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      Length‐independent telomere damage drives post‐mitotic cardiomyocyte senescence

      research-article
      1 , 2 , 1 , 2 , 3 , 4 , 2 , 4 , 1 , 2 , 1 , 2 , 1 , 2 , 1 , 2 , 1 , 2 , 5 , 1 , 2 , 1 , 2 , 1 , 2 , 1 , 2 , 6 , 4 , 7 , 8 , 3 , 3 , 8 , 4 , 4 , 8 , 8 , 8 , 9 , 10 , 9 , 11 , 8 , 8 , 3 , , 4 , , 1 , 2 , 5 ,
      The EMBO Journal
      John Wiley and Sons Inc.
      ageing, cardiomyocytes, senescence, senolytics, telomeres, Ageing, DNA Replication, Repair & Recombination, Metabolism

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          Abstract

          Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age‐related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post‐mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age‐related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent‐like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length‐independent telomere damage in cardiomyocytes activates the classical senescence‐inducing pathways, p21 CIP and p16 INK4a, and results in a non‐canonical senescence‐associated secretory phenotype, which is pro‐fibrotic and pro‐hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age‐related myocardial dysfunction and in the wider setting to ageing in post‐mitotic tissues.

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          Most cited references34

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          Extension of life-span by introduction of telomerase into normal human cells.

          Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.
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            Telomere dysfunction induces metabolic and mitochondrial compromise.

            Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.
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              Mammalian Heart Renewal by Preexisting Cardiomyocytes

