associated foci (TAF) and non‐telomere‐associated DNA damage foci (non‐TAF) in mouse embryonic CMs positive for troponin‐C and PCM1 (Fig 2A). However, only TAF were persistent, with non‐TAF numbers being significantly reduced with time (Fig 2B). Figure 2.Stress‐induced telomere‐associated DNA damage is persistent in mouse embryonic cardiomyocytes, rat neonatal cardiomyocytes and H9C2 myoblasts Representative images of mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 days following 10 Gy X‐irradiation. Left panels represent troponin‐C‐positive embryonic cardiomyocytes (troponin‐C—magenta; DAPI—light blue). Middle panels display γH2AX foci (green) and telomeres (red) in Z‐projections of 0.1 μm slices, with white arrows indicating co‐localisation. Co‐localising foci are amplified in the right‐hand panels (amplified images represent a single z‐planes where co‐localisation was observed). Scale bars represent 10 μm. Scale bars in single‐plane images 500 nm. (Left) Mean number of both TAF and non‐TAF in troponin I‐positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10 Gy X‐irradiation. Data are mean ± SEM of n = 3 independent experiments; 30–50 troponin‐positive cardiomyocytes were analysed per experiment. (Right) Mean percentage of γH2AX foci co‐localising with telomeres (% TAF) in troponin‐C‐positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10 Gy X‐irradiation. Statistical analysis performed using one‐way ANOVA (Holm–Sidak method); *P < 0.05. Significant differences were found for mean number of non‐TAF, but not for mean number of TAF. Mean number of both TAF and non‐TAF in neonatal rat cardiomyocytes at days 0, 3, 5, 10 days following treatment for 24 h with H 2 O 2 . Data are mean ± SEM of n = 3. > 50 cells were quantified per condition. Statistical analysis performed using one‐way ANOVA (Holm–Sidak method); **P < 0.01. (Left) Representative images of γH2AX immuno‐FISH in H9C2 myoblasts 3 days 10 Gy X‐irradiation (red—telo‐FISH; green—γH2AX). White arrows identify areas shown in higher magnification panels. (Right) Mean number of both TAF and non‐TAF in H9C2 myoblasts at days 3 and 5 following 10 Gy X‐irradiation. Data are mean ± SEM of n = 3. > 50 cells were quantified per condition. Statistical analysis performed using one‐way ANOVA (Holm–Sidak method); ***P < 0.001. Scale bars represent 1 μm. Scale bars in single‐plane images 500 nm. Representative time‐lapse images of H9C2 rat cardiomyoblasts expressing AcGFP‐53BP1 from 3 days after 10 Gy irradiation at the indicated times (min). Images are maximum intensity projections with a 6.7 μm focal depth. Scale bar represents 1 μm. Kaplan–Meier survival curves for AcGFP‐53BP1c DDR foci in H9C2 cells 3 days after 10 Gy irradiation at 10‐min intervals for 24 h. > 500 foci from 10 cells were tracked per condition. Gehan–Breslow test was used, P < 0.001. Schematic illustration showing 1‐month‐old C57BL/6 mice treated with 2 Gy whole‐body X‐irradiation, followed by a recovery period of 11 months before culling at 12 months of age. Mean number of TAF in α‐actinin‐positive cardiomyocytes. Data are mean ± SEM of n = 3 mice per group. Ninety α‐actinin‐positive cardiomyocytes were quantified per condition. Statistical analysis performed using two‐tailed t‐test; *P < 0.05. Cultures of rat neonatal CMs and the myoblast cell line (H9C2) treated with stressors H 2 O 2 and X‐ray irradiation, respectively, showed that the number of TAF remained persistent over time (Fig 2C and D). Three days subsequently to X‐ray irradiation, we monitored DDR in H9C2 cells using a 53BP1‐GFP fusion protein (Passos et al, 2010) and time‐lapse microscopy (Fig 2E). Over a 10‐h time‐course, the majority of individual DDR foci could be seen to be rapidly resolved with only approximately 20% of the original DNA damage foci persisting throughout the time‐course (Fig 2F), similar to the % of TAF‐positive cells for this time point (Fig 2D). To determine whether persistent TAF can also be induced in adult CMs in vivo, we exposed young mice to a whole‐body dose of 2 Gy X‐ray irradiation and measured the level of TAF in CMs after 11‐month recovery. Both the mean number of TAF and % of CMs positive for TAF were significantly higher (Fig 2G). Altogether, these data suggest that the majority of genomic DNA damage in CMs is reparable and that only telomeric DNA damage is irreparable and persistent. Double‐strand breaks can arise in telomeres due to replication errors when replication forks encounter single‐stranded breaks. In order to ascertain whether TAF could be induced in the absence of cell division, we pre‐incubated H9C2 cells with EdU for 3 h and subjected them to either X‐ray irradiation (10 Gy) or 10 μM H 2 O 2. Following a 24‐h recovery period (in the presence of EdU), the mean number of TAF was significantly increased in cells, which did not incorporate EdU (Appendix Fig S4A), indicating that TAF can be induced in the absence of DNA replication. Further, we isolated adult mouse CMs, which do not proliferate in culture, and exposed them to 5 μM H 2 O 2 . We observed that the mean number of TAF was significantly increased after treatment (Appendix Fig S4B). In addition, we did not find evidence that the shortest telomeres were specifically targeted by stressors. In fact, EdU‐negative irradiated H9C2 cells had a slightly higher median telomere length in damaged telomeres (TAF) compared to telomeres that did not co‐localise with γH2A.X (non‐TAF). However, such a difference was not found in adult murine CMs treated with H 2 O 2 (Appendix Fig S4C).

