Journal of the American College of Cardiology 2008 by the American College of Cardiology Foundation Published by Elsevier Inc.

Vol. 52, No. 6, 2008 ISSN 0735-1097/08/$34.00 doi:10.1016/j.jacc.2008.04.034

PRECLINICAL RESEARCH

Effects of Physical Exercise on Myocardial TelomereRegulating Proteins, Survival Pathways, and Apoptosis
Christian Werner, MD,* Milad Hanhoun, MD,* Thomas Widmann, MD, Andrey Kazakov, MD,* Alexander Semenov, MD,* Janine Pss, MD,* Johann Bauersachs, MD, Thomas Thum, MD, Michael Pfreundschuh, MD, Patrick Mller, MD,* Judith Haendeler, MD, Michael Bhm, MD,* Ulrich Laufs, MD* Homburg/Saar, Wrzburg, and Dsseldorf, Germany
Objectives Background Methods
The purpose of this study was to study the underlying molecular mechanisms of the protective cardiac effects of physical exercise. Telomere-regulating proteins affect cellular senescence, survival, and regeneration. C57/Bl6 wild-type, endothelial nitric oxide synthase (eNOS)decient and telomerase reverse transcriptase (TERT)decient mice were randomized to voluntary running or no running wheel conditions (n 8 to 12 per group). Short-term running (21 days) up-regulated cardiac telomerase activity to 2-fold of sedentary controls, increased protein expression of TERT and telomere repeat binding factor (TRF) 2, and reduced expression of the proapoptotic mediators cell-cyclecheckpoint kinase 2 (Chk2), p53, and p16. Myocardial and leukocyte telomere length did not differ between 3-week- and 6-month-old sedentary or running mice, but telomerase activity, TRF2 and TERT expression were persistently increased after 6 months and the expression of Chk2, p53, and p16 remained down-regulated. The exercise-induced changes were absent in both TERT / and eNOS / mice. Running increased cardiac expression of insulin-like growth factor (IGF)-1. Treatment with IGF-1 up-regulated myocardial telomerase activity 14-fold and increased the expression of phosphorylated Akt protein kinase and phosphorylated eNOS. To test the physiologic relevance of these exercise-mediated prosurvival pathways, apoptotic cardiomyopathy was induced by treatment with doxorubicin. Up-regulation of telomere-stabilizing proteins by physical exercise in mice reduced doxorubicin-induced p53 expression and potently prevented cardiomyocyte apoptosis in wild-type, but not in TERT / mice. Long- and short-term voluntary physical exercise up-regulates cardiac telomere-stabilizing proteins and thereby induces antisenescent and protective effects, for example, to prevent doxorubicin-induced cardiomyopathy. These benecial cardiac effects are mediated by TERT, eNOS, and IGF-1. (J Am Coll Cardiol 2008;52:47082) 2008 by the American College of Cardiology Foundation

Results

Conclusions

Regular physical activity is associated with a decrease of cardiovascular events (1,2). Physical training improves exercise capacity, endothelial function, and collateralization in patients with coronary artery disease and chronic heart failure (35). Physical activity is associated with improved blood pressure, insulin sensitivity, mood, body weight, and
From the *Klinik fr Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universittsklinikum des Saarlandes, Homburg/Saar, Germany; Klinik fr Innere Medizin I, Hmatologie, Onkologie und Rheumatologie Universittsklinikum des Saarlandes, Homburg, Germany; Medizinische Klinik I, Kardiologie, Universittsklinikum Wrzburg, Germany; and the Institut fr Umweltmedizinische Forschung at the Universitt Dsseldorf gGmbH, Dsseldorf, Germany. Supported by grants from the Deutsche Forschungsgemeinschaft (KFO 196) and the Universitt des Saarlandes (HOMFOR). Manuscript received February 7, 2008; revised manuscript received March 21, 2008, accepted April 14, 2008.

hemostatic and inammatory variables (6). However, despite the wealth of evidence derived from epidemiological and interventional trials, there is limited understanding of the underlying molecular mechanisms, especially in the heart (7). The prevalence, incidence, and morbidity of cardiac diseases increase with the aging human population. On the cellular level, senescence, chromosome stability, and cell viability are regulated by the telomeres and their associated proteins, deoxyribonucleic acid-protein complexes located at both ends of eukaryotic chromosomes (8). Shortening of the telomeres has been shown to be associated with increased mortality rate from heart disease (9). In men and in mice, a ribonuclear complex, called telomerase, is the central component of the telomere complex. The activity of telomerase

