Telomere attrition and Chk2 activation in human heart failure

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.

Figure 1

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.

Figure 2

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 (TRF1^ΔM) lacks the Myb telomere-binding domain; dominant-negative TRF2 (TRF2^ΔBΔM) 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.

Figure 3

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.

Figure 4

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.)