Telomere‐specific DNA damage drives a senescent‐like phenotype Having demonstrated that TAF increase with age and can be induced due to stress, we then tested whether telomere‐specific damage could directly drive a senescent‐like phenotype in the CM lineage. Rat neonatal CMs were transfected with an endonuclease that specially targets telomeres [a TRF1‐FokI fusion protein and a TRF1‐Fok1D450 inactive mutant (Dilley et al, 2016)]. In culture, we found that rat neonatal CMs had very low proliferation rates with 5% of cells incorporating EdU over a period of 24 h. Following 5 days of culture, CMs expressing TRF1‐FokI showed numerous DNA damage foci with the significant majority of damage co‐localising with telomeres (Fig 3A–C). Telomeres were targeted by the endonuclease irrespectively of their length (Fig 3D). De novo TAF formation induced a senescent phenotype in CMs characterised, in addition to TAF, by increased SA‐β‐Gal activity and upregulation of the cyclin‐dependent kinase inhibitor p21CIP (Fig 3E and F), as well as increased cellular hypertrophy (Fig 3G). Similar results were found using the H9C2 myoblasts (Fig EV2A–E). Additionally, we used the AC10 cell line derived from adult human ventricular CM (Davidson et al, 2005) stably expressing an inducible form of the TRF1‐FokI construct. These cells express CM‐specific transcription factors and contractile proteins and, while proliferative in complete media, are induced to terminal differentiation and exit the cell cycle under mitogen‐depleted conditions. We induced TRF1‐FokI expression in differentiated AC10 cells by exposure to doxycycline (DOX) for 6 h and allowed the cells to recover for an additional 36 h in the absence of DOX. Consistent with the idea that telomere damage is irreparable in differentiated CMs, we found that expression of TRF1‐FokI significantly increased the mean number of TAF and this number persisted for at least 36 h (Fig EV2F and G). Additionally, expression of TRF1‐FokI resulted in increased SA‐β‐Gal activity (Fig EV2H). In contrast, if similar numbers of non‐telomeric DNA damage foci were induced using the homing endonuclease I‐PpoI in neonatal cardiomyocytes, we observed that most DNA damage is repaired within 4 days, without induction of senescent markers or increased cell size (Fig EV2I–K). Figure 3.TRF1‐FokI fusion protein induces telomere‐specific double‐strand breaks, senescence and hypertrophy in rat neonatal cardiomyocytes A. Representative images of rat neonatal CMs 4 days following transfection with a FLAG‐tagged TRF1‐FokI‐D450A (top row) or TRF1‐FokI (middle and bottom row) fusion protein (cell treatments the same for all subsequent panels in Figure; red—telo‐FISH; green—γH2A.X). Images are z‐projections of 0.1 μm stacks taken with 100× objective. White arrows indicate co‐localisation between telomeres and γH2A.X, with co‐localising foci amplified in the right panels (taken from single z‐planes where co‐localisation was found). Scale bar represents 3.5 μm. Scale bar in magnified images showing individual co‐localisation 500 nm.

