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8/4/2019 "Effects of Physical Exercise on Myocardial TelomereRegulating Proteins, Survival Pathways, and Apoptosis". C Wer
1/13
PRECLINICAL RESEARCH
Effects of Physical Exercise on Myocardial Telomere-
Regulating Proteins, Survival Pathways, and ApoptosisChristian 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 The purpose of this study was to study the underlying molecular mechanisms of the protective cardiac effects of
physical exercise.
Background Telomere-regulating proteins affect cellular senescence, survival, and regeneration.
Methods C57/Bl6 wild-type, endothelial nitric oxide synthase (eNOS)deficient and telomerase reverse transcriptase
(TERT)deficient mice were randomized to voluntary running or no running wheel conditions (n 8 to 12 per
group).
Results Short-term running (21 days) up-regulated cardiac telomerase activity to 2-fold of sedentary controls, in-
creased 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 re-
mained down-regulated. The exercise-induced changes were absent in both TERT/ and eNOS/ mice. Run-
ning increased cardiac expression of insulin-like growth factor (IGF)-1. Treatment with IGF-1 up-regulated myo-
cardial 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.
Conclusions 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 beneficial 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
Regular physical activity is associated with a decrease of
cardiovascular events (1,2). Physical training improves ex-ercise capacity, endothelial function, and collateralization inpatients with coronary artery disease and chronic heartfailure (35). Physical activity is associated with improvedblood pressure, insulin sensitivity, mood, body weight, and
hemostatic and inflammatory variables (6). However, de-
spite the wealth of evidence derived from epidemiologicaland interventional trials, there is limited understanding ofthe underlying molecular mechanisms, especially in theheart (7).
The prevalence, incidence, and morbidity of cardiacdiseases increase with the aging human population. On thecellular level, senescence, chromosome stability, and cell
viability are regulated by the telomeres and their associatedproteins, deoxyribonucleic acid-protein complexes located atboth ends of eukaryotic chromosomes (8). Shortening of thetelomeres has been shown to be associated with increasedmortality rate from heart disease (9). In men and in mice, a
ribonuclear complex, called telomerase, is the central com-ponent of the telomere complex. The activity of telomerase
From the *Klinik fr Innere Medizin III, Kardiologie, Angiologie und InternistischeIntensivmedizin, Universittsklinikum des Saarlandes, Homburg/Saar, Germany;Klinik fr Innere Medizin I, Hmatologie, Onkologie und Rheumatologie Univer-sittsklinikum des Saarlandes, Homburg, Germany; Medizinische Klinik I, Kardi-ologie, Universittsklinikum Wrzburg, Germany; and the Institut fr Umwelt-medizinische 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.
Journal of the American College of Cardiology Vol. 52, No. 6, 2008 2008 by the American College of Cardiology Foundation ISSN 0735-1097/08/$34.00Published by Elsevier Inc. doi:10.1016/j.jacc.2008.04.034
8/4/2019 "Effects of Physical Exercise on Myocardial TelomereRegulating Proteins, Survival Pathways, and Apoptosis". C Wer
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decreases in aging mouse myocytes (10). The best-characterized function of the telomeric complex is to protectthe chromosome ends from degradation. The enzyme te-lomerase contains a catalytic subunit, the telomerase reversetranscriptase (TERT). In isolated cells, TERT gene transferreduces replicative senescence and extends the life span of
numerous cell types, including cardiac myocytes (11). Im-portant proteins that form the telomeric complex includethe telomere repeat binding factors (TRFs) 1 and 2, whichcan directly bind to the TTAGGG repeats of telomeres.
The TRF2 protein is crucial for maintaining a normalchromosomal end structure because it collaborates in theprocesses leading to T-loops (12). In addition, TRF2interacts with other factors, serving as binding platforms fora number of additional telomere-associated proteins. Thefunction of these protein complexes is not completelyknown but includes signaling to deoxyribonucleic aciddamage checkpoint controls (13,14). In addition to the
protection of chromosome ends, the components of thetelomeric complex enhance cell survival. Suppression oftelomerase enzyme activity promotes apoptosis, whereasoverexpression of TERT prevents programmed death byinterfering with a pre-mitochondrial step in the cell deathcascade (11,15). Gene products implicated in growth arrestand apoptosis, such as p53, p16, and cell-cyclecheckpointkinase 2 (Chk2), are potential downstream effectors of thetelomeric complex and increase with age in cardiac myocytes(10,13,14,1619).
Cardiomyocyte apoptosis has been shown to contributesignificantly to the pathogenesis of heart failure. In end-
stage human heart failure, myocyte apoptosis increases (20).Similarly, acute toxic cardiomyopathy, such as that inducedby doxorubicin (a frequently used anticancer drug), ischaracterized by cellular senescence and apoptosis (21).However, our understanding of potential strategies to pre-
vent cardiomyocyte senescence and apoptosis is limited (20).Here we identify and characterize voluntary exercise as apotent measure to increase telomere-stabilizing proteins inthe myocardium and to prevent doxorubicin-induced car-diac myocyte apoptosis.
