UBC Faculty Research and Publications

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice Safdar, Adeel; Khrapko, Konstantin; Flynn, James M; Saleem, Ayesha; De Lisio, Michael; Johnston, Adam P W; Kratysberg, Yevgenya; Samjoo, Imtiaz A; Kitaoka, Yu; Ogborn, Daniel I; Little, Jonathan P; Raha, Sandeep; Parise, Gianni; Akhtar, Mahmood; Hettinga, Bart P; Rowe, Glenn C; Arany, Zoltan; Prolla, Tomas A; Tarnopolsky, Mark A Jan 31, 2016

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Exercise-induced mitochondrial p53 repairsmtDNA mutations in mutator miceSafdar et al.Safdar et al. Skeletal Muscle  (2016) 6:7 DOI 10.1186/s13395-016-0075-9RESEARCH Open AccessExercise-induced mitochondrial p53 repairsmtDNA mutations in mutator miceAdeel Safdar1,2,3, Konstantin Khrapko6, James M. Flynn7, Ayesha Saleem2, Michael De Lisio1, Adam P. W. Johnston1,Yevgenya Kratysberg6, Imtiaz A. Samjoo4, Yu Kitaoka2, Daniel I. Ogborn4, Jonathan P. Little8, Sandeep Raha2,Gianni Parise1,5, Mahmood Akhtar3, Bart P. Hettinga2, Glenn C. Rowe9, Zoltan Arany10, Tomas A. Prolla11,12 andMark A. Tarnopolsky2,3*AbstractBackground: Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA(mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associatedwith greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are wellestablished, the molecular mechanisms instigating these observations remain unclear.Results: Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increaseslifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We reportevidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 iscanonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response toendurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNAmutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigatepremature mortality.Conclusions: Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome andpresent mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.Keywords: Skeletal muscle, Satellite cells, Endurance exercise, p53, Mitochondrial DNA mutations, Mutator mouse,Oxidative stress, Telomere, Apoptosis, SenescenceBackgroundThe universality of the aging phenomenon has evokedgreat interest in unveiling regenerative remedies and re-juvenation medicine designed to evade molecular insti-gators of mammalian aging. Molecular investigations ofage-related pathologies implicate mitochondrial DNA(mtDNA) mutations as one of the primary instigatorsdriving multisystem degeneration, stress intolerance, andenergy deficits [1]. It is intuitive to assume that the denovo mtDNA mutations observed during aging are dueto accumulated, unrepaired oxidative damage, but someevidence actually suggests that mtDNA replication er-rors may be the more important culprit [2]. The demon-stration that multiple aspects of aging are accelerated inmutator mice harboring error-prone mitochondrial poly-merase gamma provides support for the causal role ofmtDNA replication errors in instigating mammalianaging [3, 4]. Similar phenotypes have also been reportedin telomerase-deficient mice [5], where telomere dys-function is associated with impaired mitochondrial bio-genesis and metabolic failure resulting in progressivetissue atrophy, stem cell depletion, organ system failure,and impaired tissue injury responses as seen with aging[5]. Indeed, epidemiological studies have correlated de-creased telomere length in peripheral blood leukocytes,with higher mortality rates in individuals more than60 years old [6]. Furthermore, a recent study in* Correspondence: tarnopol@mcmaster.ca2Department of Pediatrics, McMaster University, Hamilton, ON L8N 3Z5,Canada3Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5,CanadaFull list of author information is available at the end of the article© 2016 Safdar et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Safdar et al. Skeletal Muscle  (2016) 6:7 DOI 10.1186/s13395-016-0075-9centenarians and their offspring found a positive link be-tween telomere length and longevity; in particular, thosewith longer telomeres had an overall improved healthprofile, with decreased incidence of age-associated dis-eases, better cognitive function, and improved lipid pro-files relative to controls [7].The epidemic emergence of modern chronic diseaseslargely stems from the adoption of a sedentary lifestyleand