B, C. % of γH2A.X foci co‐localising with telomeres (B) and mean number of telomere‐associated foci (TAF) (C) in FLAG‐tagged TRF1‐FokI‐D450A‐ and TRF1‐FokI‐expressing CMs. Data are mean ± SEM of n = 4 independent experiments. > 50 cells were analysed per condition. Statistical analysis was performed by two‐tailed t ‐test *** P < 0.001.

D. Histograms displaying telomere intensity for telomeres co‐localising or not co‐localising with γH2AX foci. Red dotted lines represent median. Mann–Whitney test shows no significant difference in telomere intensity between TAF and non‐TAF.

E. Mean % of FLAG‐labelled CMs positive for SA‐β‐Gal activity. Data are mean ± SEM of n = 3 independent experiments. > 100 cells were quantified per condition. Statistical analysis performed using two‐tailed t ‐test; ** P < 0.01.

F. Expression of p21 mRNA (as a function of β‐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. Statistical analysis performed using two‐tailed t ‐test; *** P < 0.001.

G. Mean ± SEM cell surface area (μm2) of FLAG‐labelled CMs expressing TRF1‐FokI‐D450A and TRF1‐FokI. Statistical analysis performed using two‐tailed t‐test; ***P < 0.001. Scale bar represents 50 μm. Click here to expand this figure. Figure EV2.TRF1‐FokI fusion protein induces telomere‐specific double‐strand breaks, senescence and hypertrophy in H9C2 cardiomyoblasts Representative images of H9C2 cardiomyoblasts 4 days following transfection with a FLAG‐tagged TRF1‐FokI‐D450A (top row) or TRF1‐FokI (middle and bottom row) fusion protein (cell treatments the same for all subsequent panels in figure; purple—FLAG; red—telo‐FISH; green—γH2A.X/53BP1). Images are z‐projections of 0.1 μm stacks taken with 100× objective. White arrows indicate co‐localisation between telomeres and γH2A.X/53BP1, with co‐localising foci amplified in the right panels (taken from single z‐planes where co‐localisation was found). Scale bars main image represent 2.5 μm. Scale bars single‐plane images 500 nm. Percentage of γH2A.X (left) or 53BP1 (right) foci co‐localising with telomeres. Data are mean ± SEM of n = 3 independent experiments. More than 50 cells were analysed per condition. Statistical analysis was performed by two‐tailed t‐test; *P < 0.05. Histograms displaying telomere intensity for telomeres co‐localising (bottom) or not co‐localising (top) with γH2AX (left) or 53BP1 (right) DDR foci. Red dotted lines represent median. Statistical analysis performed using two‐tailed t‐test. More than 50 cells were analysed per condition. Mann–Whitney tests show no significant difference in telomere intensity between TAF and non‐TAF, with either γH2A.X (left) or 53BP1 (right) DDR foci. Representative images of detection of senescent markers (purple—FLAG; red—Ki‐67; light blue—DAPI; darker cytoplasmic blue—SA‐β‐Gal). Scale bar represents 10 μm. Mean percentage of FLAG‐positive cells positive for Ki‐67, p21, SA‐β‐Gal activity and cell size. Data are mean ± SEM of n = 3 independent experiments. More than 100 cells were quantified per condition. For SA‐β‐Gal, data are presented as mean of > 100 cells representative of 1 experiment. Two additional independent experiments confirmed these findings (not shown). Statistical analysis performed using two‐tailed t‐test; *P < 0.05. Scheme depicting experimental setting: AC10 human cardiomyocyte cell line stably expressing an inducible TRF1‐FokI was cultured for 2 weeks in mitogen‐depleted media which led to terminal differentiation and loss of proliferation. Then, cells were treated for 6 h with doxycycline (DOX), after which DOX was removed and cells were allowed to recover for 36 h. Mean number of TAF following induction of TRF1‐FokI and recovery. Data are mean ± SEM of n = 4 independent experiments. Statistical analysis performed using one‐way ANOVA (Holm–Sidak method); *P < 0.05. % of SA‐β‐Gal‐positive cells expressing TRF1‐FokI 36 h after 6 h DOX treatment. Data are mean ± SEM of n = 4 independent experiments. Statistical analysis performed using two‐tailed t‐test; *P < 0.001. Forty‐eight hours post‐transfection, neonatal rat cardiomyocytes were stimulated with 4‐OH‐T (300 nM) during 6 h for nuclear re‐localisation of I‐PpoI and DSD induction. Analysis of γH2A.X was performed immediately after treatment and at day 4 post‐treatment. Data are mean ± SD from n = 50 cells per condition from two independent experiments. Scale bars represent 10 μm. % of SA‐β‐Gal‐positive neonatal cardiomyocytes transfected with inducible I‐PpoI at day 4 post‐treatment. Data are mean ± SD from n = 50 cells per condition from two independent experiments. Analysis of cell size in neonatal cardiomyocytes transfected with inducible I‐PpoI at day 4 post‐treatment. Data are mean ± SEM from n = 50 cells per condition from four independent experiments.