Methods
Animals and exercising. Animal experiments were ap-proved by the animal ethics committee of the Universittdes Saarlandes, conformed with U.S. National Institutes ofHealths Guide for the Use and Care of Laboratory Animals(NIH Pub. No. 85-23, revised 1996), and were conductedin accordance to institutional guidelines. Eight-week-oldmale 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 Lab-oratory, Bar Harbor, Maine) mice, and strain-matched
controls to the mutants were kept under usual care at 1 to 6mice per cage. Each exercising mouse was kept in an
individual cage supplied with arunning wheel (12.8-cm diame-ter) equipped with a tachometer(BC 500, Sigma Sport Europe,Neustadt, Germany) recordingthe daily running distance. Mice
ran voluntarily. The mean runningdistance was 5,100 800 m/24h and did not differ in mice afterdoxorubicin treatment as well asin eNOS/ and TERT/
mice. Transthoracic echocardi-ography was performed using aGE Systems Vivid 5 scanner(GE Healthcare, Munich, Ger-many), 13-MHz broadbandtransducer; fractional shortening,end-diastolic thickness of inter-
ventricular septum and the leftposterior wall, as well as the left
ventricular end-diastolic diame-ter was taken. Indicated mice
were treated with doxorubicin22.5 mg/kg (Medac, Wedel,Germany), administered intra-peritoneally (i.p.) for 24 h; withgrowth hormone (GH) (Sigma-Aldrich, Munich, Germany), 2.5g GH/g/day solved in 150 lphosphate-buffered saline (PBS)
once per day i.p. for 7 days; withmouse insulin-like growth factor(IGF)-1 (Sigma-Aldrich), 1.5g mouse IGF-1/g body weight solved in 150 l PBS for 3times per day i.p. for 2 days; or with 150 l PBS as controlas described (22). Telomere length analysis by flow-fluorescence in-situhybridization (FISH) assays. Telomere length was deter-mined by the FISH method (23). In brief, 600,000 pe-ripheral blood leukocytes were washed once (5% dextrose,0.1% bovine serum albumin [BSA], 10 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and resus-
pended 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 denaturingcells, 90 min incubation with either no probe (unstainedcontrol) or 0.18 g fluorescein isothiocyanate (FITC)labeled, telomere-specific (C3TA2)3 deoxyribonucleic acidprobe (Applied Biosystems, Langen, Germany) was per-formed. After 3 rounds of washing (75% deionized form-amide, 0.1% BSA, 10 mmol/l Tris, 0.1% Tween 20), cells
were resuspended in PBS, 0.1% BSA, RNAse A at 10g/ml and 0.1 g/ml LDS 751 (Exciton, Munich, Germany)for deoxyribonucleic acid (DNA) counterstain. Flow cyto-
metric analysis was carried out on a FACSCanto (BectonDickinson, Heidelberg, Germany). Telomere length was
Abbreviations
and Acronyms
BSA bovine serum
albumin
Chk2 cell-cycle
checkpoint kinase 2
eNOS endothelial nitric
oxide synthase
FISH fluorescence in-situ
hybridization
GAPDH glyceraldehyde-3-
phosphate dehydrogenase
GH growth hormone
HEK human embryonic
kidney
Ig immunoglobulin
IGF insulin-like growth
factor
i.p. intraperitoneally
mRNA messenger
ribonucleic acid
PBS phosphate buffered
saline
TERT telomerase reverse
transcriptase
TFU telomeric
fluorescence units
TRF telomere repeat
binding factor
Tris tris(hydroxymethyl)
aminomethane
WT wild-type
471JACC Vol. 52, No. 6, 2008 Werner et al.