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decreases in aging mouse myocytes (10). The bestcharacterized function of the telomeric complex is to protect the chromosome ends from degradation. The enzyme telomerase contains a catalytic subunit, the telomerase reverse transcriptase (TERT). In isolated cells, TERT gene transfer reduces replicative senescence and extends the life span of numerous cell types, including cardiac myocytes (11). Important proteins that form the telomeric complex include the telomere repeat binding factors (TRFs) 1 and 2, which can directly bind to the TTAGGG repeats of telomeres. The TRF2 protein is crucial for maintaining a normal chromosomal end structure because it collaborates in the processes leading to T-loops (12). In addition, TRF2 interacts with other factors, serving as binding platforms for a number of additional telomere-associated proteins. The function of these protein complexes is not completely known but includes signaling to deoxyribonucleic acid damage checkpoint controls (13,14). In addition to the protection of chromosome ends, the components of the telomeric complex enhance cell survival. Suppression of telomerase enzyme activity promotes apoptosis, whereas overexpression of TERT prevents programmed death by interfering with a pre-mitochondrial step in the cell death cascade (11,15). Gene products implicated in growth arrest and apoptosis, such as p53, p16, and cell-cycle checkpoint kinase 2 (Chk2), are potential downstream effectors of the telomeric complex and increase with age in cardiac myocytes (10,13,14,16 19). Cardiomyocyte apoptosis has been shown to contribute signicantly to the pathogenesis of heart failure. In endstage human heart failure, myocyte apoptosis increases (20). Similarly, acute toxic cardiomyopathy, such as that induced by doxorubicin (a frequently used anticancer drug), is characterized by cellular senescence and apoptosis (21). However, our understanding of potential strategies to prevent cardiomyocyte senescence and apoptosis is limited (20). Here we identify and characterize voluntary exercise as a potent measure to increase telomere-stabilizing proteins in the myocardium and to prevent doxorubicin-induced cardiac myocyte apoptosis. Methods Animals and exercising. Animal experiments were approved by the animal ethics committee of the Universitt des Saarlandes, conformed with U.S. National Institutes of Healths Guide for the Use and Care of Laboratory Animals (NIH Pub. No. 85-23, revised 1996), and were conducted in accordance to institutional guidelines. Eight-week-old male C57/Bl6 (Charles River Laboratories, Wilmington, Massachusetts), endothelial nitric oxide synthase (eNOS) / (B6.129/P2-Nos3, Charles River) mice, TERT / (B6.129S-Terttm1Yjc/J, mutant generation 2, Jackson Laboratory, Bar Harbor, Maine) mice, and strain-matched controls to the mutants were kept under usual care at 1 to 6 mice per cage. Each exercising mouse was kept in an

individual cage supplied with a Abbreviations running wheel (12.8-cm diameand Acronyms ter) equipped with a tachometer BSA bovine serum (BC 500, Sigma Sport Europe, albumin Neustadt, Germany) recording Chk2 cell-cycle the daily running distance. Mice checkpoint kinase 2 ran voluntarily. The mean running eNOS endothelial nitric distance was 5,100 800 m/24 oxide synthase h and did not differ in mice after FISH uorescence in-situ hybridization doxorubicin treatment as well as in eNOS / and TERT / GAPDH glyceraldehyde-3phosphate dehydrogenase mice. Transthoracic echocardiography was performed using a GH growth hormone GE Systems Vivid 5 scanner HEK human embryonic kidney (GE Healthcare, Munich, Germany), 13-MHz broadband Ig immunoglobulin transducer; fractional shortening, IGF insulin-like growth factor end-diastolic thickness of interventricular septum and the left i.p. intraperitoneally posterior wall, as well as the left mRNA messenger ventricular end-diastolic diameribonucleic acid ter was taken. Indicated mice PBS phosphate buffered saline were treated with doxorubicin 22.5 mg/kg (Medac, Wedel, TERT telomerase reverse transcriptase Germany), administered intraTFU telomeric peritoneally (i.p.) for 24 h; with uorescence units growth hormone (GH) (SigmaTRF telomere repeat Aldrich, Munich, Germany), 2.5 binding factor g GH/g/day solved in 150 l Tris tris(hydroxymethyl) phosphate-buffered saline (PBS) aminomethane once per day i.p. for 7 days; with WT wild-type mouse insulin-like growth factor (IGF)-1 (Sigma-Aldrich), 1.5 g mouse IGF-1/g body weight solved in 150 l PBS for 3 times per day i.p. for 2 days; or with 150 l PBS as control as described (22). Telomere length analysis by ow-uorescence in-situ hybridization (FISH) assays. Telomere length was determined by the FISH method (23). In brief, 600,000 peripheral blood leukocytes were washed once (5% dextrose, 0.1% bovine serum albumin [BSA], 10 mmol/l 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) and resuspended in hybridization buffer (75% deionized formamide, 20 mmol/l tris(hydroxymethyl)aminomethane [Tris] [pH 7.1], 20 mmol/l NaCl, 1% BSA) for 10 min. After denaturing cells, 90 min incubation with either no probe (unstained control) or 0.18 g uorescein isothiocyanate (FITC) labeled, telomere-specic (C3TA2)3 deoxyribonucleic acid probe (Applied Biosystems, Langen, Germany) was performed. After 3 rounds of washing (75% deionized formamide, 0.1% BSA, 10 mmol/l Tris, 0.1% Tween 20), cells were resuspended in PBS, 0.1% BSA, RNAse A at 10 g/ml and 0.1 g/ml LDS 751 (Exciton, Munich, Germany) for deoxyribonucleic acid (DNA) counterstain. Flow cytometric analysis was carried out on a FACSCanto (Becton Dickinson, Heidelberg, Germany). Telomere length was

