Association among epigenetic modifications by DNA methylation, telomere length, and physical fitness in biological aging

Association among epigenetic modifications by DNA methylation, telomere length, and physical fitness in biological aging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Association among epigenetic modifications by DNA methylation, telomere length, and physical fitness in biological aging Yasuhiro Seki, Dora Aczel, Ferenc Torma, Matyas Jokai, Anita Boros, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2151782/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Cellular senescence is greatly accelerated by telomere shortening, and the steps forward in human aging is strongly influenced by environmental and life-style factors, whether DNA methylation (DNAm) is affected by exercise training, remains unclear. In the present study we investigated the relationships between physiological functions, maximal oxygen uptake (VO2max), vertical jump, working memory, telomere length (TL) assessed by RT-PCR, DNAmethylation based estimation of TL (DNAmTL) and DNA methylation based biomarkers of aging of master rowers (N = 151) and sedentary subjects (N = 90), aged between 37–85 years. It was found that the TL inversely correlated with chronological age, while no gender dependent difference was found. We could not detect association between telomere length and VO2max, vertical jump and working memory by RT-PCR method, while these physiological test results showed correlation with DNAmTL. DNAmGrimAge and DNAmPhenoAge acceleration were inversely associated with telomere length assessed by both methods. It appears that there is no powerful beneficial effects of exercise or physiological fitness on telomere shortening, however the degree of DNA methylation is associated with telomere length. DNAm based estimation of TL shows stronger relationships with physiological functions than RT-PCR measured data. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Healthy aging with relatively high physiological function is the goal of human society. To achieve this, it would be important to understand the aging process and then develop interventions that help to reach the goals. One of the mechanisms, which known to regulate aging process is based on the limiting factors of cell proliferation, namely on telomeres (Greider 1996 ). Telomeres are protective caps on the ends of chromosomes with repeated deoxyribonucleic acid sequences rich in TTAGGG, between 3- and 20-kb- long in humans (Hande 2004 ). Oxidative stress causes accelerated telomere shortening primarily through oxidation of guanine due to its lowest oxidation potential among nucleic acid bases especially in telomere quaduplexes (Radak and Boldogh 2010). After certain number of cell division, which results in loss of telomere length, dividing cells cannot replicate anymore reaching cellular senescent (Linskens et al. 1995 ). Although the enzyme of telomerase able to add de novo base pairs to telomere (Greider and Blackburn 1989 ), the age-related shortening is well documented (Campisi 2005 ; Bize et al. 2009 ). Indeed, accumulating evidence suggest that a strong link between telomere length and aging and age-associated diseases (Morin 1997 ; Sikora 2013 ; Fyhrquist and Saijonmaa 2012 ; Sahin and DePinho 2012 ). It has been shown that telomere length (TL) is dependent on various lifestyle factors. It has been shown that on preschool children obesity was linked to reduced level of docosahexaenoic acid, and increased arachidonic acid/ docosahexaenoic acid ratio and shortened telomere in leukocytes (Liu et al. 2021 ). Short term administration of nicotinamide mononucleotide resulted in significant elongation of TL in peripheral blood mononuclear cells of C57BL/6 mice (Niu et al. 2021 ). Studies suggest that physical exercise also has beneficial effects on TL (Lee at al. 2013; Manoy et al. 2020 ; Loprinzi et al 2015 ). Even as short-term exercise program as 12 weeks, with low frequency, moderate intensity, and explosive-type resistance training could have beneficial effects on telomeres. Indeed, above mentioned explosive resistance training lessened telomere shortening and correlated with the amelioration of redox homeostasis (Dimauro et al. 2016 ). Because aging is strongly influenced by life-style Horvath and Hannum developed DNA methylation based epigenetical aging clocks, which more precisely reflect aging than chronological age (Horvath 2015 ; Hannum et al. 2013 ). Moreover, in a huge cohort (n = 5,713) it has been shown that there is an interaction between genome wide methylation, TL and epigentical aging (Lee et al. 2019 ). Based on previous studies, we hypothesize that the level of physical fitness would affect epigenetical aging clocks and TL of whole blood samples in aged individuals. Moreover, we aimed to compare the interactions between physiological test results and TL assessed by RT-PCR method and DNAmethylation based estimation (Pearce et al. 2022 ). Methods Subjects Subjects were volunteers, who participated in 2019 Masters World Rowing Championships in Venice, Hungary and aged matched sedentary individuals. The investigation was carried out on a voluntary basis with the ethics license provided by the Hungarian Scientific and Research Ethical Committee 25167-6/2019/EUIG. Our cohort consisted of a total 241 people, 108 men: 71 athletes (mean age: 60 ± 10.5 years, and 37 volunteer controls (mean age 60 ± 13.4 years); and 133 women: 80 athletes (mean age: 58 ± 8.8 years) and 53 sedentary (mean age: 63 ± 11.9 years). Physiological tests Body