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Exercise attenuates low back pain and alters epigenetic regulation in intervertebral discs in a mouse model

  • Yuya Kawarai
    Affiliations
    Faculty of Dentistry, 2001 McGill College Avenue, Suite 500, McGill University, Montreal, Quebec, H3A 1G1, Canada

    Alan Edwards Centre for Research on Pain, 740 Dr. Penfield Avenue, Suite 3100, McGill University, Montreal, Quebec, H3A 0G1, Canada

    Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba, 260-8677, Japan
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  • Seon Ho Jang
    Affiliations
    Faculty of Dentistry, 2001 McGill College Avenue, Suite 500, McGill University, Montreal, Quebec, H3A 1G1, Canada

    Alan Edwards Centre for Research on Pain, 740 Dr. Penfield Avenue, Suite 3100, McGill University, Montreal, Quebec, H3A 0G1, Canada
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  • Seunghwan Lee
    Affiliations
    Faculty of Dentistry, 2001 McGill College Avenue, Suite 500, McGill University, Montreal, Quebec, H3A 1G1, Canada

    Alan Edwards Centre for Research on Pain, 740 Dr. Penfield Avenue, Suite 3100, McGill University, Montreal, Quebec, H3A 0G1, Canada
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  • Magali Millecamps
    Affiliations
    Faculty of Dentistry, 2001 McGill College Avenue, Suite 500, McGill University, Montreal, Quebec, H3A 1G1, Canada

    Alan Edwards Centre for Research on Pain, 740 Dr. Penfield Avenue, Suite 3100, McGill University, Montreal, Quebec, H3A 0G1, Canada
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  • HyungMo Kang
    Affiliations
    Faculty of Dentistry, 2001 McGill College Avenue, Suite 500, McGill University, Montreal, Quebec, H3A 1G1, Canada

    Alan Edwards Centre for Research on Pain, 740 Dr. Penfield Avenue, Suite 3100, McGill University, Montreal, Quebec, H3A 0G1, Canada
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  • Stephanie Gregoire
    Affiliations
    Faculty of Dentistry, 2001 McGill College Avenue, Suite 500, McGill University, Montreal, Quebec, H3A 1G1, Canada

    Alan Edwards Centre for Research on Pain, 740 Dr. Penfield Avenue, Suite 3100, McGill University, Montreal, Quebec, H3A 0G1, Canada
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  • Miyako Suzuki-Narita
    Affiliations
    Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba, 260-8677, Japan
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  • Seiji Ohtori
    Affiliations
    Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba, 260-8677, Japan
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  • Laura S. Stone
    Correspondence
    Corresponding author. L. Stone, Faculty of Medicine, Department of Anesthesiology, 420 Delaware Street SE, MMC 294, University of Minnesota, Minneapolis, MN, 55455, USA. Tel.: 612-422-9880, fax: 612-626-2363.
    Affiliations
    Faculty of Dentistry, 2001 McGill College Avenue, Suite 500, McGill University, Montreal, Quebec, H3A 1G1, Canada

    Alan Edwards Centre for Research on Pain, 740 Dr. Penfield Avenue, Suite 3100, McGill University, Montreal, Quebec, H3A 0G1, Canada

    Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba, 260-8677, Japan

    Faculty of Medicine, Departments of Pharmacology and Toxicology, Anesthesiology, Neurology and Neurosurgery, 3605 de la Montagne, Montreal, Quebec, H3G 2M1, Canada

    Faculty of Medicine, Department of Anesthesiology, 420 Delaware Street SE, MMC 294, University of Minnesota, Minneapolis, MN, 55455, USA
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Open AccessPublished:June 08, 2021DOI:https://doi.org/10.1016/j.spinee.2021.06.002

      Abstract

      BACKGROUND CONTEXT

      Chronic low back pain (LBP) is a multifactorial disorder with complex underlying mechanisms, including associations with intervertebral disc (IVD) degeneration in some individuals. It has been demonstrated that epigenetic processes are involved in the pathology of IVD degeneration. Epigenetics refers to several mechanisms, including DNA methylation, that have the ability to change gene expression without inducing any change in the underlying DNA sequence. DNA methylation can alter the entire state of a tissue for an extended period of time and thus could potentially be harnessed for long-term pain relief. Lifestyle factors, such as physical activity, have a strong influence on epigenetic regulation. Exercise is a commonly prescribed treatment for chronic LBP, and sex-specific epigenetic adaptations in response to endurance exercise have been reported. However, whether exercise interventions that attenuate LBP are associated with epigenetic alterations in degenerating IVDs has not been evaluated.

      PURPOSE

      We hypothesize that the therapeutic efficacy of physical activity is mediated, at least in part, at the epigenetic level. The purpose of this study was to use the SPARC-null mouse model of LBP associated with IVD degeneration to clarify (1) if IVD degeneration is associated with altered expression of epigenetic regulatory genes in the IVDs, (2) if epigenetic regulatory machinery is sensitive to therapeutic environmental intervention, and (3) if there are sex-specific differences in (1) and/or (2).

      STUDY DESIGN

      Eight-month-old male and female SPARC-null and age-matched control (WT) mice (n=108) were assigned to exercise (n=56) or sedentary (n=52) groups. Deletion of SPARC is associated with progressive IVD degeneration and behavioral signs of LBP. The exercise group received a circular plastic home cage running wheel on which they could run freely. The sedentary group received an identical wheel secured in place to prevent rotation. After 6 months, the results obtained in each group were compared.

      METHODS

      After 6 months of exercise, LBP-related behavioral indices were determined, and global DNA methylation (5-methylcytosine) and epigenetic regulatory gene mRNA expression in IVDs were assessed. This project was supported by the Canadian Institutes for Health Research. The authors have no conflicts of interest.

      RESULTS

      Lumbar IVDs from WT sedentary and SPARC-null sedentary mice had similar levels of global DNA methylation (%5-mC) and comparable mRNA expression of epigenetic regulatory genes (Dnmt1,3a,b, Mecp2, Mbd2a,b, Tet1-3) in both sexes. Exercise attenuated LBP-related behaviors, decreased global DNA methylation in both WT (p<.05) and SPARC-null mice (p<.01) and reduced mRNA expression of Mecp2 in SPARC-null mice (p<.05). Sex-specific effects of exercise on expression of mRNA were also observed.

      CONCLUSIONS

      Exercise alleviates LBP in a mouse model. This may be mediated, in part, by changes in the epigenetic regulatory machinery in degenerating IVDs. Epigenetic alterations due to a lifestyle change could have a long-lasting therapeutic impact by changing tissue homeostasis in IVDs.

      CLINICAL SIGNIFICANCE

      This study confirmed the therapeutic benefits of exercise on LBP and suggests that exercise results in sex-specific alterations in epigenetic regulation in IVDs. Elucidating the effects of exercise on epigenetic regulation may enable the discovery of novel gene targets or new strategies to improve the treatment of chronic LBP.