              Although recent studies have revealed that heart cells are generated in adult mammals, the frequency and source of new heart cells is unclear. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes 1 . Other studies suggest that new cardiomyocytes are born at a very low rate 2,3,4 , and that they may be derived from division of pre-existing cardiomyocytes. Thus, the origin of cardiomyocytes in adult mammals remains unknown. Here we combined two different pulse-chase approaches -- genetic fate-mapping with stable isotope labeling and Multi-isotope Imaging Mass Spectrometry (MIMS). We show that genesis of cardiomyocytes occurs at a low rate by division of pre-existing cardiomyocytes during normal aging, a process that increases by four-fold adjacent to areas of myocardial injury. Cell cycle activity during normal aging and after injury led to polyploidy and multinucleation, but also to new diploid, mononucleated cardiomyocytes. These data reveal pre-existing cardiomyocytes as the dominant source of cardiomyocyte replacement in normal mammalian myocardial homeostasis as well as after myocardial injury. Despite intensive research, fundamental aspects of the mammalian heart’s capacity for self-renewal are actively debated 5,6 . Estimates of cardiomyocyte turnover range from less than 1% per year 2,3,4 to more than 40% per year 7 . Turnover has been reported to either decrease 3 or increase with age 7 , while the source of new cardiomyocytes has been attributed to both division of existing myocytes 8 and to progenitors residing within the heart 9 or in exogenous niches such as bone marrow 10 . Controversy persists regarding the plasticity of the adult heart in part due to methodological challenges associated with studying slowly replenished tissues. Toxicity attributed to radiolabeled thymidine 11 and halogenated nucleotide analogues 12 limits the duration of labeling and may produce direct biological effects. Tissue autofluorescence can reduce the sensitivity and specificity of immunofluorescent methods of detecting cell cycle activity 5,13 , including as cell cycle markers or incorporation of halogenated nucleotide analogues. The challenge of measuring cardiomyocyte turnover is further compounded by the faster rate of turnover of cardiac stromal cells relative to cardiomyocytes 14 . We used Multi-isotope Imaging Mass Spectrometry (MIMS) to study cardiomyocyte turnover and to determine whether new cardiomyocytes are derived from preexisting myocytes or from a progenitor pool (Fig 1a). MIMS uses ion microscopy and mass spectrometry to generate high resolution quantitative mass images and localize stable isotope reporters in domains smaller than one micron cubed 15,16,17 . MIMS generates 14N quantitative mass images by measuring the atomic composition of the sample surface with a lateral resolution of under 50nm and a depth resolution of a few atomic layers. Cardiomyocyte cell borders and intracellular organelles were easily resolved (Fig 1b). Regions of interest can be analyzed at higher resolution, demonstrating cardiomyocyte-specific subcellular ultrastructure, including sarcomeres (Fig 1c, Supplemental Fig 1a). In all subsequent analyses, cardiomyocyte nuclei were identified by their location within sarcomere-containing cells, distinguishing them from adjacent stromal cells. An immense advantage of MIMS is the detection of nonradioactive stable isotope tracers. As an integral part of animate and inanimate matter, they do not alter biochemical reactions and are not harmful to the organism 18 . MIMS localizes stable isotope tracers by simultaneously quantifying multiple masses from each analyzed domain; this enables the generation of a quantitative ratio image of two stable isotopes of the same element 15 . The incorporation of a tracer tagged with the rare stable isotope of nitrogen (15N) is detectable with high precision by an increase in 15N:14N above the natural ratio (0.37%). Nuclear incorporation of 15N-thymidine is evident in cells having divided during a one-week labeling period, as observed in the small intestinal epithelium, which turns over completely in 3–5 days 16 (Fig 1d); in contrast, 15N-thymidine labeled cells are rarely observed in the heart (Fig 1e) after 1 week of labeling. In subsequent studies, small intestine was used as a positive control to confirm label delivery. To evaluate for an age-related change in cell cycle activity, we administered 15N-thymidine for 8 weeks to three age groups of C57BL6 mice starting at day-4 (neonate), at 10-weeks (young adult) and at 22-months (old adult) (Supplemental Fig 2). We then performed MIMS analysis (Fig 2a, b, Supplemental Fig 3). In the neonatal group, 56% (±3% s.e.m., n=3 mice) of cardiomyocytes demonstrated 15N nuclear labeling, consistent with the well-accepted occurrence of cardiomyocyte DNA synthesis during post-natal development 19 . We observed a marked decline in the frequency of 15N-labeled cardiomyocyte nuclei (15N+CM) in the young adult (neonate= 1.00%15N+CM/day ±0.05 s.e.m. vs young adult=0.015% 15N+CM/day ±0.003 s.e.m., n=3 mice/group, p 320 cardiomyocytes entering the cell cycle; these results exclude such a high rate of turnover (expected 15N+ cardiomyocytes=321; observed=35; Fisher’s exact 2N) in the remaining cardiomyocytes as expected with compensatory hypertrophy after injury. Thus, in the 8wks after myocardial infarction, approximately 3.2% of the cardiomyocytes adjacent to the infarct had unambiguously undergone division (total 15N+ × mononucleated diploid fraction = 23% × 0.14 = 3.2%). The low rate of cardiomyocyte cell cycle completion is further supported by the absence of detectable Aurora B Kinase, a transiently expressed cytokinesis marker, which was detected in rapidly proliferating small intestinal cells but not in cardiomyocytes (Supplemental Fig 10). We also considered the possibility that a subset of 15N+ myocytes that were multinucleated and/or polyploid resulted from division followed by additional rounds of DNA synthesis without division. However, quantitative analysis of the 15N+ population did not identify a subpopulation that had accumulated additional 15N-label as would be expected in such a scenario (Supplemental Fig 11). Together, these data suggest that adult cardiomyocytes retain some capacity to reenter the cell cycle, but that the majority of DNA synthesis after injury occurs in preexisting cardiomyocytes without completion of cell division. If dilution of the GFP+ cardiomyocyte pool cannot be attributed to division and differentiation of endogenous progenitors, do these data exclude a role for progenitors in the adult mammalian heart? These data could be explained by preferential loss of GFP+ cardiomyocytes after injury, a process that we have previously considered but for which we have not found supporting evidence 23 . Such an explanation excludes a role for endogenous progenitors in cardiac repair and would be consistent with data emerging from lower vertebrates 8,26 and the neonatal mouse 27 in which preexisting cardiomyocytes are the cellular source for cardiomyocyte repletion. A second possibility to explain the dilution of the GFP+ cardiomyocyte pool is that injury stimulates progenitor differentiation without division; inevitably, this would lead to exhaustion of the progenitor pool, which if true could explain the limited regenerative potential of the adult mammalian heart. In summary, this study demonstrates birth of cardiomyocytes from preexisting cardiomyocytes at a projected rate of approximately 0.76%/year (15N+ annual rate × mononucleated diploid fraction = 4.4% × 0.17) in the young adult mouse under normal homeostatic conditions, a rate that declines with age but increases by approximately four-fold after