mass and heights were measured and the body mass index was described by body composition monitor BF214 (Omron, Japan). Relative maximum hand gripping force a measure of age-associated decline in general muscle strength (Eika 2019) was assessed by CAMRY EH101 dynamometer. Relative maximal oxygen uptake is one of the best marker a viability and higher level of VO2max is associated with decreased levels of wide range of diseases (Hawkins and Wiswell 2003 ; Carnethon et al. 2005 ). We used Chester step test to apprise the level of VO2max (Izquierdo et al. 2019 ). Digit span test was applied to assess the working memory (Martinez-Diaz et al. 2020), where larger values indicate better verbal short-term memory. Determination of hematologic and telomere length Determination of hematologic and biochemical variables blood samples were collected before the subjects performed the VO2max evaluation test, and were stored in evacuated tubes containing EDTA as an anticoagulant for determination of erythrogram. Blood samples were centrifuged and stored at − 80 C degrees. The erythrogram and biochemical tests were carried out in the Clinical Analysis Laboratory of Semmelweis University, Budapest. DNA isolation DNA was isolated from the K2-EDTA anticoagulated blood samples using a DNA isolation kit (Pure LinkTM Genomic DNA Mini kit, Thermo Fisher, Carlsbad, CA, USA), according to the manufacturer’s instructions. Measurement of telomere length : The average relative telomere length of genomic DNA was determined from whole blood samples using Cawthon’s PCR-based method (Wan et al. 2019 ) with commercially available PCR kit (ScienCell Research Laboratories inc., San Diego CA Catalog no. #8908). During this reaction telomere-specific primers recognize and amplify telomeric sequences. For each DNA sample, two consecutive reactions were performed: the first for amplification of a single-copy reference (SCR) gene and the second for the telomeric sequence. The former recognizes and amplifies a 100 bp region on human chromosome 17 and serves as a reference for calculating the telomere length of the target samples. The PCR reactions were performed in a final volume of 20 µl. We used 5 ng reference/genomic DNA sample (final concentration = 0.625 ng/µl), 2µl telomere primer and 10 µl 2XMaster Mix, PCR conditions were as follows: first 95°C for 10 min, followed by 32 cycles of 95°C for 20s, 52°C for 20s, and 72°C for 45s. All samples were tested in triplicate. In addition the telomere length was also evaluated by Horvath’s software which estimates the telomere length from methylation (Pearce et al. 2022 ). Measurement of DNA Methylation : Epigenome wide DNA methylation 85K was measured with the Infinium MethylationEPIC BeadChip (Illumina Inc., San Diego, CA) according to the manufacturer’s protocol. Briefly, 500 ng of genomic DNA was bisulfite converted using the EZ-96 DNA Methylation MagPrep Kit (Zymo Research, Irvine, CA, USA) with the KingFisher Flex robot (Thermo Fisher Scientific, Breda, Netherlands). The samples were plated in randomized order. The bisulfite conversion was performed according to the manufacturer’s protocol with the following modifications: For binding of the DNA 15 µl MagBinding Beads was used. The conversion reagent incubation was done according to the following cycle protocol: 16 cycles of 95°C for 30 seconds followed by 50°C for 1 hour. After the cycle protocol the DNA was incubated for ten minutes at 4°C. Next, DNA samples were hybridized on the Infinium MethylationEPIC BeadChip (Illumina Inc., San Diego, CA) according to the manufacturers protocol with the modification that 8 µl bisulfite treated DNA was used as start material. Quality Control of the DNA methylation data was performed using, Meffil and Ewastools packages with R version 4.0.0. Samples which failed technical controls, including extension, hybridization and bisulfite conversion, according to the criteria set by Illumina, were excluded. Samples with a call rate 0.01 in at least 10% of the samples were set as undetected. Probes with a bead number < 3 in at least 10% of the samples were excluded. We used the "noob" normalization method in R to quantify methylation level (Triche et al. 2013 ). The details on the processing of DNAm data and the calculation of the measures of aging, or pace of aging, were calculated using Horvath’s online age calculator ( https://dnamage.genetics.ucla.edu/ ). Statistics The results were subjected to statistical tests. After testing for normal distribution, the relevant parametric and non-parametric test methods were applied. The differences between groups were examined by multiway ANOVA for a given variable followed by post hoc Tukey HSD. The interdependence of the individual variables was analyzed using multivariate regression analysis. Results We have measured or estimated the TL with two different methods one based on RT-PCR measurements while the other was calculated on methylation based estimation of TL. The results from the RT-PCR measurements revealed that the TL showed a strong, significant relationship with chronological aging of the subjects (r= -0.23; p = 0.0003), and relationship was similar in women and men (women: r= -0.21; p = 0.0012 vs men: r= -0.22; p = 0.0007) (Fig. 1 A). However, we could not detect gender dependent significant difference in the TL (Fig. 1 B). Similar data was obtained by DNAm based estimation (Fig. 1 C, D). According to results measured by RT-PCR there are no relationship between BMI, (r= -0.02; p = 0.7450, Fig. 2 A), vertical jump results (r = 0.12; p = 0.0689, Fig. 2 B), the scores