Increased shelterin mRNA expression in peripheral blood ...

J Appl Physiol 112: 773–781, 2012. First published December 8, 2011; doi:10.1152/japplphysiol.00997.2011.

Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event Matthew J. Laye, Thomas P. J. Solomon, Kristian Karstoft, Karin K. Pedersen, Susanne D. Nielsen, and Bente K. Pedersen The Center of Inflammation and Metabolism, Department of Infectious Diseases and Copenhagen Muscle Research Center, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Submitted 5 August 2011; accepted in final form 2 December 2011

Laye MJ, Solomon TP, Karstoft K, Pedersen KK, Nielsen SD, Pedersen BK. Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event. J Appl Physiol 112: 773–781, 2012. First published December 8, 2011; doi:10.1152/japplphysiol.00997.2011.—Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects (n ⫽ 8, mean age ⫽ 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 (P ⬍ 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P ⬍ 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4⫹ or CD8⫹ cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle. marathon; aging; mononucleocytes

chromosomes, telomeres are formed by repeats of TTAGGG associated with a number of proteins. Telomeres are progressively shortened with each replication of DNA. Shortened telomeres are associated with biological aging, increased oxidative stress, and chronic diseases such as insulin resistance and hypertension (11, 14). Telomere length is commonly measured in peripheral blood mononuclear cells (PBMCs) in large cohort or association studies. However, telomere lengths in different cell types may better reflect specific diseases, tissue-specific aging, or cell-specific adaptations. For instance, endothelial cell telomere length is shorter in patients who have had cardiovascular risk factors for a longer period of time (43). Telomere length can be controlled directly by the telomerase enzyme, htert, and indirectly by a group of proteins called the

LOCATED AT THE END OF

Address for reprint requests and other correspondence: M. J. Laye, Centre of Inflammation and Metabolism, Rigshospitalet M7641, Blegdamsvej 9, DK2100 Copenhagen, Denmark (e-mail: [email protected]). http://www.jappl.org

shelterin complex. Telomerase knockout mice have shortened lifespan and increased susceptibility to many age-related diseases (12, 17), which is prevented and also reversed following reactiviation of telomerase (19). The shelterin complex interacts directly with the telomere and can modulate telomere length through allowing access to positive and negative regulators of telomere length in both telomerase-dependent and -independent mechanisms. The shelterin complex consists of the following six proteins: telomere-related factors 1 (TRF1) and 2 (TRF2), TRF1-interacting protein 2 (TIN2), protection of telomeres 1 (Pot-1), the Pot-1- and Tin2-organizing protein repressor/activator protein 1 (RAP1), and tripeptidyl peptidase I (TPP1) (reviewed in Ref. 28). A major role of the Ku70/80 complex is as DNA damage response enzymes, but they are also believed to be important for telomere health, where they can help the binding of TRF2 to DNA (13) and decrease telomere length when knocked down in cells (24). While some roles of the shelterin complex have been determined through knockout (6, 21) and overexpression (18, 29, 30) mouse models, a cross-sectional study of older endurance athletes revealed increased shelterin expression vs. older sedentary controls (44). However, how the shelterin complex changes in other tissues or in response to acute physiological stimuli or stress is unknown. Acute physical activity is a stress that results in beneficial homeostatic adaptations to better deal with subsequent bouts of stress. Stress in general is associated with shorter telomeres, but regular physical activity can prevent such stress-induced loss of mean telomere length (35). While several studies have examined telomere length in the PBMCs of physically active individuals, results are mixed, showing either no difference (45), an increased telomere length in older active individuals (22, 44), or an increase only in a moderately active group (27). PBMCs are a proliferative cell type in which replication leads to continued decrease of telomere length, and thus correlates well with aging. On the other hand, skeletal muscle, which is modified by physical activity, is a mainly postmitotic tissue with satellite cells and nonmyocytes being the only proliferative cell types. Still, telomere length in skeletal muscle remains informative and modifiable. For instance, minimum telomere length is lower in skeletal muscle of older highly endurance trained individuals (36) and overtrained individuals (8) but not altered in normally active older individuals (33). However, data examining changes in the shelterin complex in skeletal muscle in response to physical activity are lacking. Thus, we took advantage of a unique set of individuals that were running one marathon a day for 7 days to examine whether an extreme physiological stress alters expression of members of the shelterin complex, expression of the Ku70/80

