To begin to understand the early adaptive response to endurance‐type exercise of the telomere maintenance system in rodent cardiac tissue, we determined the effects of an acute bout of treadmill running on the gene expression and protein content of telomere‐related proteins in mouse cardiac muscle. Furthermore, we investigated MAPK (p38, extracellular signal‐regulated kinases (ERK) and C‐Jun N‐Terminal Kinase/Stress‐Activated Protein Kinase 1 (JNK)) activation as potential pathways through which exercise may result in telomere‐length maintenance in cardiac tissue. We hypothesized that acute exercise would increase the expression of telomere‐length‐maintaining proteins in parallel with increased activation of MAPKs in mouse myocardium.

Previously, we showed that long‐term (44 weeks) voluntary wheel running in rodents maintained telomeres at a similar length compared with young animals and significantly longer compared with age‐matched sedentary animals in heart tissue (Ludlow et al . 2012 b ). In a follow‐up study, we showed that treadmill running induces an early adaptive response of the telomere‐length‐maintenance system in the highly activated skeletal muscles (plantaris) but not the lesser activated muscles (tibialis anterior), driven in part by activation of p38 mitogen‐activated protein kinase (MAPK; Ludlow et al . 2012 a ). Whether a similar phenomenon of upregulation of genes related to maintenance of telomeres occurs in the cardiac tissue following an acute bout of endurance‐type exercise is currently unknown.

Telomere length is maintained in some cells by the reverse transcriptase enzyme telomerase, which consists of two main components, a protein component, telomerase reverse transcriptase (TERT), and an RNA template, telomerase RNA component (TERC; Shay & Wright, 2007 ). Telomere length and genome stability are also regulated by a DNA‐binding complex found at the ends of chromosomes called shelterin (de Lange, 2010 ), which consists of six proteins that bind to telomere DNA and regulate telomerase at the telomeres (de Lange, 2010 ). Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind to double‐stranded telomere DNA and regulate telomere length, while protection of telomeres 1 (POT1) regulates telomerase action at the telomere. Additionally, DNA‐damage‐response proteins that transiently associate with telomeres (KU70/80, CHK2 and p53) can aid in genome stability and modify the telomere end structure.

A hallmark of molecular ageing is the progressive shortening of telomeres with advancing age (Lopez‐Otin et al . 2013 ; Bär et al . 2014 ). Telomeres are repetitive DNA elements (5′‐TTAGGG n ) at the ends of linear chromosomes that when sufficiently long prevent DNA ends from being recognized as DNA double‐strand breaks (Shay & Wright, 2010 ). Telomeres shorten with each cell division until the protective effect of telomere DNA is diminished, thus telomere length limits the regenerative capacity of a cell or tissue (Bodnar et al . 1998 ). Short telomere length is associated with many age‐related diseases and is an independent risk factor for mortality (Ludlow & Roth, 2011 ; Blackburn et al . 2015 ). Short telomeres in human and mouse tissues and cells are also associated with increased genome instability and tissue dysfunction (O'Sullivan & Karlseder, 2010 ). Importantly, several lines of evidence indicate that human telomeres shorten at nearly equal rates across tissues with ageing, even in tissues with a low turnover, such as the heart (Daniali et al . 2013 ). A recently identified additional function of telomere length in cells is that of telomere‐length‐dependent chromosome looping that regulates gene expression or telomere position effect over long distances (Robin et al . 2014 ). Telomeres can also shorten as a result of unrepaired DNA damage at the telomeres caused by oxidative stress or other genotoxic insults (Ludlow et al . 2014 ). Thus, understanding how exercise can maintain telomere length in cardiac tissue is important from several vantage points, including genome stability and regulation of gene expression.

Certain cell types in the adult heart can regenerate and are proliferative (van Berlo & Molkentin, 2014 ; Bergmann et al . 2015 ). Neonatal mouse hearts can fully regenerate after partial surgical resection or induced myocardial infarction (Porrello et al . 2011 ). However, the ability of the heart to regenerate in this fashion is rapidly lost in the first week of postnatal life and is concurrent with massive changes in gene expression of the heart tissue (Porrello et al . 2011 ). Furthermore, the regenerative capacity of the heart is reduced with age (Bergmann et al . 2015 ), and the mutational load of the heart cells also increases with age, leading to genome instability and tissue dysfunction (Dollé et al . 2000 ). Thus, understanding how age and ageing per se influence the heart and finding means to slow, prevent or reverse age‐related changes in the heart tissue are paramount.