of working memory did not correlate well with telomere length (r = 0.03; p = 0.622, Fig. 2 C) and TL. The DNAm based estimation of TL showed similar results with BMI (Fig. 2 D) and memory (Fig. 2 F), while the jumping test results correlated significantly with TL (p < 0.0001, Fig. 2 E) The maximal oxygen uptake (VO2max) which was calculated from the Chester step tests results showed no relationship with RT-PCR based TL (r = 0.011; p = 0.82, Fig. 3 A-C). On the other hand, DNAm based estimated resulted significant correlation between VO2max and TL in all subject case (p < 0.0045, Fig. 3 D) and in male (p < 0.0028, Fig. 3 E) and female subjects as well (p < 0.002, Fig. 3 F). When the possible association between RT-PCR measurement originated data and DNA methylation based epigenetical aging was examined, it turned out that in all subject case both DNAmPhenoAge and DNAmGrimAge related to TL (r= -0.76; p < 0.001; and r= -0.78; p < 0.0007) respectively (Fig. 4 A, B). The relationship between DNAm based TL estimation with DNAmPhenoAge and DNAmGrimAge showed even more powerful relationship (p < 0.0001, Fig. 4 C, D). When the relationship of DNAmPhenoAge acceleration (r= -0.21; p = 0.0012) and DNAmGrimAge acceleration (r= -0.25; p < 0.0001) with telomere length were evaluated, the results revealed that longer telomeres were related to decelerated aging (Table 1). Discussion In present study, our data revealed that TL was associated with DNA methylation based epigenetic aging biomarkers. It has been known that higher level of cardiovascular fitness, the VO2max is associated with longer TL in wide range of age group (18–72 years old) (LaRocca et al. 2010 ). It is clear that one of the striking effect of aging is suppressed physiological function, however it is also known that the progress of aging is depends on environmental and life-style factors, including physical fitness (Radak et al. 2019). Indeed, the DNA methylation based epigenetic biomarkers reflect the individual aging more precisely than chronological aging and related to telomere length (Lee et al. 2019 ). Here we further report that exercise induced DNA methylation based calculation of DNAmPhenoAge and DNAmGrimAge are associated with longer telomere, hence exercise-induced DNA methylation delay cellular senescence. Moreover, both DNAmPhenoAge acceleration and DNAmGrimAge acceleration showed that higher level of physical fitness suppresses the progress of aging. The health promoting effects of exercise are well documented (Hortobágyi et al. 2022 ; Quan et al. 2020 ; Radak et al. 2012), but this is the first study, to our knowledge, which demonstrates that these changes are based on DNA methylation, hence could be inherited in a certain degree. Surralles et al (Surrallés et al. 1999 ) demonstrated that telomere shortening could be affected by histone acetylation, therefore influenced by epigenetics. Moreover, the same study found that longer lived cell lineages have an active X chromosome with longer telomere than the inactive X, indicating that telomere maintenance alleles on X chromosome impact survival. It was also suggested that in male Y chromosome is less protected and more prone to telomere shortening, but the chromosome dependent telomere shortening is under debate (Genovesi et al. 2021 ). The results of the current study could not demonstrate that at the given population females have longer telomere than males. It was suggested that there is a gender difference in the length of telomere, based on the telomerase activity, which could add base pairs to telomere is influenced by estrogen (Kyo et al. 1999 ), however the study of Lin et al reported that postmenopausal women who had longer endogenous estrogen therapy had longer telomere length with lower telomerase activity (Lin et al. 2011 ). Therefore, the mechanism of telomere shortening with gender bias appears to be complex, which requires further investigations (Barrett and Richardson 2011 ). The associations between TL and physiological functions are very important (Nordfjäll et al. 2008 ; Colon et al. 2019 ; Buttet et al. 2022 ). The age-related loss of cognitive and physical performance is very normal, but the degree of loss and values are very much related to the level of physical fitness (Booth and Roberts 2008 ). Genetics can influence the trainability, but regular exercise can greatly improve the level of physical fitness (Radak and Taylor 2022). In the present study we have shown that DNAm based estimation is more sensitive method to examine the relationship between TL and physiological function, especially with VO2max than RT-PCR based method. In conclusion, the results of this study further emphasize the importance of the level of physical fitness in aging process. The beneficial effects of exercise or physiological fitness on telomere shortening is, at least a part, mediated through changing DNA methylation levels. Declarations Acknowledgements: ZR acknowledge support from the National Excellence Program (126823) and the Scientific Excellence Program, TKP2020-NKA-17 and TKP2021-EGA-37, at the Hungarian University of Sport Science, Innovation and Technology Ministry, Hungary. Authors’ contributions YS, EK, DA, FT, MJ, AB, KS, MH, KT and ZR contributed at the measurements of the study. ZR, IB, SH drafted the final version of the paper, but all contributed to the manuscript writing. All authors have read and approved the final version of the manuscript, and agree with the order of presentation of the authors. Competing interest The authors declare that they have no competing interests. References Barrett E, Richardson DS (2011) Sex differences in telomeres and lifespan. Aging Cell 10:913–21 Bize P, Criscuolo F, Metcalfe N B, et al (2009) Telomere dynamics rather than age predict life expectancy in the wild. Proc Biol Sci 276:1679–83 Booth FW, Roberts CK (2008) Linking performance and chronic disease risk: indices of physical performance are surrogates for health. Br J Sports Med 42:950–2 Buttet M, Bagheri R, Ugbolue UC, et al (2022) Effect of a lifestyle intervention on telomere length: A systematic review and meta-analysis. Mech Ageing Dev 206:111694 Campisi J (2005) Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. 