August 5, 2008:47082 Exercise Regulates Cardiac Telomere Biology
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expressed as mean fluorescent signal intensity. To controlfor interday variation, FITC-labeled beads (Quantum 24,Caltag, Hamburg, Germany) with defined amounts offluorescence (molecules of equivalent soluble fluorochrome)
were run in parallel each day. Telomere length was con-verted into base pairs according to a previously established
standard curve (23).Myocardial telomere length analysis by FISH. Telomerelength is directly related to its integrated fluorescenceintensity when marked with a specific telomere peptidenucleic acid probe using quantitative FISH as described(24). We fixed 5-m cryosections of the hearts in 4%paraformaldehyde for 30 min at room temperature followedby treatment with 0.1% Pepsine solution (Sigma-Aldrich)for 10 min at 37C. After washing and dehydration, 25 lof the hybridization mix (70% formamide, 0.13 g Cy3-conjugated telomere-specific 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 wascarried out at 80C for 3 min and preceded incubation for2 h in a humid chamber and washing with wash buffer (70%formamide, 10 mmol/l Tris [pH 7.2], 0.1% BSA), Tris-buffered saline (1% Tween) and PBS for a total of 1 h.Nuclei were then counterstained with 4,=6-Diamidino-2-phenylindoldihydrochloride (DAPI). All sections were an-alyzed using a Nikon E600 epifluorescence microscope(Nikon, Dsseldorf, Germany) and 1,000 magnificationby a blinded investigator. At least 3 representative highpower fields of the Cy3 and DAPI channel from each slide
were recorded with Lucia G software, version 4.81 (Labo-ratory Imaging for Nikon, Prague, Czech Republic) withidentical exposure time. Telomere fluorescence intensity wascalculated with TFL-Telo freeware version 2.2.07.04182002 (25,26).Telomerase activity. Telomerase activity was quantified us-ing a telomerase repeat amplification protocol (23,27,28).Protein extracts (1g) of mouse left ventricle, 0.1g of primer
TS (5=-ATCGCTTCTCGGCCTTTT-3=) (template), 0.05g primer ACX (5=-GCGCGG [CTTACC]3CTAACC-3=)in 20 l LightCycler FastStart SYBR Green PCR MasterMix (Roche) containing 1.5 mmol/l MgCl2 were incubated
at 30C for 30 min to allow template elongation bytelomerase activity. After immediate transfer to the LightCyclerinstrument (Roche), telomerase activity was terminated andhot start DNA polymerase activated by incubation at 95Cfor 10 min. Forty cycles of amplification were carried out
with 20 s at 95C, 30 s at 60C, and 50 s at 72C. Telomerase activity was displayed as relative telomeraseactivity to 1,000 human embryonic kidney (HEK 293) cells(Gibco, Karlsruhe, Germany). A standard titration curve ofHEK 293 cells was established from 0 to 1,000 cells toensure linearity of the assay (R2 0.99). A positive result ofthe telomerase assay was considered if the quantity of
telomerase activity was 3 times above the standard deviationof the mean negative controls background level (heat-
inactivated lysate from 1,000 HEK 293 cells). A positivecontrol (protein extracts from 1,000 HEK 293 cells) was runin every experiment.Immunofluorescence analysis of Ki-67 in cardiomyo-cytes. Coimmunostaining for the proliferation markerKi-67 (Novocastra, Leica Biosystems, Newcastle, United
Kingdom) and alpha-sarcomeric actin (clone5c5, Sigma-Aldrich) was performed in 3-m thick myocardial sectionsas previously described (29). The percentage of Ki-67expression was calculated from the total number of Ki-67positive cardiomyocytes and the total number of cardiomy-ocytes per area.Western blot analysis. Left ventricular tissue was homog-enized with 500 l lysis buffer (100 mmol/l Tris [pH 6.8],4% sodium dodecyl sulfate [SDS], 20% glycerol) containingthe protease inhibitor M phenylmethanesulfonyl fluoride0.1 mmol/l, leupeptin 0.5 l, and aprotinin 0.5 l. Weseparated 50 g of proteins on SDSpolyacrylamide gel
electrophoresis 10%. Proteins were transferred to nitrocel-lulose membrane (162-0112, Bio-Rad Laboratories, Her-cules, California) blocked with 5% dry milk or blockingsolution for Western blot (Roche) and exposed to rabbitpolyclonal immunoglobin G (IgG) TRF2 (H-300: sc-9143,Santa Cruz Biotechnology, Santa Cruz, California; dilution1:200), mouse monoclonal IgG p16 (F-12: sc-1661, SantaCruz Biotechnology; dilution 1:250), rabbit polyclonal IgGanti-p53 (FL-393: sc-6243, Santa Cruz Biotechnology;dilution 1:500 in dry milk 1%), mouse monoclonal IgGChk2 (A-11: sc-17747, Santa Cruz Biotechnology; dilution1:1,000 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-phosphatedehydrogenase (GAPDH) (6C5: sc-32233, Santa CruzBiotechnology; dilution 1:1,000). For analysis of TERT,immunoprecipitation was performed using polyclonalIgG anti-TERT (H-231: sc-7212, Santa Cruz Biotech-nology) and agarose-A protein goat antirabbit IgG(Sigma-Aldrich). Immunodetection was accomplishedusing goat anti-rabbit IgG (Sigma-Aldrich) and goatanti-mouse (170-6516, Bio-Rad) secondary antibodies
(1:4,000 dilution), and an enhanced chemiluminescencekit (Amersham Biosciences) (30).Reverse transcriptionpolymerase chain reaction. Re-
