Received Date : 31-Jan-2017

Accepted Article Revised Date : 20-Feb-2017

Accepted Date : 21-Feb-2017

Article type : Editorial

Editorial

Telomeres, exercise, and cardiovascular disease: finding the means to justify

the ends

Warrick L Chilton1, Brendan J. OBrien1, Fergal Grace1, Fadi J. Charchar2*

1 Faculty of Health Sciences, Federation University Australia, Victoria, Australia

2 Faculty of Science and Engineering, Federation University Australia, Victoria,

Australia

*corresponding author:

Federation University Australia - Science

University Drive, Ballarat, Victoria 3350
Australia

e-mail: f.charchar@federation.edu.au

Telomeres are repetitive tandem DNA sequences (TTAGGG) located at

chromosomal ends where they protect genomic DNA from enzymatic degradation.
Telomeres are scaffolded upon six regulatory proteins, collectively known as the
shelterin complex. A state of replicative senescence is triggered when telomeres
reach a critically shortened threshold. This senescent state elicits a sequela of
oxidative stress and inflammation.1 Telomerase is an RNA-dependent DNA
polymerase that reverse transcribes telomeric repeats to chromosomal ends during
genome replication, slowing but not preventing eventual telomere erosion.

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/apha.12862
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 Observational associations between leukocyte telomere length (LTL) and
cardiovascular disease (CVD) abound, yet mechanistic clarity is lacking. Accelerated
Accepted Article
LTL shortening was initially regarded as a consequential epiphenomenon; however,
genetic and prospective studies have garnered support for a causal role.

Peripheral artery disease (PAD) is a significant health burden typically resulting in

restricted lower limb mobility. This functional limitation restricts exercise capacity and
the subsequent vascular adaptations from enhanced distal blood flow (increased
fluid shear). In Acta physiologica, Zietzer and colleagues (doi: 10.1111/apha.12820)
investigated acute telomere homeostasis in the context of PAD (Fontane Stage II), in
a small cohort of ageing patients. To achieve this, the authors undertook two
separate studies. The first study compared a single bout of exercise to a single 45
min exposure of individual counter-propulsion shear rate therapy (ISRT) in young
healthy participants. The second study compared a single 45 min of ISRT to a 5
week program totalling 30 hours of ISRT in PAD patients. The authors reported
increased peripheral blood mononuclear cell (PBMC) telomerase expression (i)
following a single acute bout of exercise in a healthy young cohort, and (ii) following
daily external counter-pulsation therapy over the course of 5 weeks. Neither group
responded to the single 45 min session of ISRT. Using multiple lines of association,
Zeitzer and colleagues (2016) link shear stress enhancing therapy with the
stimulation of therapeutic arteriogenesis and telomere biology. Whilst providing an
interesting snapshot, the equivocal associations between LTL and PAD challenge
the validity of PBMC telomerase as a PAD clinical outcome measure.2 Current
evidence posits a cardio-protective role for increased telomerase in mice;3 however,
direct evidence in humans is lacking.

The significant association between LTL and CVD implies a physiological benefit
from increased telomerase expression. The maxim if some is good, more must be
better seems to be tacitly accepted in popular discourse. However, it remains to be
determined whether an acute increase in PBMC telomerase expression represents a
positive pro-telomeric adaptation or is an indicator of cellular stress. Low telomerase
activity is often inferred by the presence of shortened telomeres; however, it does
not reliably correlate with average telomere length.4 Low telomerase activity is
independently associated with major CVD risk factors in humans,5 yet increased
telomerase activity, accompanied by shortened telomeres, is paradoxically

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 associated with progression of subclinical coronary atherosclerosis.6 Acute increases
in telomerase activity may indicate a stressed system attempting to stabilize critically
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shortened telomeres;5 a hypothesis evidenced in several pathological models.6

The majority of factors that negatively modulate LTL are also established CVD risk
factors.1 Habitual physical activity appears to confer a measure of telomeric
protection, with longer LTL routinely reported amongst the physically active.7 Despite
the ever-increasing associations between physical activity and telomere length, a
clear mechanistic explanation is lacking. A small yet growing number of studies have
investigated exercise-induced modulation of shelterin genes8-10 and the rate-limiting
catalytic subunit TERT.8, 10 These studies have produced some conflicting results
and issues such as sample size, precise expression time courses, functional
consequences, and cell-type specificity are unresolved.