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Nucleic Acids Res 41:e90 Wan ES, Goldstein RL, Fan VS, et al (2019) Telomere length in COPD: Relationships with physical activity, exercise capacity, and acute exacerbations. PLoS One 14:e0223891 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1telomerTLDNAmTL.pptx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 26 Oct, 2022 Reviews received at journal 21 Oct, 2022 Reviewers agreed at journal 12 Oct, 2022 Reviewers invited by journal 12 Oct, 2022 Editor assigned by journal 12 Oct, 2022 Submission checks completed at journal 11 Oct, 2022 First submitted to journal 10 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2151782","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":143443279,"identity":"d755ce7e-6bef-468c-92a0-4f4de49e7371","order_by":0,"name":"Yasuhiro Seki","email":"","orcid":"","institution":"Waseda University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasuhiro","middleName":"","lastName":"Seki","suffix":""},{"id":143443280,"identity":"c6b105c0-fa44-40fd-92ed-e67f1572baf9","order_by":1,"name":"Dora Aczel","email":"","orcid":"","institution":"Hungarian University of Sport 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17:54:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe association between telomere length and chronological age\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTelomere length measured by RT-PCR negatively correlated with chronological age (Panel A, N=241) but gender dependent significant association was not found (Panel B). Panel C and D shows that data obtained by the estimation of TL by DNA methylation. (male N=108, female N=133).\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-2151782/v1/fedae52be634f6d8b87dc67b.png"},{"id":27791391,"identity":"9c54a912-a4de-47b7-8e8d-4f662d9bae62","added_by":"auto","created_at":"2022-10-14 17:59:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between physiological test results and telomere length\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant relationship was not present between RT-PCR based telomere length, body mass index (BMI, Panel A), maximal vertical jump height (Jump max, Panel B) and working memory (Panel D). Panel E, F, G show DNAm based results. N=241\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-2151782/v1/765279ddcfdf6f80bd61686a.png"},{"id":27790809,"identity":"b25cd750-1b76-45a1-b060-fe6b1e2120ef","added_by":"auto","created_at":"2022-10-14 17:54:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between cardiovascular fitness (VO2max) and telomere length\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe maximal oxygen uptake was estimated from the step test results and significant relationship was neither found in the total number of subjects when assessed by RT-PCR gained results (N=241, Panel A) nor in males (N=108 Panel B) and females (Panel C). DNAm based TL calculation on the other hand, showed significant relationships (N=133, Panel D, E, F).\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-2151782/v1/1b2c0ce29d0e0633bfd470bd.png"},{"id":27790807,"identity":"ffd5edac-03b7-4907-b064-3cd653a1eb64","added_by":"auto","created_at":"2022-10-14 17:54:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe correlation between telomere length and DNAmPhenoAge and DNAmGrimAge.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe epigenetical aging was calculated on the DNA methylation pattern based on the description of DNAmPhenoAge and DNAmGrimAge. Both the DNAmPhenoAge (Panel A) and DNAmGrimAge (Panel B) showed significant relationship with RT-PCR based TL. DNAm assessed TL showed even stronger relationship with DNAmPhenoAge and DNAmGrimAge (Panel D, E, F). N=231\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-2151782/v1/c0f5d54279205bd4288e6896.png"},{"id":27791519,"identity":"40da4559-fa19-4264-858e-f0c431072bcd","added_by":"auto","created_at":"2022-10-14 18:00:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":796631,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2151782/v1/776abe8a-ebba-4382-95ce-799003fc9dd5.pdf"},{"id":27790806,"identity":"f0892a09-b145-4622-904b-01b300810728","added_by":"auto","created_at":"2022-10-14 17:54:52","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34013,"visible":true,"origin":"","legend":"","description":"","filename":"Table1telomerTLDNAmTL.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2151782/v1/83e4d17a53cd14600d3713ea.