      Keywords

      Introduction

      Chronic pain is a serious and disabling condition that impacts more than 25% of adults in the United States [
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      ]. Chronic low back pain (LBP) is the second most prevalent reason for impairment in adults in the United States [
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      ]. LBP is a complicated and multifactorial disease, and existing treatments are often ineffective or limited by adverse effects [
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      ]. Despite persistent efforts, satisfactory pain management for chronic LBP has yet to be achieved. The urgency of this unfulfilled medical need is highlighted by the ongoing opioid crisis. New treatments are needed to reduce suffering and to end the growing epidemic of opioid misuse.
      Intervertebral disc (IVD) pathology is one of the major contributors to chronic LBP [
      • Buchbinder R
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      Interleukin-8 as a therapeutic target for chronic low back pain: upregulation in human cerebrospinal fluid and pre-clinical validation with chronic reparixin in the SPARC-null mouse model.
      ,
      • James G
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      • Stone LS
      • Hodges PW
      ISSLS Prize in Basic science 2019: Physical activity attenuates fibrotic alterations to the multifidus muscle associated with intervertebral disc degeneration.
      ,
      • Miyagi M
      • Millecamps M
      • Danco AT
      • Ohtori S
      • Takahashi K
      • Stone LS
      ISSLS Prize winner: Increased innervation and sensory nervous system plasticity in a mouse model of low back pain due to intervertebral disc degeneration.
      ,
      • Millecamps M
      • Tajerian M
      • Sage EH
      • Stone LS
      Behavioral signs of chronic back pain in the SPARC-null mouse.
      ]. Secreted Protein, Acidic, Rich in Cysteine (SPARC)-null mice display progressive age-dependent disc degeneration as well as pathological disc innervation. In addition, decreased SPARC protein levels are linked with painful disc degeneration in humans [
      • Gruber HE
      • Ingram JA
      • Leslie K
      • Hanley Jr, EN
      Cellular, but not matrix, immunolocalization of SPARC in the human intervertebral disc: decreasing localization with aging and disc degeneration.
      ] and decreased mRNA expression of SPARC is associated with normal aging in mouse IVDs [
      • Tajerian M
      • Alvarado S
      • Millecamps M
      • et al.
      DNA methylation of SPARC and chronic low back pain.
      ]. Thus, this model mirrors the impact of normal aging on SPARC protein expression. Both healthy and degenerated discs are normally observed in the same animal, which is consistent with what is observed in humans. By 6 months of age, most SPARC-null mice have multiple moderately degenerated and at least one severely degenerated lumbar IVD.
      It has been demonstrated that epigenetics is involved in the pathology of IVD degeneration [
      • Tajerian M
      • Alvarado S
      • Millecamps M
      • et al.
      DNA methylation of SPARC and chronic low back pain.
      ,
      • Hou Y
      • Shi G
      • Guo Y
      • Shi J
      Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration.
      ]. Epigenetics describes several mechanisms that modify gene expression without generating changes to the underlying DNA sequence [
      • Henikoff S
      • Smith MM
      Histone variants and epigenetics.
      ]. These mechanisms include DNA methylation, histone modification, and chromatin reorganization. Moreover, post-transcriptional mechanisms such as microRNA are also typically described alongside epigenetic mechanisms [
      • Pritchard CC
      • Cheng HH
      • Tewari M
      MicroRNA profiling: approaches and considerations.
      ]. DNA methyltransferases (DNMTs) are enzymes that add a methyl group to cytosine at the 5ʹ­carbon position of the pyrimidine in DNA while enzymes of the ten–eleven translocation (TET) family catalyze the step-by-step oxidation reaction of 5-methylcytosine (5-mC) in DNA to 5-hydroxymethylcytosine and further oxidation products. Given DNA methylation most commonly leads to inhibition of gene transcription, these enzymes modify gene transcription [
      • Niederberger E
      • Resch E
      • Parnham MJ
      • Geisslinger G
      Drugging the pain epigenome.
      ]. MeCP2 and MBD1-3 are members of the methyl-CpG binding domain (MBD) protein family. As transcriptional repressors, MBD proteins are crucial in coordinating interactions between DNA methylation, histone modifications, and chromatin structure to attain a systematic transcriptional program [
      • Du Q
      • Luu PL
      • Stirzaker C
      • Clark SJ
      Methyl-CpG-binding domain proteins: readers of the epigenome.
      ].
      The application of epigenetic concepts to pain and pain therapy has attracted increasing attention over the past decade [
      • Ligon CO
      • Moloney RD
      • Greenwood-Van Meerveld B
      Targeting epigenetic mechanisms for chronic pain: a valid approach for the development of novel therapeutics.
      ,
      • Liang L
      • Lutz BM
      • Bekker A
      • Tao YX
      Epigenetic regulation of chronic pain.
      ,
      • Descalzi G
      • Ikegami D
      • Ushijima T
      • Nestler EJ
      • Zachariou V
      • Narita M
      Epigenetic mechanisms of chronic pain.
      ,
      • Bai G
      • Ren K
      • Dubner R
      Epigenetic regulation of persistent pain.
      ,
      • Burridge S
      Target watch: drugging the epigenome.
      ]. Indeed, an individual's epigenetic status can be modified by environmental influences, including nutrition, exposure to chemicals, emotional challenges, or physical activity. For example, both acute and chronic exercise significantly impact DNA methylation in a highly tissue- and gene-specific manner [
      • Voisin S
      • Eynon N
      • Yan X
      • Bishop DJ
      Exercise training and DNA methylation in humans.
      ]. Studies also indicate long-term changes in DNA methylation can be driven by exposure to nutritional, behavioral, or other environmental stimuli [
      • Vineis P
      • Chatziioannou A
      • Cunliffe VT
      • et al.
      Epigenetic memory in response to environmental stressors.
      ]. Therefore, the application of epigenetics to pain therapy could result in novel therapeutics that are long-lasting compared with the current treatments. To date, the role of epigenetics in LBP, IVD degeneration and physical activity remain unexplored.
      Exercise therapy is one of the fundamental treatments for chronic LBP, and it is recommended in clinical guidelines based on strong evidence [
      • Oliveira CB
      • Maher CG
      • Pinto RZ
      • et al.
      Clinical practice guidelines for the management of non-specific low back pain in primary care: an updated overview.
      ,
      • Qaseem A
      • Wilt TJ
      • McLean RM
      • Forciea MA
      Noninvasive treatments for acute, subacute, and chronic low back pain: A Clinical Practice Guideline from the American College of Physicians.
      ]. Exercise reduces pain in animals [
      • Lima LV
      • Abner TSS
      • Sluka KA
      Does exercise increase or decrease pain? Central mechanisms underlying these two phenomena.
      ,
      • Sluka KA
      • O'Donnell JM
      • Danielson J
      • Rasmussen LA
      Regular physical activity prevents development of chronic pain and activation of central neurons.
      ] and in humans [
      • Babatunde OO
      • Jordan JL
      • Van der Windt DA
      • Hill JC
      • Foster NE
      • Protheroe J
      Effective treatment options for musculoskeletal pain in primary care: A systematic overview of current evidence.
      ]. Exercise not only enhances back strength, flexibility, range of motion, and physical fitness, but also provides acute improvement in mood and protection from anxiety and depression [
      • Hoffman MD
      • Hoffman DR
      Does aerobic exercise improve pain perception and mood? A review of the evidence related to healthy and chronic pain subjects.
      ,
      • Rainville J
      • Hartigan C
      • Martinez E
      • Limke J
      • Jouve C
      • Finno M
      Exercise as a treatment for chronic low back pain.
      ,
      • van Tulder M
      • Koes B
      • Bombardier C
      Low back pain.
      ]. However, the underlying molecular mechanism for pain relief is still uncertain. Studies have recognized that males and females adapt differently in response to exercise [
      • Tarnopolsky MA
      • Rennie CD
      • Robertshaw HA
      • Fedak-Tarnopolsky SN
      • Devries MC
      • Hamadeh MJ
      Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity.
      ,
      • Carter SL
      • Rennie C
      • Tarnopolsky MA
      Substrate utilization during endurance exercise in men and women after endurance training.
      ]. One study discovered sex differences in the timing of the modulation of several biological processes, such as tissue remodeling and metabolism. Significantly, gene expression changes were sustained in males but were swiftly restored in females [
      • Liu D
      • Sartor MA
      • Nader GA
      • et al.
      Skeletal muscle gene expression in response to resistance exercise: sex specific regulation.
      ]. Although accumulating scientific evidence has revealed the effects of exercise on epigenetic regulation of gene and protein expression [
      • McGee SL
      • Hargreaves M
      Epigenetics and Exercise.
      ], whether exercise interventions that attenuate LBP are associated with epigenetic alterations in degenerating IVDs has not been evaluated.
      We hypothesize that the therapeutic efficacy of physical activity is mediated at the epigenetic level. We therefore used the SPARC-null mouse model of LBP associated with IVD degeneration to investigate (1) the impact of disc degeneration on epigenetic regulatory genes in the IVDs, (2) the sensitivity of the epigenetic regulatory machinery to therapeutic environmental change, and (3) the effects of sex on these processes.