excess energy intake [8]. There is incontrovertibleevidence that endurance exercise extends life expectancyand reduces the risk of chronic diseases in both rodentsand humans [9, 10]. We have previously shown that en-durance exercise effectively rescued progeroid aging inmutator mice concomitant with a reduction in mtDNAmutations, despite an inherent defect in mitochondrialpolymerase gamma (POLG1) proofreading function [11].Exercise has also been shown to increase telomerase ac-tivity and reduce senescence markers [12]. These find-ings suggest a link between exercise-mediated metabolicadaptations and genomic (nuclear and mitochondrial)stability; however, the identity of this metabolic link re-mains unknown. In this study, we have utilized PolGmice to investigate the mitochondrial-telomere dysfunc-tion axis in the context of progeroid aging, and toelucidate how exercise counteracts mitochondrialdysfunction and mtDNA mutation burden throughmitochondrial localization of the tumor suppressorprotein p53.MethodsMice breedingHeterozygous mice (C57Bl/6J, PolgA+/D257A) for themitochondrial polymerase gamma knock-in mutationwere a kind gift from Dr. Tomas A. Prolla, Universityof Wisconsin-Madison, USA [4]. We generated homo-zygous knock-in mtDNA mutator mice (PolG; Pol-gAD257A/D257A) and littermate wild-type (WT; PolgA+/+) from heterozygous mice-derived colony main-tained at the McMaster University Central Animal Fa-cility as previously described [11]. Muscle-specific p53knock-out mice (p53 MKO) were bred by crossingp53 flox mice (Trp53tm1Brn/J) with muscle-creatinekinase Cre recombinase mice (Tg(Ckmm-cre)5Khn/J)purchased from Jackson Laboratories. We generatedgenetically modified homozygous knock-in mtDNAmutator mice with muscle-specific p53 knockout(PolG-p53 MKO), by crossing heterozygous mice(PolgA+/D257A) with p53 MKO mice. During breeding,all animals were housed three to five per cage in a12-h light/dark cycle and were fed ad libitum(Harlan-Teklad 8640 22/5 rodent diet) after weaning.The presence of the polymerase gamma homozygousknock-in mutation was confirmed as previouslydescribed [4].Endurance exercise protocolEndurance exercise protocol and tissue harvesting wascarried out, as previously described, using an independ-ent cohort of mice [11]. Briefly, at 3 months of age, micewere housed individually in micro-isolator cages in atemperature- and humidity-controlled room and main-tained on a 12-h light–dark cycle with food and waterad libitum [13]. PolG mice and PolG-p53 MKO micewere randomly assigned to sedentary (PolG-SED orPolG-p53 MKO-SED) or forced-endurance (PolG-ENDor PolG-p53 MKO-END) exercise groups (n = 5–20/group; ♀ =♂). None of the mice had been previouslysubjected to a structured exercise regiment. One weekof pre-training was allowed to acclimatize mice in en-durance exercise groups to the treadmill. Mice in endur-ance exercise groups were subjected to forced treadmillexercise (Eco 3/6 treadmill; Columbus Instruments,Columbus, Ohio) three times per week at 15 m/min for45 min for 6 months. A 5-min warm-up and cool-downat 8 m/min were also included. PolG mice were age- andsex-matched with sedentary littermate WT mice (n = 20;♀ =♂), which served as controls for the study to assessif endurance exercise intervention can molecularly bringPolG mice to normalcy. At 8 months of age, animalswere euthanized and tissues were collected for molecularanalyses. The study was approved by the McMasterUniversity Animal Research and Ethics Board under theglobal Animal Utilization Protocol # 12-03-09, and theexperimental protocol strictly followed guidelines putforth by the Canadian Council of Animal Care.Endurance stress testThe mice were subjected to four separate endurancestress tests over to indirectly assess improvements inaerobic capacity with exercise as previously described[11]. Briefly, animals from all groups were placed in indi-vidual lanes on the treadmill and allowed to acclimatizefor 30 min to eliminate any confounding effects due tostress or anxiety related to a new environment. The testbegan with a 5-min warm-up session at 8 m/min,followed by +1 m/min increase in speed every 2 minuntil the mouse reached exhaustion. A low-intensityelectrically stimulus was provided to ensure compliance.Time to exhaustion (min) was recorded