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Association among epigenetic modifications by DNA methylation, telomere length, and physical fitness in biological aging","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHealthy aging with relatively high physiological function is the goal of human society. To achieve this, it would be important to understand the aging process and then develop interventions that help to reach the goals. One of the mechanisms, which known to regulate aging process is based on the limiting factors of cell proliferation, namely on telomeres (Greider \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Telomeres are protective caps on the ends of chromosomes with repeated deoxyribonucleic acid sequences rich in TTAGGG, between 3- and 20-kb- long in humans (Hande \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Oxidative stress causes accelerated telomere shortening primarily through oxidation of guanine due to its lowest oxidation potential among nucleic acid bases especially in telomere quaduplexes (Radak and Boldogh 2010). After certain number of cell division, which results in loss of telomere length, dividing cells cannot replicate anymore reaching cellular senescent (Linskens et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Although the enzyme of telomerase able to add de novo base pairs to telomere (Greider and Blackburn \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), the age-related shortening is well documented (Campisi \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bize et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Indeed, accumulating evidence suggest that a strong link between telomere length and aging and age-associated diseases (Morin \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Sikora \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fyhrquist and Saijonmaa \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sahin and DePinho \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has been shown that telomere length (TL) is dependent on various lifestyle factors. It has been shown that on preschool children obesity was linked to reduced level of docosahexaenoic acid, and increased arachidonic acid/ docosahexaenoic acid ratio and shortened telomere in leukocytes (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Short term administration of nicotinamide mononucleotide resulted in significant elongation of TL in peripheral blood mononuclear cells of C57BL/6 mice (Niu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studies suggest that physical exercise also has beneficial effects on TL (Lee at al. 2013; Manoy et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Loprinzi et al \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Even as short-term exercise program as 12 weeks, with low frequency, moderate intensity, and explosive-type resistance training could have beneficial effects on telomeres. Indeed, above mentioned explosive resistance training lessened telomere shortening and correlated with the amelioration of redox homeostasis (Dimauro et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBecause aging is strongly influenced by life-style Horvath and Hannum developed DNA methylation based epigenetical aging clocks, which more precisely reflect aging than chronological age (Horvath \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hannum et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Moreover, in a huge cohort (n\u0026thinsp;=\u0026thinsp;5,713) it has been shown that there is an interaction between genome wide methylation, TL and epigentical aging (Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Based on previous studies, we hypothesize that the level of physical fitness would affect epigenetical aging clocks and TL of whole blood samples in aged individuals. Moreover, we aimed to compare the interactions between physiological test results and TL assessed by RT-PCR method and DNAmethylation based estimation (Pearce et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eSubjects were volunteers, who participated in 2019 Masters World Rowing Championships in Venice, Hungary and aged matched sedentary individuals. The investigation was carried out on a voluntary basis with the ethics license provided by the Hungarian Scientific and Research Ethical Committee 25167-6/2019/EUIG. Our cohort consisted of a total 241 people, 108 men: 71 athletes (mean age: 60\u0026thinsp;\u0026plusmn;\u0026thinsp;10.5 years, and 37 volunteer controls (mean age 60\u0026thinsp;\u0026plusmn;\u0026thinsp;13.4 years); and 133 women: 80 athletes (mean age: 58\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 years) and 53 sedentary (mean age: 63\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9 years).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological tests\u003c/h2\u003e \u003cp\u003eBody mass and heights were measured and the body mass index was described by body composition monitor BF214 (Omron, Japan). Relative maximum hand gripping force a measure of age-associated decline in general muscle strength (Eika 2019) was assessed by CAMRY EH101 dynamometer. Relative maximal oxygen uptake is one of the best marker a viability and higher level of VO2max is associated with decreased levels of wide range of diseases (Hawkins and Wiswell \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Carnethon et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). We used Chester step test to apprise the level of VO2max (Izquierdo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Digit span test was applied to assess the working memory (Martinez-Diaz et al. 2020), where larger values indicate better verbal short-term memory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of hematologic and telomere length\u003c/h2\u003e \u003cp\u003eDetermination of hematologic and biochemical variables blood samples were collected before the subjects performed the VO2max evaluation test, and were stored in evacuated tubes containing EDTA as an anticoagulant for determination of erythrogram. Blood samples were centrifuged and stored at \u0026minus;\u0026thinsp;80 C degrees. The erythrogram and biochemical tests were carried out in the Clinical Analysis Laboratory of Semmelweis University, Budapest.