      Materials and methods

      Animals

      All research protocols were approved by our Institutional Animal Care Committee (McGill University, AUP #2007-5405) and adhered to the ethical guidelines of the Canadian Council on Animal Care and the guidelines of the International Association for the Study of Pain Committee for Research and Ethical Issues [
      • Zimmermann M
      Ethical guidelines for investigations of experimental pain in conscious animals.
      ].
      SPARC-null and age-matched control animals (wildtype, WT), both reproduced in-house, were used. SPARC-null mice were developed on a mixed C57BL/6 × 129 SVJ background and backcrossed to C57BL/6 [
      • Norose K
      • Clark JI
      • Syed NA
      • et al.
      SPARC deficiency leads to early-onset cataractogenesis.
      ]. C57BL/6 mice (Charles River Laboratories, Montreal, Quebec, Canada) were utilized as WT as in previous studies [
      • Millecamps M
      • Tajerian M
      • Sage EH
      • Stone LS
      Behavioral signs of chronic back pain in the SPARC-null mouse.
      ,
      • Millecamps M
      • Tajerian M
      • Naso L
      • Sage EH
      • Stone LS
      Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice.
      ,
      • Brekken RA
      • Puolakkainen P
      • Graves DC
      • Workman G
      • Lubkin SR
      • Sage EH
      Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM.
      ]. The animals were housed with corncob bedding (7097, Teklad Corncob Bedding, Envigo, UK) and cotton nesting squares for enrichment in same-sex groups of 3 to 5 per pathogen-free ventilated polycarbonate cage (Allentown, Inc, Allentown, NJ, USA). They had unrestricted access to food (2092X Global Soy Protein-Free Extruded Rodent Diet, Irradiated) and water and were on a 12-hour light-dark cycle. Testing apparatus was cleaned with diluted Windex and rinsed with water between animals.
      The animals in the running wheel assay (Fig. 1B,F) and the IVD mRNA extracts (Fig. 3, Fig. 4, Fig. 5) used in this study overlap with those reported by Jang [
      • Jang SH
      • Lee S
      • Millecamps M
      • et al.
      Effect of voluntary running activity on mRNA expression of extracellular matrix genes in a mouse model of intervertebral disc degeneration.
      ].
      Fig 1
      Fig. 1Effect of home cage wheels on body weight, voluntary running and behavioral indices of chronic low back pain.
      No changes in body weight before and after exercise (Ex) were observed in SPARC-null and WT mice in either sex (A, E). Voluntary running during a 60 min trial was significantly higher in all exercise groups compared with the sedentary controls (B, F). Mechanical sensitivity was not affected by strain or exercise in either male (C) or female (G) mice. SPARC-null sedentary mice were more sensitive to cold stimuli compared with WT sedentary mice (D, H). Cold sensitivity in SPARC-null exercise groups was significantly attenuated compared with SPARC-null sedentary groups in both sexes (D, H). Data are presented as mean ± SEM, n=7–16/group. Data were analyzed by two-way ANOVA followed by Sidak's post hoc test. *= p<.05, **= p<.01, ***= p<.001, ****= p<.0001, Sed vs Ex group. ¶¶¶¶=p<.0001, WT vs SPARC-null. Ex, exercise; Sed, sedentary.

      Exercise

      Male and female SPARC-null and WT mice were separated into two groups: sedentary (Sed; n=52) and exercise (Ex; n=56). Starting at 8 months of age, the exercise group was housed with an Innowheel and Innodome (Bio-serve, Flemington, NJ, USA) for 6 months that consisted of an igloo-style hut underneath a rotating wheel for voluntary exercise. Voluntary exercise was selected over treadmill protocols to prevent stress-related confounders and changes in circadian rhythm related to forced exercise [
      • Sasaki H
      • Hattori Y
      • Ikeda Y
      • et al.
      Phase shifts in circadian peripheral clocks caused by exercise are dependent on the feeding schedule in PER2::LUC mice.
      ]. The sedentary groups received the same Innodome and Innowheel, except the wheel was screwed in place to not rotate, thus preventing voluntary exercise. At the end of the 6-month period of intervention, the 14 month old mice were individually weighed.