Aged cardiomyocytes activate senescence pathways but not a typical SASP Within the heart, CMs comprise approximately only 30% of the total cells (Nag & Zak, 1979). To overcome this heterogeneity and obtain an accurate representation of the CM transcriptome during ageing, we devised a new method for isolation and enrichment of CMs. In short, we conducted a retrograde Langendorff perfusion for dissociation of cardiomyocytes, followed by removal of CD31+/CD45+/ScaI+ interstitial cells via magnetic bead sorting (Fig 4A). This method allowed us to obtain a highly enriched cardiomyocyte population (Fig EV3A). RT–PCR quantification of mRNAs encoding the cyclin‐dependent kinase inhibitors p16Ink4a, p21CIP and p15Ink4b in 3‐ and 20‐month‐old animals demonstrated an age‐dependent increase in expression of all three genes (Fig 4B). Immunohistochemistry on tissue sections from ageing mice validated the increase of p21CIP at the protein level, specifically in CMs (Fig 4C). Furthermore, we detected increased activity of SA‐β‐Gal in old mice (Fig 4D). While SA‐β‐Gal positivity was rare, we could detect it in CMs but no other cell types from old mice. By centromere‐FISH in CMs, we also observed an age‐dependent increase of senescence‐associated distension of satellites (SADS), a marker of senescence (Swanson et al, 2013; Fig 4E). Global transcription analysis of purified CM populations by RNA‐sequencing revealed 416 differentially expressed genes between young (3 months) and old (20 months; Fig EV3B) mice. Principal component analysis revealed that both cohorts separate well with the first and second components, accounting for 80.1 and 7.8% of the cumulative variance across the data set (Fig EV3C). Consistent with rare CM proliferation in vivo, we did not observe any significant changes in the expression of proliferation genes (Fig EV3D). However, genes involved in the regulation of myofilaments, contraction and CM hypertrophy were enriched in old mice (Myh7, Capn3, Ankrd1, Myom3, Myot, Mybpc2). Confirmatory qRT–PCR showed age‐dependent increase in hypertrophy genes Myh7 and Acta1, but not Anf (Fig EV3E and F). Interestingly, pro‐inflammatory genes associated with the SASP (Coppé et al, 2008), such as Il‐1α, Il‐1β, Il‐6, Cxcl1 and Cxcl2, were not differentially expressed between CMs from young and aged animals (Fig 4F). To determine whether SASP factors were secreted by CMs, we collected conditioned medium from isolated CMs from young and old mice. In accordance with the RNA‐seq results, we did not find any significant differences in the levels of secreted proteins using a cytokine array (Fig 4F). Figure 4.Aged cardiomyocytes activate senescence pathways but not a typical SASP Scheme illustrating CM isolation and purification procedure. Real‐time PCR gene expression analysis in isolated mouse CMs from C57BL/6 mice. Data are mean ± SEM of n = 3–4 per age group. Statistical analysis performed by one‐way ANOVA (Holm–Sidak method); *P < 0.05. Mean % of p21‐positive CM nuclei from 3‐, 15‐ and 30‐month‐old C57BL/6 mice by immunohistochemistry (IHC). Data are mean ± SEM of n = 4 per age group. > 100 CMs were quantified per age group. Statistical analysis performed using one‐way ANOVA (Holm–Sidak method); *P < 0.05. Mean % of 3‐ and 24‐month‐old mouse CMs staining positive for SA‐β‐Gal in vivo with representative images above (blue—SA‐β‐Gal; green—troponin‐C; red—WGA). Black arrows indicate SA‐β‐Gal expression in a troponin‐C‐expressing CM. Statistical analysis performed using two‐tailed t‐test; *P < 0.05. Data are mean ± SEM from the analysis of > 500 CMs per mouse, four mice per age group. Scale bar 20 μm. Mean % of SADS‐positive CM nuclei from 3‐ and 30‐month‐old mouse CMs positive for SADS in vivo, as detected by centromere‐FISH. Data are mean ± SEM of n = 4 per age group. > 200 CMs per mouse were quantified. Statistical analysis performed using two‐tailed t‐test; *P < 0.05. SASP heatmap: 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). The colour intensity represents column Z‐score, where red indicates highly and blue lowly expressed. Click here to expand this figure. Figure EV3.Aged cardiomyocytes have no alterations in proliferation gene expression but show increased expression of genes associated with hypertrophy