Age is the number one risk factor for both males and females in the development of cardiovascular diseases (Dominguez et al . 2006 ). Prevention of cardiovascular diseases is associated with lifestyle choices such as cessation of smoking, reduction in body mass and maintenance of physical activity (Dominguez et al . 2006 ). The long‐recognized positive benefits or ‘cardioprotective’ effects of exercise on reduction of cardiovascular disease risk include reduced resting blood pressure, lowered basal heart rate, reduced cholesterol concentrations and improved vascular health (e.g. reduced atherosclerosis; Neufer et al . 2015 ). However, these improvements following regular endurance exercise training do not fully explain the cardioprotective effects of exercise (Neufer et al . 2015 ), and questions about the biological underpinnings of the effects of exercise remain unanswered.

Telomerase enzyme activity was measured with a commercially available kit using the telomere repeat amplification protocol (TRAP; Quantitative Telomerase Detection Kit; US Biomax, Rockville, MD, USA). Protein concentration was determined (as above), and 1 μg of protein was added to the reaction according to the recommendations of the manufacturer and as previously performed in our laboratory (Ludlow et al . 2008 ). In addition to the standards provided in the kit, we assayed heat‐treated samples as a negative control. Heat‐treated samples were concluded to be telomerase negative if the mean of the critical threshold ( Ct ) for the heat‐treated sample duplicates was three standard deviations above that of the telomerase‐positive sample (this criterion was also used to determine a telomerase‐positive sample). We also assayed a human cancer cell line known to be telomerase positive (HeLa, ATCC, CCL‐2, Manassas, VA, USA) to ensure sensitivity of the assay. Owing to sample processing and limitations in the assay (the heat‐treated negative control was not three standard deviations different from lysate), we had to exclude two animals from the postexercise groups. This exclusion reduced the power of our analysis and may have hindered the detection of biologically relevant and statistically significant differences.

Total RNA was extracted from the frozen samples using TRIzol reagent (Invitrogen Technologies, San Diego, CA, USA) on the basis of previously described techniques (Chomczynski & Sacchi, 1987 ). One microgram of total RNA was reverse transcribed using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). Gene‐expression analyses were performed for all target genes using 1 μl of each reverse transcription reaction product and normalized to 18S and glyceraldehyde 3‐phosphate dehydrogenase ( Gapdh ); however, expression of the target genes did not differ based on normalization to Gapdh or to 18S (data not shown). Specific primer sequences are shown in Table 2 . The PCR products were separated on a 2.0% agarose gel and visualized using ethidium bromide. Band intensities were analysed by densitometry using ImageJ software (Rasband, 1997 –2011).

Forty‐eight hours after the incremental exercise test, the mice were exposed to 30 min of treadmill running at ∼70% of their peak speed and were euthanized immediately (TP1; n = 8) or 1 h after the running bout (TP2; n = 8). The duration and intensity of the treadmill exercise for the mice were chosen to mimic the current exercise recommendations of the American College of Sports Medicine for maintenance of human health (Balady et al . 2000 ). The treadmill was kept at a 7 deg incline throughout the experiment. In order to encourage the animals to continue to run, a shock grid at the back of the treadmill was used (details shown in Table 1 ). All animals completed the 30 min treadmill bout without stopping.

Forty‐eight hours after the last acclimation session, animals from the exercise groups were subjected to an incremental exercise test for assessment of their peak treadmill running speed. First, the mouse was placed for 2 min on the stationary treadmill belt. Thereafter, the treadmill speed was set at 6 m min −1 and increased 3 m min −1 every 2 min until refusal to run (animal seated on the shock pad for >30 s). The speed of the last stage completed was recorded as the peak treadmill running speed.