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Table 1. qPCR primers Gene

18S TRF1 TRF2 Pot-1 Ku70 Ku80 hTerc Telomere 36B4

Primer Sequence

Concentration, nM

Fwd 5=-GCAATTATTCCCCATGAACG-3= Rev 5=-GGCCTCACTAAACCATCCAA-3= Fwd 5=-CGAGTGCCAGGTGCAGGTGG-3= Rev 5=-ATAATAGCCTCTGCGCTGTTGCGG-3= Fwd 5=-GGGTAGCCGGTACGGGGACT-3= Rev 5=-GGATTCCGTAGCTGCCTTGCG-3= Fwd 5=-TTGTTCGCTTTCACAGGCTGAAGA-3= Rev 5=-TGTCCTGGTGCCATCCCATACCTTT-3= Fwd 5=-GCGCCAAAGTGAGCAGTAGCCA-3= Rev 5=-CTGCTTCTTCATCGCCCTCGGT-3= Fwd 5=-AAGCGAGTTGCGACACGGCA-3= Rev 5=-ACAGCTGCCTTATTCCCCGACCG-3= Fwd 5=-CCCACTGCCACCGCGAAGAG-3= Rev 5=-GGGTGCACGTCCCACAGCTC-3= Fwd 5=-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3= Rev 5=-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3= Fwd 5=-CAGCAAGTGGGAAGGTGTAATCC-3= Rev 5=-CCCATTCTATCATCAACGGGTACAA-3=

500 500 150 150 300 600 300 600 300 300 500 500 500 500 270 900 300 500

Fwd, forward; Rev, reverse; TRF, telomere-related factor; Pot-1, protection of telomeres 1.

complex, mean telomere length, or telomerase activity in PBMCs and skeletal muscle. We hypothesized that no change in mean telomere length would occur, but telomerase activity and proteins within the shelterin complex would increase at the mRNA and protein levels. MATERIALS AND METHODS

Subjects. Nine (8 males, 1 female) participants in the “2010 Bornholm Ultramarathon 7xMarathon” race (results located at the bottom of the webpage: http://www.tejnif-lob.dk/www/index.php/ 62448timer/resultaterultramarathonforside/res2010ultramarathon) volunteered to participate in the study. After medical screening, all volunteers were included. Written informed consent was obtained from all participants. The study was approved by the ethical committee of the Capital Region of Denmark. Event description. The ultramarathon race took place on the Danish island, Bornholm. Seven consecutive daily marathons were run in one week, a total distance of 183 miles. One subject was unable to complete all seven marathons and was not included in the analysis, resulting in an n ⫽ 8 (7 males, 1 female). Subject characteristics are in Table 2. The temperature and humidity ranged 10 –18°C and 60 –90%. No precipitation fell. Pre race clinical testing. Within two weeks before the race, subjects came to the laboratory for pre race testing after an overnight fast and having refrained from vigorous physical activity for the 2 days prior. A venous blood sample was obtained from an antecubital vein for analysis of standard clinical parameters and the isolation of PBMCs by density gradient centrifugation, as previously described (40). A full blood count (including monocytes) was performed using an automated fluorescence flow-cytometry platform (Sysmex XE2100; TOA Medical Electronics, Kobe, Japan). In addition, skeletal muscle biopsies were taken from the vastus lateralis muscle via the percutaneous Bergstrom needle technique under local anesthesia. Muscle tissue was immediately frozen in liquid nitrogen and stored at ⫺80°C until further analysis. Body composition was assessed by dual-energy X-ray absorptiometry (DXA, Lunar Prodigy Advance; GE Healthcare, Madison, WI). Furthermore, each subject’s fitness level was determined via a treadmill test. In brief, running speed was set at the subject’s individual lactate threshold, and the grade was increased 2% every 2 min starting at 1% until volitional exhaustion, while maximal oxygen consumption (VO2max) was measured using an automated indirect calorimetry system (Cosmed Quark b2, Rome, Italy).

