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Telomere Shortening Is a Hallmark of Genetic Cardiomyopathies

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Telomere Shortening Is a Hallmark of Genetic Cardiomyopathies Telomere shortening is a hallmark of genetic cardiomyopathies Alex C. Y. Changa,b,c,d,e,1, Andrew C. H. Changa,b, Anna Kirillovaa,b, Koki Sasagawaa,b, Willis Sua,b, Gerhard Webera,b,c, Jue Linf, Vittavat Termglinchanc, Ioannis Karakikesc,g, Timon Seegerc, Alexandra M. Dainisc,h, John T. Hinsoni,j, Jonathan Seidmank, Christine E. Seidmani,k,l, John W. Daym, Euan Ashleyc,d,e, Joseph C. Wuc,n, and Helen M. Blaua,b,c,1 aBaxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; bInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; cStanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305; dStanford Center for Inherited Cardiovascular Disease, Stanford University School of Medicine, Stanford, CA 94305; eDivision of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305; fDepartment of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, CA 94158; gDepartment of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 94305; hDepartment of Genetics, Stanford University, Stanford, CA 94305; iDivision of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, MA 02115; jCardiology Division, UConn Health and The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032; kDepartment of Genetics, Harvard Medical School, Boston, MA 02115; lHoward Hughes Medical Institute, Chevy Chase, MD 20815; mDepartment of Neurology, Stanford University, Stanford, CA 94304; and nDepartment of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Helen M. Blau, July 23, 2018 (sent for review August 17, 2017; reviewed by Dirk Hockemeyer and Charles E. Murry) This study demonstrates that significantly shortened telomeres are We first implicated telomere attrition in DCM in studies of a hallmark of cardiomyocytes (CMs) from individuals with end-stage Duchenne muscular dystrophy (DMD) (7, 8). We found that, hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy although the mdx transgenic mouse, like patients with DMD, (DCM) as a result of heritable defects in cardiac proteins critical to lacks dystrophin, it does not exhibit cardiac symptoms typical of contractile function. Positioned at the ends of chromosomes, DCM or premature death, a major conundrum for testing ther- telomeres are DNA repeats that serve as protective caps that apeutic strategies for DMD. We postulated that mice are pro- shorten with each cell division, a marker of aging. CMs are a tected from the disease by the lengths of their telomeres, which, known exception in which telomeres remain relatively stable for unknown reasons, are substantially longer than in humans. In throughout life in healthy individuals. We found that, relative to support of this hypothesis, we “humanized” mdx mice to have healthy controls, telomeres are significantly shorter in CMs of shortened telomeres by breeding with the mTR-KO mouse that genetic HCM and DCM patient tissues harboring pathogenic muta- lacks telomerase activity as a result of the absence of the RNA tions: TNNI3, MYBPC3, MYH7, DMD, TNNT2,andTTN. Quantitative component TERC (TR). Strikingly, the humanized mdx mice FISH (Q-FISH) of single cells revealed that telomeres were signifi- developed the severe skeletal muscle phenotype and heart fail- cantly reduced by 26% in HCM and 40% in DCM patient CMs in ure seen in patients with DMD (7). We corroborated the findings fixed tissue sections compared with CMs from age- and sex- in our mouse model (mdx4cv/mTRG2) in CMs of the hearts of matched healthy controls. In the cardiac tissues of the same patients, patients with DMD, which exhibited, on average, a 48% decrease telomere shortening was not evident in vascular smooth muscle cells that do not express or require the contractile proteins, an important Significance control. Telomere shortening was recapitulated in DCM and HCM CMs differentiated from patient-derived human-induced pluripotent We find that telomere shortening, which usually accompanies stem cells (hiPSCs) measured by two independent assays. This study cell division in the course of aging, occurs in cardiomyocytes reveals telomere shortening as a hallmark of genetic HCM and DCM (CMs) of individuals with genetic hypertrophic cardiomyopathy and demonstrates that this shortening can be modeled in vitro by (HCM) or dilated cardiomyopathy (DCM). HCM and DCM CMs using the hiPSC platform, enabling drug discovery. differentiated from human-induced pluripotent stem cells (hiPSCs) also exhibit significant telomere shortening relative to telomere | dilated cardiomyopathy | hypertrophy cardiomyopathy | healthy controls. By contrast, no telomere shortening was hiPSC-CM detected in vascular smooth muscle cells in tissue or hiPSC- derived cells, a cell type that does not express the mutant enetic cardiomyopathy—hypertrophic and dilated—affects