Twenty‐two female C57Bl/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed at 25°C on a 12 h–12 h light–dark cycle with access to standard laboratory mouse chow (Prolab RMH 3000, 5P00, LabDiet by Purina, Nestlé S.A., Vevey, Switzerland) and water ad libitum . We choose to use only female C57Bl/6J mice because they have a greater propensity to run compared with male mice (Lightfoot et al . 2004 ; Turner et al . 2005 ). Mice from the two exercise groups were acclimated to the treadmill for 2 weeks before the tests were performed, beginning at 6 weeks of age. The acclimation period was composed of seven progressive exercise sessions, the first involving a 5 min running bout at 2 m min −1 with no shock and the last one involving a 10 min running bout at 15–20 m min −1 with moderate shock (details shown in Table 1 ). All mice were euthanized at 8 weeks of age to limit the amount of influence that age could have on telomere‐related variables. Data on the skeletal muscles from these animals have been published (Ludlow et al . 2012 a ).

The procedures used in the present study were approved by the University of Maryland Institutional Animal Care and Use Committee and conformed to the National Institutes of Health's Guide for the Use and Care of Laboratory Animals (NIH publication no. 85‐23, revised in 1996). The authors of this study understand the ethical principles of the journal and made every attempt to ensure that this work complies with the animal ethics checklist. C57Bl/6J mice were assigned to a baseline (BL) group that was not exposed to exercise or to one of two groups exposed to an acute treadmill running bout and euthanized immediately (TP1) or 1 h after the exercise (TP2). Animals were kept on an ad libitum diet and on a standard 12 h light–12 h dark schedule. Animals were anaesthetized by inhalation of isoflurane and euthanized by cardiac excision. Cardiac gene expression of telomere‐related proteins ( Trf1 , Trf2 , Pot1a , Pot1b , Tert , Ku70 , Ku80 , p53 and Chk2 ) was analysed via RT‐PCR, and TRF1, TRF2 and p38 MAPK protein content was analysed via immunoblotting.

To test whether MAPKs were activated during acute exercise in parallel with alterations in shelterin components, we measured the phosphorylation status of three MAPKs, namely p38 MAPK, ERK1/2 and JNK1/2. Phosphorylation levels of ERK1 (p44) were significantly reduced immediately after exercise ( P = 0.02; Fig. 5 ) and were similar to BL at TP2, whereas phosphorylation levels of ERK2 (p42) were not significantly different at any time point ( P = 0.16). The p38 MAPK phosphorylation levels were greater in TP1 animals compared with BL animals ( P = 0.04) and tended to be greater in TP2 compared with BL animals ( P = 0.10), but were not different between TP1 and TP2 ( P = 0.63; Fig. 5 ). Phosphorylated levels of JNK1 (p46) were not different at any time point ( P = 0.59), but levels of phosphorylated JNK2 (p54) were significantly reduced at TP2 following exercise compared with both BL and TP1 ( P = 0.001).

Previous literature has implicated the accumulation of DNA damage and p53 signalling in the ageing heart; therefore, we tested the influence of acute exercise on DNA‐damage and ‐repair and DNA‐damage‐signalling genes. The expression of DNA‐damage‐repair protein Ku70 was not different ( P = 0.08; Fig. 4 A ), whereas Ku80 gene expression was significantly increased in TP1 and TP2 animals compared with BL ( P = 0.01 and P = 0.02, respectively; Fig. 4 B ). Expression of the mRNA of DNA‐damage‐response protein p53 was not different between groups ( P = 0.17; Fig. 4 C ), but Chk2 mRNA expression was significantly increased at TP1 compared with BL animals ( P = 0.03; Fig. 4 D ).

Discussion

Researchers in our laboratory have previously determined that chronic voluntary wheel‐running exercise in mice resulted in telomere‐length maintenance and increased expression of shelterin and DNA‐damage‐response and ‐repair genes in cardiac tissue (Ludlow et al. 2012a,b). The purpose of the present study was to begin to elucidate the acute effects of exercise on cardiac tissue gene expression of shelterin proteins and DNA‐damage‐response and ‐repair genes. We describe for the first time in C57Bl/6J mice, that an acute bout of treadmill exercise results in upregulation of cardiac telomere‐length‐maintaining proteins that occurs in parallel with activation of the p38 MAPK signalling pathway. These data provide important insights into a potential pathway mediating telomere‐length maintenance and genome stability in response to chronic exercise in cardiac tissue. We also show that key DNA‐damage‐response and ‐repair proteins are increased after acute exercise. Together, these results support a ‘telomere‐length‐protective’ effect of exercise in cardiac tissue and indicate that the initial adaptation may be associated with altered p38 MAPK signalling (Spallarossa et al. 2009). Maintenance of longer telomeres in cardiac tissues or cell types may result in a ‘youthful’ gene‐expression programme and longer healthy heart function with chronological ageing (Robin et al. 2014, 2015). We hypothesize that the chronic effects of exercise on telomere length may be the cumulative effect of each bout and that understanding the pathways associated with the adaptive response to exercise may lead to improved therapeutics and/or exercise prescription for the prevention of cardiovascular disease.