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expressed as mean uorescent signal intensity. To control for interday variation, FITC-labeled beads (Quantum 24, Caltag, Hamburg, Germany) with dened amounts of uorescence (molecules of equivalent soluble uorochrome) were run in parallel each day. Telomere length was converted into base pairs according to a previously established standard curve (23). Myocardial telomere length analysis by FISH. Telomere length is directly related to its integrated uorescence intensity when marked with a specic telomere peptide nucleic acid probe using quantitative FISH as described (24). We xed 5- m cryosections of the hearts in 4% paraformaldehyde for 30 min at room temperature followed by treatment with 0.1% Pepsine solution (Sigma-Aldrich) for 10 min at 37C. After washing and dehydration, 25 l of the hybridization mix (70% formamide, 0.13 g Cy3conjugated telomere-specic peptide nucleic acid probe (C2TA3)3 (Applied Biosystems), blocking reagent (Roche, Mannheim, Germany), MgCl2 buffer, and 1 mmol/l Tris (pH 7.2) was added to the sections. Denaturation was carried out at 80C for 3 min and preceded incubation for 2 h in a humid chamber and washing with wash buffer (70% formamide, 10 mmol/l Tris [pH 7.2], 0.1% BSA), Trisbuffered saline ( 1% Tween) and PBS for a total of 1 h. Nuclei were then counterstained with 4,=6-Diamidino-2phenylindoldihydrochloride (DAPI). All sections were analyzed using a Nikon E600 epiuorescence microscope (Nikon, Dsseldorf, Germany) and 1,000 magnication by a blinded investigator. At least 3 representative high power elds of the Cy3 and DAPI channel from each slide were recorded with Lucia G software, version 4.81 (Laboratory Imaging for Nikon, Prague, Czech Republic) with identical exposure time. Telomere uorescence intensity was calculated with TFL-Telo freeware version 2.2.07.0418 2002 (25,26). Telomerase activity. Telomerase activity was quantied using a telomerase repeat amplication protocol (23,27,28). Protein extracts (1 g) of mouse left ventricle, 0.1 g of primer TS (5=-ATCGCTTCTCGGCCTTTT-3=) (template), 0.05 g primer ACX (5=-GCGCGG [CTTACC]3CTAACC-3=) in 20 l LightCycler FastStart SYBR Green PCR Master Mix (Roche) containing 1.5 mmol/l MgCl2 were incubated at 30C for 30 min to allow template elongation by telomerase activity. After immediate transfer to the LightCycler instrument (Roche), telomerase activity was terminated and hot start DNA polymerase activated by incubation at 95C for 10 min. Forty cycles of amplication were carried out with 20 s at 95C, 30 s at 60C, and 50 s at 72C. Telomerase activity was displayed as relative telomerase activity to 1,000 human embryonic kidney (HEK 293) cells (Gibco, Karlsruhe, Germany). A standard titration curve of HEK 293 cells was established from 0 to 1,000 cells to ensure linearity of the assay (R2 0.99). A positive result of the telomerase assay was considered if the quantity of telomerase activity was 3 times above the standard deviation of the mean negative controls background level (heat-

inactivated lysate from 1,000 HEK 293 cells). A positive control (protein extracts from 1,000 HEK 293 cells) was run in every experiment. Immunouorescence analysis of Ki-67 in cardiomyocytes. Coimmunostaining for the proliferation marker Ki-67 (Novocastra, Leica Biosystems, Newcastle, United Kingdom) and alpha-sarcomeric actin (clone5c5, SigmaAldrich) was performed in 3- m thick myocardial sections as previously described (29). The percentage of Ki-67 expression was calculated from the total number of Ki-67 positive cardiomyocytes and the total number of cardiomyocytes per area. Western blot analysis. Left ventricular tissue was homogenized with 500 l lysis buffer (100 mmol/l Tris [pH 6.8], 4% sodium dodecyl sulfate [SDS], 20% glycerol) containing the protease inhibitor M phenylmethanesulfonyl uoride 0.1 mmol/l, leupeptin 0.5 l, and aprotinin 0.5 l. We separated 50 g of proteins on SDSpolyacrylamide gel electrophoresis 10%. Proteins were transferred to nitrocellulose membrane (162-0112, Bio-Rad Laboratories, Hercules, California) blocked with 5% dry milk or blocking solution for Western blot (Roche) and exposed to rabbit polyclonal immunoglobin G (IgG) TRF2 (H-300: sc-9143, Santa Cruz Biotechnology, Santa Cruz, California; dilution 1:200), mouse monoclonal IgG p16 (F-12: sc-1661, Santa Cruz Biotechnology; dilution 1:250), rabbit polyclonal IgG anti-p53 (FL-393: sc-6243, Santa Cruz Biotechnology; dilution 1:500 in dry milk 1%), mouse monoclonal IgG Chk2 (A-11: sc-17747, Santa Cruz Biotechnology; dilution 1:1,000 in dry milk 1%), rabbit polyclonal IgG anti-p-Akt (Ser473) (#9271, Cell Signaling Technology, Danvers, Massachusetts; dilution 1:1,500), mouse IgG anti-p-eNOS (pS1177) (#612392, Becton Dickinson; dilution 1:1,000), and mouse monoclonal IgG glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (6C5: sc-32233, Santa Cruz Biotechnology; dilution 1:1,000). For analysis of TERT, immunoprecipitation was performed using polyclonal IgG anti-TERT (H-231: sc-7212, Santa Cruz Biotechnology) and agarose-A protein goat antirabbit IgG (Sigma-Aldrich). Immunodetection was accomplished using goat anti-rabbit IgG (Sigma-Aldrich) and goat anti-mouse (170-6516, Bio-Rad) secondary antibodies (1:4,000 dilution), and an enhanced chemiluminescence kit (Amersham Biosciences) (30). Reverse transcriptionpolymerase chain reaction. Reverse transcriptionpolymerase chain reaction standardized to GAPDH was performed using the following primers: TRF1-for 5=-CATGGACTACACAGACTTAC-3= TRF1-rev 5=-ATCTGGCCTATCCTTAGACG-3= 55C, 27 cycles TRF2-for 5=- TGTCTGTCGCGCATTGAAGA-3= TRF2-rev 5=-GCTGGAAGACCTCATAGGAA-3= 55C, 26 cycles Ku70-for 5=-GAGCATCCAGTGTATCCAGA-3= Ku70-rev 5=-CAGCATGATCCTCTTGTGAC-3= 55C, 26 cycles

