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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2003 Apr 17;100(9):5378–5383. doi: 10.1073/pnas.0836098100

Telomere attrition and Chk2 activation in human heart failure

Hidemasa Oh *,†, Sam C Wang *,†, Arun Prahash *,†, Motoaki Sano *,†, Christine S Moravec ‡, George E Taffet †,§, Lloyd H Michael †,§, Keith A Youker †,§, Mark L Entman †,§, Michael D Schneider *,†,¶,‖,**
PMCID: PMC154353  PMID: 12702777

Abstract

The “postmitotic” phenotype in adult cardiac muscle exhibits similarities to replicative senescence more generally and constitutes a barrier to effective restorative growth in heart disease. Telomere dysfunction is implicated in senescence and apoptotic signaling but its potential role in heart disorders is unknown. Here, we report that cardiac apoptosis in human heart failure is associated specifically with defective expression of the telomere repeat- binding factor TRF2, telomere shortening, and activation of the DNA damage checkpoint kinase, Chk2. In cultured cardiomyocytes, interference with either TRF2 function or expression triggered telomere erosion and apoptosis, indicating that cell death can occur via this pathway even in postmitotic, noncycling cells; conversely, exogenous TRF2 conferred protection from oxidative stress. In vivo, mechanical stress was sufficient to down-regulate TRF2, shorten telomeres, and activate Chk2 in mouse myocardium, and transgenic expression of telomerase reverse transcriptase conferred protection from all three responses. Together, these data suggest that apoptosis in chronic heart failure is mediated in part by telomere dysfunction and suggest an essential role for TRF2 even in postmitotic cells.

The emerging concept of heart failure as a myocyte-deficiency disease is predicated on the limited regenerative capacity of mammalian cardiac muscle, which is inadequate to maintain pump function after cell death (1–4). Conceptually, approaches to augment cardiac myocyte number include cell grafting (5), driving nonmuscle cells to a cardiac “fate” (6), potentiating repair by endogenous stem cells (7), and alleviating apoptosis (8). A rational approach to such interventions encompasses identifying endogenous molecules that contribute to cell survival in the heart (9–12).

Telomere maintenance is one mechanism through which cell viability is preserved (13–21). Telomeres consist of tandem T2AG3 repeats at chromosome ends, maintained by telomerase reverse transcriptase (TERT), and bound by specific telomere repeat-binding factors (TRFs) including TRF1 and TRF2 (17, 20, 22, 23). We have shown that TERT and telomerase activity are down-regulated in adult mouse myocardium [unlike some other adult tissues in the mouse (24)], and that forced expression of TERT in transgenic mice can delay the timing of the cell cycle exit of cardiac myocytes (3). At later ages, continued expression of TERT at the level found in embryonic hearts had two other effects with possible therapeutic significance. First, TERT induced myocyte enlargement (hypertrophic growth), after the cessation of cycling. Second, TERT suppressed cardiac myocyte apoptosis both in vitro (serum starvation) and in vivo (ischemia-reperfusion injury).

In end-stage human heart failure, myocyte apoptosis increases typically to an incidence of 0.5–1% (25) but surprisingly little is known of the instigating or signal transducing events for cell death in this remarkably common disorder. We demonstrate here that failing human hearts have telomeres that are 25% shorter on average than age-matched controls, with decreased expression of TRF2 and marked activation of the DNA damage kinase, checkpoint kinase 2 (Chk2). Suppressing TRF2 function in cultured cardiac myocytes provoked telomere erosion, Chk2 kinase activation, and apoptosis, and an antisense “knockdown” of TRF2 did the same. Conversely, exogenous TRF2 conferred protection from oxidative stress. In mouse myocardium, biomechanical stress (partial aortic constriction) reduced telomere length within 1 week, provoked the loss of TRF2, and triggered Chk2 activation, all as seen in failing human hearts. Forced expression of telomerase prevented telomere erosion, down-regulation of TRF2, activation of Chk2, and myocyte apoptosis. Together, the results suggest a role for telomere dysfunction in heart failure via stress-induced down-regulation of TRF2.

Materials and Methods

Patient Samples and Controls.