The regenerative capacity of the heart cell types, particularly the cardiomyocyte, is limited (van Berlo & Molkentin, 2014); however, recent reports indicate that rare neonatal‐like adult cardiomyocytes can divide (Porrello et al. 2011; Canseco et al. 2015; Kimura et al. 2015). DNA‐damage signalling (p53, p21 and p16) and mitochondrial dysfunction occur with ageing in the heart and lead to pathological conditions, such as heart failure and myocardial infarcts, indicating that the turnover of heart cells is not sufficient to replace cells under physiological wear and tear (Keller & Howlett, 2016; Narasimhan & Rajasekaran, 2016). Furthermore, it has been shown that telomere length in the adult human heart and in mouse heart tissue does shorten with ageing, suggesting that some level of cellular turnover and/or telomeric DNA damage is occurring with age (Werner et al. 2008, 2009; Wong et al. 2010; Ludlow et al. 2012b). Exercise training is associated with substantial cardiovascular health benefits, and we recently showed that long‐term wheel running in mice maintained telomere length and increased the expression of telomere‐length‐maintaining proteins in heart tissue of active animals compared with control mice (Ludlow et al. 2012b).

Previous research has indicated that exercise training is associated with increased gene expression of shelterin in cardiac tissue (Werner et al. 2008, 2009; Ludlow et al. 2012b). In our previous chronic exercise study, we observed statistically significant decreases in Trf1, Trf2, Pot1a and Pot1b mRNA levels in hearts of aged mice that were attenuated by exercise training (Ludlow et al. 2012b). Both TRF1 and TRF2 are double‐stranded telomere‐binding proteins that regulate telomere length (de Lange, 2010). Both POT1a and POT1b interact with the single‐stranded G‐rich overhang of telomeres and interact with another shelterin protein, TPP1, that together recruit telomerase to telomeres and control telomere repeat processivity (the ability of telomerase to add telomere repeats successively to chromosome ends; Nandakumar et al. 2012; Nandakumar & Cech, 2013; Schmidt & Cech, 2015). Maintenance of expression levels of shelterin components is crucial to maintaining telomere‐length homeostasis and to prevent DNA‐damage signalling at the telomere. Here, consistent with our previous data showing that shelterin adapts to exercise training, we show that a single bout of exercise is able to increase the expression of TRF1, TRF2 (both protein and mRNA) and Pot1a (mRNA), but not Pot1b. These data indicate that exercise‐stress‐related signalling results in an upregulation of shelterin components in cardiac muscle and that with training (cumulative effect of each individual exercise bout) the mRNA levels may be maintained over time. The immediate response of an increase in TRF1 and TRF2 protein levels is likely to be attributable to an increase in protein stability or a reduction in protein degradation, whereas the increase in mRNA levels is an adaptive response caused by early adaptive signalling events. Briefly, studies of masters athletes have shown increased TRF2 levels (protein and mRNA) compared with age‐matched healthy, sedentary individuals (Werner et al. 2009). A study of white blood cells (WBCs) of ultra‐endurance athletes 24 h after seven marathons in 7 days showed an increase in TRF1 and TRF2 mRNA levels compared with baseline (Laye et al. 2012). On the contrary, an acute endurance exercise bout (30 min cycling, 80% of maximal heart rate) was associated with reduced TRF2 mRNA levels in human WBCs 60 min after exercise (Chilton et al. 2014). The authors also measured mitochondrial RNAs and found several upregulated microRNAs that could target TRF2, indicating that the exercise‐induced regulation of TRF2 could be very tightly controlled and depend on the timing of measurement and the intensity and duration of the exercise stimulus. Levels of Pot1a mRNA increased 1 h after exercise. In rodents, POT1a suppresses the Ataxia Telangiectasia and Rad3‐Related Protein (ATR) dependent DNA‐damage response at telomeres (Palm et al. 2009). These findings also extend our previous results showing that chronic voluntary wheel running resulted in increased Pot1a gene expression in hearts of 1‐year‐old animals compared with sedentary age‐matched animals (Ludlow et al. 2012b). Laye et al. (2012) also showed an increase in POT1 levels compared with baseline levels in WBCs following the ultra‐marathon event described above. Thus, the upregulation of shelterin with acute exercise may accumulate over time (i.e. a training effect or repeated bout effect), resulting in increased shelterin and aiding in the telomere‐length‐maintenance phenotype observed in chronically trained animals.