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Ku80-for 5=-TCACAGTGTGCAGACACCTG-3= Ku80-rev 5=-AACTGCAGAGAGATGCCAGA-3= 56C, 26 cycles IGF-1-for 5=-CTTCACATCCTCTCTACCT-3= IGF-1-rev 5=-ATTCTGTAGGTCTTGTTTCC-3= 54C, 27 cycles P16-for 5=-ACGGTGCAGATTCGAACTGC-3= P16-rev 5=-TACACAAAGACCACCCAGCG-3= 53C, 40 cycles P53-for 5=-GGGACAGCCAACTCTGTTATG TGC-3= P53-rev 5=-CTGTCTTCCAGATACTCGGGA TAC-3= 62C, 30 cycles Chk2-for 5=-GCTGTCCTCTGAGTAACAAC-3= Chk2-rev 5=-GAAGTAGAGCTTACAGGTGG-3= 53C, 40 cycles GAPDH-for 5=-ACCACAGTCCATGCCATCAC-3= GADPH-rev 5=-TCCACCACCCTGTTGCTGTA-3= 60C, 27 cycles Quantication of apoptosis by hairpin oligonucleotide assay. To detect apoptosis, 3- m thick parafn sections of formalin-xed mice heart tissue were examined using the ApopTag Peroxidase In Situ Oligo Ligation Kit (Chemicon International, Millipore, Billerica, Massachusetts) (31). The in situ oligo ligation assay specically detects apoptosis by staining only cells that contain double-stranded breaks that are blunt-ended or have a 1 base 3= overhang (cells containing nicked, gapped, 3=-recessed, 3=-overhanging ends longer than 1 base and single-stranded ends are not detected). Unlike conventional terminal transferase-based labeling (terminal deoxynucleotidyl transferase mediated 2=-deoxyuridine 5=triphosphate nick end labeling [TUNEL]), the assay stains apoptotic but not necrotic or transiently damaged cells (31). To distinguish between cardiomyocytes and other cardiac cells, eosin staining was performed after the in situ oligo ligation assay. Statistical analysis. Band intensities were analyzed by densitometry. All values are expressed as mean standard error of the mean. Unpaired Student t tests and analysis of variance for multiple comparisons were applied. Post-hoc comparisons were performed with the Bonferroni adjustment test. The Mann-Whitney U test was used for the analysis of the Ki-67 positive cardiomyocytes because the data were not normally distributed. SPSS software, version 12.0 (SPSS Inc., Chicago, Illinois), was used. Differences were considered signicant at p 0.05. Results Voluntary physical exercise increases telomere-stabilizing proteins. The mean voluntary running distance was 5,100 800 m/24 h. Voluntary exercise for 21 days, n 8 to 12 per group, did not change heart or body weight or the ratio of heart weight to tibia length (Fig. 1A). Echocardiography of mouse hearts showed no change of fractional shortening, end-diastolic and end-systolic thickness of the interventric-

ular septum and the left posterior wall, as well as the left ventricular diameters. Flow-FISH assays showed equal mean length of telomeres compared with sedentary controls (not shown). At the same time, telomerase repeat amplication protocol assays revealed that 3 weeks of exercising up-regulated cardiac telomerase activity to 230.7 21%, p 0.01 (Fig. 1B). Exercising increased the expression of TERT in the heart to 165 4%, p 0.05 (Fig. 1C). Next, the effects of voluntary running on cardiac T-loop stabilizing proteins were assessed. Three weeks of running had no effect on the messenger ribonucleic acid (mRNA) expression of TRF1, but up-regulated TRF2 mRNA to 145 12% and TRF2 protein expression to 168.7 8.4%, p 0.05. Exercise up-regulated mRNA expression of the 80kDa subunit but not the 70-kDa subunit of the repair protein Ku (Figs. 1D to 1F). Exercise decreases markers of cellular aging in the heart. Compared with sedentary controls, the left ventricles of the mice that were supplied with a running wheel for 21 days were characterized by a decreased expression of the aging marker protein p16 (52.6 3.3% of control, p 0.01). Similarly, expression of Chk2, which mediates cellcycle arrest and apoptosis, was reduced to 78.7 2.7%, p 0.05. The cardiac expression of the proapoptotic transcription factor p53 was reduced by one-half (56.4 2.7%, p 0.01). Data are shown in Figure 2. Reverse transcription polymerase chain reaction analysis showed that p16 and Chk2 mRNA expression were signicantly down-regulated (53.3 7.2% of control, p 0.01 and 75.7 10.8% of control, p 0.01, respectively), but p53 mRNA levels were not altered after running (101 12% of control). Effects of long-term voluntary exercise on the telomere complex and cellular aging. To test if the regulation observed after 21 days was a transient effect, mice were equipped with individual running wheels or no running wheel for the duration of 6 months (n 8 per group). As expected, long-term running induced a mild myocardial hypertrophy that did not impair left ventricular fractional shortening (Fig. 3A). Compared with the 21-day training, the long-term training exerted qualitatively and quantitatively similar effects and signicantly increased the activity of telomerase and the expression TRF2. The expression of the markers of reduced cellular life span p16, Chk2, and p53was markedly inhibited by 29 3%, 34.8 5%, and 43 4.3%, respectively (p 0.05) (data depicted in Fig. 3). The experiments show that exercising exerts a continuous and persistent effect on regulators of cellular survival. Flow-FISH assays detected no shortening of telomeres in leukocytes from 6-month-old mice compared with 3-weekold mice (Fig. 4A). Consequently, mean telomere length did not differ between mice running for 6 months (21.6 kb) and sedentary animals (21.4 kb). A group of 18-month-old C57/Bl6 animals was studied as a positive control that showed reduction of telomere length (17.5 kb in 18-month-