myocardial injury in the border region. This study shows that cardiac progenitors do not play a significant role in myocardial homeostasis in mammals and suggests that their role after injury is also limited. Online Methods Mice All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and approved by the Harvard Medical School Standing Committee on Animals. C57Bl/6 male mice were obtained from Charles River. We generated double transgenic Mer-CreMer-ZEG male mice by crossbreeding cardiomyocyte-specific MerCreMer mice and ZEG mice (Jackson Laboratory). β-galactosidase-GFP is under the control of a cytomegalovirus (CMV) enhancer/chicken actin promoter (Actb); the background strain was C57BL/6J (N7). The background strain of the MerCreMer mice was C57Bl/6SV129. Genotyping was performed by PCR on tail DNA using the following primers: MerCreMer forward: 5′-GTCTGAC TAGGTGTCCTTCT-3′; MerCreMer backward: 5′-CGTCCTCCTGCTGGTA TAG-3′; ZEG forward: 5′-AAGTTCATCTGCACCACCG-3′; ZEG backward: 5′-TCCTTGAAGAAGATGGTGCG-3′; ZEG control forward: 5′-CTAGGCCA CAGAATTGAAAGATCT-3′; and ZEG control backward: 5′-GTAGGTG GAAATTCTAGCATCATCC-3′. To induce Cre recombination and GFP labeling in cardiomyocytes, we injected 4-OH-tamoxifen (provided by a generous gift from Laboratoires Besins), dissolved in peanut oil (Sigma) at a concentration of 5 mg/ml, intraperitoneally into 8-week-old MerCreMer-ZEG mice daily at a dosage of 0.5 mg/day for 14 days. Experimental myocardial infarction Mice were subjected to experimental myocardial infarction as described. Surgeries were performed by a single operator with more than 20 years of experience in the performance of coronary ligation in rodents. In brief, the left coronary artery was permanently ligated approximately 2 mm below the left atrial appendage. For sham operations, the thoracic cavity was opened and the heart exposed, but no intramyocardial sutures were placed. 15N-thymidine (Cambridge Isotopes) was administered at a rate of 20μg/hr via osmotic minipump (Alzet), implanted subcutaneously at the time of experimental myocardial infarction after a single intraperitoneal bolus dose of 500μg. MIMS data acquisition The factory prototype of the NanoSIMS50 as well as a standard NanoSIMS 50 and a large radius NanoSIMS 50L (Cameca, Gennevilliers, France) was used for MIMS analysis as previously described 15 . A focused beam of Cs+ ions was used to sputter a few atomic layers and generate secondary ions from the left ventricular free wall of heart section samples. The Cs+ primary ions were scanned over a raster pattern of either 256 × 256 pixels or 512 × 512 pixels. At each pixel location, the secondary ion intensities for 12C−, 13C−, 12C14N− and 12C15N−were recorded in parallel from the same sputtered volume. The detection of nitrogen requires the use of cluster ions 12C14N− and 12C15N− for 14N and 15N, respectively, due to the low ionization efficiency of nitrogen as N−. MIMS data analysis From a single field image acquisition, we first extracted four image files: the four original quantitative mass images (QMIs; 12C, 13C, 12C14N, 12C15N) and the two ratio images (13C/12C and 12C15N/12C14N), derived from the pixel-by-pixel division of the 13C QMI by the 12C QMI and of the 12C15N QMI by the 12C14N QMI, respectively. We then used a hue saturation intensity transformation (HSI) of the ratio image to map 15N-labeled regions. The hue corresponds to the ratio value, and the intensity at a given hue is an index of statistical reliability. 15N-thymidine labeling For the neonatal cohort in the aging experiment, labeling was starting at post-natal day 4 with subcutaneous injections of 50μg/g 15N-thymidine (Cambridge Isotopes) every 12h and continued through post-natal week 4. Starting at age 4 wks – and in all other experiments using adult mice – osmotic minipumps (Alzet) were implanted subcutaneously, delivering 15N-thymidine (Cambridge Isotopes) at a rate of 20 μg/hour. Multinucleation Analysis Serial adjacent sections (0.5μm) were stained to identify cardiomyocyte borders. A given cardiomyocyte was tracked in the vertical axis by locating it in serial sections. Uninjured hearts were stained using a modified PAS protocol with standard solutions (Electron Microscopy Services), but with longer incubation times optimized for LR white embedding. Slides were incubated in xylene at 37C, 1 hour, rehydrated through graded alcohols, incubated in periodic acid for two hours, and Schiff’s reagent for two nights. Sections were counterstained in hematoxylin and Scott’s Bluing for 1 hour each. Injured hearts were stained using a modified Trichrome staining protocol with standard solutions (Fisher Scientific), but with longer incubation times. Slides were incubated in xylene at 37C, 1 hour, rehydrated through graded alcohols, incubated in bouin’s fluid at 56C, 1 hour, rinsed in tap water, incubated in weigert’s iron hematoxylin stain 1 hour, rinsed in tap water, incubated in scarlet-acid fuchsin solution, 1 hour, rinsed in DI water, incubated in phophotungistic-phophomolybdic acid solution, 30 minutes, incubated in aniline blue stain solution, 30 minutes, and incubated in 1% acetic acid, 20 minutes. Fluorescent in situ hybridization Sections were incubated in proteinase K (50ug/ml) at 60C for 15min. After a PBS/MgCl2 (45mM) wash, slides were post-fixed in 4% paraformaldehyde (PBS/MgCl2), dehydrated through graded alcohols. Biotinylated-labeled chromosome Y paint (Star-FISH, Cambio) in hybridization mix was applied to sections, and sealed under glass with rubber cement (note some samples were analyzed with chromosome-18 paint due to product discontinuation of Y-paint). Samples were heated to 90C for 15 min. After an overnight incubation at 37C, slides were washed three times with 50% formamide/2x standard saline citrate at 45C, three washes with 2x standard saline citrate at room temperature, and two washes with 4x standard saline citrate/0.1% Tween at room temperature. Samples were blocked 10 minutes with 4x standard saline citrate/0.1% Tween/ 0.05% milk and incubated for 2 hr in streptavidin-conjugated Alexa Fluor 488 (Invitrogen) prior to washing and mounting. An observer unaware of the MIMS images or 15N-thymidine labeling status of the nuclei assigned ploidy status. Immunofluorescent staining Sections were incubated in glycine/tris (50mM glycine/0.05M Tris) at room temperature for 5min. After a brief wash with Tris, sections were incubated with chicken anti-GFP (Abcam), rabbit anti-ckit (Abcam), rat anti-sca1 (Abcam) with fresh 0.1%BSA in TBST (TBS/0.1%Tween) overnight at 4C. After a brief wash with TBS, sections were incubated with anti-chicken Alexa Fluor 488 (Invitrogen) prior to TBS wash and mounting. An observer unaware of the MIMS images or 15N-thymidine labeling status of the nuclei assigned GFP status. Fluorescence Image analysis We used a custom-written script in IP Lab version 4.0 (Scanalytics) imaging software for serial image acquisition. Tissue sections were auto-imaged using an Olympus IX-70 microscope with a digital charge-coupled device camera (CoolSNAP EZ, Roper Scientific), an automated stage with a piezoelectric objective positioner (Polytec PI, Auburn MA) to compensate for deviations in the z-axis. Images were compressed and stitched into a mosaic using stitching software (Canon Photostitch). Multichannel images were merged in ImageJ prior to stitching. Statistical analysis Statistical testing was performed using Prism 3.0 (Graphpad). Results are presented as mean ± s.e.m. and were compared using T-tests (significance was assigned for p<0.05). Data comparing event rates were tested with a Fisher-Exact test. Supplementary Material 1
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                Author and article information