The major telomere‐length‐maintenance enzyme complex, telomerase, has also been shown to be upregulated following short‐term (3 weeks) and long‐term (6 months) exercise training in rodent heart tissue (Werner et al. 2008, 2009). We observed a slight but not statistically significant increase in telomerase activity and in mTert (mouse telomerase reverse transcriptase) gene expression after exercise. Our data indicate that a single acute bout of exercise does not significantly increase telomerase activity levels and that the effect of exercise on telomerase may be a repeat bout effect, with telomerase adapting after several bouts of exercise. Several human studies to date have observed mixed results concerning the acute effects of exercise on hTERT gene expression. In a study of human WBCs, hTERT mRNA levels increased 60 min after a bout of cycling compared with resting levels (Chilton et al. 2014), whereas in the ultra‐marathon study mentioned above, no change in hTERT mRNA levels was observed in WBCs (Laye et al. 2012). Most recently, a study of WBCs in young healthy individuals after a 30 min treadmill run showed an increase in telomerase activity compared with the rested state (Zietzer et al. 2016). These mixed results point to the need for more research on exercise and telomerase. Several considerations about how exercise could be influencing telomerase and hTERT should be made, as follows. In order for telomerase to maintain telomeres, two different steps must occur. First, there must be active enzyme (i.e. TERT must be expressed), and second, the active enzyme must be recruited to the telomeres. Thus, an increase in either process could initiate telomere‐length maintenance. These data could suggest that the initial adaptation to exercise may be to increase telomerase recruitment to telomeres and the training adaptation is to increase telomerase enzyme activity, as observed in previous studies (Werner et al. 2008, 2009). However, further research into the activation, assembly and recruitment of telomerase (i.e. TPP1 levels) and mTert/hTERT gene‐expression regulation as well as the cell cycle/division kinetics and types of cells expressing telomerase following exercise on telomere‐length maintenance in cardiac tissue of rodents and humans is warranted.

Given that longer telomeres are associated with increased genome stability and we have previously shown an upregulation of DNA‐damage‐response and ‐repair genes in cardiac tissue of trained animals, we investigated the effects of acute exercise on DNA‐damage‐response and ‐repair genes. We observed that Ku80 and Chk2 gene expression increased immediately after exercise and returned to baseline levels by 1 h postexercise. Ku80 is a heterodimeric protein that has been shown to associate transiently with telomeres and is involved in the DNA‐damage response (Lopez et al. 2011; Pfingsten et al. 2012). Furthermore, Laye et al. (2012) observed increased gene expression of Ku70/Ku80 in both immune cells and skeletal muscle in humans after running seven marathons in 7 days. Moreover, we have previously observed greater Ku70/Ku80 gene expression in cardiac tissue of 1‐year‐old mice that voluntarily ran for 44 weeks compared with sedentary aged‐matched animals (Ludlow et al. 2012b). Combined, these data indicate that exercise training and acute exercise in both trained and untrained individuals increase gene expression related to enhanced DNA‐damage response and repair. We hypothesize that improved DNA‐damage response and repair, in concert with increased shelterin expression and telomerase activity, would result in telomere‐length maintenance and an anti‐apoptotic and anti‐senescent cellular environment in exercise‐trained compared with sedentary individuals.