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDNA isolation\u003c/strong\u003e \u003cp\u003eDNA was isolated from the K2-EDTA anticoagulated blood samples using a DNA isolation kit (Pure LinkTM Genomic DNA Mini kit, Thermo Fisher, Carlsbad, CA, USA), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eMeasurement of telomere length\u003c/em\u003e: The average relative telomere length of genomic DNA was determined from whole blood samples using Cawthon\u0026rsquo;s PCR-based method (Wan et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) with commercially available PCR kit (ScienCell Research Laboratories inc., San Diego CA Catalog no. #8908). During this reaction telomere-specific primers recognize and amplify telomeric sequences. For each DNA sample, two consecutive reactions were performed: the first for amplification of a single-copy reference (SCR) gene and the second for the telomeric sequence. The former recognizes and amplifies a 100 bp region on human chromosome 17 and serves as a reference for calculating the telomere length of the target samples. The PCR reactions were performed in a final volume of 20 \u0026micro;l. We used 5 ng reference/genomic DNA sample (final concentration\u0026thinsp;=\u0026thinsp;0.625 ng/\u0026micro;l), 2\u0026micro;l telomere primer and 10 \u0026micro;l 2XMaster Mix, PCR conditions were as follows: first 95\u0026deg;C for 10 min, followed by 32 cycles of 95\u0026deg;C for 20s, 52\u0026deg;C for 20s, and 72\u0026deg;C for 45s. All samples were tested in triplicate.\u003c/p\u003e \u003cp\u003eIn addition the telomere length was also evaluated by Horvath\u0026rsquo;s software which estimates the telomere length from methylation (Pearce et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMeasurement of DNA Methylation\u003c/em\u003e: Epigenome wide DNA methylation 85K was measured with the Infinium MethylationEPIC BeadChip (Illumina Inc., San Diego, CA) according to the manufacturer\u0026rsquo;s protocol. Briefly, 500 ng of genomic DNA was bisulfite converted using the EZ-96 DNA Methylation MagPrep Kit (Zymo Research, Irvine, CA, USA) with the KingFisher Flex robot (Thermo Fisher Scientific, Breda, Netherlands). The samples were plated in randomized order. The bisulfite conversion was performed according to the manufacturer\u0026rsquo;s protocol with the following modifications: For binding of the DNA 15 \u0026micro;l MagBinding Beads was used. The conversion reagent incubation was done according to the following cycle protocol: 16 cycles of 95\u0026deg;C for 30 seconds followed by 50\u0026deg;C for 1 hour. After the cycle protocol the DNA was incubated for ten minutes at 4\u0026deg;C. Next, DNA samples were hybridized on the Infinium MethylationEPIC BeadChip (Illumina Inc., San Diego, CA) according to the manufacturers protocol with the modification that 8 \u0026micro;l bisulfite treated DNA was used as start material. Quality Control of the DNA methylation data was performed using, Meffil and Ewastools packages with R version 4.0.0. Samples which failed technical controls, including extension, hybridization and bisulfite conversion, according to the criteria set by Illumina, were excluded. Samples with a call rate\u0026thinsp;\u0026lt;\u0026thinsp;96% or at least with 4% of undetected probes were also excluded. Probes with a detection p-value\u0026thinsp;\u0026gt;\u0026thinsp;0.01 in at least 10% of the samples were set as undetected. Probes with a bead number\u0026thinsp;\u0026lt;\u0026thinsp;3 in at least 10% of the samples were excluded. We used the \"noob\" normalization method in R to quantify methylation level (Triche et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The details on the processing of DNAm data and the calculation of the measures of aging, or pace of aging, were calculated using Horvath\u0026rsquo;s online age calculator (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dnamage.genetics.ucla.edu/\u003c/span\u003e\u003cspan address=\"https://dnamage.genetics.ucla.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStatistics\u003c/p\u003e \u003cp\u003eThe results were subjected to statistical tests. After testing for normal distribution, the relevant parametric and non-parametric test methods were applied. The differences between groups were examined by multiway ANOVA for a given variable followed by post hoc Tukey HSD. The interdependence of the individual variables was analyzed using multivariate regression analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eWe have measured or estimated the TL with two different methods one based on RT-PCR measurements while the other was calculated on methylation based estimation of TL.