Post race clinical testing. The day following the ultramarathon race (22–24 h following completion of the last marathon), all participants returned to the laboratory following an overnight fast for further blood sampling, PBMC isolation, body composition assessment (DXA scan), and a muscle biopsy. RNA isolation and qPCR. Muscle samples were homogenized (⬃30 mg skeletal muscle tissue) using a Tissuelyser (Qiagen). PBMCs were lysed (⬃1 ⫻ 106 cells) in TRIzol (Invitrogen, Carlsbad, CA). Total RNA was isolated from muscle and PBMCs according to the manufacturer’s protocol (Invitrogen). Total RNA was dissolved in RNase-free water and quantified using a Nanodrop ND 1000 (Saveen Biotech). Equal amounts of RNA were reverse transcribed using the High-Capacity Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer’s protocol. Ten microliters of reaction mixture containing 15 ng of cDNA in triplicate and SYBR green master mix (Applied Biosystems) for all genes of interest (except hTert, which utilized a primer-probe set Hs00972656_m1; Applied Biosystems) were loaded into 384-well plates. PCR was performed on an ABI-PRISM 7900 Sequence Detection system (Applied Biosystems) with 40 cycles (two steps: 95°C for 15 s followed by 60°C for 30 s). Standard curves and dissociation curves for each primer set (sequences listed in Table 1) were done to ensure for equal efficiencies and single product formation. Genes of interest were normalized to 18S using the ⌬⌬Ct method (User Bulletin no. 2; ABI PRISM 7700 Sequence Detection System). Mean telomere length. DNA for telomere length was isolated from muscle tissue and PBMCs using the Promega Wizard SV kit (Promega, Madison, WI) according to manufacturer’s instructions. DNA

Table 2. Subject characteristics Subject Characteristics

n Age, yr BM, kg Height, cm BMI, kg/m2 Body fat, % ˙ O2max, ml · kg body mass⫺1 · min⫺1 V No. of marathons last 12 mo Running quantity last 12 mo, km/wk

8 (7 male, 1 female) 44 ⫾ 2 74.4 ⫾ 2.5 178.3 ⫾ 3.1 23.5 ⫾ 0.5 16.7 ⫾ 3.2 58.2 ⫾ 3.1 34 ⫾ 8 68 ⫾ 8

Data are means ⫾ SE; n, no. of subjects. BM, body mass; BMI, body mass ˙ O2max, maximal oxygen uptake during exhaustive exercise. index; V