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Figure 1

Voluntary Physical Exercise Increases Telomere-Stabilizing Proteins

Effects of voluntary exercise on running wheels on C57/Bl6 mice for 21 days compared with sedentary controls on (A) the ratio of heart weight to tibia length, (B) cardiac telomerase activity determined by telomerase repeat amplication protocol, (C) expression of the telomerase reverse transcriptase (TERT), (D) the messenger ribonucleic acid (mRNA) expression of telomere repeat binding factors (TRFs) 1 and 2, (E) TRF2 protein expression, and (F) the mRNA expression 80 kDa subunit but not the 70 kDa subunit of the repair protein Ku. Standardized for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *p 0.05, **p 0.01, n 8 to 12 per group.

old animals compared with 21.8 kb in 3-week-old animals, p 0.001). Telomere length in the hearts of corresponding left ventricular myocardial sections was directly examined by FISH using a Cy3-conjugated telomere-specic PNA probe (Figs. 4B and 4C). There was no signicant difference in the myocardial telomere length between mice after 3 weeks and 6 months of exercise or sedentary condition (3 weeks sedentary: 4,495.0 159.7 individual telomeric uorescence units [TFU]; 6 months sedentary: 5,172.0 361.1 TFU; and 6 months running: 4,766.0 468.3 TFU). The

18-month-old mice exhibited a reduced telomere length (2,687.3 146.7 TFU, p 0.001 vs. all other groups). Inuence of exercise on Ki-67 expression in cardiomyocytes. Coexpression of alpha-sarcomeric actin and Ki-67 was not detected in myocardial sections (n 8) of control mice. However, there was a marked increase to 0.023 0.01% Ki-67 positive cardiomyocytes in the hearts of mice after 3 weeks of running (p 0.02) (Fig. 4D). Effect of voluntary exercise in TERT / mice. To test if the effects of physical training on the expression of Chk2, p16, and p53 are coincidence or mediated by the telomere

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Figure 2

Exercise Decreases Markers of Cellular Aging in the Heart

Representative Western blot analysis and quantication of the effects of voluntary exercise for 21 days on the left ventricular expression of (A) the senescence marker protein p16, (B) the cell-cycle checkpoint kinase 2 (Chk2), and (C) the proapoptotic transcription factor p53. *p 0.05, **p 0.01, n 8 to 12 per group. Abbreviation as in Figure 1.

complex, second-generation TERT / mice and their strain-matched wild-type (WT) were subjected to 21 days of voluntary running. Wild-types exhibited a signicant up-regulation of TRF2 protein expression to 466 121% and a down-regulation of p16, p53, and Chk2 expression to 68 8.7%, 50.6 9.0%, and 56.4 10.6%, respectively, n 8 per group, p 0.05. However, these exercise-induced changes were absent in the TERT / mice (Fig. 5).

IGF-1 and eNOS mediate the effects of exercising on survival proteins. Physical exercise is associated with increased serum concentrations of IGF-1 (32). IGF-1 is decreased in elderly patients and has been shown to enhance telomerase delaying cellular aging and death (10). The effect of exercising on cardiac IGF-1 is not known. Voluntary running induced up-regulation of myocardial IGF-1 expression to 169.7 13% (Fig. 6A). To provide evidence for a potential role of IGF-1 as mediator of exercise-induced cardiac telomerase activity, mice were treated with IGF-1 and GH, which increases endogenous IGF-1 levels (22). Treatment with GH led to an 8-fold increase of telomerase activity and IGF-1 up-regulated telomerase activity by 14-fold, p 0.001 (Fig. 6B). The IGF-1 treatment of animals resulted in 2-fold increased expression of p-Akt (p 0.01) and p-eNOS (p 0.05) in the myocardium (Figs. 6C to 6E). Running is known to up-regulate the expression and function of eNOS, which has been conrmed in our model of voluntary running at several time points (3335). To test a potential role of eNOS as a mediator of the observed effects, the experiments were repeated in eNOS / mice as a head-tohead comparison with strain-matched WT animals (n 8 per group, 21 days). The running distance was not different between groups. The results are summarized in Figures 6F to 6J. Sedentary eNOS / mice showed a decrease of TRF2 expression to 70.4 2.4% and an increase of p16 expression to 130 5.1% compared with WT sedentary mice (p 0.05); telomerase activity and p53 expression were not signicantly altered. The eNOS-decient micein contrast to the WT mice did not show that exercise modied the cardiac telomerase, TRF2, p16, p53, or Chk2. Exercise prevents doxorubicin-induced cardiac apoptosis. Doxorubicin is a commonly used antineoplastic drug with frequent cardiotoxicity. The acute doxorubicin-induced cardiomyopathy is characterized by cellular senescence and apoptosis (21). To test the functional relevance of the observed exerciseinduced up-regulation of telomere stabilizing and downregulation of proapoptotic genes, we exposed C57/Bl6 and TERT / mice with and without running wheels to a single injection with 22.5 mg/kg i.p. of doxorubicin (n 8 per group). We measured TRF2 expression 24 h after injection and found that it was moderately decreased and telomerase activity was not changed in sedentary control mice (Figs. 7A and 7B). Voluntary running was able to increase TRF2 as well as telomerase activity in the presence of doxorubicin. Doxorubicin-induced p53 expression in WT mice by 2-fold to 213 7%, p 0.01. The effect was signicantly blunted in mice that had been running voluntarily for 21 days. Exercise prevented the doxorubicininduced down-regulation of TRF2 and markedly reduced up-regulation of p53 (163 25% of control, p 0.05 vs. doxorubicin group) (Figs. 7C and 7D). Apoptosis was quantitated by hairpin oligonucleotide assays. Interestingly, 21 days of voluntary exercising decreased apoptotic cardiomyocytes by 5-fold (0.03 0.01 vs.