\u003c/p\u003e \u003cp\u003eThe results from the RT-PCR measurements revealed that the TL showed a strong, significant relationship with chronological aging of the subjects (r= -0.23; p\u0026thinsp;=\u0026thinsp;0.0003), and relationship was similar in women and men (women: r= -0.21; p\u0026thinsp;=\u0026thinsp;0.0012 vs men: r= -0.22; p\u0026thinsp;=\u0026thinsp;0.0007) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, we could not detect gender dependent significant difference in the TL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similar data was obtained by DNAm based estimation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to results measured by RT-PCR there are no relationship between BMI, (r= -0.02; p\u0026thinsp;=\u0026thinsp;0.7450, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), vertical jump results (r\u0026thinsp;=\u0026thinsp;0.12; p\u0026thinsp;=\u0026thinsp;0.0689, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), the scores of working memory did not correlate well with telomere length (r\u0026thinsp;=\u0026thinsp;0.03; p\u0026thinsp;=\u0026thinsp;0.622, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and TL. The DNAm based estimation of TL showed similar results with BMI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), while the jumping test results correlated significantly with TL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe maximal oxygen uptake (VO2max) which was calculated from the Chester step tests results showed no relationship with RT-PCR based TL (r\u0026thinsp;=\u0026thinsp;0.011; p\u0026thinsp;=\u0026thinsp;0.82, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). On the other hand, DNAm based estimated resulted significant correlation between VO2max and TL in all subject case (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0045, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and in male (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0028, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and female subjects as well (p\u0026thinsp;\u0026lt;\u0026thinsp;0.002, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen the possible association between RT-PCR measurement originated data and DNA methylation based epigenetical aging was examined, it turned out that in all subject case both DNAmPhenoAge and DNAmGrimAge related to TL (r= -0.76; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; and r= -0.78; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0007) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The relationship between DNAm based TL estimation with DNAmPhenoAge and DNAmGrimAge showed even more powerful relationship (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). When the relationship of DNAmPhenoAge acceleration (r= -0.21; p\u0026thinsp;=\u0026thinsp;0.0012) and DNAmGrimAge acceleration (r= -0.25; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with telomere length were evaluated, the results revealed that longer telomeres were related to decelerated aging (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn present study, our data revealed that TL was associated with DNA methylation based epigenetic aging biomarkers. It has been known that higher level of cardiovascular fitness, the VO2max is associated with longer TL in wide range of age group (18\u0026ndash;72 years old) (LaRocca et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is clear that one of the striking effect of aging is suppressed physiological function, however it is also known that the progress of aging is depends on environmental and life-style factors, including physical fitness (Radak et al. 2019). Indeed, the DNA methylation based epigenetic biomarkers reflect the individual aging more precisely than chronological aging and related to telomere length (Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Here we further report that exercise induced DNA methylation based calculation of DNAmPhenoAge and DNAmGrimAge are associated with longer telomere, hence exercise-induced DNA methylation delay cellular senescence. Moreover, both DNAmPhenoAge acceleration and DNAmGrimAge acceleration showed that higher level of physical fitness suppresses the progress of aging. The health promoting effects of exercise are well documented (Hortob\u0026aacute;gyi et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Quan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Radak et al. 2012), but this is the first study, to our knowledge, which demonstrates that these changes are based on DNA methylation, hence could be inherited in a certain degree.\u003c/p\u003e \u003cp\u003eSurralles et al (Surrall\u0026eacute;s et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) demonstrated that telomere shortening could be affected by histone acetylation, therefore influenced by epigenetics. Moreover, the same study found that longer lived cell lineages have an active X chromosome with longer telomere than the inactive X, indicating that telomere maintenance alleles on X chromosome impact survival. It was also suggested that in male Y chromosome is less protected and more prone to telomere shortening, but the chromosome dependent telomere shortening is under debate (Genovesi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The results of the current study could not demonstrate that at the given population females have longer telomere than males. It was suggested that there is a gender difference in the length of telomere, based on the telomerase activity, which could add base pairs to telomere is influenced by estrogen (Kyo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), however the study of Lin et al reported that postmenopausal women who had longer endogenous estrogen therapy had longer telomere length with lower telomerase activity (Lin et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, the mechanism of telomere shortening with gender bias appears to be complex, which requires further investigations (Barrett and Richardson \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe associations between TL and physiological functions are very important (Nordfj\u0026auml;ll et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Colon et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Buttet et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The age-related loss of cognitive and physical performance is very normal, but the degree of loss and values are very much related to the level of physical fitness (Booth and Roberts \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Genetics can influence the trainability, but regular exercise can greatly improve the level of physical fitness (Radak and Taylor 2022). In the present study we have shown that DNAm based estimation is more sensitive method to examine the relationship between TL and physiological function, especially with VO2max than RT-PCR based method.