Representative images of cardiomyocytes isolations before (left) and after (right) purification to remove SCA1‐, CD31‐, and CD45‐positive cells. Scale bar represents 20 μm. Counts of differentially expressed genes calculated using DESeq2 at % 5 FDR. Principal component analysis (PCA) of FPKM expression for young (red) and old (blue) cardiomyocytes. Components one and two account for 80.1 and 7.8% of the total variance, respectively. Correlation clustered heatmap of a curated list of known proliferation genes in young and old cardiomyocytes. The colour intensity represents column Z‐score, with red indicating high expression and blue low expression. Note that there is no enrichment for differential expression in this subset of pro‐proliferation genes. Trace plots and heatmap of the relative expression of Ankrd1, Capn3, Mybcp2, Myot, Myom3 and their associated FDR‐corrected q‐values derived from DESeq2. Confirmatory RT–PCRs showing changes in cardiac hypertrophy genes Acta1 and Myh7, but not Anf. Data were normalised to Gapdh. Data are mean ± SEM of n = 4–5 mice per age group. Two‐tailed t‐test was used. **P < 0.01; *P < 0.05. Our data are in contrast with a previous report that shows increased expression of SASP components in murine heart with age using whole heart homogenates (Ock et al, 2016). We therefore compared the expression of previously reported SASP components in non‐purified and purified CM populations. Consistently, we found that following a simple Langendorff digestion that collects a heterogeneous population of CMs and stromal cells, we found significant differences in expression of SASP components such as Il‐6 and Cxcl1 between young and old mice (Appendix Fig S5A). However, the population of purified CMs demonstrated no such differences, suggesting that cell types other than CMs could explain previous observations (Appendix Fig S5A). Interestingly, RNA sequencing led to the identification of three secreted proteins, not commonly categorised as SASP components, which were confirmed to be significantly increased at the mRNA level in aged purified CMs: Edn3, Tgfb2 and Gdf15 (Fig 5A). Of these, only Edn3 was increased exclusively in aged CMs, but neither in cardiac stromal cells nor in other organs, suggesting that increased Edn3 levels in plasma is a good indicator of CM ageing (Appendix Fig S5B–D). The SASP has been shown to impact on proliferation of neighbouring cells, ultimately inducing senescence (Acosta et al, 2013). Consistently, we found that conditioned medium isolated from old adult CMs reduced proliferation of neonatal fibroblasts (measured by EdU incorporation) and increased the expression of α‐smooth muscle actin (α‐SMA), an indicator of myofibroblast activation (Fig 5B–E). We then examined in more detail the role of the newly identified CM senescence‐associated proteins in terms of their ability to induce bystander effects, particularly their role in fibrosis, proliferation and hypertrophy. We found that Edn3, Tgfb2 and Gdf15 induced expression of α‐SMA in fibroblasts; however, only Tgfb2 reduced cellular proliferation as measured by EdU incorporation (Fig 5F–H). Furthermore, treatment of neonatal rat CMs with Edn3 or Tgfb2, but not Gdf15, resulted in a significant increase in the cell surface area, supporting their involvement in the induction of hypertrophy (Fig 5I). Consistent with previous reports indicating that the SASP can induce senescence in neighbouring cells (Acosta et al, 2013), we found that conditioned medium from old adult CMs was able to induce an increase in SA‐β‐Gal and a reduction in EdU incorporation in fibroblasts. No significant increase in DNA damage foci was observed in fibroblasts (Appendix Fig S5E–H). Figure 5.Cardiomyocyte SASP induces fibrosis and reduces proliferation in fibroblasts and induces hypertrophy in cardiomyocytes A. (Above) RNA sequencing of purified CMs from four mice per age group reveals age‐dependent increased expression of three secreted proteins Edn3, Tgfb2 and Gdf15; the colour intensity represents column Z ‐score, where red indicates highly and blue lowly expressed. (Below) mRNA expression of Edn3, Tgfb2 and Gdf15 was independently validated by RT–PCR in young and old isolated adult CMs. Data are mean ± SEM of n = 5 mice per group.

