Effects of Physical Exercise on Myocardial Telomere-Regulating Proteins, Survival Pathways, and Apoptosis
Christian Werner, MD*,
Milad Hanhoun, MD*,
Thomas Widmann, MD ,
Andrey Kazakov, MD*,
Alexander Semenov, MD*,
Janine Pöss, MD*,
Johann Bauersachs, MD ,
Thomas Thum, MD,
Michael Pfreundschuh, MD,
Patrick Müller, MD*,
Judith Haendeler, MD ,
Michael Böhm, MD* and
Ulrich Laufs, MD*,*
* Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, Homburg/Saar, Germany
Klinik für Innere Medizin I, Hämatologie, Onkologie und Rheumatologie Universitätsklinikum des Saarlandes, Homburg, Germany
Medizinische Klinik I, Kardiologie, Universitätsklinikum Würzburg, Germany
Institut für Umweltmedizinische Forschung at the Universität Düsseldorf gGmbH, Düsseldorf, Germany.
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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 amplification 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.
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Figure 2 Exercise Decreases Markers of Cellular Aging in the Heart
Representative Western blot analysis and quantification 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.
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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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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-fluorescence 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 fluorescence units per high-power field 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 fields 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, 40x magnification. (D) Quantification (n = 8) and representative fluorescence microscopic image of Ki-67–positive nuclei (red) in cardiomyocytes identified by alpha-sarcomeric actin coimmunostaining (green) after 3 weeks of voluntary running. Nuclei are stained blue by DAPI. 100x magnification. *p < 0.05. LV = left ventricle.
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Figure 5 No Effect of Voluntary Exercise in TERT–/– Mice
Representative Western blot analysis and quantification 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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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 µg/g once per day for 7 days) and mouse IGF-1 (1.5 µg/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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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 amplification 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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