J Appl Physiol • doi:10.1152/japplphysiol.00997.2011 • www.jappl.org


was quantified using a Nanodrop ND 1000 (Saveen Biotech). Mean telomere length was determined essentially as described and validated by Cawthon (5). Briefly, 15 ng of DNA were amplified in 10 ␮l of reaction mixture containing SYBR green master mix and primers either targeting the telomere or the single copy gene 36B4. PCR conditions for telomere amplification were 40 cycles of 95°C for 15 s, 54°C for 2 min and for 36B4 were 40 cycles of 95°C for 15 s, 58°C for 1 min. Standard curves and dissociation curves for each primer set (sequences listed in Table 1) were done to ensure for equal efficiencies and single product formation. Relative telomere-to-36B4 ratios were calculated using the ⌬⌬Ct method (User Bulletin no. 2, ABI PRISM 7700 Sequence Detection System). 36B4 did not differ pre and post (P ⬎ 0.05). This method correlates well (r2 ⫽ 0.677) with traditional Southern blot methods (5). Telomerase activity. Maximal telomerase activity was measured with the TRAPeze RT Telomerase Detection Kit (Millipore) as per the manufacturer’s instructions. Briefly, this kit measures the ability of telomerase enzyme in a sample lysate to add telomeric repeats (GGTTAG) to the 3=-end of an oligonucleotide, which is then amplified by qPCR using fluorescent-labeled primers. A standard curve was generated with an oligonucleotide identical to the telomerase-extended ogilonucleotide, which allowed for quantification of the amount of telomeric repeats in attomoles. The assay is sensitive enough to detect 0.004 amol of standard curve substrate. PBMC protein lysate (0.5 ␮g) was determined to be within the linearity of the assay. Skeletal muscle protein lysate (0.2–20 ␮g) was tested but failed to generate a signal despite amplification of the internal control, which indicated that the lysate did not inhibit the Taq enzyme. Immunoblotting. Immunoblots were completed as previously reported (15). Briefly, skeletal muscle biopsies were homogenized using a Tissuelyser (Qiagen), and PBMCs were lysed directly in lysis buffer (50 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM NaP, and 0.2% Ipegal-CA-630) supplemented with complete protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma). Protein concentrations were measured with the Bradford (2) assay. Equal amounts of proteins were subjected to SDSPAGE using Invitrogen 8% precast gels and an I-blot dry transfer machine according to the manufacturer’s instructions. Multiple amounts of skeletal muscle and PBMC lysate were tested to determine the amount of protein necessary to obtain a signal in each tissue. All pre and post comparisons for each tissue type were loaded on separate gels, with 30 ␮g loaded for the skeletal muscle gels (Fig. 3A) and 12.5 ␮g of protein for the PBMC gels (Fig. 3B). To directly compare the concentration of proteins between skeletal muscle and PBMCs, a separate gel containing 12.5 ␮g of the pre sample for each tissue was run (Fig. 3C). Polyvinylidene fluoride membranes were probed with primary antibodies at the following concentrations: Ku70 (no. 4103; Cell Signaling) 1:1,000, Ku80 (no. 2180; Cell Signaling) 1:1,000, TRF1 (no. 3529; Cell Signaling) 1:1,000, and TRF2 (no. 9143; Santa Cruz) 1:1,000. Detection of primary antibodies was performed using either a mouse (Pierce) or rabbit (Dako) peroxidase-conjugated IgG, and protein signals were visualized using FEMTO-enhanced chemiluminescence and a Bio-Rad Chemidoc XRS imager. To obtain a quantifiable signal, skeletal muscle pre and post comparison immunoblots were exposed for a longer time than PBMC immunoblots. Quantification of the immunoblots was done using Image J (National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij) and corrected for reactive brown-stained total protein. Skeletal muscle and PBMC pre and post immunoblots (Fig. 3, A and B) were quantified separately and were not compared. Direct comparisons of skeletal muscle vs. PBMC pre samples (Fig. 3C) were not quantified due to the weak relative signal of skeletal muscle and the consistency of the strong differences observed. Flow cytometry. Subpopulations of lymphocytes were determined using flow cytometry. CD4⫹ and CD8⫹/CD3⫹ T-lymphocyte subpopulations were defined as follows: early activated (CD69⫹), chronic activated (CD38⫹HLR⫺DR⫹), naïve (CD45RA⫹CD27⫹CCR7⫹), central

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memory (CD45RA⫺CD27⫹CCR7⫹), effector memory (CD45RA⫺ CD27⫹CCR7⫺), terminal differentiated (CD45RA⫹CD27⫺CCR7⫺), senescent (CD8⫹CD28⫺CD57⫹), apoptotic (CD28⫺CD95⫹), and recent thymic emigrants (CD4⫹CD45RA⫹CD31⫹). PBMCs were defrosted and stained with antibodies for 20 min, washed with PBS (Sigma), and then resuspended in FACS Flow from Becton-Dickinson Biosciences (BD). The monoclonal antibodies used were all purchased from BD and included isotype control IgG1/IgG2a phycoerythin (PE), IgG1 peridinin chlorophyll cyanine 5.5 (PerCP-Cy5.5), IgM/IgG1 fluorescein isothiocyanate (FITC), IgG1/IgG2b allophycocyanin (APC), IgG1 PE-Cy7, IgG1 APC-Cy7, CD28-PE, CCR7-PE, CD8-PerCP-Cy5.5, CD3-FITC, CD57FITC, CD31-FITC, HLA-DR-APC, CD95-APC, CD45RA-APC, CD38PE-Cy7, CD69-PE-Cy7, CD27-PE-Cy7, and CD4-APC-Cy7. Six-color FACS Canto was used, and data were processed using FACS Diva software (BD). Fifty thousand cells were analyzed for each sample. Statistics. Pre and post intervention means were compared by paired t-tests using GraphPad Prism (version 5.00 for Windows; GraphPad Software, San Diego, CA, www.graphpad.com). Statistical significance was accepted if P ⬍ 0.05. RESULTS