Human myocardium was obtained through the Methodist DeBakey Heart Center and the Human Heart Tissue Transplant Core of the Cleveland Clinic. Tissue procurement was based on patient-informed consents and approved by the respective institutional review boards. Heart failure tissue (idiopathic and ischemic dilated cardiomyopathy) was obtained from explanted hearts at the time of therapeutic transplantation. Normal hearts were obtained from unmatched organ donors and victims of motor vehicle accidents. Hypertrophic obstructive cardiomyopathy (HOCM), a heterogenous primary disorder of heart growth without ventricular pump failure, was also used for comparison.

Cell Culture and Viral Gene Transfer.

Ventricular myocytes from 2-d-old Sprague–Dawley rats were purified and cultured (3, 26). By this age, ventricular myocytes become refractory to serum-induced G1 exit, after initial serum starvation in vitro (26). Plasmids for human TRF1, TRF2, and the corresponding dominant-negative truncations (TRF1ΔM and TRF2ΔBΔM) were provided by T. de Lange (The Rockefeller University, New York; ref. 17). Adenoviruses coexpressing enhanced GFP were generated by using pAdTrack-cytomegalovirus and pShuttle-cytomegalovirus (provided by B. Volgelstein, Johns Hopkins Oncology Center, Baltimore; refs. 3 and 27). Myocytes were infected at a multiplicity of infection of 20. To visualize TRF1/2 after gene transfer, myocytes were fixed in 70% ethanol then incubated sequentially with tetramethyl rhodamine isothiocyanate-conjugated MF-20 Ab to sarcomeric myosin-heavy chains (MHCs) to confirm cell type (University of Iowa Hybridoma Bank, Iowa City), rabbit Abs to TRF1 and TRF2 (1:500; nos. 581420 and 581425, Calbiochem), and FITC-conjugated goat Ab to rabbit IgG (1:1,000, Sigma). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured with a Zeiss Axioplan 2 epifluorescence microscope.

Antisense Oligonucleotides.

Three antisense phosphorothioate oligonucleotides for mouse TRF2 were generated (Molecula Research Laboratories, Herndon, VA), one of which inhibited endogenous TRF2 expression effectively in NIH 3T3 cells (data not shown). The sequences used were antisense TRF2 (asTRF2), 5′-CCTGGGCTGCCGGCTCGAGC-3′; sense TRF2 (sTRF2), 5′-CGAGCTCGGCCGTCGGGTCC-3′; and antisense GFP (28), 5′-CGTTTACGTCGCCGTCCAGC-3′. Oligonucleotides were transfected into 1- to 2-d-old C57BL/6 mouse cardiomyocytes, cultured as above, with Oligofectamine (Invitrogen).

Animal Models.

Cardiac-specific TERT transgenic mice (αMHC-TERT; ref. 3) and wild-type littermates (10–12 weeks old and 18–22 g) were subjected for 1 week to partial occlusion of the transverse aorta (2). The control “sham” operation comprised anesthesia, thoracotomy, and ligature placement without constriction. The presence and severity of obstruction were corroborated by Doppler flow studies; only mice in which severe load was confirmed (a right to left carotid artery velocity ratio >3.5) were analyzed further. Doppler echocardiography and staining with Sirius red were performed 7 d after surgery (3).

Apoptosis.

For myocardium, terminal transferase-mediated dUTP-biotin nick end-labeling assays were performed with the Oncor ApopTaq Direct In Situ Apoptosis Detection kit (2), MF20 Ab to sarcomeric MHC, and Texas red-conjugated Ab to mouse IgG. For cultured cardiomyocytes, hypodiploid DNA was detected with two-color flow cytometry by using propidium iodide for DNA content and FITC-conjugated MF20 (3, 26).

Telomere Length.

DNA was digested with RsaI, resolved by electrophoresis in 0.5% agarose, transferred to Hybond-N+ membranes (Amersham Biosciences), and hybridized with a 32P-labeled (TTAGGG)4 telomeric probe (3, 29). Mean telomere length was ascertained by PhosphorImager scanning (Molecular Dynamics).