verse transcriptionpolymerase chain reaction standardizedto GAPDH was performed using the following primers:
TRF1-for 5=-CATGGACTACACAGACTTAC-3= TRF1-rev 5=-ATCTGGCCTATCCTTAGACG-3=
55C, 27 cyclesTRF2-for 5=- TGTCTGTCGCGCATTGAAGA-3= TRF2-rev 5=-GCTGGAAGACCTCATAGGAA-3=
55C, 26 cyclesKu70-for 5=-GAGCATCCAGTGTATCCAGA-3=
Ku70-rev 5=-CAGCATGATCCTCTTGTGAC-3 =55C, 26 cycles
472 Werner et al. JACC Vol. 52, No. 6, 2008
Exercise Regulates Cardiac Telomere Biology August 5, 2008:47082
8/4/2019 "Effects of Physical Exercise on Myocardial TelomereRegulating Proteins, Survival Pathways, and Apoptosis". C Wer
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Ku80-for 5=-TCACAGTGTGCAGACACCTG-3=Ku80-rev 5=-AACTGCAGAGAGATGCCAGA-3=
56C, 26 cyclesIGF-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 cyclesP53-for 5=-GGGACAGCCAACTCTGTTATG
TGC-3=P53-rev 5=-CTGTCTTCCAGATACTCGGGA
TAC-3= 62C, 30 cyclesChk2-for 5=-GCTGTCCTCTGAGTAACAAC-3=Chk2-rev 5=-GAAGTAGAGCTTACAGGTGG-3=
53C, 40 cyclesGAPDH-for 5=-ACCACAGTCCATGCCATCAC-3=GADPH-rev 5=-TCCACCACCCTGTTGCTGTA-3=
60C, 27 cyclesQuantification of apoptosis by hairpin oligonucleotideassay. To detect apoptosis, 3-m thick paraffin sections offormalin-fixed mice heart tissue were examined using theApopTag Peroxidase In Situ Oligo Ligation Kit (ChemiconInternational, Millipore, Billerica, Massachusetts) (31). Thein situ oligo ligation assay specifically detects apoptosis bystaining only cells that contain double-stranded breaks thatare blunt-ended or have a 1 base 3= overhang (cells contain-ing nicked, gapped, 3=-recessed, 3=-overhanging ends longerthan 1 base and single-stranded ends are not detected). Unlikeconventional terminal transferase-based labeling (terminal de-
oxynucleotidyl transferase mediated 2=-deoxyuridine 5=-triphosphate nick end labeling [TUNEL]), the assay stainsapoptotic but not necrotic or transiently damaged cells (31).
To distinguish between cardiomyocytes and other cardiaccells, eosin staining was performed after the in situ oligoligation assay.Statistical analysis. Band intensities were analyzed bydensitometry. All values are expressed as mean standarderror of the mean. Unpaired Student t tests and analysis of
variance for multiple comparisons were applied. Post-hoccomparisons were performed with the Bonferroni adjust-ment test. The Mann-Whitney U test was used for the
analysis of the Ki-67 positive cardiomyocytes because thedata were not normally distributed. SPSS software, version12.0 (SPSS Inc., Chicago, Illinois), was used. Differences
were considered significant at p 0.05.
Results
Voluntary physical exercise increases telomere-stabilizingproteins. The mean voluntary running distance was 5,100 800 m/24 h. Voluntary exercise for 21 days, n 8 to 12 pergroup, did not change heart or body weight or the ratio ofheart 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 equalmean length of telomeres compared with sedentary controls(not shown). At the same time, telomerase repeat amplifi-cation protocol assays revealed that 3 weeks of exercisingup-regulated cardiac telomerase activity to 230.7 21%,
p 0.01 (Fig. 1B). Exercising increased the expression ofTERT in the heart to 165 4%, p 0.05 (Fig. 1C). Next,the effects of voluntary running on cardiac T-loop stabiliz-ing proteins were assessed. Three weeks of running had noeffect on the messenger ribonucleic acid (mRNA) expres-sion 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 80-kDa subunit but not the 70-kDa subunit of the repairprotein Ku (Figs. 1D to 1F).Exercise decreases markers of cellular aging in theheart. Compared with sedentary controls, the left ventri-cles of the mice that were supplied with a running wheel for21 days were characterized by a decreased expression of theaging marker protein p16 (52.6 3.3% of control, p 0.01). Similarly, expression of Chk2, which mediates cell-cycle arrest and apoptosis, was reduced to 78.7 2.7%, p0.05. The cardiac expression of the proapoptotic transcrip-tion factor p53 was reduced by one-half (56.4 2.7%, p 0.01). Data are shown in Figure 2. Reverse transcriptionpolymerase chain reaction analysis showed that p16 andChk2 mRNA expression were significantly 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 werenot altered after running (101 12% of control).Effects of long-term voluntary exercise on the telomerecomplex and cellular aging. To test if the regulationobserved after 21 days was a transient effect, mice wereequipped with individual running wheels or no running
wheel for the duration of 6 months (n 8 per group). Asexpected, long-term running induced a mild myocardialhypertrophy that did not impair left ventricular fractionalshortening (Fig. 3A). Compared with the 21-day train-ing, the long-term training exerted qualitatively andquantitatively similar effects and significantly increased
the activity of telomerase and the expression TRF2. Theexpression of the markers of reduced cellular life spanp16, 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 thatexercising exerts a continuous and persistent effect onregulators of cellular survival.