B. CMs were isolated by Langendorff heart perfusion, purified and cultured for 48 h. Conditioned medium (CM) was collected and added to cultures of neonatal fibroblasts in the presence of 10 μM EdU.

C. Representative images of immunofluorescent staining against α‐SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs.

D, E. Quantification of % of EdU incorporation (D) and % of a‐SMA‐positive cells (E) in neonatal fibroblasts after treatment for 48 h with CM from young and old CM. Data are mean ± SEM of n = 3–4 mice per age group; > 200 cells were quantified per condition.

F. Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins: Tgfb2, Edn3 and Gdf15 for 48 h and immunostained against α‐SMA and EdU (fibroblasts) and α‐actinin (CMs).

G–I. Quantification of α‐SMA‐ (G) and EdU‐positive neonatal fibroblasts (H) and surface area (μm2) (I) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ± SEM of n = 3–4 independent experiments. Data information: Asterisks denote statistical significance with one‐way ANOVA (multiple comparisons) (G, H, I) or two‐tailed t‐test (A, D, E). ***P < 0.001; **P < 0.01; *P < 0.05. For all panels, scale bars represent 50 μm. Data information: Asterisks denote statistical significance with one‐way ANOVA (multiple comparisons) (G, H, I) or two‐tailed‐test (A, D, E). ***< 0.001; **< 0.01; *< 0.05. For all panels, scale bars represent 50 μm. We conclude that aged CMs in vivo activate a number of senescence effector pathways, including hypertrophy and a non‐typical SASP, which may mediate both autocrine and paracrine effects.

Mitochondrial ROS drive TAF in mouse cardiomyocytes in vivo Mitochondrial dysfunction has been described as both a driver and consequence of cellular senescence (Passos et al, 2007, 2010; Correia‐Melo et al, 2016). Gene Set Enrichment Analysis (GSEA) of RNA‐seq data revealed that the most negatively enriched Gene ontology term in old CMs is the mitochondrial inner membrane (Fig EV4A). Consistently, in CMs from old mice we observed an overall decline in expression of most mitochondrial genes, particularly those genes involved in the electron transport chain (ETC; Fig 6A) and mitochondrial ultrastructural defects by transmission electron microscopy (Fig EV4B). Click here to expand this figure. Figure EV4.Mitochondrial dysfunction induces senescence and hypertrophy in cardiomyocytes Normalised GSEA enrichment scores for the top 20 positively and negatively enriched GO ontologies. Those negatively enriched ontologies associated with mitochondrial structure and function are highlighted in red. Transmission electron microscopy to detect mitochondrial ultrastructural defects in young (3 months) and old (20 months) mice. Scale bars represent 500 nm. Cardiomyocyte length analysis on WT, MAO‐A or MAO‐A mice with or without drinking water supplemented with 1.5 g/kg/day NAC from the age of 4–24 weeks. Data are mean ± SEM of n = 5–10 mice per group. Representative images of mitochondrial complex IV (red) and WGA (blue) in 12‐month‐old WT and Polgmut/mut mice. Graphs represent complex IV intensity (left), p21 positivity by immunohistochemistry and mean cardiomyocyte area (right). Data are mean ± SEM of n = 3–5 mice per group. More than 100 cardiomyocytes were quantified per group. Scale bar represents 20 μm. Data information: Asterisks denote a statistical significance at P < 0.05 using two‐tailed t‐test. Data information: Asterisks denote a statistical significance at< 0.05 using two‐tailed‐test. Figure 6.Mitochondrial dysfunction is a feature of cardiomyocyte senescence and drives TAF in mouse cardiomyocytes in vivo A. Mito and ETC genes with GSEA analysis: Clustered heatmap showing all genes associated with the “Mitochondrion” GO term in young and old, mouse CMs as observed by the GSEA pre‐ranked list enrichment analysis (normalised enrichment score: −1.70; FDR q ‐value < 0.05). Alongside this is a column clustered heatmap displaying a list of genes from the electron transport chain (ETC) GO ontology. In both instances, genes are by column and samples by row with the colour intensity representing column Z ‐score, where red indicates highly and blue lowly expressed.