when the mousesat at the lower end of the treadmill, near a shock bar,for >10 s and was unresponsive to further stimulation tocontinue running.Survival analysisAn independent cohort of animals from all groups wasused to carry out survival analyses as previously de-scribed [11], and Kaplan–Meier survival curves were cal-culated using GraphPad Prism 4.0.Safdar et al. Skeletal Muscle  (2016) 6:7 Page 2 of 17Tissue harvestingTissues were collected at the time of euthanasia as previ-ously described [11]. Immediately following cervical dis-location, the chest cavity was exposed and the heart wasremoved rapidly, followed by the skeletal muscle (quad-riceps femoris). The skeletal muscle (quadriceps femoris,tibialis anterior, and soleus) and heart were either (i) col-lected in RNase-free cryovials, immediately immersed inliquid nitrogen, and stored at −80 °C for later analysis ofDNA, RNA, protein, and enzyme activity or (ii) immedi-ately rinsed with phosphate buffer saline (PBS) and usedfor skeletal muscle and heart mitochondrial and nuclearfractionations.Hematopoietic stem and progenitor cell isolationMouse hematopoietic stem and progenitor cells (HSC)were isolated according to the method of Ema et al. withminor modifications [14]. Marrow was flushed from thefemur and tibia using a 25-g needle, passed through a 50-μm sieve and counted with a hemocytometer. Cells wereincubated with primary antibodies for 90 min at 4 °Cfollowed by 20 min incubation in the appropriate second-ary antibody at 4 °C. Lineage negative, and Sca-1 and c-Kit positive (LSK) population enriched for stem cellswere sorted using the EPICS ALTRA™ fluorescence-activated cell sorter (Beckman Coulter, Mississauga,ON) with gating strategies established using single-stained controls. The following antibodies were used:lineage panel (BD Pharmingen™, Mississauga, ON),anti-mouse Sca-1 Clone: E13-161.7 (BD Pharmingen™,Mississauga, ON), anti-mouse c-Kit Clone: 2B8(eBioscience, San Diego, CA), and streptavidin (Bio-Source, Burlington, ON).Satellite cell isolationPrimary skeletal muscle satellite cells (SC) were isolatedfrom WT, PolG-SED, and PolG-END mice using themethods described previously [15] and subsequentlypurified by fluorescence-activated cell sorting. Briefly,the hind limb skeletal muscles were carefully dissected,cleaned of fat and washed in cold PBS. Cells were re-leased by mulching the tissue with scissors and incuba-tion in a collagenase/dispase solution three times,12 min each, at 37 °C with further mechanical disruptionusing a pipette between incubations. Following passagethrough 70 and 30 μm filters, cells were stained usingprimary antibody to c-met conjugated to PE (1:200,eBioscience, San Diego, CA) and subjected to FACSsorting (EPICS ALTRA™, Beckman Coulter, Mississauga,ON). SC were pelleted in RNase-free cryovials, immedi-ately immersed in liquid nitrogen, and stored at −80 °Cfor later analyses.Mouse embryonic fibroblast isolation and reporter assayMouse embryonic fibroblasts (MEFs) were generatedusing standard techniques from WT (p53+/+) and p53knockout (KO) mice (p53−/−). Cell used in the experi-ments were from passages 4–5. Promoter sequence forPGC-1α was amplified by PCR from mouse muscle gen-omic DNA and cloned into the pGL4 luciferase reportervector (Promega, Madison, WI). The pG13-luc plasmidcontaining 13 copies of a synthetic p53 DNA bindingsite was used as a positive control (which has been com-prehensively characterized in Jackson et al., 2001 andKern et al., 1991). A GFP expressing plasmid was usedto normalize transfection efficiency. p53+/+ and p53−/−MEFs were transfected (Lipofectamine 2000, Invitrogen,Burlington, ON) with either empty pGL4, pG13-luc(positive control), or pGL4-PGC-1α vectors. p53 tran-scriptional activity was measured using Bright-Glo™ lu-ciferase reporter assay system (Promega, Madison, WI).Total RNA isolation from skeletal muscle and heartTotal RNA was isolated from ~25 mg of the skeletalmuscle (quadriceps femoris) and heart using theQiagen total RNA isolation kit (Qiagen, Mississauga,ON) [11, 13]. RNA samples were treated withRNase-free DNase on Qiagen spin-columns (Qiagen,Mississauga, ON) to remove DNA contamination.RNA integrity and concentration were assessed usingthe Agilent 2100 Bioanalyzer (Agilent Technologies,Palo