Flow-FISH assays detected no shortening of telomeres inleukocytes from 6-month-old mice compared with 3-week-old mice (Fig. 4A). Consequently, mean telomere lengthdid 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 thatshowed reduction of telomere length (17.5 kb in 18-month-
473JACC Vol. 52, No. 6, 2008 Werner et al.
August 5, 2008:47082 Exercise Regulates Cardiac Telomere Biology
8/4/2019 "Effects of Physical Exercise on Myocardial TelomereRegulating Proteins, Survival Pathways, and Apoptosis". C Wer
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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 byFISH using a Cy3-conjugated telomere-specific PNA probe(Figs. 4B and 4C). There was no significant difference in themyocardial telomere length between mice after 3 weeks and6 months of exercise or sedentary condition (3 weekssedentary: 4,495.0 159.7 individual telomeric fluores-
cence units [TFU]; 6 months sedentary: 5,172.0 361.1TFU; 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).Influence of exercise on Ki-67 expression in cardiomyo-cytes. Coexpression of alpha-sarcomeric actin and Ki-67
was not detected in myocardial sections (n 8) of controlmice. However, there was a marked increase to 0.023 0.01% Ki-67 positive cardiomyocytes in the hearts of miceafter 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
Control Running
Heartweight/tibialength
[m
g/mm]
0
50
100
150
200
Control Running
*
TE
RTexpression
[%c
ontrol]
TERT
GAPDH
0
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RNA[%
control]
*
Control Running
TRF2
GAPDH
TRF1
0
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Control Running
*
*
TRF2
GAPDH
TRF2protein
[%c
ontrol]
0
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250
Control Ku 70
Run
Ku 80
Run
KumRNA[%
control]
A
B
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D
E
F
0
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34
5
6
7
8
9
Telomeraseactivity[%
control]
0
50
100
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250
300
Control Running
* *
Control
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) car-
diac telomerase activity determined by telomerase repeat amplification protocol,(C) expression of the telomerase reverse transcriptase (TERT),(D) the messenger ribo-
nucleic 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.
474 Werner et al. JACC Vol. 52, No. 6, 2008
Exercise Regulates Cardiac Telomere Biology August 5, 2008:47082
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complex, second-generation TERT/ mice and theirstrain-matched wild-type (WT) were subjected to 21days of voluntary running. Wild-types exhibited a significantup-regulation of TRF2 protein expression to 466 121% anda 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-inducedchanges were absent in the TERT/ mice (Fig. 5).
IGF-1 and eNOS mediate the effects of exercising onsurvival proteins. Physical exercise is associated withincreased serum concentrations of IGF-1 (32). IGF-1 isdecreased in elderly patients and has been shown toenhance telomerase delaying cellular aging and death(10). The effect of exercising on cardiac IGF-1 is not
known. Voluntary running induced up-regulation ofmyocardial IGF-1 expression to 169.7 13% (Fig. 6A).
To provide evidence for a potential role of IGF-1 asmediator of exercise-induced cardiac telomerase activity,mice were treated with IGF-1 and GH, which increasesendogenous IGF-1 levels (22). Treatment with GH ledto an 8-fold increase of telomerase activity and IGF-1up-regulated telomerase activity by 14-fold, p 0.001(Fig. 6B). The IGF-1 treatment of animals resulted in 2-foldincreased expression of p-Akt (p 0.01) and p-eNOS (p 0.05) in the myocardium (Figs. 6C to 6E). Running isknown to up-regulate the expression and function of eNOS,
which has been confirmed in our model of voluntaryrunning at several time points (3335). To test a potentialrole of eNOS as a mediator of the observed effects, theexperiments were repeated in eNOS/ mice as a head-to-head comparison with strain-matched WT animals (n 8per group, 21 days). The running distance was not differentbetween groups. The results are summarized in Figures 6Fto 6J. Sedentary eNOS/mice showed a decrease of TRF2expression to 70.4 2.4% and an increase of p16 expressionto 130 5.1% compared with WT sedentary mice (p 0.05); telomerase activity and p53 expression were notsignificantly altered. The eNOS-deficient micein contrast
to the WT micedid not show that exercise modified thecardiac telomerase, TRF2, p16, p53, or Chk2.Exercise prevents doxorubicin-induced cardiac apoptosis.Doxorubicin is a commonly used antineoplastic drug withfrequent cardiotoxicity. The acute doxorubicin-inducedcardio-myopathy is characterized by cellular senescence and apoptosis(21). To test the functional relevance of the observed exercise-induced up-regulation of telomere stabilizing and down-regulation of proapoptotic genes, we exposed C57/Bl6 and
TERT/ mice with and without running wheels to a singleinjection with 22.5 mg/kg i.p. of doxorubicin (n 8 pergroup). We measured TRF2 expression 24 h after injection
and found that it was moderately decreased and telomeraseactivity 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 ofdoxorubicin. Doxorubicin-induced p53 expression in WTmice by 2-fold to 213 7%, p 0.01. The effect wassignificantly blunted in mice that had been running volun-tarily for 21 days. Exercise prevented the doxorubicin-induced down-regulation of TRF2 and markedly reducedup-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 de-creased apoptotic cardiomyocytes by 5-fold (0.03 0.01 vs.