A common methodological inconsistency in exercise-based telomere research is the

use of self-reported physical activity and discrepant exercise intensities. Such
inconsistencies likely misrepresent the magnitude of reported stimuli and responses.
Without quantitative physiological data to accurately measure or individualize training
intensity, participants within and between studies are invariably exposed to different
stimuli. This is significant as the magnitude of the immune response, and therefore
the cellular composition of the immune pool, closely scales with exercise intensity.11
Crucially, the cellular composition at any given time point will determine the mean
LTL and telomerase activity.12

A notable limitation that belies telomere research is the varied methodology used to
measure telomeres and telomerase. At present, ten different protocols can be used
to measure telomere length. These range from the original gold standard Southern
blot analysis of terminal restriction fragment (TRF), quantitative polymerase chain
reaction-based techniques (qPCR), through to single telomere length analysis
(STELA), and quantitative fluorescence in situ hybridization (Q-FISH). Each has its
own strengths, limitations, and coefficients of variation, making comparisons
between studies potentially inaccurate. Preceding the actual quantification of

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 telomere length, differences in DNA extraction protocol are also known to
significantly influence telomere length. Similarly, telomerase can be assessed using
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more than 30 protocols and variants thereof, either directly measuring telomerase
products or signals from telomerase-mediated DNA.13

It is unclear in Zietzer (2016) whether telomerase is investigated in the context of an

atherosclerotic disease risk factor or biomarker. The difference is subtle yet
important. Telomere research should first establish the requisite criteria for
telomere/telomerase system to be considered a risk factor; namely it must be
unequivocally located within an independent causal pathway that precedes a CVD
outcome. At present, LTL is variably associated with several established CVD
pathways; however, the independence from each of these pathways remains to be
established. Consequently, studies that measure telomere length and/or telomerase
expression may simply be measuring the downstream consequence of an unrelated
pathway.

The need to distinguish between statistical and clinical significance is apposite in

telomere and telomerase research. In the absence of a known minimal clinically
important difference (MCID) in telomerase activity (currently an unexplored avenue
of investigation), the summary dismissal of non-significant results may be a case of
throwing the baby out with the bath water. We encourage telomere biology
researchers to investigate the minimal threshold of telomerase activity required to
mitigate telomere-dependent senescence and the associated inflammatory
phenotype. Comparison between obtained results and the MCID might indicate
whether the results are clinically trivial, beneficial, or harmful.

Telomere research would benefit from returning to methodological first principles.

This will require uniformity in measurement protocols including cell type, extraction,
and measurement techniques. Furthermore, measurement variability should be
clearly established and stated to prevent spurious associations and false positive
findings. Exercise-based studies should use clearly established and physiologically
justified exercise intensities. Additionally, future research must strive to establish
expression time courses and dose-response curves for acutely measured variables.
Longitudinal studies are needed that systematically measure telomere length,
telomerase expression, oxidative stress, and inflammatory burden whilst accounting

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 for changes in cell type. The relative influence and independence of each index can
then be determined via regression analysis. Future studies in at risk populations
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should further establish a tangible degree of risk amelioration mediated by the
telomere/telomerase system. True clinical significance will be achieved when
positive telomeric adaptations directly reduce pathology in those with established
CVD.

Future telomere research will establish the causal, not merely the correlational
influence of the telomere/telomerase system. This will necessitate an evolved
understanding of the underpinning mechanisms. The incidence and subsequent
influence of exercise-induced epigenetic regulation such as microRNA, long non-
coding RNA, and methylation changes need to be determined. A universal
consensus must be established regarding the role of telomeres and telomerase as
biomarkers, risk factors, or both. To do this, researchers will need to establish a
standardized methodological approach. The issue of cause or consequence should
be addressed as a matter of priority.

Conflict of interest

None declared.

