Telomere shortening is a hallmark of genetic cardiomyopathies

Abstract

This study demonstrates that significantly shortened telomeres are a hallmark of cardiomyocytes (CMs) from individuals with end-stage hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) as a result of heritable defects in cardiac proteins critical to contractile function. Positioned at the ends of chromosomes, telomeres are DNA repeats that serve as protective caps that shorten with each cell division, a marker of aging. CMs are a known exception in which telomeres remain relatively stable throughout life in healthy individuals. We found that, relative to healthy controls, telomeres are significantly shorter in CMs of genetic HCM and DCM patient tissues harboring pathogenic mutations: TNNI3, MYBPC3, MYH7, DMD, TNNT2, and TTN Quantitative FISH (Q-FISH) of single cells revealed that telomeres were significantly reduced by 26% in HCM and 40% in DCM patient CMs in fixed tissue sections compared with CMs from age- and sex-matched healthy controls. In the cardiac tissues of the same patients, telomere shortening was not evident in vascular smooth muscle cells that do not express or require the contractile proteins, an important control. Telomere shortening was recapitulated in DCM and HCM CMs differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs) measured by two independent assays. This study reveals telomere shortening as a hallmark of genetic HCM and DCM and demonstrates that this shortening can be modeled in vitro by using the hiPSC platform, enabling drug discovery.

Keywords: dilated cardiomyopathy; hiPSC-CM; hypertrophy cardiomyopathy; telomere.

Conflict of interest statement

Conflict of interest statement: The sponsor declares a conflict of interest. J.L. is a cofounder and consultant to Telomere Diagnostics. The company played no role in this research.

Copyright © 2018 the Author(s). Published by PNAS.

Figures

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).

Fig. 2.

HCM and DCM VSMCs do not exhibit telomere shortening. (A) Paraffin-embedded cardiac samples were used for telomere Q-FISH quantification of VSMCs. (B) Patient VSMCs [smooth muscle actin (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 = 30–150 nuclei per patient tissue) within three to four regions of interest in two nonconsecutive sections, and (C) telomere signal intensity per DAPI-stained nucleus is plotted as mean ± SEM.

Fig. 3.

HCM and DCM hiPSC-derived CMs recapitulate telomere shortening. (A) Patient PBMCs were used to generate patient-specific hiPSC lines and used to differentiate into hiPSC-CMs. (B) Cardiomyopathic patient and control day 30 hiPSC-CMs were used to assess telomere levels. Representative hiPSC-CMs (cardiac Troponin-T; green) were stained for telomeres (red) and nuclear DAPI (blue). (Scale bars, 10 μm.) (C) Telomere signal intensity per DAPI-stained nucleus is shown for HCM, DCM, and control hiPSCs (day 0) and hiPSC-CMs (day 30). hiPSC differentiation was performed in two independent experiments per line from the same passage cells, and telomere levels were scored in a blinded fashion (n = 40–330 nuclei per hiPSC or hiPSC-CM). (D) FACS gating for TMRM sorting. (E) TMRM+ population enriched for CM markers whereas TMRM− population enriched for non-CM markers by RT-qPCR (TMRM+, n = 5; TMRM−, n = 3). (F) Telomere T/S ratio nucleus is shown for HCM (n = 6), DCM (n = 8), and control (n = 8) TMRM+ hiPSC-CMs (day 30). All data plotted as mean ± SEM (*P < 0.05, ***P < 0.001, and ****P < 0.0001).

Fig. 4.

HCM and DCM hiPSC-derived VSMCs do not exhibit telomere shortening. (A) Patient PBMCs were used to generate patient-specific hiPSC lines and used to differentiate into hiPSC-VSMCs. (B) Cardiomyopathic patient and control day 14 hiPSC-VSMCs were used to assess telomere signals. Representative hiPSC-VSMCs (smooth muscle actin; green) were stained for telomere signal (red) and nuclear DAPI (blue). (Scale bars, 10 μm.) (C) Telomere signal intensity per DAPI-stained nucleus is shown for HCM, DCM, and control hiPSC-VSMCs. hiPSC-VSMC differentiation was performed in two independent experiments from the same passage cells, and telomere TFUs were scored in a blinded fashion (n = 36–49 nuclei per group) and plotted as mean ± SEM.