proteins. Our findings provide evidence for accelerated ag- G1 in 500–2,500 people worldwide and is the leading in- ing in CMs with familial cardiomyopathy. The potential to dication for heart transplantation. Genetic hypertrophic cardio- monitor the dynamics of telomere attrition in hiPSC-CMs myopathy (HCM) and dilated cardiomyopathy (DCM) are over time will enable future mechanistic studies and screens caused by mutations in proteins with a diverse range of functions for novel therapeutic agents to arrest telomere shortening (1, 2). Genetic HCM is characterized by ventricular wall thick- and disease progression. ening, whereas DCM is characterized by dilation of the ven- tricular chamber. Patient outcomes range from reduced quality Author contributions: A.C.Y.C. and H.M.B. designed research; A.C.Y.C., A.C.H.C., A.K., K.S., W.S., G.W., J.L., V.T., I.K., T.S., A.M.D., and J.T.H. performed research; A.C.Y.C., A.C.H.C., of life as a result of heart failure to sudden cardiac death be- A.K., K.S., W.S., G.W., J.L., V.T., I.K., T.S., A.M.D., J.T.H., and H.M.B. analyzed data; and tween the ages of 20 and 60 y (3). Given this dismal prognosis, A.C.Y.C., A.C.H.C., A.K., K.S., W.S., G.W., J.L., V.T., I.K., T.S., A.M.D., J.T.H., J.S., C.E.S., there is a great need for biomarkers and therapeutic targets J.W.D., E.A., J.C.W., and H.M.B. wrote the paper. common to HCM and DCM of diverse genetic etiologies. Reviewers: D.H., Whitehead Institute for Biomedical Research; and C.E.M., University of Here, we show that telomere length constitutes such a bio- Washington. marker. Telomeres are DNA sequences composed of (TTAGGG) Conflict of interest statement: The sponsor declares a conflict of interest. J.L. is a co- founder and consultant to Telomere Diagnostics. The company played no role in repeats that serve as protective caps at the ends of chromosomes. this research. Telomeres shorten at every cell division as a result of replication This open access article is distributed under Creative Commons Attribution-NonCommercial- insufficiency and are markers of cellular aging. In contrast to most NoDerivatives License 4.0 (CC BY-NC-ND). cell types, in healthy postnatal cardiomyocytes (CMs), telomeres 1To whom correspondence may be addressed. Email: [email protected] or hblau@ are largely maintained throughout life (4, 5), presumably because stanford.edu. postnatal CMs are relatively nonproliferative (6). Published online August 27, 2018. 9276–9281 | PNAS | September 11, 2018 | vol. 115 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1714538115 Downloaded by guest on September 26, 2021 Fig. 1. HCM and DCM CMs exhibit telomere shortening. (A) Paraffin-embedded cardiac samples were used for telomere Q-FISH quantification of CMs. (B) Patient CMs [cardiac Troponin-T (green) indicated by white arrowhead] were stained for telomere (red) and for nuclear DAPI (blue) in patient and control cardiac tissue sections. (Scale bars, 10 μm.) Telomere levels were scored in a blinded fashion (n = 40–220 nuclei per patient tissue) within five to six regions of interest in two nonconsecutive sections, and (C) telomere signal intensity per DAPI-stained nucleus is plotted as mean ± SEM (*P < 0.05 and ****P < 0.0001). in average telomere lengths relative to healthy controls. Notably, To investigate whether the telomere shortening seen in CMs in contrast to CMs, contractile vascular smooth muscle cells in tissues also occurs in a disease model of HCM and DCM (VSMCs) that do not express dystrophin had telomere lengths in vitro, we compared telomere lengths in human-induced plu- similar to control VSMCs in the mouse DMD model and Du- ripotent stem cells (hiPSCs) differentiated into CMs (hiPSC- chenne heart tissues, suggesting that the observed telomere CMs). We generated hiPSCs from HCM and DCM patients’ shortening was specific to CMs and caused by the stress of con- peripheral blood mononuclear cells using the four Yamanaka traction in the absence of the structural protein dystrophin (7, 8). factors (13) and used a sequential regimen of growth factors to In this report, we test the hypothesis that telomere shortening differentiate these cells into beating CMs (Fig. 3 A and B)by is a general hallmark of genetic HCM and DCM as a result of using well-established protocols (13, 14). Control hiPSC lines mutations in contractile proteins essential to cardiac function. were from a healthy individual (Con#1), healthy family members We measured telomeres in CMs from end-stage patient heart (Con#2, Con#3 for TNNT2#1–3; Con#5 for TTN#2), or a tissues at the time of heart transplantation and CMs derived CRISPR/Cas9-mediated corrected isogenic line (Con#4 for from patient-induced pluripotent stem cells. Patient mutations TTN#1; Table 1). Importantly, in all cases, we measured telo- included three genes associated with HCM [troponin I (TNNI3), meres in hiPSC-CMs at day 30 of differentiation.
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