      Behavioral measurements

      Animals were habituated to the testing room for 60 minutes and to the testing apparatus (von Frey and acetone tests only) for an additional 60 minutes prior to testing. All testing was carried out between 9:00 am and 3:00 pm. The experimenter was blind to strain and treatment group. Behavioral assessments were performed 6 months after wheel placement to evaluate the effect of long-term exercise (n=7–10/group). All behavioral measures have been extensively described in previous studies [
      • Miyagi M
      • Millecamps M
      • Danco AT
      • Ohtori S
      • Takahashi K
      • Stone LS
      ISSLS Prize winner: Increased innervation and sensory nervous system plasticity in a mouse model of low back pain due to intervertebral disc degeneration.
      ,
      • Millecamps M
      • Tajerian M
      • Sage EH
      • Stone LS
      Behavioral signs of chronic back pain in the SPARC-null mouse.
      ,
      • Tajerian M
      • Alvarado S
      • Millecamps M
      • et al.
      DNA methylation of SPARC and chronic low back pain.
      ,
      • Millecamps M
      • Tajerian M
      • Naso L
      • Sage EH
      • Stone LS
      Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice.
      ,
      • Millecamps M
      • Czerminski JT
      • Mathieu AP
      • Stone LS
      Behavioral signs of axial low back pain and motor impairment correlate with the severity of intervertebral disc degeneration in a mouse model.
      ,
      • Tajerian M
      • Millecamps M
      • Stone LS
      Morphine and clonidine synergize to ameliorate low back pain in mice.
      ].

      Measurement of voluntary running with counting wheels

      After the 6 months of exercise, mice were individually placed in a clean polycarbonate cage with fresh bedding containing a low-profile wireless running wheel (ENV-047, Med-Associates Inc). After 5-minute habitation to the enclosure, the total number of wheel's rotation was recorded during one hour to assess animal's running capability. A Wireless USB Hub (DIG-807, Med Associates, Inc.) and Wheel Manager Software (SOF-860, Med Associates, Inc.) were used for data collection [
      • Falkenhain K
      • Ruiz-Uribe NE
      • Haft-Javaherian M
      • et al.
      A pilot study investigating the effects of voluntary exercise on capillary stalling and cerebral blood flow in the APP/PS1 mouse model of Alzheimer's disease.
      ,
      • Giorgetti E
      • Panesar M
      • Zhang Y
      • et al.
      Modulation of microglia by voluntary exercise or CSF1R inhibition prevents age-related loss of functional motor units.
      ]. The wheel was cleaned with soap and water before each new user.

      Measurement of mechanical sensitivity

      Mechanical sensitivity was determined by making use of von Frey filaments (Stoelting Co, Wood Dale, IL, USA) on the plantar surface of the left hindpaw using an up-down technique [
      • Chaplan SR
      • Bach FW
      • Pogrel JW
      • Chung JM
      • Yaksh TL
      Quantitative assessment of tactile allodynia in the rat paw.
      ]. The von Frey filaments were applied for 4 seconds or until paw withdrawal. Results are expressed as the 50% threshold to withdraw in grams as previously described [
      • Millecamps M
      • Tajerian M
      • Sage EH
      • Stone LS
      Behavioral signs of chronic back pain in the SPARC-null mouse.
      ]. The stimulus intensity ranged from 0.008 to 4.0 g.

      Measurement of cold sensitivity

      Cold sensitivity was assessed using the acetone test. Mice were investigated for cold sensitivity for 1 min after a 25 μL drop of acetone was applied to the plantar surface of the left hindpaw. The total duration of time spent in acetone-evoked behaviors (ie, flinching, biting, licking, or scratching) was measured in seconds [
      • Millecamps M
      • Tajerian M
      • Sage EH
      • Stone LS
      Behavioral signs of chronic back pain in the SPARC-null mouse.
      ,
      • Millecamps M
      • Tajerian M
      • Naso L
      • Sage EH
      • Stone LS
      Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice.
      ].

      Global DNA methylation analysis

      At the age of 14 months, after 6 months of voluntary running, the animals were euthanized by decapitation under isoflurane anesthesia, and 5 IVDs (L1/L2–L5/L6) were isolated per mouse (n=4–5/group). Harvested IVDs were immediately flash frozen in liquid nitrogen and stored at −80°C. They were homogenized in liquid nitrogen with a mortar and pestle for DNA extraction. Genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Global DNA methylation was measured with a MethylFlash Methylated DNA Quantification kit (Epigentek, Farmingdale, NY, USA). In 100 ng of genomic disc-derived DNA, methylated DNA was identified using capture and detection antibodies to 5-mC and then measured colorimetrically by reading absorbance at 450 nm using a Spectramax M2e Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of methylated DNA is proportional with the optical density determined. Relative quantification was used to determine the percentage of 5-mC (%5-mC) in total DNA complying with the supplier's instructions. Each sample was run in duplicate.

      RNA extraction and quantitative real-time polymerase chain reaction

      Relative transcript expression levels were determined with quantitative real-time PCR (qPCR) based on SYBR Green technology. Five IVDs (L1/L2–L5/L6) were harvested in the same manner as the DNA extraction. They were immediately crushed in liquid nitrogen using a mortar and pestle. Crushed IVDs were placed in tubes with QIAzol buffer (Qiagen, Hilden, Germany) and completely homogenized with a pellet pestle on ice at all times. Chloroform was added to each tube, and samples were centrifuged for 15 min at 12,000 xg at 4°C. The aqueous layer at the top was carefully obtained. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) in accordance with the supplier's method. The quality of RNA was assessed with Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and RNA Pico Chip Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The range of RNA integrity number of all samples was 6.8 to 9.2. A total of 50 ng of RNA from each sample was reverse-transcribed into single-strand using SuperScript II Reverse Transcriptase (Invitrogen, Waltham, MA, USA). A cDNA template was mixed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), and qPCR was performed with the Rotor-Gene Q real-time PCR cycler (QIAGEN, n=4–6/group in females, n=7/group in males). The primers used for the qPCR analysis are described (Supplemental Table 1) and each qPCR was conducted in duplicate for each sample. The mRNA expression was normalized to the average Ct values of glyceraldehyde 3-phosphate dehydrogenase, β-actin, and β-glucuronidase using the ΔΔCt method [
      • Bustin SA
      • Benes V
      • Garson JA
      • et al.
      The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.
      ,
      • Livak KJ
      • Schmittgen TD
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
      ]. The mean Ct values of the average of the reference genes was equivalent across all treatment groups (Supplemental Table 2). The fold-change relative expression was calculated with the WT-Sedentary group as a reference group to clarify the exercise effects (Results 3.4-1, 3.4-2, 3.4-3). In the comparison of the mRNA expression between males and females (Results 3.4-4), values were calculated by the ΔΔCt method with the exercise group in comparison to the corresponding sedentary group as a reference.

      Statistical analysis

      All data were analyzed by using GraphPad Prism Version 8 (GraphPad Software, San Diego, CA, USA) with p ≤ 0.05 considered statistically significant. Data are presented as mean ± standard error of the mean (SEM). The data were analyzed as indicated in the figure legends.

      Results

      Effect of home cage wheels on body weight and voluntary running

      No changes in body weight after 6 months of voluntary running were observed in SPARC-null or WT mice in either sex (Fig. 1A, 1E). Mice with home cage-running wheels (exercise groups) voluntarily ran more during a 60 min trial compared to sedentary groups in both WT and SPARC-null mice of both sexes. (Fig. 1B, 1F).