Physiological stress increases mRNA levels of shelterin complex. A number of proteins make up the shelterin complex, and, following the ultramarathon event, mRNA levels of TRF1, TRF2, pot-1, Ku70, and Ku80 in PBMCs increased 25, 67, 45, 13, and 12%, respectively (all P ⬍ 0.05, Figs. 1 and 2). The increase was extremely consistent between subjects and could not be accounted for by changes in the reference gene 18S (P ⬎ 0.05). In contrast, in skeletal muscle, only levels of Ku70 and Ku80 mRNA increased significantly (27 and 30%, respectively; P ⬍ 0.05, Fig. 1), whereas TRF1 mRNA tended to increase (P ⫽ 0.10, Fig. 2). Shelterin complex and associated proteins are present in skeletal muscle. While the shelterin complex is well appreciated in PBMCs, its role in skeletal muscle is less understood. We compared the relative amount of shelterin complex proteins TRF1 and TRF2, as well as Ku70 and Ku80 levels in skeletal muscle vs. PBMCs (Fig. 3C). While TRF1, Ku70, and Ku80 protein concentrations were all significantly higher in the PBMC lysate, TRF2 was consistently higher in the skeletal muscle lysate. Within skeletal muscle (Fig. 3A) and PBMCs (Fig. 3B), there was no significant difference in relative protein expression pre vs. post multiple marathons. However, in the PBMCs, the Ku70 and Ku80 protein levels showed trends (P ⫽ 0.11) toward an increase in post vs. pre samples. Physiological stress does not alter telomere length or telomerase activity. Next we wanted to determine whether increases in the mRNA levels of the shelterin complex were associated with increased levels of active components of the telomerase complex. However, neither the RNA-mediated telomerase subunit hTerc mRNA level (Fig. 4B), telomere catalytic subunit hTert mRNA level (Fig. 4C), nor the telomerase activity (Fig. 4D) was altered by the multiple marathons. While directional changes in telomere length and telomerase activity varied between subjects, these two variables showed the same direction of change within subjects, as demonstrated by a significant correlation between the change in telomere length and the change in telomerase activity (r2 ⫽ 0.50, P ⬍ 0.05, data not shown). Surprisingly, skeletal muscle did not express detectable mRNA levels of the catalytic subunit of telomerase, hTert (Fig. 4C), but did express hTerc at detectable, albeit unchanged, mRNA levels. Similar to the lack of hTert mRNA,


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In this study, we sought to determine the consequences of an extreme physiological stress, running seven marathons in 7 days, on telomere biology. The major findings of this study are that the mRNA levels of three proteins within the shelterin complex were upregulated at the mRNA level in PBMCs, and two proteins that can associate with shelterin complex were also upregulated at the mRNA level in both the PBMCs and skeletal muscle biopsies. To our knowledge, this is the first study to show that a physiological intervention can alter shelterin complex mRNA levels in PBMCs and suggests that shelterin complex is present at the protein level in human skeletal muscle. Additionally, mean telomere length was unaltered in either tissue by the stress of completing seven marathons in 7 days. In the PBMCs, the lack of change in telomere

Fig. 1. mRNA levels of telomere-related proteins Ku70 (A) and Ku80 (B) were measured in the skeletal muscle (SkM) and peripheral blood mononuclear cells (PBMC) before (Pre) and after (Post) each runner completed 7 marathons in 7 days. AU, arbitrary units; ddct, ⌬⌬cycle threshold. Data are presented relative to skeletal muscle, normalized to 18S. Each line represents one runner (n ⫽ 8) with the open circles indicating males (n ⫽ 7) and the closed circle indicating the single female (n ⫽ 1). *P ⬍ 0.05 for Pre vs. Post comparisons, as measured with paired t-test.

skeletal muscle contained no telomerase activity (Fig. 3D), consistent with the concept of skeletal muscle as a mostly post mitotic tissue and suggesting that there was no active satellite cell recruitment or immune cell infiltration following the multiple marathon event. Physiological stress does not alter the Subpopulations of CD4⫹ or CD8⫹ cells. Absolute monocyte, but not lymphocyte, number increased following the multiple marathon event (Fig. 5, A and B). We determined that no significant changes in early activated (CD69⫹) or apoptotic (CD28⫺CD95⫹) CD4⫹ and CD8⫹ cells occurred (Fig. 5, C and D), nor were any differences in chronically activated (CD38⫹HLR⫺DR⫹), naïve (CD45RA⫹CD27⫹CCR7⫹), central memory (CD45RA⫺CD27⫹ CCR7⫹), effector memory (CD45RA⫺CD27⫹CCR7⫺), terminal differentiated (CD45RA⫹CD27⫺CCR7⫺), senescent (CD8⫹ CD28⫺CD57⫹), or recent thymic emigrants (CD4⫹CD45RA⫹ CD31⫹) found (data not shown).