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Figure 3

Effects of Long-Term Voluntary Exercise on the Telomere Complex and Cellular Aging

Effects of long-term voluntary exercise (6 months) on (A) the ratio of heart weight to tibia length, (B) cardiac telomerase activity, (C) expression of TRF2, (D) p16, (E) Chk2, and (F) p53. *p 0.05, **p 0.01, n 8 to 12 per group. Abbreviations as in Figures 1 and 2.

0.17 0.03%, p 0.01). As expected, doxorubicin increased cardiomyocyte apoptosis in the inactive animals (0.29 0.08%, p 0.05). However, mice supplied with running wheels were completely protected from doxorubicin-induced programmed cell death of cardiomyocytes (Fig. 7D). Cardiomyocyte apoptosis rate of sedentary versus running TERT / B6.129S mice was in the same range of the corresponding C57/Bl6 strain as discussed previously (0.51 0.21% and 0.21 0.03%, p 0.03 for control vs. control doxorubicin). In contrast, sedentary TERT / mice of the same strain showed an increased susceptibility to doxorubicin-induced apoptosis (0.73 0.14%, p 0.05 vs. WT doxorubicin control) (Fig. 7F). In these mice, exercise was not able to render a signicant protection (p NS for TERT / doxorubicin control vs.

runner; p 0.001 vs. WT doxorubicin runner) demonstrating the importance of TERT for the exercise-mediated effects on apoptosis. Discussion The study identies a novel effect of physical exercise on the heart. Compared with mice kept under the regular conditions of laboratory animals, voluntary running exercise up-regulated telomere-stabilizing proteins, reduced cellular senescence, and prevented doxorubicin-induced apoptotic cell death. Further characterization shows that the effect depends on TERT expression and is mediated by IGF-1 and eNOS. The effect was observed after 3 weeks of voluntary running and persisted for at least 6 months.

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(7). Here we observe that exercising increases the activity of the telomerase, the reverse transcriptase responsible for the extension of telomeric repeat sequences, as well as the expression of its catalytic subunit, TERT. Both telomerase and TERT have been shown to regulate cardiac muscle cell growth and survival (10,11,16,37), and telomerase ribonucleic acid knockout mice (Terc / ) develop cardiac dysfunction, increased expression of p53, and increased apoptosis (16). Overexpression of TERT causes hypertrophy in cultured cardiac myocytes and protects from apoptosis in vitro and in vivo (11). In addition to the effects on telomerase and TERT, exercising mice showed up-regulation of TRF2 mRNA and protein expression. Telomere repeat binding

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Figure 4

Telomere Length in Sedentary and Running Mice

**

(A) Telomere length of blood leukocytes in 3-week (wk)-, 6-month (mo)-, and 18-month-old sedentary mice and in mice with running wheels exercising for 6 months (run) determined by Flow-uorescence in-situ hybridization (FISH) assays displayed in base pairs (bp) and expressed as box plots, indicating the median as horizontal lines and boxes as 25th and 75th percentiles as well as whiskers as 10th and 90th percentiles. ***p 0.001 versus 3 wk and 6 mo, n 8 to 12 per group. (B) Effects of 6 months of running wheel exercise compared with 3-week-, 6-month, and 18-month-old sedentary condition on murine cardiomyocyte telomere length as determined by quantitative (Q) FISH. Results are presented in mean telomere uorescence units per high-power eld as box plots. ***p 0.001 versus every other condition (n 4 per condition, with each n consisting of 2 myocardial sections and 3 high-power elds captured from each section). (C) Exemplary images of murine cardiomyocyte telomeres (QFISH, red dots) and the corresponding nuclei (4,=6-Diamidino-2-phenylindoldihydrochloride [DAPI], blue) for 3-week- and 18-month-old sedentary condition, 40 magnication. (D) Quantication (n 8) and representative uorescence microscopic image of Ki-67positive nuclei (red) in cardiomyocytes identied by alpha-sarcomeric actin coimmunostaining (green) after 3 weeks of voluntary running. Nuclei are stained blue by DAPI. 100 magnication. *p 0.05. LV left ventricle.

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Figure 5

No Effect of Voluntary Exercise in TERT

Mice

Physical activity is associated with cardiovascular protection and longevity (13,5,6,8,9,36). However, the underlying molecular mechanisms remain only partially understood

Representative Western blot analysis and quantication of the effects of voluntary exercise for 21 days on the left ventricular expression of (A) TRF2, (B) p16, (C) Chk2, and (D) p53. *p 0.05, **p 0.01, n 6 to 8 per group. Contr control; WT wild-type; other abbreviations as in Figures 1 and 2.

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Telomerase activity [% control]

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Figure 6

IGF-1 and eNOS Mediate the Effects of Exercising on Survival Proteins

(A) Effects of 21 days voluntary running on cardiac expression of insulin-like growth factor (IGF) mRNA (n 8). (B) Regulation of telomerase activity by treatment with growth hormone (GH) (2.5 g/g once per day for 7 days) and mouse IGF-1 (1.5 g/g 3 times per day for 2 days) (n 4). (C) Representative Western blots and (D) quantication of phosphorylated Akt (pAkt) and (E) phosphorylated endothelial nitric oxide synthase (peNOS) (n 4 per group). Effects of 21 days of running in B6.129S WT and eNOS-decient (eNOS / ) mice compared with sedentary controls on (F) cardiac telomerase activity, (G) expression of TRF2, (H) p16, (I) Chk2, and (J) p53 (n 8 per group). Standardization for GAPDH. *p 0.05, **p 0.01, ***p 0.001. Abbreviations as in Figures 1, 2, and 5.