      Effect of long-term exercise on behavioral indices of LBP

      No strain-, intervention- or sex-differences were observed in mechanical sensitivity (Fig. 1C, 1G). Sedentary SPARC-null mice of both sexes were more sensitive to cold stimuli compared to WT mice (Fig. 1D, 1H). Hypersensitivity to cold was significantly reduced in the SPARC-null exercise groups compared with the sedentary controls.

      Effect of long-term exercise on global DNA methylation in intervertebral discs

      Global methylation for whole genomic DNA was measured in five discs/mouse and includes CpG sites from promoter, coding sequences, and non-coding sequences. Significant decreases in global methylation were observed after exercise in WT and SPARC-null male mice and SPARC-null female mice (Fig. 2). There was no impact of strain in either males or females, regardless of exercise status (Fig. 2).
      Fig 2
      Fig. 2Effect of exercise on global DNA methylation in intervertebral discs.
      There were significant reductions in the % methylation in exercise (Ex) compared with sedentary (Sed) controls in male and female SPARC-null mice and in male WT mice. Data are presented as mean ± SEM, n=4–5/ group. Data were analyzed by two-way ANOVA followed by Sidak's post hoc test. *= p<.05, **= p<.01, Sed vs Ex group. Ex, exercise; Sed, sedentary.

      Effect of long-term exercise on mRNA expression of epigenetic regulatory genes in the mouse intervertebral discs

      DNA methyltransferases

      We measured mRNA expression of the genes encoding Dnmt1, Dnmt3a and Dnmt3b (Fig. 3). Exercise resulted in a significant decrease in Dnmt3a mRNA expression in male WT and Dnmt3b in female WT mice. No other statistically significant strain- or exercise effects were observed.
      Fig 3
      Fig. 3Effect of exercise on mRNA expression of DNA methyltransferases in intervertebral discs.
      Significant reductions were observed in mRNA expression in WT exercise (Ex) compared to WT sedentary (Sed) groups in Dnmt3a in males (B) and Dnmt3b in females (F). No other significant differences were observed in Dnmt1 (A,D), Dnmt3a (B,E) and Dnmt3b (C,F)). Data are presented as mean ± SEM, n=4–7/ group. Data were analyzed by two-way ANOVA followed by Sidak's post hoc test. *= p<.05, ***= p<.001, Sed vs Ex group. Ex, exercise; Sed, sedentary.

      Methyl-CpG binding domain proteins

      We measured mRNA expression for the genes encoding Mecp2, Mbd2a and Mbd2b (Fig. 4). No significant differences were observed in mRNA expression comparing sedentary WT to sedentary SPARC-null mice in either sex. Exercise resulted in significant reductions in gene expression of Mecp2 in SPARC-null male and female mice. In females, significant increases in mRNA expression of Mbd2a and Mbd2b were noted in the WT exercise group compared with the WT sedentary group.
      Fig 4
      Fig. 4Effect of exercise on mRNA expression of Methyl-CpG binding domain protein family in intervertebral discs.
      There were significant reductions in mRNA expression of Mecp2 in the SPARC-null exercise (Ex) groups compared with the SPARC-null sedentary (Sed) groups in both sexes (A,D). In females, significant increases in gene expression of Mbd2a and Mbd2b were noted in the WT exercise group compared with the WT sedentary group (E, F). Data are presented as mean ± SEM, n=4–7/ group. Data were analyzed by a two-way ANOVA followed by Sidak's post hoc test. *= p<.05, **= p<.05, Sed vs Ex group. Ex, exercise; Sed, sedentary.

      Ten–eleven translocation family

      We measured mRNA expression for the genes encoding Tet1, Tet2 and Tet3 (Fig. 5). Exercise decreased the mRNA expression of Tet1 in WT and SPARC-null males and Tet3 in WT males. No differences were observed in females.
      Fig 5
      Fig. 5Effect of exercise on mRNA expression of Ten–eleven translocation family in intervertebral discs.
      Significant decreases in mRNA expression Tet1 and Tet3 were observed in male WT exercise (Ex) compared to WT sedentary (Sed) groups (A, C). In males, a significant decrease in Tet1 between the SPARC-null sedentary and SPARC-null exercise groups was also observed (A). Data are presented as mean ± SEM, n=4–7/ group. Data were analyzed by two-way ANOVA followed by Sidak's post hoc test. *= p<.05, **= p<.05, Sed vs Ex group. Ex, exercise; Sed, sedentary.

      Sex differences in the effect of long-term exercise on mRNA expression

      The magnitude and direction of methylation changes in males and females in response to exercise are summarized in Fig. 6. In each heatmap, the data are normalized to the same-sex WT sedentary group. This representation illustrates that there were differences between two sexes in gene expression after exercise in Dnmt3a, Mbd2b, and Tet1 (p<.0001, p<.001, p<.001, respectively, 2-way ANOVA, sex effect).
      Fig 6
      Fig. 6Heatmaps of altered mRNA expression of epigenetic regulatory genes in intervertebral discs in males and females.
      To provide an overview of altered mRNA expression in different groups, the magnitude and direction of altered mRNA expression are normalized to the WT sedentary group within each sex on a log2-fold scale. n=4–7/ group. Data were analyzed by a two-way ANOVA followed by Sidak's post hoc test. *= p<.05, **= p<.01, ***= p<.001, WT Sed vs WT Ex group. §= p<.05, SPARC Sed vs SPARC Ex group. ¶= p<.05, WT Sed vs SPARC Sed. Ex, exercise; Sed, sedentary.

      Discussion

      In the present study, we demonstrate that long-term voluntary running exercise improved sensory symptoms associated with LBP, reduced global DNA methylation in healthy and pathologic IVDs, and produced sex-specific differences in the mRNA expression of epigenetic regulatory genes. Indeed, discs extracted from active mice had reduced mRNA expression of Dnmt3a, Mecp2, Tet1 and Tet3 in males and Dnmt3b and Mecp2 in females. Activity increased the expression of Mbd2a and Mbd2b in females. In the comparison between males and females, the effect of exercise on mRNA expression of Dnmt3a, Mbd2b, and Tet1 was sex-specific.

      Impact of degeneration on global DNA methylation and epigenetic regulatory gene expression in intervertebral discs

      This is the first report to describe the impact of IVD degeneration on mRNA expression of the epigenetic regulatory machinery in IVD. In our condition, global DNA methylation and mRNA expression of epigenetic regulatory gene expression was not affected in a model of IVD degeneration in male and female sedentary mice. This suggests that IVD degeneration does not significantly alter the epigenetic regulatory machinery in IVDs. To date, a few studies have reported epigenetic alterations of individual genes in degenerating IVDs in humans or animals. The first report demonstrated that DNA methylation of the SPARC promoter in IVDs is involved in chronic LBP in both mice and humans and that it is increased with age and IVD degeneration, leading to reduced expression of a gene that protects against the progression of disc degeneration [
      • Tajerian M
      • Alvarado S
      • Millecamps M
      • et al.
      DNA methylation of SPARC and chronic low back pain.
      ]. A genome-wide DNA methylation profile study in human LBP patients has identified more than 200 differentially methylated loci that consisted of 187 individual genes, disclosing the early versus advanced stages of degenerated nucleus pulposus tissues in humans display significantly different methylomes [
      • Ikuno A
      • Akeda K
      • Takebayashi SI
      • Shimaoka M
      • Okumura K
      • Sudo A
      Genome-wide analysis of DNA methylation profile identifies differentially methylated loci associated with human intervertebral disc degeneration.
      ]. These studies demonstrated that alterations in gene-specific DNA methylation may be involved in IVD degeneration. Our study, in contrast, suggests that IVD degeneration is not associated with changes in epigenetic regulatory mechanisms as a whole. This observation does not exclude epigenetically-mediated changes in the expression of individual genes in degenerating IVDs, but it does suggest that the epigenetic regulatory machinery remains operational in the degenerating IVDs as in the normal IVDs.