0
20
40
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p16
GAPDH
p16[%
control]
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p53
GAPDH
p53[%
control]
Control Running
A
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C
* *
* *
Figure 2Exercise Decreases Markers
of Cellular Aging in the Heart
Representative Western blot analysis and quantification of the effects of volun-
tary exercise for 21 days on the left ventricular expression of(A) the senes-
cence marker protein p16, (B) the cell-cyclecheckpoint 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.
475JACC Vol. 52, No. 6, 2008 Werner et al.
August 5, 2008:47082 Exercise Regulates Cardiac Telomere Biology
8/4/2019 "Effects of Physical Exercise on Myocardial TelomereRegulating Proteins, Survival Pathways, and Apoptosis". C Wer
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0.17 0.03%, p 0.01). As expected, doxorubicin in-
creased cardiomyocyte apoptosis in the inactive animals(0.29 0.08%, p 0.05). However, mice supplied
with running wheels were completely protected fromdoxorubicin-induced programmed cell death of cardiomyo-cytes (Fig. 7D). Cardiomyocyte apoptosis rate of sedentary
versus running TERT/ B6.129S mice was in the samerange of the corresponding C57/Bl6 strain as discussedpreviously (0.51 0.21% and 0.21 0.03%, p 0.03 forcontrol vs. control doxorubicin). In contrast, sedentary
TERT/ mice of the same strain showed an increasedsusceptibility 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 significantprotection (p NS for TERT/ doxorubicin control vs.
runner; p 0.001 vs. WT doxorubicin runner) demonstrat-
ing the importance of TERT for the exercise-mediatedeffects on apoptosis.
Discussion
The study identifies a novel effect of physical exercise on theheart. Compared with mice kept under the regular condi-tions of laboratory animals, voluntary running exerciseup-regulated telomere-stabilizing proteins, reduced cellularsenescence, and prevented doxorubicin-induced apoptoticcell death. Further characterization shows that the effectdepends on TERT expression and is mediated by IGF-1
and eNOS. The effect was observed after 3 weeks ofvoluntary running and persisted for at least 6 months.
*
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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.
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Physical activity is associated with cardiovascular protec-
tion and longevity (13,5,6,8,9,36). However, the underly-ing molecular mechanisms remain only partially understood
(7). Here we observe that exercising increases the activity ofthe telomerase, the reverse transcriptase responsible for theextension of telomeric repeat sequences, as well as theexpression of its catalytic subunit, TERT. Both telomeraseand TERT have been shown to regulate cardiac muscle cellgrowth and survival (10,11,16,37), and telomerase ribonu-
cleic acid knockout mice (Terc/
) develop cardiac dysfunc-tion, increased expression of p53, and increased apoptosis (16).Overexpression of TERT causes hypertrophy in culturedcardiac myocytes and protects from apoptosis in vitro andin vivo (11). In addition to the effects on telomerase and
TERT, exercising mice showed up-regulation of TRF2mRNA and protein expression. Telomere repeat binding
QFISH, 3 wk; 40x QFISH, 18 mo; 40x
om81kw3 6 mo
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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 (1.5g/g 3 times per day for 2 days) (n 4). (C) Representative Western blots and(D)
quantification 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-deficient (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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factor 2 can bind to the TTAGGG repeats of telomeres andcontributes to the formation of the chromosome-protecting
T-loops (12). Importantly, TRF2 serves as the bindingplatform for additional telomere-associated proteins andmediates signaling to DNA damage checkpoint controls(13,14). Interestingly, cardiac apoptosis in human heartfailure was associated specifically with defective expressionof TRF2 and activation of the DNA damage checkpointkinase, Chk2 (17). In addition, exogenous TRF2 was shownto confer protection from oxidative stress (17). In circulatingprogenitor cells, TRF2 was identified as a regulator of
clonogenic potential and migratory capacity that can bemodified by pharmacological treatment (38,39). Here, vol-
untary exercise resulted in up-regulation of telomerase,TERT, and TRF2 after only 3 weeks and the regulationpersisted for at least 6 months. Therefore, physical exerciserepresents a potent regulator of the telomere complex.