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A
TRF2 [% control]

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p53 GAPDH 100 50 0 Contr Run Doxo Doxo Contr Run

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1 0.8 0.6 0.4 0.2 0 WT WT Doxo Doxo Contr Run

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TERT-/-TERT-/Doxo Doxo Contr Run

Figure 7

Exercise Prevents Doxorubicin-Induced Cardiac Apoptosis

Effects of doxorubicin (Doxo) (22.5 mg/kg intraperitoneally for 24 h) in sedentary mice and in mice supplied with running wheels for 21 days (n 10 per group) on (A) protein expression of TRF2 (*p 0.05 and **p 0.01 vs. vehicle-treated sedentary control), (B) telomerase activity as determined by telomerase repeat amplication protocol assays, and (C, D) p53 protein expression (quantication and representative Western blots). (E) Quantication of cardiomyocyte apoptosis in C57/Bl6 and (F) in B6.129S TERT / and TERT / mice by hairpin oligonucleotide assays. *p 0.05, **p 0.01, ***p 0.001 versus vehicle-treated sedentary control, p 0.05 versus Doxo-treated sedentary control. Abbreviations as in Figures 1 and 5.

factor 2 can bind to the TTAGGG repeats of telomeres and contributes to the formation of the chromosome-protecting T-loops (12). Importantly, TRF2 serves as the binding platform for additional telomere-associated proteins and mediates signaling to DNA damage checkpoint controls (13,14). Interestingly, cardiac apoptosis in human heart failure was associated specically with defective expression of TRF2 and activation of the DNA damage checkpoint kinase, Chk2 (17). In addition, exogenous TRF2 was shown to confer protection from oxidative stress (17). In circulating progenitor cells, TRF2 was identied as a regulator of clonogenic potential and migratory capacity that can be modied by pharmacological treatment (38,39). Here, vol-

untary exercise resulted in up-regulation of telomerase, TERT, and TRF2 after only 3 weeks and the regulation persisted for at least 6 months. Therefore, physical exercise represents a potent regulator of the telomere complex. Relatively little is known about the physiologic development of telomere length in healthy untreated WT C57/Bl6 mice over time (8,14,40). After 6 months, we observed no shortening of telomere length, and telomere length did not differ between running and sedentary animals both in blood leucocytes as well as in the myocardium. A control group of 18-month-old C57/Bl6 mice exhibited signicantly shorter telomeres. This nding may be interpreted in several ways. Six months may be too short a time, and signicantly longer

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observation, such as 18 months, may be necessary to potentially observe a protective effect of exercising on telomere length. On the other hand, it is possible that the enhanced telomerase activity may counteract a putative telomerase-independent negative effect of exercising. However, the interesting aspect of these data is that the regulation of the telomere-regulating proteins by exercisemediated survival signaling seems to be independent of telomere length. Indeed, recent evidence suggests a direct telomerase-dependent transcriptional regulation of genes involved in cell growth, which has been recently suggested as an additional mechanism by which telomerase promotes cell proliferation. The regulation of telomerase activity and subsequent replicative potential is reported to occur rapidly and independently of telomere length in several cell types (8,22,41). Defects in mice lacking the ribonucleic acid component of telomerase involve apoptosis, not just proliferation defects, and telomerase directly protects cells against programmed cell death (8,16,42,43). Similarly, TRF2 was suggested to mediate proapoptotic signaling in post-mitotic, noncycling cardiomyocytes (17) and was shown to signal independently of telomere length in endothelial progenitor cells (39). It therefore seems likely that the telomeric complex serves as a regulator of cellular aging and function beyond and potentially independently of protecting telomere length. Several gene products implicated in senescence and cell death, such as transformation-related protein p53, p16, and Chk2, have been identied downstream of the telomeric complex (10,13,14,16 19). The tumor suppressor protein p53 has been shown to mediate telomere dysfunction (16,19). Protein p53 modulates apoptosis and senescence by increasing the expression of specic proteins, including Bax, Bad, and p21. Importantly, p53 was recently identied as a key mediator of maladaptive cardiac remodeling essential for the transition from cardiac hypertrophy to heart failure (18). Here, the data identify voluntary running as a novel and potent inhibitor of cardiac Chk2, p16, and p53. To test whether the exercise-induced regulation of the telomere complex and the regulation of p53 represent a coincidence or may be causally related, the experiments were repeated in TERT-decient mice. Running induced very similar regulation of the transformation-related proteins in TERT / WT and C57/Bl6 mice, but the exercisemediated effects were absent in the TERT / mice. These data support the concept that the regulation of the telomeric complex by exercising mediates the downstream effects on apoptosis. Physical exercise is associated with increased serum concentrations of IGF-1 (32,44). IGF-1 enhances telomerase, delaying cellular aging and death (10,22). Cardiac overexpression of IGF-1 in transgenic mice increases the heart weight (45,46). In IGF-1 transgenic mice, cardiac stem cell division is increased, which is accompanied by enhanced telomerase activity and delayed senescence (10). Similarly, age-dependent impairment of endothelial progenitor cells is