      Effect of long-term voluntary exercise on behavioral indices of low back pain

      Male and female 14 month-old SPARC-null sedentary mice presented cold but not mechanical hypersensitivity that is consistent with our previous reports [
      • Miyagi M
      • Millecamps M
      • Danco AT
      • Ohtori S
      • Takahashi K
      • Stone LS
      ISSLS Prize winner: Increased innervation and sensory nervous system plasticity in a mouse model of low back pain due to intervertebral disc degeneration.
      ,
      • Millecamps M
      • Tajerian M
      • Naso L
      • Sage EH
      • Stone LS
      Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice.
      ,
      • Sasaki H
      • Hattori Y
      • Ikeda Y
      • et al.
      Phase shifts in circadian peripheral clocks caused by exercise are dependent on the feeding schedule in PER2::LUC mice.
      ]. The cold sensitivity observed in the SPARC-null mice is analogous to radiating pain in humans. We previously also reported that IVDs in SPARC-null mice at this age were degenerated [
      • Miyagi M
      • Millecamps M
      • Danco AT
      • Ohtori S
      • Takahashi K
      • Stone LS
      ISSLS Prize winner: Increased innervation and sensory nervous system plasticity in a mouse model of low back pain due to intervertebral disc degeneration.
      ,
      • Millecamps M
      • Tajerian M
      • Naso L
      • Sage EH
      • Stone LS
      Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice.
      ,
      • Sasaki H
      • Hattori Y
      • Ikeda Y
      • et al.
      Phase shifts in circadian peripheral clocks caused by exercise are dependent on the feeding schedule in PER2::LUC mice.
      ] and that the severity of disc degeneration correlated with measures of LBP [
      • Sasaki H
      • Hattori Y
      • Ikeda Y
      • et al.
      Phase shifts in circadian peripheral clocks caused by exercise are dependent on the feeding schedule in PER2::LUC mice.
      ].
      Long-term voluntary running exercise significantly attenuated hypersensitivity to cold in SPARC-null mice in males and females. This is consistent with other studies in chronic LBP in animals. In one study, animals receiving swimming exercise exhibited higher pressure pain thresholds than animals in the control group [
      • de Azambuja G
      • Hortscht U
      • Hoheisel U
      • Oliveira Fusaro MC
      • Mense S
      • Treede RD
      Short-term swimming exercise attenuates the sensitization of dorsal horn neurons in rats with NGF-induced low back pain.
      ]. In another study, running exercise alleviated the mechanical allodynia induced by intradiscal Freund's Adjuvant injection via disc repair and cell proliferation in rats [
      • Luan S
      • Wan Q
      • Luo H
      • et al.
      Running exercise alleviates pain and promotes cell proliferation in a rat model of intervertebral disc degeneration.
      ]. In clinical practice, exercise therapy is one of the fundamental treatments for chronic LBP patients and is recommended in many clinical guidelines based on strong evidence [
      • Oliveira CB
      • Maher CG
      • Pinto RZ
      • et al.
      Clinical practice guidelines for the management of non-specific low back pain in primary care: an updated overview.
      ,
      • Qaseem A
      • Wilt TJ
      • McLean RM
      • Forciea MA
      Noninvasive treatments for acute, subacute, and chronic low back pain: A Clinical Practice Guideline from the American College of Physicians.
      ]. However, a consensus has yet to be reached regarding what type of exercise protocol is the most effective and how long is appropriate. In this study, 6 months of voluntary running ameliorated radiating LBP in SPARC-null mice, suggesting the clinical utility of moderate voluntary exercise. This implies an active lifestyle with regular exercise is beneficial for chronic LBP regardless of sex. Given the age of mice used for this study, an active lifestyle may be beneficial for the middle-aged or older populations. This study supports the validity of exercise therapy for patients with chronic LBP, which is common among the elderly, from the molecular biological aspect.

      Effect of long-term voluntary exercise on global DNA methylation in intervertebral discs

      A decrease in global DNA methylation was observed in IVDs from the exercise groups compared with sedentary groups in WT males and SPARC-null males and females. A trend in the same direction was observed in WT females but statistical significance was not reached. This is the first report on the effects of exercise on global DNA methylation in IVDs in human or rodent. However, it is known that exercise decreases global DNA methylation in other tissues. Barres et al. investigated DNA methylation in vastus lateralis biopsies after exercise and reported an exercise-mediated reduction in global DNA methylation in human participants. Their results demonstrate that exercise-induced DNA hypomethylation has generally been associated with increased gene expression, and with greater effects for higher exercise intensities [
      • Barres R
      • Yan J
      • Egan B
      • et al.
      Acute exercise remodels promoter methylation in human skeletal muscle.
      ]. Denham et al. had revealed that global DNA methylation levels in the sperm of healthy young adult men were decreased by aerobic exercise twice a week for 3 months [
      • Denham J
      • O'Brien BJ
      • Harvey JT
      • Charchar FJ
      Genome-wide sperm DNA methylation changes after 3 months of exercise training in humans.
      ]. The results of the present research are also concordant with other previous reports on the DNA hypomethylation following exercise training in blood leukocytes [
      • Hunter DJ
      • James L
      • Hussey B
      • Wadley AJ
      • Lindley MR
      • Mastana SS
      Impact of aerobic exercise and fatty acid supplementation on global and gene-specific DNA methylation.
      ,
      • Dimauro I
      • Scalabrin M
      • Fantini C
      • Graziol E
      • Beltran Valls MR
      • Mercatelli N
      • et al.
      Resistance training and redox homeostasis: correlation with age-associated genomic changes.
      ] and plasma [
      • da Silva IRV
      • de Araujo CLP
      • Dorneles GP
      • et al.
      Exercise-modulated epigenetic markers and inflammatory response in COPD individuals: a pilot study.
      ].

      Effect of long-term voluntary exercise on epigenetic regulatory gene expression in intervertebral discs

      This is the first study investigating the mRNA expression of epigenetic regulatory genes in IVDs after voluntary running exercise. Voluntary running exercise significantly reduced mRNA expression of Dnmt3a, Tet1 and Tet3 in WT males, Mecp2 and Tet1 in SPARC-null males, Dnmt3b in female WT and Mecp2 in SPARC-null females. Running significantly increased mRNA of Mbd2a and Mbd3b in female WT mice.