Telomerase Expression and Activity.

Telomerase activity was measured by a PCR-based telomerase repeat amplification protocol assay and 1 μg of cell or tissue extract (3). TERT, the RNA component of telomerase, and GAPDH were analyzed by RT-PCR in the log-linear range of amplification (30–32).

Western Blot and Immune Complex Kinase Assays.

Proteins were resolved by electrophoresis in 10% SDS-polyacrylamide gels and transferred to membranes by electroblotting. Abs were human and mouse TRF2 (Calbiochem), human and mouse TRF1 (Calbiochem), phospho-Chk2 (Thr-68; Cell Signaling Technology, Beverly, MA), sarcomeric α-actin and myc (Sigma), FLAG epitope (M2, Kodak), GFP (CLONTECH), Chk2 (Santa Cruz Biotechnology), and poly (ADP-ribose) polymerase (PARP, Oncogene). To detect exogenous TRF2 in virus-infected cardiomyocytes, goat and rabbit Abs to TRF2 were used (C-16, H-300; Santa Cruz Biotechnology), and endogenous rat TRF2 was detected by using rabbit Ab to TRF2 (Alpha Diagnostic International, San Antonio, TX). After blocking with 5% nonfat milk plus 0.1% Tween 20, blots were incubated with primary Abs (1:500), horseradish peroxidase-conjugated secondary Abs (1:3,000; Amersham Biosciences), and enhanced chemiluminescence reagents (Amersham Biosciences).

To assay Chk2 activity, samples were lysed in 20 mM Tris⋅HCl (pH 8.0), 0.1% Triton X-100, 10 mM NaF, 1 mM NaVa3VO4, 10 μg/ml of aprotinin, and 1 mM PMSF and then incubated for 1 h with Ab to Chk2 and protein A/G-Sepharose (Amersham Biosciences). Immunoprecipitates were washed and assayed in the presence of 30 μM CHKtide substrate peptide (KKKVSRSGLYRSPSMPENLNRPR; Upstate Biotechnology, Lake Placid, NY), 40 μM adenosine triphosphate, and 15 μCi [γ-32P]ATP for 30 min at 30°C (1 Ci = 37 GBq). Proteins were resolved by electrophoresis in SDS-polyacrylamide gels and visualized by autoradiography. Aliquots of Chk2 immunoprecipitates were also used for Western blotting, allowing activity and content to be compared in the same samples.

Statistical Analysis.

Data, reported as the mean ± SE, were analyzed by ANOVA and Scheffé's test using a significance level of P ≤ 0.05.

Results

Telomere Attrition, Loss of TRF2, and Checkpoint Kinase Activation in Human Heart Failure.

To address the expression and function of telomeric proteins in human heart disease, we analyzed cardiac muscle from patients with end-stage heart failure at the time of transplantation, HOCM undergoing therapeutic partial resection of the septum, and normal myocardium. The prevalence of apoptosis (Fig. 1A) increased markedly in heart failure (0.70 ± 0.04% by terminal transferase-mediated dUTP-biotin nick end-labeling assay; normal <0.005%, P = 0.0001; HOCM 0.04 ± 0.001%, P = 0.0001; n = 8 for each group), comparable to recent reports (25). We next examined telomere length, telomerase activity, and TRF1/2 expression using heart samples well matched for age and sex. Mean telomere length (Fig. 1B Left) was reduced 25% in failing hearts (6.5 ± 0.2 kb), compared with normal samples (7.8 ± 0.2 kb, P = 0.0001) or patients with HOCM (7.7 ± 0.1 kb, P = 0.0001). Although the RNA component of telomerase was present in all three groups without significant difference, neither telomerase activity nor TERT expression was detected, in any of the three groups, by using a telomeric repeat amplification protocol (data not shown) and RT-PCR for 30 cycles, respectively (Fig. 1B Right). The paucity of telomerase activity in adult human myocardium concurs with our prior findings in mice (3) and suggests a mechanism other than defective telomerase activity for the loss of telomere length in failing hearts.

Figure 1.