Fig. 2. mRNA levels of telomere-related proteins telomere-related factor 1 (TRF1, A), telomere-related factor 2 (TRF2, B), and protection of telomeres 1 (pot-1, C) were measured in the skeletal muscle and PBMC before (Pre) and after (Post) each runner completed 7 marathons in 7 days. Data are presented relative to skeletal muscle, normalized to 18S. Each line represents one runner (n ⫽ 8) with the open circles indicating males (n ⫽ 7) and the closed circle indicating the single female (n ⫽ 1). *P ⬍ 0.05 for Pre vs. Post comparisons, as measured with paired t-test.



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Fig. 3. Protein was isolated from skeletal muscle and PBMC before (Pre) and after (Post) each runner completed 7 marathons in 7 days. Separate gels containing either 30 ␮g of skeletal muscle (A) or 12.5 ␮g PBMC (B) lysate were subjected to SDS-PAGE and immunoblotted for TRF1, TRF2, Ku70, or Ku80. Data are presented normalized to total protein as measured by reactive brown staining. Each line represents one runner (n ⫽ 8) with the open circles indicating males (n ⫽ 7) and the closed circle indicating the single female (n ⫽ 1). In C, equal amounts (12.5 ␮g) of Pre skeletal muscle and PBMC lysate were run on the same SDS-PAGE to compare the relative protein expression of TRF1, TRF2, Ku70, or Ku80 between skeletal muscle and PBMCs. These data were not quantified.

length was not due to an increase in telomerase activity, the appearance of new lymphocytes, or the removal of older lymphocytes. On the other hand, skeletal muscle telomere length was maintained despite undetectable amounts of telomerase activity and hTert mRNA. Thus, the completion of seven marathons in 7 days results in an increase in the mRNA level of proteins involved in protecting telomere length without directly altering telomere length in PBMCs. The roles of the individual proteins within the shelterin complex in physiological conditions remain difficult to elucidate. While many elegant molecular and animal studies that genetically manipulate components of the shelterin complex have been done, few studies examining the shelterin complex in vivo with regard to human physiology exist. In contrast, in pathological conditions, such as cancer, there have been a

number of studies looking at members of the shelterin complex. For instance, in cancerous gastric surgery tissue, TRF1, TRF2, and TIN2 proteins were increased relative to precancerous samples (18), with similar findings in hepatocarcinogenesis (31). Furthermore, in coronary artery disease, internal mammary arterial segments had lower TRF1 protein expression in isolated and endothelial cells, a finding maintained in cultured endothelial cells (43). Conversely, increased TRF2 expression resulted from statin treatment of cultured endothelial cells, originating from healthy donors (41). In skeletal muscle of the mouse model of Duchenne muscular dystrophy, muscle TRF1 expression is increased and associated with shortened telomeres (1). However, we saw no alteration in any shelterin complex protein mRNA (TRF1, TRF2, Pot-1) or protein level (TRF1, TRF2) in skeletal muscle following seven marathons in


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7 days. This suggests that the transcriptional regulation of the shelterin complex in muscle may respond to pathological insults (disease) rather than physiological stimuli (exercise). Conversely, voluntary wheel running in mice increases TRF1 and TRF2 mRNA expression in the aorta and splanchnic mononuclear cells of mice (44). Furthermore, regardless of age, endurance athletes have increased TRF1 and TRF2 mRNA in circulating PBMCs, which was also associated with maintained telomere length in older subjects (44). We extend this finding by showing that mRNA levels of TRF1 and TRF2 in PBMCs can be increased by prolonged endurance exercise. Although the mRNA concentrations of TRF2 were not different between PBMCs and skeletal muscle, the relative protein expressions of TRF2 were much higher in skeletal muscle, suggesting differences in posttranscriptional mechanisms between PBMCs and skeletal muscle. The subjects in this study