Alto, CA) [13]. The average RIN (RNA integritynumber) value for all samples was 9.64 ± 0.20 (scale1–10), ensuring a high quality of isolated RNA.RNA, DNA, and protein isolation from HSC and SCTotal RNA, DNA, and protein were isolated from HSCand SC using the Qiagen AllPrep DNA/RNA Mini Kit(Qiagen, Mississauga, ON) according to the manufac-turer’s instructions.Microarray analysisTotal RNA was extracted from skeletal muscle (quadri-ceps femoris) using the Qiagen RNeasy Micro kit(Qiagen, Mississauga, ON) and processed on Qiagen’sQIAcube (Qiagen, Mississauga, ON) using the standardmanufacturer’s protocol. The samples were then checkedfor quality using Nanodrop 2000 (Thermo Scientific,Wilmington, DE) and Agilent 2100 Bioanalyzer (AgilentTechnologies, Palo Alto, CA). TransPlex Whole Tran-scriptome Amplification kit (Sigma-Aldrich®, Oakville,ON) was used to amplify complementary (cDNA) fromthe muscle RNA samples according to the manufac-turer’s instructions. Samples were amplified for 25 cyclesusing the recommended cycling parameters. All sampleswere subsequently purified using Qiagen’s QIAquickPCR Purification kit (Qiagen, Mississauga, ON) andSafdar et al. Skeletal Muscle  (2016) 6:7 Page 3 of 17processed on Qiagen’s QIAcube (Qiagen, Mississauga,ON) using the standard “Cleanup QIAquick PCR foramplification reactions” (Version 4) protocol. Sampleswere purified and examined using Nanodrop 2000(Thermo Scientific, Wilmington, DE) and Agilent 2100Bioanalyzer DNA 7500 chip (Agilent Technologies, PaloAlto, CA) to ensure proper yield and quality of amplifi-cation. To perform the microarray hybridization, 2 μg ofcDNA from each sample was labeled using NimbleGen’sOne Color Labeling kit (Cat.# 05223555001; RocheNimbleGen Inc., Madison, WI) according to the manu-facturer’s protocol. Five micrograms of Cy3 labeledsamples were hybridized to Mus musculus 12x135k,NimbleGen Gene Expression Arrays (Cat.# 05543797001;Roche NimbleGen Inc., Madison, WI), washed, andscanned according to manufacturer’s protocol. NimbleGengene expression arrays were scanned using an AxonGenePix 4200A scanner (Molecular Devices Inc., Down-ingtown, PA) with settings of 100 POW and 300–350photomultiplier (PMT). Pair files were generated for eacharray using NimbleScan software (Roche NimbleGen Inc.,Madison, WI). Resulting array data was analyzed with Bio-conductor software (Bioconductor, Seattle, WA) in whichthe data were normalized and tested for significantly dif-ferentially expressed genes which were assessed basedupon a 5 % false discovery rate (FDR). The gene array datareported here is deposited in Gene Expression Omnibus(Accession Number: GSE75869) public functional genom-ics data repository. The resulting data were input into In-genuity Pathway Analysis (Ingenuity® Systems, RedwoodCity, CA) to determine the over-represented gene categor-ies using strict association. The normalized expression inthese categories was plotted in a heat map using R scriptand Bioconductor software (Bioconductor, Seattle, WA).Real-time quantitative PCRThe messenger RNA (mRNA) expression of peroxisomeproliferator-activated receptor gamma co-activator 1alpha (PGC-1α), mitochondrial transcription factor A(TFAM), estrogen-related receptor alpha (ERRα), 5-aminolevulinate synthase (ALAS), cytochrome c oxidasesubunit-I (COX-I), cytochrome c oxidase subunit-IV(COX-IV), complex I NADH dehydrogenase subunit 1(ND1), complex V subunit ATPase 6 (ATPase 6), cyclin-dependent kinase inhibitor 1A (p21WAF1), cyclin-dependent kinase inhibitor 2A (p16INK4A), and growtharrest and DNA-damage-inducible beta (GADD45B)were quantified using 7300 Real-time PCR System (Ap-plied Biosystems Inc., Foster City, CA) and SYBR® Greenchemistry (PerfeCTa SYBR® Green Supermix, ROX,Quanta BioSciences, Gaithersburg, MD) as previouslydescribed [11, 13]. First-strand cDNA synthesis from1 μg of total RNA was performed with random primersusing a high-capacity cDNA reverse transcription kit(Applied Biosystems Inc., Foster City, CA) [11]. Forwardand reverse primers for the aforementioned genes(Additional file 1: Table S2) were designed based on se-quences available in GenBank using the online MITPrimer 3 designer software (developed at Whitehead In-stitute and Howard Hughes Medical Institute by SteveRozen and Helen Skaletsky) and