B. Real‐time PCR gene expression analysis of MAO‐A, MnSOD and catalase in isolated mouse CMs from young (3 months) and old (20 months) mice. Data are mean ± SEM of n = 4–5 per age group. Asterisks denote a statistical significance at P < 0.05 using two‐tailed t ‐test.

C. Mean % of 4‐HNE‐ (top) or 8‐oxodG‐positive (bottom) CMs from 3 month (young) or 30 month (old) mice. Data are mean ± SEM of n = 4 per age group. 100 CMs were quantified per age group. Asterisks denote a statistical significance at P < 0.05 using two‐tailed t ‐test.

D–G. Mean % of TAF‐positive nuclei (left graphs) or mean % of TAF (right graphs) in wild‐type (control) compared to MAO‐A transgenic mice with or without drinking water supplemented with 1.5 g/kg/day NAC from the age of 4–24 weeks, MnSOD +/+ vs MnSOD −/+ , Catalase +/+ vs Catalase −/− , WT vs POLG double‐mutant mice. Data are mean ± SEM of n = 3–4 per group. > 100 CMs were quantified per age group. Statistical analysis was performed using two‐tailed t ‐test (E–G) or one‐way ANOVA (for multiple comparisons) (D); * P < 0.05.

H. Scheme depicting isolated mouse adult CMs isolated from four animals were treated with or without 100 nM rotenone either in the presence of 5 mM NAC or vehicle control (pre‐treated for 30 min before rotenone treatment), for 24 h before fixation. Mean number of TAF (top graph) and mean % of TAF‐positive nuclei (bottom graph). Data are mean ± SEM from four separate CM cultures isolated from 3‐month‐old mice. Fifty CMs were quantified per condition. Asterisks denote a statistical significance at P < 0.05 using one‐way ANOVA (Holm–Sidak method). We then speculated whether mitochondrial ROS could be a driver of telomere dysfunction in CMs in vivo. Previous data indicate that mitochondrial ROS can drive telomere shortening in vitro (Passos et al, 2007) and that telomere regions are particularly sensitive to oxidative damage (Hewitt et al, 2012). Consistent with this hypothesis, we found increased mRNA expression of the pro‐oxidant enzyme monoamine oxidase A (MAO‐A) and decreased expression of antioxidant enzymes mitochondrial MnSOD and catalase in isolated CMs from old mice (Fig 6B). Furthermore, we found increased lipid peroxidation marker, 4‐HNE, and DNA oxidation marker, 8‐oxodG, in hearts with age (Fig 6C). To address whether these age‐associated changes are causal in TAF induction, we utilised a model of CM‐specific overexpression of MAO‐A (MHC‐MAO‐A tg), which displayed enhanced levels of mitochondrial ROS specifically in CMs (Villeneuve et al, 2013). In this transgenic mouse, both the mean number and % of CMs positive for TAF were significantly increased compared to age‐matched controls (Fig 6D). Critically, when transgenic animals were treated with the antioxidant N‐acetyl cysteine (NAC), both the increase in TAF (Fig 6D) and CM hypertrophy were significantly rescued (Fig EV4C). Complementary studies in old MnSOD+/− and Catalase−/− mice also revealed that these had a higher number of TAF than age‐matched controls (Fig 6E and F). Furthermore, we found a significant increase in TAF in CMs from Polgmut/mut mice, a model of accelerated ageing due to mitochondrial dysfunction (Trifunovic et al, 2004; Fig 6G). Increased telomere dysfunction in these mice was associated with decreased expression of mitochondrial proteins, increased expression of p21, and cardiac hypertrophy (Fig EV4D). Finally, treatment of isolated adult CMs with rotenone, a mitochondrial complex I inhibitor, induced TAF, which could be rescued by treatment with the antioxidant NAC (Fig 6H). In summary, CMs from aged heart exhibit downregulation of mitochondrial inner membrane and ETC. genes, and this is associated with indicators of increased ROS metabolism that is causative of telomere‐associated DNA damage.