Fig. 4. DNA, RNA, or protein was isolated from skeletal muscle and PBMC before (Pre) and after (Post) each runner completed 7 marathons in 7 days. A: mean telomere length was measured by PCR on gDNA, normalized to the single copy gene 36B4 expressed relative to skeletal muscle. mRNA levels of hTERC (B) and hTERT (C) were measured by qPCR and are presented normalized to 18S. hTERC was expressed relative to skeletal muscle levels and hTERT relative to PBMC levels, since skeletal muscle displayed undetectable (UD) levels of hTERT. D: telomerase protein activity was measured in both skeletal muscle (UD) and PBMCs. Each line represents one runner (n ⫽ 8) with the open circles indicating males (n ⫽ 7) and the closed circle indicating the single female (n ⫽ 1).

Fig. 5. Absolute numbers of monocytes (A) and lymphocytes (B) were determined before (Pre) and after (Post) each runner completed 7 marathons in 7 days. FACS analysis determined the early activated (CD69⫹, C) and apoptotic (CD28⫺CD95⫹, D) as a percentage of CD4⫹ and CD8⫹ cells, respectively. Each line represents one runner (n ⫽ 8) with the open circles indicating males (n ⫽ 7) and the closed circle indicating the single female (n ⫽ 1).



may already have elevated TRF1 and TRF2 mRNA in their PBMCs due to their extremely active lifestyle, but the inclusion of healthy normally active individuals would be needed to test this. However, whether the effect we saw was an acute effect due to the last marathon being completed 24 h previous is unknown. In addition to TRF1 and TRF2 mRNA, we made the novel observation that Pot-1 mRNA also increases in PBMCs following seven marathons in 7 days. While Pot-1, which binds single-stranded telomeric DNA, can act either as a positive or negative regulator of telomere length and telomerase activity depending on the experimental conditions (16, 34), relatively little is known about its response to physiological stress. Our data in PBMCs suggest that multiple components of the shelterin complex are upregulated at the mRNA level in response to physiological stresses such as exercise, but future studies determining how the shelterin complex allows access to modulators of telomere length under physiological conditions in different tissues are needed. Not within the shelterin complex, but interacting with it, is the Ku70/80 protein complex. Here we make the novel observation that, in PBMCs as well as in skeletal muscle, Ku70 and Ku80 mRNA levels increase following seven marathons in 7 days. One of the main functions of the Ku70/Ku80 complex is to help repair double-stranded DNA breaks in response to DNA damage. Whether the increased mRNA of Ku70/80 in our paradigm is in response to DNA damage or acts to protect DNA and/or telomeres in humans is not possible to ascertain in the current study. Animal models suggest that the Ku70/80 act to protect against future DNA damage. For instance, the deletion of the Ku70/80 complex decreases lifespan in mice without altering cancer incidence (25). Similarly, in the rat, Ku70 protein and DNA binding activity decreases with aging in the kidney and testes, but not in the liver (42). Interestingly, caloric restriction, which results in some similar adaptations as increased physical activity, is unable to increase Ku70 protein levels with aging (42). Alternatively, voluntary wheel running in mice is able to increase mRNA Ku70 in circulating leukocytes and aortic vessels (44). These same authors determined an association of increased Ku70 and Ku80 mRNA expression with increased mRNA levels of lifetime physical activity in humans by showing increased Ku70/80 mRNA expression in the leukocytes of older endurance athletes relative to older controls (44). Thus, our data are in agreement that endurance exercise can increase Ku70/80 mRNA expression in circulating PBMCs and in specific cells types required for adaptations to physical activity such as endothelial cells or in the case of the present study skeletal muscle. A potential mechanism for the increase in Ku70/80 mRNA in PBMCs and skeletal muscle and shelterin complex proteins following endurance exercise is the activation of endothelial nitric oxide (eNOS). Werner et al. (44) demonstrate that eNOS⫺/⫺ and Tert⫺/⫺ mice were unable to upregulate components of the shelterin complex in aortas of voluntary wheel running mice. Consistent with a role for eNOS, endurance exercise training increases nitric oxide availability in the plasma (37) and eNOS expression in skeletal muscle (23). However, our intervention is not capable of suggesting a mechanism due to the multitude of physiological stresses that seven marathons in 7 days elicit. Thus, the mechanism regulating human shelterin complex and telomere length in human PBMCs or skeletal muscle will require future studies that limit