References

1. Fyhrquist, F, Saijonmaa, O, Strandberg, T: The roles of senescence and telomere

shortening in cardiovascular disease. Nat Rev Cardiol, 10: 274-283, 2013.

2. Raschenberger, J, Kollerits, B, Hammerer-Lercher, A, Rantner, B, Stadler, M,

Haun, M, Klein-Weigel, P, Fraedrich, G, Kronenberg, F: The association of

relative telomere length with symptomatic peripheral arterial disease: results

from the CAVASIC study. Atherosclerosis, 229: 469-474, 2013.

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 3. Br, C, de Jesus, BB, Serrano, R, Tejera, A, Ayuso, E, Jimenez, V, Formentini, I,
Accepted Article Bobadilla, M, Mizrahi, J, de Martino, A, Gomez, G, Pisano, D, Mulero, F,

Wollert, KC, Bosch, F, Blasco, MA: Telomerase expression confers

cardioprotection in the adult mouse heart after acute myocardial infarction.

Nature Communications, 5: 5863, 2014.

4. Zalli, A, Carvalho, LA, Lin, J, Hamer, M, Erusalimsky, JD, Blackburn, EH, Steptoe,

A: Shorter telomeres with high telomerase activity are associated with raised

allostatic load and impoverished psychosocial resources. Proceedings of the

National Academy of Sciences, 111: 4519-4524, 2014.

5. Epel, ES, Lin, J, Wilhelm, FH, Wolkowitz, OM, Cawthon, R, Adler, NE, Dolbier, C,

Mendes, WB, Blackburn, EH: Cell aging in relation to stress arousal and

cardiovascular disease risk factors. Psychoneuroendocrinology, 31: 277-287,

2006.

6. Kroenke, CH, Pletcher, MJ, Lin, J, Blackburn, E, Adler, N, Matthews, K, Epel, E:

Telomerase, telomere length, and coronary artery calcium in black and white

men in the CARDIA study. Atherosclerosis, 220: 506-512, 2012.

7. Cherkas, LF, Hunkin, JL, Kato, BS, Richards, JB, Gardner, JP, Surdulescu, GL,

Kimura, M, Lu, X, Spector, TD, Aviv, A: The association between physical

activity in leisure time and leukocyte telomere length. Archives of Internal

Medicine, 168: 154-158, 2008.

8. Chilton, WL, Marques, FZ, West, J, Kannourakis, G, Berzins, SP, OBrien, BJ,

Charchar, FJ: Acute exercise leads to regulation of telomere-associated

genes and microRNA expression in immune cells. PLoS ONE, 9: e92088,

2014.

This article is protected by copyright. All rights reserved.

 9. Laye, MJ, Solomon, TPJ, Karstoft, K, Pedersen, KK, Nielsen, SD, Pedersen, BK:
Accepted Article Increased shelterin mRNA expression in peripheral blood mononuclear cells

and skeletal muscle following an ultra-long-distance running event. Journal of

applied physiology, 112: 773-781, 2012.

10. Zietzer, A, Buschmann, EE, Janke, D, Li, L, Brix, M, Meyborg, H, Stawowy, P,

Jungk, C, Buschmann, I, Hillmeister, P: Acute physical exercise and long-term

individual shear rate therapy increase telomerase activity in human peripheral blood

mononuclear cells. Acta Physiologica (Oxford, England), 2016. Version of Record

online: 22 NOV 2016 | DOI: 10.1111/apha.12820

11. Simpson, RJ: The effects of exercise on blood leukocyte numbers. In: Exercise

Immunology. edited by GLEESON, M., BISHOP, N., WALSH, N., Hoboken,

Taylor and Francis, 2013, pp 64-105.

12. Lin, J, Epel, E, Cheon, J, Kroenke, C, Sinclair, E, Bigos, M, Wolkowitz, O,

Mellon, S, Blackburn, E: Analyses and comparisons of telomerase activity and

telomere length in human T and B cells: insights for epidemiology of telomere

maintenance. Journal of immunological methods, 352: 71-80, 2010.

13. Skvortsov, DA, Zvereva, ME, Shpanchenko, OV, Dontsova, OA: Assays for

detection of telomerase activity. Acta Naturae, 3: 48-68, 2011.

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