      DNA methyltransferases

      The mRNA expression of Dnmt1 in IVDs tended to be reduced by exercise in WT males (p=.08) and showed a trend in the same direction in females. This finding supports the evidence by Yang Hou et al. that inhibition of Dnmt1 could be related to relief of chronic LBP in mice [
      • Hou Y
      • Shi G
      • Guo Y
      • Shi J
      Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration.
      ]. Specifically, they showed that inhibition of Dnmt1 at the promotor region in macrophages in IVDs significantly lowered pro-inflammatory cytokines such as TNFα and IL-6, increased anti-inflammatory cytokines, enhanced anti-inflammatory M2 macrophage polarization, decreased cell apoptosis in the degenerated discs, and attenuated LBP [
      • Hou Y
      • Shi G
      • Guo Y
      • Shi J
      Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration.
      ].
      In addition, exercise significantly reduced the mRNA expression of Dnmt3a in males and Dnmt3b in females in our study. Both DNMT3a and DNMT3b regulate the addition of methyl groups to cytosine bases during de novo methylation; this is in contrast to DMNT1, which maintains methyl marks during subsequent cell divisions [
      • Jacques M
      • Hiam D
      • Craig J
      • Barres R
      • Eynon N
      • Voisin S
      Epigenetic changes in healthy human skeletal muscle following exercise- a systematic review.
      ]. Cells in degenerating IVDs undergo less cell division than in healthy discs due to an increase in senescence, in which disc cells are arrested in the G1 or G0 stage of the cell cycle [
      • Feng C
      • Liu H
      • Yang M
      • Zhang Y
      • Huang B
      • Zhou Y
      Disc cell senescence in intervertebral disc degeneration: causes and molecular pathways.
      ]. It is therefore reasonable to propose that DNMT3a and DNMT3b, the de novo DNA methyltransferases, might play a greater role in degenerative IVDs than DNMT1. It has been proposed that exercise also alters gene and protein expression of Dnmt3a and Dnmt3b in human blood. Hunter et al. revealed that the mRNA expression of both Dnmt3a and Dnmt3b in human peripheral blood mononuclear cells was reduced by aerobic exercise [
      • Hunter DJ
      • James L
      • Hussey B
      • Wadley AJ
      • Lindley MR
      • Mastana SS
      Impact of aerobic exercise and fatty acid supplementation on global and gene-specific DNA methylation.
      ]. Another study described exercise-conditioned plasma attenuates nuclear concentrations of DNMT3b protein in human peripheral blood mononuclear cells [
      • Horsburgh S
      • Todryk S
      • Toms C
      • Moran CN
      • Ansley L
      Exercise-conditioned plasma attenuates nuclear concentrations of DNA methyltransferase 3B in human peripheral blood mononuclear cells.
      ]. These are consistent with our finding that voluntary exercise reduced the mRNA expression of Dnmt3a and Dnmt3b.

      Methyl-CpG binding domain proteins

      In the SPARC-null mice, exercise significantly reduced the mRNA expression of Mecp2 in both sexes. Accumulating evidence suggests that MeCP2 may be associated with chronic pain. MeCP2 is expressed in mature neurons and glial cells [
      • Skene PJ
      • Illingworth RS
      • Webb S
      • et al.
      Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state.
      ] and loss of function of the MeCP2 protein causes Rett syndrome, a neurodevelopmental disease that is primarily caused by mutations of MeCP2 locus [
      • Cuddapah VA
      • Pillai RB
      • Shekar KV
      • et al.
      Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity in Rett syndrome.
      ]. Pain hypoalgesia has been reported in Rett syndrome patients [
      • Cuddapah VA
      • Pillai RB
      • Shekar KV
      • et al.
      Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity in Rett syndrome.
      ,
      • Downs J
      • Geranton SM
      • Bebbington A
      • et al.
      Linking MECP2 and pain sensitivity: the example of Rett syndrome.
      ] and MeCP2 mutant mice displayed decreased sensitivity to noxious heat [
      • Samaco RC
      • McGraw CM
      • Ward CS
      • Sun Y
      • Neul JL
      • Zoghbi HY
      Female Mecp2(+/-) mice display robust behavioral deficits on two different genetic backgrounds providing a framework for pre-clinical studies.
      ]. Infiltration of sensory nerve fibers from the dorsal root ganglia into degenerating discs is hypothesized to be a primary neural mechanism driving discogenic back pain [
      • Yang G
      • Liao W
      • Shen M
      • Mei H
      Insight into neural mechanisms underlying discogenic back pain.
      ]. We previously reported increased nerve fiber density in IVDs of aging SPARC-null mice [
      • Miyagi M
      • Millecamps M
      • Danco AT
      • Ohtori S
      • Takahashi K
      • Stone LS
      ISSLS Prize winner: Increased innervation and sensory nervous system plasticity in a mouse model of low back pain due to intervertebral disc degeneration.
      ] and increased expression of Nerve Growth Factor (NGF) in degenerating human discs drives neurite outgrowth [
      • Krock E
      • Rosenzweig DH
      • Chabot-Doré AJ
      • et al.
      Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors.
      ]. Interestingly, impaired function of MeCP2 in Rett syndrome has been linked to reduced expression of NGF [
      • Calamandrei G
      • Aloe L
      • Hajek J
      • Zappella M
      Developmental profile of serum nerve growth factor levels in Rett complex.
      ,
      • Lipani JD
      • Bhattacharjee MB
      • Corey DM
      • Lee DA
      Reduced nerve growth factor in Rett syndrome postmortem brain tissue.
      ]. It is therefore possible that reduced levels of MeCP2 after therapeutic exercise might attenuate NGF expression in IVDs, leading to decreased pathological innervation and reduced pain.
      Manners et al. revealed that the expression of Mecp2 was increased in mouse dorsal root ganglia after spared nerve injury [
      • Manners MT
      • Tian Y
      • Zhou Z
      • Ajit SK
      MicroRNAs downregulated in neuropathic pain regulate MeCP2 and BDNF related to pain sensitivity.
      ]. Our data showing exercise-induced reductions in mRNA expression of Mecp2 in IVDs is consistent with that previous study. In addition, Manners et al. reported that MeCP2-null mice had reduced levels of Dnmt1, proposing that Mecp2 is linked to DNMT1-mediated maintenance of DNA methylation [
      • Manners MT
      • Ertel A
      • Tian Y
      • Ajit SK
      Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury.
      ]. Given that exercise reduced both mRNA expression of Mecp2 and global DNA methylation in IVDs, this study supports the hypothesis that a decrease in Mecp2 expression mediates global DNA methylation via DNMT-mediated maintenance of DNA methylation.
      Voluntary running exercise increased mRNA expression of Mbd2a and Mbd2b in WT females in the current study. MBD2 is the only member of the family of methyl-CpG-binding proteins that has been reported to be both a transcriptional repressor and a DNA demethylase [
      • Detich N
      • Theberge J
      • Szyf M
      Promoter-specific activation and demethylation by MBD2/demethylase.
      ]. Interestingly, WT females were the only group where exercise did not significantly alter global DNA methylation. Thus, the role Mbd2 mRNA expression and exercise-mediated decreases in global DNA methylation requires further investigation.