Relatively little is known about the physiologic develop-ment of telomere length in healthy untreated WT C57/Bl6mice over time (8,14,40). After 6 months, we observed noshortening of telomere length, and telomere length did notdiffer between running and sedentary animals both in bloodleucocytes as well as in the myocardium. A control group of18-month-old C57/Bl6 mice exhibited significantly shorter
telomeres. This finding may be interpreted in several ways.Six months may be too short a time, and significantly longer
A
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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 amplifica-
tion protocol assays, and (C, D) p53 protein expression (quantification and representative Western blots).(E) Quantification 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.
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observation, such as 18 months, may be necessary topotentially observe a protective effect of exercising ontelomere length. On the other hand, it is possible that theenhanced telomerase activity may counteract a putativetelomerase-independent negative effect of exercising. How-ever, the interesting aspect of these data is that the regula-
tion of the telomere-regulating proteins by exercise-mediated survival signaling seems to be independent oftelomere length. Indeed, recent evidence suggests a directtelomerase-dependent transcriptional regulation of genesinvolved in cell growth, which has been recently suggestedas an additional mechanism by which telomerase promotescell proliferation. The regulation of telomerase activity andsubsequent replicative potential is reported to occur rapidlyand independently of telomere length in several cell types(8,22,41). Defects in mice lacking the ribonucleic acidcomponent of telomerase involve apoptosis, not just prolif-eration defects, and telomerase directly protects cells against
programmed cell death (8,16,42,43). Similarly, TRF2 wassuggested to mediate proapoptotic signaling in post-mitotic,noncycling cardiomyocytes (17) and was shown to signalindependently of telomere length in endothelial progenitorcells (39). It therefore seems likely that the telomericcomplex serves as a regulator of cellular aging and functionbeyond and potentially independently of protecting telo-mere length.
Several gene products implicated in senescence and celldeath, such as transformation-related protein p53, p16, andChk2, have been identified downstream of the telomericcomplex (10,13,14,1619). The tumor suppressor protein
p53 has been shown to mediate telomere dysfunction(16,19). Protein p53 modulates apoptosis and senescence byincreasing the expression of specific proteins, including Bax,Bad, and p21. Importantly, p53 was recently identified as akey mediator of maladaptive cardiac remodeling essentialfor the transition from cardiac hypertrophy to heart failure(18). Here, the data identify voluntary running as a noveland potent inhibitor of cardiac Chk2, p16, and p53.
To test whether the exercise-induced regulation of thetelomere complex and the regulation of p53 represent acoincidence or may be causally related, the experiments wererepeated in TERT-deficient mice. Running induced very
similar regulation of the transformation-related proteins inTERT/ WT and C57/Bl6 mice, but the exercise-mediated effects were absent in the TERT/ mice. Thesedata support the concept that the regulation of the telomericcomplex by exercising mediates the downstream effects onapoptosis.
Physical exercise is associated with increased serum con-centrations of IGF-1 (32,44). IGF-1 enhances telomerase,delaying cellular aging and death (10,22). Cardiac overex-pression of IGF-1 in transgenic mice increases the heart
weight (45,46). In IGF-1 transgenic mice, cardiac stem celldivision 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-1and its ligand may have the potential to regulate the re-entryof adult ventricular myocytes into the cell cycle (46,47). Incontrast to the systemic effects of exercising on IGF-1, theeffect of exercise on local cardiac IGF-1 is not known. Here,the data show that voluntary running increases the expres-
sion of IGF-1 in the heart. To test the role of IGF-1 inexercise-induced telomerase regulation, mice were treated
with recombinant GH and IGF-1, which resulted, respec-tively, in powerful 8- and 14-fold increases of telomeraseactivity. The effects of IGF-1 on telomerase have beenshown to be associated with the regulation of endothelialnitric oxide and phosphatidylinositol 3-kinaseAkt-p70S6Ksignaling, a pathway that plays an important role in regu-lating cardiac hypertrophy, viability, and homeostasis(22,48). In line with these data, treatment with recombinantIGF-1 led to significant increases in myocardial expressionof p-Akt and p-eNOS. Indeed, one of the best characterized
molecular effects of exercising in the model of voluntaryrunning is the up-regulation of eNOS (33,35). Therefore,the experiments were repeated in eNOS/mice showingthat the effects of exercising on telomere-regulating proteinsand subsequent survival signaling are mediated via eNOS.
Taken together, the data identify IGF-1, Akt, and eNOS asimportant mediators of the exercise-induced up-regulationof telomerase activity.