were confirmed for spe-cificity using the basic local alignment search tool. β-2microglobulin was used as a control house-keeping gene,as its expression was not affected with the experimentalintervention (data not shown). All samples were run induplicate simultaneously with a negative control whichcontained no cDNA. Melting point dissociation curvesgenerated by the instrument were used to confirm thespecificity of the amplified product.Tissue total DNA isolationTotal DNA (genomic and mtDNA) was isolated from~15 mg of the skeletal muscle (soleus) and heart usingthe QIAamp DNA Mini kit (Qiagen, Mississauga, ON)[11, 16]. DNA samples were treated with RNase(Fermentas, Mississauga, ON) to remove RNA contam-ination. DNA concentration and quality were assessedusing Nanodrop 2000 (Thermo Scientific, Wilmington,DE).mtDNA copy number analysisMitochondrial DNA copy number, relative to the diploidchromosomal DNA content, was quantitatively analyzedfrom the skeletal muscle (soleus), heart, primaryhematopoietic stem cells, primary satellite cells, and pri-mary fibroblasts using ABI 7300 real-time PCR (AppliedBiosystems, CA) [11, 16]. Primers were designed aroundCOX-II region of the mitochondrial genome (Additionalfile 1: Table S2). Nuclear β-globin gene was used as ahousekeeping gene (Additional file 1: Table S2).Average telomere lengthAverage telomere length was measured in heart, primaryhematopoietic stem cells, and primary satellite cell gen-omic DNA using a real-time quantitative PCR methodas previously described [17]. The premise of this assay isto measure an average telomere length ratio by quantify-ing telomeric DNA with specially designed primer se-quences and dividing that amount by the quantity of asingle-copy gene [17]. All samples were run using a 7300Real-time PCR System (Applied Biosystems Inc., FosterCity, CA) and SYBR® Green chemistry (PerfeCTa SYBR®Green Supermix, ROX, Quanta BioSciences, Gaithers-burg, MD). A single-copy gene, 36B4, which encodes forthe acidic ribosomal phosphoprotein PO, was used as acontrol for amplification for every sample performed[17, 18]. Each PCR reaction for the telomere and 36B4included 12.5 μL of 1x SYBR® Green master mixSafdar et al. Skeletal Muscle  (2016) 6:7 Page 4 of 17(PerfeCTa SYBR® Green Supermix, ROX, Quanta BioSci-ences, Gaithersburg, MD), 300 nM each of the forwardand reverse telomere or 36B4 primers (Additional file 1:Table S2), 20 ng genomic DNA, and enough DNase/RNase-free H2O (Applied Biosystems Inc., Foster City,CA) to yield a 25-μL reaction. Cycling conditions fortelomere are as follows: 95 °C for 10 min followed by30 cycles of data collection at 95 °C for 15 s and a 56 °Canneal-extend step for 1 min. Cycling conditions for36B4 are as follows: 95 °C for 10 min followed by 35 cy-cles of data collection at 95 °C for 15 s, with 52 °C an-nealing for 20 s, followed by extension at 72 °C for 30.Each sample was analyzed in duplicate, and the ratio oftelomere:36B4 was calculated. The average of these ra-tios was reported as the average telomere length ratio(ATLR).Whole tissue lysateTotal protein was extracted from tissue samples as pre-viously described [11]. Briefly, ~30 mg of the skeletalmuscle (quadriceps femoris) and heart were homoge-nized on ice in a 2-mL Wheaton glass homogenizer(Fisher Scientific, Ottawa, ON) with 25 volumes of phos-phate homogenization buffer [50 mM KPi, 5 mM EDTA,0.5 mM DTT, 1.15 % KCl supplemented with aComplete Mini, ETDA-free protease inhibitor cocktailtablet and a PhosSTOP, phosphatase inhibitor cocktailtablet (Roche Applied Science, Mannhein, Germany) per10 mL of buffer]. The lysate was centrifuged at 600g for15 min at 4 °C to pellet cellular debris. The supernatantwas aliquoted, snap frozen in liquid nitrogen, and storedat −80 °C until further analysis.Nuclear fractionationNuclear fractions were prepared from 40 mg of thefreshly obtained skeletal muscle (quadriceps femoris),heart, primary satellite cells, and primary fibroblastsusing a commercially available nuclear extraction kit(Pierce NE-PER®, Rockford, IL) as previously described[11, 16]. Briefly, samples were homogenized in CER-Ibuffer containing protease inhibitor cocktail Complete,ETDA-free (Roche Applied Science, Mannheim,Germany) using an electronic homogenizer (Pro 250,Pro Scientific, Oxford, CT, USA). Pellets containing nu-clei were obtained by centrifugation at 16,000g for10 min at 4 °C and were subsequently washed four timesin PBS to remove cytosolic contaminating proteins. Nu-clear proteins were extracted in NER buffer supple-mented with protease inhibitors [11]. Enrichment andpurity of nuclear fractions were confirmed by the abun-dance of nuclear histone H2B and absence of the cyto-solic protein lactate dehydrogenase in Western blotanalyses as previously shown by our group [16].Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was per-formed using an EZ-ChIP™ kit (Millipore, Billerica, MA)as previously described [11]. Twenty-milligram piece ofthe quadriceps femoris muscle was cross-linked in 5 mL ofphosphate-buffered saline containing 1 % formaldehydefor 10 min at room temperature. One milliliter of 10X gly-cine was added to stop fixation. Muscles were then ho-mogenized in 1 mL of SDS lysis buffer supplemented withprotease inhibitor cocktail Complete, ETDA-free (RocheApplied Science, Mannheim, Germany). Chromatin wassheared by sonicating each sample on ice using a BransonDigital Sonifier® S-450D (output 20 %, 4 times for 20 s,with a 20-s pause each time; Branson Ultrasonics Corpor-ation, Danbury, CT). Following centrifugation at 10,000×gat 4 °C for 10 min, the supernatant containing 1 mg ofprotein was diluted to 1 mL with dilution buffer. Ten mi-crograms of anti-p53 (FL-393) antibody (Santa Cruz Bio-technology Inc., Santa Cruz, CA) was added per sampleand incubated overnight at 4 °C. Anti-IgG antibody wasused as a non-specific control. Sixty microliters of proteinG-agarose was added, and the sample was mixed for 1 hat 4 °C with rotation. Precipitated complexes were elutedin 100 μL of elution buffer, and cross-linking was reversedby the addition of 8 μL of 5 M NaCl per sample followedby incubation at 65 °C for 10 h. Co-immunoprecipitatedDNA was purified according to the manufacturer’s in-structions. Primers were designed to amplify the p53 bind-ing regions (−564 and −954) of the PGC-1α promoter(Additional file 1: Table S2). The amount of PGC-1α pro-moter immunoprecipitated with p53 was quantified usingthe 7300 Real-time PCR System (Applied Biosystems Inc.,Foster City, CA) and SYBR® Green chemistry (PerfeCTaSYBR® Green Supermix, ROX, Quanta BioSciences,Gaithersburg, MD). Purified DNA from the input samplethat did not undergo immunoprecipitation was PCR-amplified using of β-globin primers (Additional file 1:Table S2) and was used to normalize signals from ChIPassays.Mitochondrial fractionationMitochondrial fractions were isolated using differentialcentrifugation as previously outlined [11]. Briefly, theskeletal muscle (quadriceps femoris and tibialis anterior),heart, primary satellite cells, and primary fibroblastswere finely minced and homogenized on ice in 1:10 (wt/vol) ice-cold isolation buffer A (10 mM sucrose, 10 mMTris/HCl, 50 mM KCl, and 1 mM EDTA, and 0.2 % fattyacid-free BSA, pH 7.4, supplemented with protease in-hibitor cocktail Complete, ETDA-free [Roche AppliedScience, Mannheim, Germany]) using a Potter-Elvehjemglass homogenizer. The resulting homogenates werecentrifuged for 15 min at 700g, and the subsequent su-pernatants were centrifuged for 20 min at 12,000g. TheSafdar et al. Skeletal Muscle  (2016) 6:7 Page 5 of 17mitochondrial pellets from 12,000g spin were washedand then re-suspended in a small volume of ice-cold iso-lation buffer B (10 mM sucrose, 0.1 mM EGTA/Tris,and 10 M Tris/HCl, pH 7.4, supplemented with proteaseinhibitor cocktail Complete, ETDA-free [Roche AppliedScience, Mannheim, Germany]). All centrifugation stepswere carried out at 4 °C. The mitochondrial pellets wereimmediately frozen at −80 °C for further biochemicalanalyses. Enrichment and purity of mitochondrial frac-tions were confirmed by the abundance of mitochondrialcytochrome c oxidase subunit IV protein and absence ofthe nuclear histone H2B and the cytosolic protein lactatedehydrogenase in Western blot analyses as previouslyshown by our group [16].Mitochondrial co-immunoprecipitation assayMitochondrial co-immunoprecipitation assay was per-formed on isolated mitochondrial fractions using PierceCo-Immunoprecipitation Kit (Pierce, Rockford, IL) aspreviously described [16]. Briefly, mitochondrial frac-tions were homogenized