      Ten–eleven translocation family

      In healthy mammalian cells, methylation patterns are the result of the competing activities of DNMTs and TET enzymes. Tet gene expression generally correlates with genomic methylation levels. For example, if the genomic methylation level is high, the TET enzyme activity increases to maintain homeostasis in methylation. On the other hand, if genomic methylation is low, TET activity decreases, indicating negative feedback regulation [
      • Lopez-Moyado IF
      • Tsagaratou A
      • Yuita H
      • et al.
      Paradoxical association of TET loss of function with genome-wide DNA hypomethylation.
      ,
      • Yang L
      • Yu SJ
      • Hong Q
      • Yang Y
      • Shao ZM
      Reduced expression of TET1, TET2, TET3 and TDG mRNAs are associated with poor prognosis of patients with early breast cancer.
      ,
      • Yang H
      • Liu Y
      • Bai F
      • et al.
      Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation.
      ]. Accordingly, the mRNA expression of Tet family members decreases with decreased DNA methylation, which is consistent with our finding that exercise resulted in reduced global DNA hypomethylation and reduced mRNA expression of Tet1 and Tet3.

      Sex-specificity of long-term voluntary exercise on global DNA methylation and epigenetic regulatory machinery in intervertebral discs

      In the currently study. significant sex-specificity in mRNA gene expression in response to exercise were observed in Dnmt3a, Mbd2b, and Tet1. It is uncertain why global methylation after exercise is similar between males and females despite the difference in mRNA expression of epigenetic regulatory genes. One hypothesis is other epigenetic mechanisms such as histone modifications influence the global DNA methylation. Indeed, histone modifications are a key epigenetic regulator of DNA methylation. For example, the Dnmt3a enzyme is not able to bind its DNA sequence target when the histone H3 lysine 4 is tri-methylated [
      • Dhayalan A
      • Rajavelu A
      • Rathert P
      • et al.
      The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation.
      ]. To date, no studies have reported alterations in histone methylation in response to exercise, yet alone in a sex-specific fashion. Further research on sex-specific differences in DNA methylation and interactions with other epigenetic mechanisms such as histone modifications in response to exercise are needed to better understand the adaptive response to exercise.
      Studies have reported that males and females adapt differently to exercise [
      • Tarnopolsky MA
      • Rennie CD
      • Robertshaw HA
      • Fedak-Tarnopolsky SN
      • Devries MC
      • Hamadeh MJ
      Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity.
      ,
      • Carter SL
      • Rennie C
      • Tarnopolsky MA
      Substrate utilization during endurance exercise in men and women after endurance training.
      ,
      • Aguiar Jr., AS
      • Speck AE
      • Amaral IM
      • Canas PM
      • Cunha RA
      The exercise sex gap and the impact of the estrous cycle on exercise performance in mice.
      ]. A few studies have proposed that exercise may cause sex-specific alterations in DNA methylation of skeletal muscle. One study indicated that approximately 5,000 loci were differentially methylated in men and women after 3 months of knee extensor exercise, and sex was identified as a significant contributing factor to variability in DNA methylation [
      • Lindholm ME
      • Marabita F
      • Gomez-Cabrero D
      • et al.
      An integrative analysis reveals coordinated reprogramming of the epigenome and the transcriptome in human skeletal muscle after training.
      ]. Since sex was dealt with as a confounder, no direct statistical comparison between methylation changes in men and women in response to exercise was performed. Also, a meta-analysis study stated that larger effect sizes in DNA methylation were observed in women than men following exercise, proposing sex differences in the epigenetic response to exercise [
      • Brown WM
      Exercise-associated DNA methylation change in skeletal muscle and the importance of imprinted genes: a bioinformatics meta-analysis.
      ]. However, specific differences between sexes were not examined because it was not the aim of the study. Therefore, future studies should aim for sample sizes large enough to discover potential sex-differences and perform sex-specific analyses.

      Limitations

      First, it is uncertain how much each animal ran during 6 months since the amount of exercise was not controlled. However, voluntary running during a 60 min trial was significantly higher in the exercise groups compared with the sedentary groups in both WT and SPARC-null mice of both sexes. We opted for this voluntary approach because treadmill running might provoke stress-related confounders. Second, the samples used in this study are from mouse IVDs. While acquiring IVDs from humans is challenging, further research with human IVDs is necessary to develop these findings. Third, RNA integrity numbers varied from 6.8 to 9.2 among RNA samples, and the number of biological repeats ranged from 4 to 7. This variability in quality and limited ‘n’ increases the possibility of false positive or negative results; these results therefore need to be replicated in future studies. Fourth, these data do not directly link the epigenetic changes in the IVDs to the therapeutic improvement in LBP. Indeed, voluntary exercise might attenuate LBP through other mechanisms in other systems (eg, immune and central nervous systems). Finally, the DNA methylation and transcriptional status of individual genes that contribute directly to IVD inflammation or nociceptor sensitization should be examined in a future study. We are currently exploring the DNA methylation status in the promoter areas of pain-related genes in IVDs.

      Clinical relevance

      Epigenetic modifications are at the interface between environmental influence and genetics, generating a mechanism through which lifestyle or life experiences can embed long-lasting changes in gene expression. Exercise therapy is a fundamental treatment for chronic LBP patients, nevertheless, its molecular biological mechanism is still uncertain. This study confirmed the therapeutic benefits of exercise on LBP in a pre-clinical model and suggests that exercise results in sex-specific alterations in epigenetic regulation in IVDs. While further studies are required to link improved outcomes to altered DNA methylation, targeting epigenetic mechanisms with lifestyle change could have a long-lasting therapeutic impact by changing tissue homeostasis in IVDs.

      Conclusions

      Long-term voluntary running exercise improved sensory symptoms associated with LBP, reduced global DNA methylation in healthy and pathologic IVDs, and produced sex-specific differences in the mRNA expression of DNA methylation regulatory genes. Elucidating epigenetic changes after exercise will provide increased understanding of the underlying mechanisms of physical activity on chronic LBP.

      Declarations of competing interests

      Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article.

      Acknowledgments

      We thank the staff of McGill University's Comparative Medicine and Animal Resources Center, the Alan Edwards Centre for Research on Pain, and Dr. Moshe Syzf's lab for their support. This work was supported by the Canadian Institutes for Health Research Projects Grants MOP‐142291 to LSS and MM and PJT-148636 to LSS. YK was supported by the Uehara Memorial Foundation Overseas postdoctoral fellowships and postdoctoral training grant from the Fonds de Recherche du Québec Santé (No dossier 284676) . SL was supported by the Catherine Bushnell postdoctoral fellowship from the Louise and Alan Edwards Foundation .

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