Apoptosis has been implicated in both acute and chronicheart diseases causing progressive loss of cardiac myocytes.End-stage human heart failure is characterized by increasedmyocyte apoptosis (20). Similarly, several forms of acute
toxic cardiomyopathy are characterized by cellular senes-cence and apoptosis; however, our understanding of poten-tial strategies to prevent cardiomyocyte senescence andapoptosis is limited (20). Doxorubicin is a potent, widelyused antineoplastic agent in cancer chemotherapy. However,its clinical application is compromised by its dose-dependentcardiotoxicity mediated by cardiomyocyte apoptosis (21).Here, we applied the well-characterized model of acutedoxorubicin-induced cardiac apoptosis to test if the ob-served effects of exercising on telomere-regulating proteinsand survival markers are able to confer protection fromcardiomyocytes apoptosis. After acute exposure to doxoru-
bicin, mice showed a small reduction of TRF2 expression.However, in mice that had access to a running wheel duringthe 3 weeks prior to doxorubicin exposure, TRF2 wassignificantly up-regulated. Similarly, the doxorubicin-induced increase of cardiac p53, a key mediator of cardiaccell death (16,18,19), was markedly blunted in mice of therunning group. Cardiomyocyte apoptosis was assessed bythe hairpin oligonucleotide assay that specifically detectsapoptosis by selectively staining cells that contain double-stranded DNA breaks that are blunt-ended or have a 1 base3= overhang whereas cells containing nicked, gapped, 3=-recessed, 3=-overhanging ends longer than 1 base, and
single-stranded ends are not detected. Unlike conventionalterminal transferase-based labeling, the assay stains apopto-
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tic but not necrotic or transiently damaged cells (31). Thehairpin oligonucleotide experiments revealed that providingmice with a running wheel to allow voluntary running forthe relatively short period of 3 weeks represents a powerfulintervention to protect from doxorubicin-induced cardio-myocyte apoptosis.
Somewhat unexpectedly, voluntary running decreased thebasal rate of cardiomyocyte apoptosis compared with sed-entary animals. Ascenso et al. (49) observed prevention ofdoxorubicin-induced increase in Bax, Bax-to-Bcl-2 ratio,and tissue caspase-3 activity by a 14-week training protocolin rats but no effect of exercise on the basal rate of thesemarkers 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 inthe absence of doxorubicin; however, the latter effect did notreach statistical significance. None of these studies hasdirectly assessed cardiomyocyte specific apoptosis; in addi-
tion, there were significant differences in the protocols,species, and modalities of training. In agreement withprevious studies, long-term voluntary running (e.g., 6months) is associated with increased heart weight, anadaptive change without impairment of cardiac function. Inthe light of these data, it is interesting to speculate that theopportunity to exercise resembles the natural habitat of micemore closely than cages without running wheels. Conse-quently, the inactivity of regular laboratory mice could beconsidered the experimental intervention in this study. Theprovocative hypothesis would be that the observed cardiacmorphology in voluntary exercise is normal and that
sedentary animals exhibit an adaptive cardiac hypotrophy.The notion of an anti-aging effect of exercising on theheart is supported by earlier reports in the literature (51)that survival rates decrease in sedentary as opposed toexercising rodents.
The effects of physical activity and inactivity are notlimited to the regulation of telomere-regulating proteins.Improved cardiac antioxidant capacity and beneficial effectson both circulating progenitor cells and cardiac residentstem cells are likely to contribute to the molecular andcellular 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 charac-terize both the quantitative and the cell-type specific con-tribution of the exercise-induced mechanisms with the aimto develop more specific therapeutic interventions. Specifi-cally, the effects of exercise on telomere biology in cardiacstem cells may be of functional significance (10).
A significant proportion of elderly heart failure patientsshow no other confounding variables suggesting that age assuch may be a primary cause of cardiac decompensation anddiastolic dysfunction (53,54). Shortening of the telomereshas recently been shown to predict cardiac morbidity and
mortality, and telomere length of circulating leukocytes isdecreased in patients with chronic heart failure (9,14,17,55).
In addition, recent evidence suggests that telomere biologyrepresents an indicator for the effect of a pharmacologicintervention (56). Here, we identify physical exercise as apotent antisenescent intervention to up-regulate telomere-stabilizing proteins and to reduce cardiomyocyte apoptosis,improving the molecular understanding of the beneficial
cardiovascular effects of exercise. Furthermore, the data setthe stage to prospectively investigate these novel cardiopro-tective effects in specific clinical situations, for example, forthe prevention of doxorubicin-induced cardiomyopathy.
Acknowledgments
The authors thank Sascha Jakob, Ellen Becker, and SimoneJger for excellent technical assistance.
Reprint requests and correspondence: Dr. Ulrich Laufs, Klinikfr Innere Medizin III, Kardiologie, Angiologie und InternistischeIntensivmedizin, Universittsklinikum des Saarlandes, 66424
Homburg/Saar, Germany. E-mail:[email protected].
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Key Words: exercise y myocardium y aging y prevention y telomere.
482 Werner et al. JACC Vol. 52, No. 6, 2008
Exercise Regulates Cardiac Telomere Biology August 5, 2008:47082
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