corrected by GH-mediated increase of IGF-1 (22). IGF-1 and its ligand may have the potential to regulate the re-entry of adult ventricular myocytes into the cell cycle (46,47). In contrast to the systemic effects of exercising on IGF-1, the effect of exercise on local cardiac IGF-1 is not known. Here, the data show that voluntary running increases the expression of IGF-1 in the heart. To test the role of IGF-1 in exercise-induced telomerase regulation, mice were treated with recombinant GH and IGF-1, which resulted, respectively, in powerful 8- and 14-fold increases of telomerase activity. The effects of IGF-1 on telomerase have been shown to be associated with the regulation of endothelial nitric oxide and phosphatidylinositol 3-kinaseAkt-p70S6K signaling, a pathway that plays an important role in regulating cardiac hypertrophy, viability, and homeostasis (22,48). In line with these data, treatment with recombinant IGF-1 led to signicant increases in myocardial expression of p-Akt and p-eNOS. Indeed, one of the best characterized molecular effects of exercising in the model of voluntary running is the up-regulation of eNOS (33,35). Therefore, the experiments were repeated in eNOS / mice showing that the effects of exercising on telomere-regulating proteins and subsequent survival signaling are mediated via eNOS. Taken together, the data identify IGF-1, Akt, and eNOS as important mediators of the exercise-induced up-regulation of telomerase activity. Apoptosis has been implicated in both acute and chronic heart diseases causing progressive loss of cardiac myocytes. End-stage human heart failure is characterized by increased myocyte apoptosis (20). Similarly, several forms of acute toxic cardiomyopathy are characterized by cellular senescence and apoptosis; however, our understanding of potential strategies to prevent cardiomyocyte senescence and apoptosis is limited (20). Doxorubicin is a potent, widely used antineoplastic agent in cancer chemotherapy. However, its clinical application is compromised by its dose-dependent cardiotoxicity mediated by cardiomyocyte apoptosis (21). Here, we applied the well-characterized model of acute doxorubicin-induced cardiac apoptosis to test if the observed effects of exercising on telomere-regulating proteins and survival markers are able to confer protection from cardiomyocytes apoptosis. After acute exposure to doxorubicin, mice showed a small reduction of TRF2 expression. However, in mice that had access to a running wheel during the 3 weeks prior to doxorubicin exposure, TRF2 was signicantly up-regulated. Similarly, the doxorubicininduced increase of cardiac p53, a key mediator of cardiac cell death (16,18,19), was markedly blunted in mice of the running group. Cardiomyocyte apoptosis was assessed by the hairpin oligonucleotide assay that specically detects apoptosis by selectively staining cells that contain doublestranded DNA breaks that are blunt-ended or have a 1 base 3= overhang whereas cells containing nicked, gapped, 3=recessed, 3=-overhanging ends longer than 1 base, and single-stranded ends are not detected. Unlike conventional terminal transferase-based labeling, the assay stains apopto-

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tic but not necrotic or transiently damaged cells (31). The hairpin oligonucleotide experiments revealed that providing mice with a running wheel to allow voluntary running for the relatively short period of 3 weeks represents a powerful intervention to protect from doxorubicin-induced cardiomyocyte apoptosis. Somewhat unexpectedly, voluntary running decreased the basal rate of cardiomyocyte apoptosis compared with sedentary animals. Ascenso et al. (49) observed prevention of doxorubicin-induced increase in Bax, Bax-to-Bcl-2 ratio, and tissue caspase-3 activity by a 14-week training protocol in rats but no effect of exercise on the basal rate of these markers in left ventricular homogenates. Chicco et al. (50) show a decrease of caspase-3 activity in the rats left ventricles induced by exercising both in the presence and in the absence of doxorubicin; however, the latter effect did not reach statistical signicance. None of these studies has directly assessed cardiomyocyte specic apoptosis; in addition, there were signicant differences in the protocols, species, and modalities of training. In agreement with previous studies, long-term voluntary running (e.g., 6 months) is associated with increased heart weight, an adaptive change without impairment of cardiac function. In the light of these data, it is interesting to speculate that the opportunity to exercise resembles the natural habitat of mice more closely than cages without running wheels. Consequently, the inactivity of regular laboratory mice could be considered the experimental intervention in this study. The provocative hypothesis would be that the observed cardiac morphology in voluntary exercise is normal and that sedentary animals exhibit an adaptive cardiac hypotrophy. The notion of an anti-aging effect of exercising on the heart is supported by earlier reports in the literature (51) that survival rates decrease in sedentary as opposed to exercising rodents. The effects of physical activity and inactivity are not limited to the regulation of telomere-regulating proteins. Improved cardiac antioxidant capacity and benecial effects on both circulating progenitor cells and cardiac resident stem cells are likely to contribute to the molecular and cellular actions of exercise (7,10,22,35,52). In agreement with this hypothesis, Ki-67positive cardiomyocytes were detected in the hearts of running but not in sedentary mice. In our opinion, future research is needed to further characterize both the quantitative and the cell-type specic contribution of the exercise-induced mechanisms with the aim to develop more specic therapeutic interventions. Specically, the effects of exercise on telomere biology in cardiac stem cells may be of functional signicance (10). A signicant proportion of elderly heart failure patients show no other confounding variables suggesting that age as such may be a primary cause of cardiac decompensation and diastolic dysfunction (53,54). Shortening of the telomeres has recently been shown to predict cardiac morbidity and mortality, and telomere length of circulating leukocytes is decreased in patients with chronic heart failure (9,14,17,55).

In addition, recent evidence suggests that telomere biology represents an indicator for the effect of a pharmacologic intervention (56). Here, we identify physical exercise as a potent antisenescent intervention to up-regulate telomerestabilizing proteins and to reduce cardiomyocyte apoptosis, improving the molecular understanding of the benecial cardiovascular effects of exercise. Furthermore, the data set the stage to prospectively investigate these novel cardioprotective effects in specic clinical situations, for example, for the prevention of doxorubicin-induced cardiomyopathy.
Acknowledgments

The authors thank Sascha Jakob, Ellen Becker, and Simone Jger for excellent technical assistance.
Reprint requests and correspondence: Dr. Ulrich Laufs, Klinik fr Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universittsklinikum des Saarlandes, 66424 Homburg/Saar, Germany. E-mail: ulrich@laufs.com.
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50. 51. 52. 53. 54. 55. 56.

Key Words: exercise y myocardium y aging y prevention y telomere.