To address possible signalling mechanisms, we measured a common stress‐response pathway in relationship to the observed changes in gene expression. We measured activation of p38 MAPK, ERK1/2 and JNK1/2, which are known to be a part of cardiac growth pathways (Rose et al. 2010). We observed a significant upregulation in p38 MAPK phosphorylation after exercise in conjunction with gene‐expression changes, whereas we observed decreased activation (phosphorylation) of ERK1/2 and JNK1/2. We recently reported that activation of p38 MAPK in skeletal muscle was directly related to altered levels of shelterin components (Ludlow et al. 2012a). Similar to our skeletal muscle data and other reports in cardiac muscle, we observed significant p38 MAPK phosphorylation after an acute bout of exercise. Activation of these three MAPKs has been associated with cardiac disease and pathophysiological hypertrophy (Asrih et al. 2013). Furthermore, MAPKs have been associated with exercise and ageing responses, as well as expression of telomere‐binding proteins in cardiac tissue (Iwasa et al. 2003; Iemitsu et al. 2006; Collado et al. 2007; Spallarossa et al. 2009). In a model of artificially selected rats bred for low or high ability to run, an acute treadmill running bout activated all three MAPKs (Hunter et al. 2008). These discrepant results may be attributable to the exercise stress or the rodent model used; however, both interventions resulted in significant activation of p38 MAPK. Activation of p38 MAPK results in changes in activation and localization (i.e. cytoplasmic versus nuclear) of a variety transcription factors (Rose et al. 2010) that are likely to be related to the altered gene expression of shelterin components. Although only descriptive in nature in the present experiment, we and others have previously shown links between p38 MAPK and shelterin gene expression (Spallarossa et al. 2009; Ludlow et al. 2012a). The response of p38 MAPK is likely to be attributable to transient increases in calcium concentrations, oxidative stress and the local concentration of trophic factors (growth hormone and Insulin‐like growth factor‐1) in the cardiac tissue and cells (Rose et al. 2010). Although activation of the MAPKs has been implicated as being related to pathophysiological cardiac remodelling of the heart, we propose that activation of p38 MAPK alone might trigger an early adaptive response to increase genome‐stabilizing gene expression. Future experiments are necessary to investigate the exercise‐associated activation of p38 MAPK, which isoform is specifically activated, and the regulatory roles that the p38 MAPK stress‐response pathway plays in cardiac telomere‐length‐maintenance‐related gene expression.

This study is not without limitations that must be considered for appropriate interpretation of the presented data. We did not separate out the different p38 MAPK isoforms to determine which specific isoform is responsible for the observed changes in shelterin expression after acute exercise. To help prevent acute bout effects from carrying over from the acclimation or testing period, animals were placed in their normal cages for 48 h before the acute exercise bout and euthanized, which should minimize the effects of the acclimation and testing, but these effects cannot be ruled out. Additionally, we were limited by sample availability and assay sensitivity (qPCR‐based TRAP assay); thus, although we were close to significance in several instances, we failed to detect differences because of these limitations. Future studies with larger sample sizes and more robust measures of telomerase activity might detect significant differences. Furthermore, translation of these data from the rodent model to humans should be done with caution, because laboratory mice have significantly longer telomeres compared with human telomeres. That being said, the regulation of shelterin in vivo in humans and mice appears to be similar, thus providing evidence for the translational value of shelterin and telomere data obtained in mice (Werner et al. 2008, 2009; Laye et al. 2012). Again, we emphasize that this is the first report describing a novel gene‐expression phenotype and potential association between shelterin and p38 MAPK in cardiac tissue. We did not measure telomere length, because we would not anticipate such a short‐term exercise stimulus to alter telomere length in a meaningful or detectable fashion.

Understanding how telomere length is regulated in cardiac tissue with exercise training and how each bout of exercise results in a cardioprotective phenotype is important research for improving the healthspan of humans. Telomere length is one aspect of ageing tissues that regulates genomic stability and, as elucidated recently, chromatin structure and gene expression. How exercise results in longer telomeres and perhaps influences gene expression with ageing in cardiac tissue and how the altered gene‐expression affects specific cell types and the increase in fibrotic cells in the heart with ageing makes these findings and future research particularly important to human cardiovascular health. Ultimately, understanding the positive benefits of exercise training on these phenotypes may lead to novel therapies in preventative and personalized medicine.