in lysis buffer (0.025 M Tris,0.15 M NaCl, 0.001 M EDTA, 1 % NP-40, 5 % glycerol,pH 7.4) supplemented with protease inhibitor cocktailComplete, ETDA-free (Roche Applied Science,Mannheim, Germany). Two milligrams of mitochondrialfraction was pre-cleared by incubation with 100 μL ofcontrol agarose resin to minimize non-specific binding.Forty micrograms of anti-p53 (FL-393) antibody (SantaCruz Biotechnology Inc., Santa Cruz, CA) was covalentlycoupled onto an amine-reactive resin. The pre-clearedlysates were subsequently incubated with antibody-coupled beads overnight at 4 °C. Co-immunoprecipitateswere collected by centrifugation, boiled in 50 μL ofLaemmli sample buffer, and used for immunoblot ana-lysis for POLG1 (a kind gift of Dr. William C. Copeland,National Institutes of Health) or anti-Tfam (sc23588, A-17; Santa Cruz Biotechnology Inc., Santa Cruz, CA) anti-body. Anti-IgG antibodies were used as a non-specificcontrol.mtDNA immunoprecipitation assaymtDNA immunoprecipitation was performed on skeletalmuscle mitochondrial fraction that was cross-linked andsonicated as previously described [16, 19]. One milli-gram of mitochondrial fraction was pre-cleared in 25 %v/v pre-clearing matrix F (Santa Cruz Biotechnology,Santa Cruz, CA) overnight at 4 °C. The supernatant wasthen incubated with 20 μg of anti-p53 (FL-393) antibody(Santa Cruz Biotechnology Inc., Santa Cruz, CA) andExactaCruz™ F matrix (Santa Cruz Biotechnology, SantaCruz, CA) [20] with mixing by end-over-end inversionovernight at 4 °C in the presence of 5 μg of shared sal-mon sperm DNA (Sigma-Aldrich®, Oakville, ON) to re-duce non-specific DNA-bead interactions. Anti-IgGantibodies were used as a non-specific control. Thematrix was centrifuged at 16,000g for 30 s, and the pelletmatrix-immune complex precipitate was washed fourtimes under stringent conditions (50 mM Tris-HCl, pH7.4, 500 mM NaCl, 2 mM EDTA) and incubated over-night at 65 °C in the presence of 1 % SDS for cross-linking reversion. DNA was extracted from supernatantsusing the QIAamp DNA Mini kit (Qiagen, Mississauga,ON) according to the manufacturer’s instructions.mtDNA COX-II and cytochrome b regions (Additionalfile 1: Table S2) were quantified using 7300 Real-timePCR System (Applied Biosystems Inc., Foster City, CA)and SYBR® Green chemistry (PerfeCTa SYBR® GreenSupermix, ROX, Quanta BioSciences, Gaithersburg,MD), as previously described [16].Western blotting and markers of oxidative damageProtein concentrations of whole tissue lysates, and mito-chondrial and nuclear fractions were determined using acommercial assay (BCA Protein Assay, Pierce, Rockford,IL). Proteins were resolved on 10 or 12.5 % SDS-PAGEgels depending on the molecular weight of the protein ofinterest. The gels were transferred onto Hybond® ECLnitrocellulose membranes (Amersham, Piscataway, NJ)and immunoblotted using the following commerciallyavailable primary antibodies: MitoProfile® Total OXPHOSRodent cocktail (MS604) antibody (MitoSciences, Eugene,OR); anti-PGC-1α (2178), anti-VDAC (4866), and anti-α/β-tubulin (2148) antibodies (Cell Signaling Technology,Denver, MA); anti-p53 (MABE283-PAb421) antibody(EMD Millipore); anti-Tfam (sc-23588) and anti-NRF-1(sc-33771) antibodies (Santa Cruz Biotechnology Inc.,Santa Cruz, CA); anti-POLG1 antibody (a kind gift of Dr.William C. Copeland, National Institutes of Health, USA);anti-citrate synthase antibody (a kind gift of Dr. Brian H.Robinson, The Hospital for Sick Children, Canada); anti-4-HNE (ab48506), anti-SOD2 (ab13533), anti-catalase(ab1877), and anti-p21WAF1 (ab7960) antibodies (Abcam,Cambridge, MA); anti-Pax7 (Developmental StudiesHybridoma Bank, University of Iowa, Iowa City, IO); andanti-ERRα (EPR46Y) and anti-actin (NB600-535) anti-bodies (Novus Biologicals, Littleton, CO) [11, 16]. Thecarbonylated protein content in whole tissue lysates andmitochondrial fractions was quantified by Western blotusing OxyBlot Protein Detection kit (S7150; Millipore,Bedford, MA) as per manufacturer’s instructions. All anti-bodies were used at 1:1000 dilution, except for anti-actin(1:10,000). Membranes were then incubated with theappropriate anti-mouse or anti-rabbit horse radishperoxidase-linked secondary antibody (1:10,000) and visu-alized by enhanced chemiluminescence detection reagent(Amersham, Piscataway, NJ). Relative