                Contributors
                Jeanne.Perez@inserm.fr
                Gavin.Richardson@ncl.ac.uk
                Passos.Joao@mayo.edu
                Journal
                EMBO J
                EMBO J
                10.1002/(ISSN)1460-2075
                EMBJ
                embojnl
                The EMBO Journal
                John Wiley and Sons Inc. (Hoboken )
                0261-4189
                1460-2075
                08 February 2019
                01 March 2019
                08 February 2019
                : 38
                : 5 ( doiID: 10.1002/embj.v38.5 )
                : e100492
                Affiliations
                [ 1 ] Ageing Research Laboratories Institute for Ageing Newcastle University Newcastle upon Tyne UK
                [ 2 ] Institute for Cell and Molecular Biosciences Newcastle University Newcastle upon Tyne UK
                [ 3 ] INSERM Institute of metabolic and cardiovascular diseases University of Toulouse Toulouse France
                [ 4 ] Cardiovascular Research Centre Institute for Genetic Medicine Newcastle University Newcastle upon Tyne UK
                [ 5 ] Department of Physiology and Biomedical Engineering Mayo Clinic Rochester MN USA
                [ 6 ] The Bio‐Imaging Unit Newcastle University Newcastle upon Tyne UK
                [ 7 ] Wellcome Trust Centre for Mitochondrial Research Centre for Ageing and Vitality Newcastle University Newcastle upon Tyne UK
                [ 8 ] Robert and Arlene Kogod Center on Aging Mayo Clinic Rochester MN USA
                [ 9 ] Institute of Cancer Sciences CR‐UK Beatson Institute University of Glasgow Glasgow UK
                [ 10 ] Institute of Cellular Medicine Newcastle University Newcastle upon Tyne UK
                [ 11 ] Sanford Burnham Prebys Medical Discovery Institute La Jolla CA USA
                Author notes
                [*] [* ] Corresponding author. Tel: +33 5 61325643; E‐mail: Jeanne.Perez@ 123456inserm.fr