Figure 1

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Telomere dysfunction in human heart failure. (A) Cardiomyocyte apoptosis, shown by terminal transferase-mediated dUTP-biotin nick end-labeling and sarcomeric MHC staining, was comparable to the incidence in recent reports (25). *, P = 0.0001. (Bar = 10 μm.) (B) Cardiac telomere erosion. (Left) Southern blot using a telomere-specific probe. (Center) Telomere length as a function of age. *, P = 0.0001. (Right) Telomere erosion occurred without overt change in cardiac TERT or RNA component of telomerase (TERC) mRNA levels. (C) Loss of cardiac TRF2 protein in heart failure, shown by Western blot. *, P = 0.0001. (D) Activation of Chk2 (Thr-68 phosphorylation) in heart failure. *, P = 0.002. (Lower) Patient no. 6 illustrates the one counterexample without Chk2 activation despite decreased TRF2.

To test one alternative mechanism for telomere dysfunction (17, 33), TRF1 and TRF2 were examined (Fig. 1C). Both proteins were readily detected in normal adult human myocardium, with no change in HOCM. By contrast, in patients with heart failure, TRF2 was down-regulated 50 ± 8% (P = 0.0001; range 25–75%). Interference with endogenous TRF2 activates apoptosis via the ataxia-telangiectasia mutated (ATM) protein kinase (17), and partial loss of TRF2 is the earliest event in some forms of telomere shortening (33). Consistent with this reported pathway, phosphorylation of Chk2 at Thr-68, the principal site for activation by ATM (34), was apparent in 12 of 14 failing hearts but in none of the normal controls or patients with HOCM (Fig. 1D and data not shown). Chk2 levels were unaffected.

Interference with Endogenous TRF2 Triggers Telomere Dysfunction and Apoptosis in Postmitotic Cardiomyocytes.

To ascertain whether the inferred pathway from TRF2 to Chk2 is operative in postmitotic cardiomyocytes (which might differ from cycling cells), we expressed epitope-tagged dominant-negative and wild-type TRF2 and TRF1 in primary culture using adenoviral vectors (Fig. 2A). At the stage tested, cardiomyocytes are already growth-arrested in vivo and refractory to mitogenic serum (3, 26). All four constructs were expressed uniformly. Staining was most intense in the nuclei, with a heterogenous intranuclear distribution similar to that of endogenous TRF1/2 (Fig. 2A). Myc-tagged dominant-negative TRF2 induced telomere erosion (Fig. 2B), accompanied by Chk2 activation (Fig. 2C), PARP cleavage (indicative of caspase-3 activity; Fig. 2E), and apoptosis (Fig. 2D). Myc-tagged wild-type TRF2, FLAG-tagged wild-type TRF1, and FLAG-tagged dominant-negative TRF1 had no effect (Fig. 2 B–E).

Figure 2.

Figure 2

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Dominant-negative TRF2 triggers telomere dysfunction and apoptosis in cardiomyocytes. (A) Viral vectors. (Upper Left) TRF1 and TRF2 tagged with FLAG and myc epitopes, respectively (17). Dominant-negative TRF1 (TRF1ΔM) lacks the Myb telomere-binding domain; dominant-negative TRF2 (TRF2ΔBΔM) lacks the Myb domain and N-terminal basic domain (17). (Lower Left) Western blots confirming expression of the exogenous proteins in cardiomyocytes. dnTRF2 is detected with Ab H-300 (against amino acids 49–300) but not Ab C-16 (against the C terminus). (Right) Immunocytochemistry for the exogenous proteins in cardiomyocytes. TRF1/2, FITC; MF20, tetramethyl rhodamine isothiocyanate; nuclei, 4′,6-diamidino-2-phenylindole (DAPI). (Bar = 5 μm.) (B) Telomere shortening, shown by Southern blot. *, P = 0.002. (C) Activation of Chk2, shown by immune complex kinase assays. (D) Apoptosis, shown as hypodiploid DNA by flow cytometry. n = 7; *, P = 0.0001. (E) PARP cleavage, shown by Western blotting.