\u003c/p\u003e \u003cp\u003eIn conclusion, the results of this study further emphasize the importance of the level of physical fitness in aging process. The beneficial effects of exercise or physiological fitness on telomere shortening is, at least a part, mediated through changing DNA methylation levels.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZR acknowledge support from the National Excellence Program (126823) and the Scientific Excellence Program, TKP2020-NKA-17 and TKP2021-EGA-37, at the Hungarian University of Sport Science, Innovation and Technology Ministry, Hungary.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYS, EK, DA, FT, MJ, AB, KS, MH, KT and ZR contributed at the measurements of the study. ZR, IB, SH drafted the final version of the paper, but all\u0026nbsp;contributed to the manuscript writing. All authors have read and approved the final version of the manuscript,\u0026nbsp;and agree with the order of presentation of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarrett E, Richardson DS (2011) Sex differences in telomeres and lifespan. Aging Cell 10:913\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBize P, Criscuolo F, Metcalfe N B, et al (2009) Telomere dynamics rather than age predict life expectancy in the wild. Proc Biol Sci 276:1679\u0026ndash;83\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBooth FW, Roberts CK (2008) Linking performance and chronic disease risk: indices of physical performance are surrogates for health. 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PLoS One 14:e0223891\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biogerontology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Biogerontology](https://www.springer.com/journal/10522)","snPcode":"10522","submissionUrl":"https://submission.nature.com/new-submission/10522/3","title":"Biogerontology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2151782/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2151782/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCellular senescence is greatly accelerated by telomere shortening, and the steps forward in human aging is strongly influenced by environmental and life-style factors, whether DNA methylation (DNAm) is affected by exercise training, remains unclear. In the present study we investigated the relationships between physiological functions, maximal oxygen uptake (VO2max), vertical jump, working memory, telomere length (TL) assessed by RT-PCR, DNAmethylation based estimation of TL (DNAmTL) and DNA methylation based biomarkers of aging of master rowers (N\u0026thinsp;=\u0026thinsp;151) and sedentary subjects (N\u0026thinsp;=\u0026thinsp;90), aged between 37\u0026ndash;85 years. It was found that the TL inversely correlated with chronological age, while no gender dependent difference was found. We could not detect association between telomere length and VO2max, vertical jump and working memory by RT-PCR method, while these physiological test results showed correlation with DNAmTL. DNAmGrimAge and DNAmPhenoAge acceleration were inversely associated with telomere length assessed by both methods. It appears that there is no powerful beneficial effects of exercise or physiological fitness on telomere shortening, however the degree of DNA methylation is associated with telomere length. DNAm based estimation of TL shows stronger relationships with physiological functions than RT-PCR measured data.\u003c/p\u003e","manuscriptTitle":"Association among epigenetic modifications by DNA methylation, telomere length, and physical fitness in biological aging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-14 17:54:50","doi":"10.21203/rs.3.rs-2151782/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-10-26T10:09:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-10-21T08:10:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"cef37cfc-30c8-4490-aef4-e43aa92e1326","date":"2022-10-12T11:13:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-12T09:48:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-12T09:44:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-10-11T13:53:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biogerontology","date":"2022-10-10T16:00:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biogerontology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Biogerontology](https://www.springer.com/journal/10522)","snPcode":"10522","submissionUrl":"https://submission.nature.com/new-submission/10522/3","title":"Biogerontology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"69caf68b-fa3b-4200-a8af-6d4f8c0f159f","owner":[],"postedDate":"October 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-12-15T11:44:27+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-14 17:54:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2151782","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2151782","identity":"rs-2151782","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}