                Corresponding author. Tel: +44 0 1912418615; E‐mail: Gavin.Richardson@ 123456ncl.ac.uk

                Corresponding author. Tel: +1507 293 9785; E‐mail: Passos.Joao@ 123456mayo.edu

                [†]

                These authors contributed equally to this work

                Author information
                https://orcid.org/0000-0002-3647-7383
                https://orcid.org/0000-0002-1786-496X
                https://orcid.org/0000-0002-1765-0283
                https://orcid.org/0000-0002-2310-9987
                https://orcid.org/0000-0001-8765-1890
                Article
                EMBJ2018100492
                10.15252/embj.2018100492
                6396144
                30737259
                852b7e0c-de3c-4b28-bab3-4654655486da
                © 2019 The Authors. Published under the terms of the CC BY 4.0 license

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 16 August 2018
                : 18 December 2018
                : 02 January 2019
                Page count
                Figures: 13, Tables: 0, Pages: 21, Words: 16629
                Funding
                Funded by: Connor Group
                Funded by: Noaber Foundation
                Funded by: Région Occitanie, France
                Funded by: Medical Research Council and Arthritis UK funded centre for integrated Musculoskeletal Ageing (CIMA)
                Award ID: MR/K0063121/1
                Funded by: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)
                Award ID: BB/H022384/1
                Award ID: BB/K017314/1
                Funded by: British Heart Foundation (BHF)
                Award ID: PG/15/85/31744
                Funded by: Foundation for the National Institutes of Health (FNIH)
                Award ID: AG013925
                Categories
                Article
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                Custom metadata
                2.0
                embj2018100492
                1 March 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.6.1 mode:remove_FC converted:01.03.2019

                Molecular biology
                ageing,cardiomyocytes,senescence,senolytics,telomeres,dna replication, repair & recombination,metabolism

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