Because dominant-negative mutations are not formally equivalent to reduced expression, we confirmed the above findings by using an antisense oligonucleotide for TRF2 vs. the sense strand TRF2 control and an irrelevant antisense oligonucleotide against GFP. In cardiomyocytes, TRF2 and GFP were specifically reduced by the respective antisense oligonucleotides (Fig. 3A). Reduction of endogenous TRF2 provoked the same responses as did the dominant inhibitor: telomere shortening, Chk2 activation, PARP cleavage, and apoptosis (Fig. 3 B–E). Thus, interference with TRF2 causes apoptosis and activation of Chk2 even in postmitotic, noncycling cells.

Figure 3.

Figure 3

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Down-regulation of endogenous TRF2 in cardiomyocytes by antisense (as) oligonucleotide or oxidative stress. (A–E) Mouse cardiomyocytes were transfected as indicated for 48 h. (A) Reduction of TRF2 specifically by asTRF2 (Western blot). Adenoviral delivery of GFP was used for all myocytes (Upper). (B) Chk2 activation (immune complex kinase assay). (C) Telomere shortening (Southern blot). (D) Cardiomyocyte apoptosis (flow cytometry). n ≥ 5; P = 0.0001. (E) PARP cleavage (Western blot). (F–I) Rat cardiomyocytes infected with the viruses shown were treated 48 h later with 100 μM H2O2 for 8 h. (F) Western blot showing rapid down-regulation of TRF2 by H2O2. Telomere shortening (G), PARP cleavage (H), and apoptosis (I) were each induced by H2O2 and rescued by viral delivery of TRF2 or TERT. n ≥ 6; P < 0.02.

TRF2 and TERT Protect Cardiomyocytes from Pathophysiological Stress.

Endogenous TRF2 in cardiomyocytes decreased within 2 h of oxidative stress (100 μM H2O2; Fig. 3F). Compared with a viral control expressing GFP alone, either TRF2 or TERT rescued the adverse effect of H2O2 on telomere length, PARP cleavage, and apoptosis (Fig. 3 G–I), consistent with earlier evidence for cardioprotection by TERT (3). Dominant-negative TRF2 markedly potentiated the effect of H2O2 on apoptosis (Fig. 3I) but not on telomere length (Fig. 3G); thus, telomere attrition does not simply reflect the extent of apoptosis.

Mechanical load activates signaling cascades including oxidative stress (35), predisposes cardiac muscle to late-onset apoptosis (36), and can trigger apoptosis acutely, especially in susceptible backgrounds (9, 11). To test whether mechanical load might induce telomere dysfunction in myocardium, adult mice were subjected to severe aortic constriction. By comparison to littermate controls undergoing the control procedure, telomere length was reduced 3 kbp by increased load for 7 d (n = 4; P ≤ 0.01; Fig. 4A). Under the conditions tested, mechanical load also triggered down-regulation of TRF2 by 52 ± 2% (P ≤ 0.001; Fig. 4B), induced Chk2 kinase activity (P = 0.002; Fig. 4C), and induced apoptosis (0.32 ± 0.06%; P = 0.0003; Fig. 4D).

Figure 4.

Figure 4

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TERT protects adult mouse myocardium from telomere shortening, apoptosis, fibrosis, and systolic dysfunction after biomechanical stress. αMHC-TERT mice and nontransgenic littermates (ntg) were analyzed 7 d after severe aortic constriction. Telomere length (A), TRF2 levels (B), and Chk2 kinase activation (C) were measured as in Fig. 2. *, P ≤ 0.01 vs. ntg without banding; †, P = 0.0001 vs. ntg with banding. (D Upper) Representative terminal transferase-mediated dUTP-biotin nick end-labeling and Sirius red staining, in banded mice. (D Lower) Mean results ± SE are shown for apoptosis (Left), fibrosis (Center), and peak aortic ejection velocity by Doppler echocardiography (Right). *, P = 0.0001. (Bar = 20 μm.)