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use interventions to target specific exercise or physiological pathways. While 80 –90% of cancers have increased telomerase activity (39), physiological aging is associated with decreased telomerase activity and telomere length in leukocytes (38) and skeletal muscle in some (4, 10), but not all, studies (33). While aging can be slowed by increased levels of exercise, whether exercise is protective or detrimental to telomere length is dependent upon the intensity, duration of the exercise, and tissue examined. On one hand, excessively trained athletes with fatigued athlete myopathic syndrome (8) or children with muscular dystrophies (9) have shorter skeletal muscle telomeres. Furthermore, lifetime training volume is inversely proportional to skeletal muscle minimum telomere length (36), potentially related to the increased recruitment and proliferation of muscle stem cells to repair damaged tissue. On the other hand, a study of 2,400 subjects (7) and of 69 subjects (27) showed positive relationships between physical activity and leukocyte telomere length. However, our data demonstrate no change in mean telomere length of circulating PBMCs or skeletal muscle following the extreme endurance event of seven marathons in 7 days. One potential explanation is that these subjects are more prepared for this kind of event than most, having completed an average of 34 marathons the past year, and are likely less affected by the stress of such an event. Thus, although it was certainly a physiological stress, it was not as extreme as it would be for most runners. Another potential explanation is that we were unable to measure minimum telomere length in skeletal muscle, which is considered a better marker of muscle turnover than mean telomere length (20, 26). To see whether any changes in telomere length or shelterin complex protein in leukocytes was due to increased relative appearance of newly formed lymphocytes or decreased older lymphocytes removed via apoptosis, we examined several different CD4⫹ and CD8⫹ cell types. Previously, it has been ˙ O2max test reduced mean terminal restricshown that a single V tion fragment length in PBMCs and CD4⫹ cells of young subjects and CD8⫹ cells in older subjects (3), which was suggested to be a consequence of the decrease in naïve cells following exercise. In contrast, we find no change in mean telomere length in PBMCs, which is consistent with lack of change in naïve CD4⫹ or CD8⫹ we observed following seven marathons is 7 days. It is possible that by examining the subjects 22–24 h after their last marathon we have missed transient alterations in lymphocyte subpopulations (32). There are several limitations to the study that should be noted. For instance, skeletal muscle biopsies contain many different cell types and thus we cannot absolutely conclude whether the changes that occurred are from skeletal myocytes per se. However, the undetectable level of telomerase activity and htert mRNA in skeletal muscle suggests that the more proliferative cell types where hTert is known to be expressed (endothelial cells, immune cells, fibroblasts, and satellite cells) do not significantly contribute the gene expression within skeletal muscle. Second, the majority of our significant findings are at the mRNA level, while changes at the protein level were nonsignificant. The lack of change at the protein level may be attributable to the semiquantitative nature of immunoblotting, or the increase in mRNA is insufficient to increase protein expression. While our data are descriptive, they provide


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a rationale to undertake future studies in humans to explore the physiological role and regulation of the shelterin complex and telomere length in low proliferative organs such as skeletal muscle, adipose tissue, or liver. In conclusion, we have provided the first evidence that a physiological stress represented by endurance exercise in humans leads to upregulation at the mRNA level of components of the shelterin complex in PBMCs and of the Ku70/80 complex in both PBMCs and skeletal muscle, without concomitant changes in mean telomere length.

12. 13. 14.

15.

ACKNOWLEDGMENTS Noemi Galicki James, Ruth Rousing, and Hanne Villumsen are acknowledged for technical assistance.

16.

GRANTS The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National Research Foundation (no. 02-512-55). This study was further supported by the Danish Council for Independent ResearchMedical Sciences, and the Commission of the European Communities (Grant Agreement no. 223576-MYOAGE). CIM is part of the UNIK Project: Food, Fitness & Pharma for Health and Disease, supported by the Danish Ministry of Science, Technology, and Innovation. CIM is a member of DD2, the Danish Center for Strategic Research in Type 2 Diabetes (the Danish Council for Strategic Research, grants no. 09-067009 and 09-075724). M. J. Laye is supported by the Danish Ministry of Science-Health.

17.

18.

19.

DISCLOSURES No conflicts of interest are declared by the authors. REFERENCES

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