In culture, TERT largely prevented the loss of endogenous TRF2 provoked by oxidative stress (Fig. 3H). Forced expression of TERT in adult myocardium maintains telomere length and confers protection from apoptosis after ischemia-reperfusion injury (3). Hence, we tested whether TERT might attenuate or rescue telomere dysfunction induced by severe mechanical load. As reported previously (3), telomere length was 21.5 ± 0.5 kbp in the αMHC-TERT mice and 3 kbp longer than the length in wild-type littermates (n = 4; P ≤ 0.01; Fig. 4A). By contrast to the sequelae of biomechanical stress in wild-type animals, αMHC-TERT mice were refractory to telomere erosion (Fig. 4A), loss of TRF2 (Fig. 4B), Chk2 kinase activation (Fig. 4C), and apoptosis (Fig. 4D). Consistent with the inhibition of cardiomyocyte death, αMHC-TERT mice had less replacement fibrosis after banding and better preservation of left ventricular ejection velocity, a measure of systolic function (Fig. 4D).

Discussion

In summary, telomere shortening and down-regulation of the telomere end-capping protein, TRF2, occur in end-stage human heart failure. Neither telomere erosion nor loss of TRF2 was observed in normal hearts at the ages tested, and neither occurred in hypertrophy if failure was absent. Thus, telomere dysfunction in myocardium is not, at least overtly, a feature of normal aging, as in proliferating cells, and is not a mere result of hypertrophic growth. The fraction of actively cycling myocytes in adult myocardium is minuscule at best (1, 4), and the prevalence of apoptosis in failing hearts was six to seven per thousand. Hence, the 25% loss of telomere length here can neither be explained by the “end-replication” problem nor by generalized DNA fragmentation (37). In cultured cardiac myocytes, interference with endogenous TRF2 triggered rapid telomere shortening, activation of Chk2, and apoptosis, without cell cycle reentry. Importantly, antisense reduction of endogenous TRF2 had comparable adverse effects, and exogenous TRF2 was protective against oxidative stress, at least in culture. In adult mouse myocardium, severe mechanical load for just 1 week down-regulated endogenous TRF2, shortened telomeres, and activated Chk2. Forced expression of TERT, at levels normal for the embryonic heart, rescued all three responses. Our results suggest the likelihood of active telomere erosion in human heart failure, possibly contingent on the down-regulation of TRF2 in diseased myocardium, and implicate oxidative stress as one signal for this loss. Interestingly, telomere loss is likewise associated with heart failure in fifth-generation mice lacking the RNA component of telomerase (38).

Preservation of TRF2 levels and suppression of Chk2 activity by TERT, after mechanical stress, together provide further genetic evidence for the potential use of telomerase in cardiac protection and repair (3). Telomere structure, not length per se, is the prerequisite for normal telomere function (23), and a decrease in TRF2 is thought to be the rate-limiting step for some forms of apoptosis (33). TRF2 is essential to form the lariat-like loop at chromosome ends that protects the 3′ single-stranded overhang from degradation (23, 39). One pathway that might be expected to promote telomere shortening in this context is the unopposed action of TRF1 and TRF1-binding proteins. Also, TRF2 recruits a number of proteins to the telomere including Ku (the regulatory component of DNA-dependent protein kinase), Nijmegen breakage syndrome 1 (a component of the Mre11 DNA repair complex), and the Werner syndrome helicase, which all are required for telomere maintenance (40, 41). Predisposition to apoptosis is a known consequence of defects in each of these proteins, whose importance in heart failure is untested.

Acknowledgments

We thank the physicians of the Methodist Hospital DeBakey Heart Center, Cleveland Clinic Heart Transplant Teams, and Life Banc of Northeast Ohio; L. Shirley, J. Pocius, A. Aldape, and C. Scott for technical assistance; and V. Lundblad and S. Elledge for helpful suggestions. This work was supported by National Institutes of Health grants (to M.D.S., L.H.M., and M.L.E.), the Howard Hughes Medical Institute Research Training Fellowship for Medical Students (to S.C.W.), and the M. D. Anderson Foundation Professorship (to M.D.S.).

Abbreviations

MHC

myosin heavy chain

Chk2

checkpoint kinase 2

HOCM

hypertrophic obstructive cardiomyopathy

PARP

poly (ADP-ribose) polymerase

TERT

telomerase reverse transcriptase

TRF

telomere repeat-binding factor

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

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