intensities of theprotein bands were digitally quantified by using NIHSafdar et al. Skeletal Muscle  (2016) 6:7 Page 6 of 17Image J, version 1.37, analysis software (Scion Image,NIH).ROS assayMitochondrial H2O2 production was measured using theAmplex® Red Hydrogen Peroxide assay (A22188; Invitro-gen, Burlington, ON) as per manufacturer’s instructions.Briefly, 40 μg of mitochondrial fraction was diluted in50 μL reaction buffer (125 mM KCl, 10 mM HEPES,5 mM MgCl2, 2 mM K2HPO4, pH 7.44) to determinemitochondrial respiratory chain complex I (5 mM pyru-vate/malate) or complex II (5 mM succinate) drivenH2O2 production with and without inhibitors (0.5 μMrotenone, complex I inhibitor, and 0.5 μM antimycin A,complex III inhibitor). Mitochondrial H2O2 productionwas measured after the addition of 50 μL of reactionbuffer containing horseradish peroxidase and Amplex®Red. Fluorescence was followed at an excitation wave-length of 545 nm and an emission wavelength of 590 nmfor 5 min using fluorescence microplate reader (TecanSafire, MTX Lab Systems, Inc., Vienna, VA). The slopeof the increase in fluorescence is converted to the rate ofH2O2 production with a standard curve. All of the assayswere performed at 25 °C. The results are expressed aspmoles.min−1.mg protein−1.Mitochondrial respiratory chain complex I and IV enzymeactivityMitochondrial ETC complex I and complex IV activitieswere determined in tissue lysates following establishedprotocols [11, 21–23]. All samples were analyzed in du-plicates on the Cary UV-vis spectrophotometer (Varion,Inc., Palo Alto, CA).Superoxide dismutase and catalase enzyme activityMuscle total superoxide dismutase (Mn-SOD and Cu/Zn-SOD) activity was determined in muscle lysates by meas-uring the kinetic consumption of superoxide radical (O2−)by SOD in a competitive reaction with cytochrome c, aspreviously described [20]. Absorption was recorded at550 nm and was observed every 15 s for 2 min at 37 °C.One unit (U) of SOD activity was defined as the amountof enzyme that caused a 50 % inhibition of the reductionof cytochrome c. Total SOD activity was expressed inU.mg of protein−1. In a separate cuvette, the same samplewas analyzed under identical conditions in the presence of0.2 M KCN (pH 8.5–9.5), a potent inhibitor of cytosolicCu/Zn-SOD [24], for determination of mitochondrial Mn-SOD activity. Cu/Zn-SOD activity was approximated bysubtracting Mn-SOD activity from total SOD activity.Both Mn-SOD and Cu/Zn-SOD activity were expressed inU.mg protein−1. Catalase activity was determined bymeasuring the kinetic decomposition of H2O2 as previ-ously described [25]. Catalase activity was measured bycombining 960 μL of K2HPO4 buffer (50 mM with50 mM EDTA and 0.01 % Triton X-100, pH 7.2–7.4) with30 μL of muscle homogenate. Ten microliters of H2O2(1 M) was added to the cuvette and mixed by inversion toinitiate the reaction. Absorbance was measured at 240 nmevery 15 s for 2 min. Catalase activity was calculated andreported in μmol · min−1 · mg protein−1. All samples wereanalyzed in duplicates on the Cary UV-vis spectrophotom-eter (Varion, Inc., Palo Alto, CA).Caspase-3 and caspase-9 enzyme activityCaspase-3 and caspase-9 enzyme activity was measuredusing fluorometric protease assays caspase-3/CPP32 andcaspase-9/Mch6, respectively (Biovision, Mountain View,CA) according to manufacturer’s instructions. Briefly,the assays are based on the detection of cleavage of thesubstrate DEVD-AFC (AFC: 7-amino-4-trifluoromethylcoumarin) by caspase-3 and LEHD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin) by caspase-9. UncleavedDEVD-AFC and LEHD-AFC fluoresce at λmax = 400 nm,upon cleavage of the respective substrate by caspase-3or caspase-9, free AFC emits a yellow-green fluorescence(λmax = 505 nm), which was quantified using a fluores-cence microplate reader (Tecan Safire, MTX Lab Sys-tems, Inc., Vienna, VA). Results were expressed as rawfluorescence units per milligram of cytosolic protein.Apoptosis cell death detection ELISAApoptotic DNA fragmentation was quantified in the skel-etal muscle (quadriceps femoris), heart, primaryhematopoietic stem cells, and primary satellite cells bymeasuring the amount of cytosolic mono- and oligo-nucleosomes using a Cell Death detection ELISAPLUSassay (Roche Applied Science, Laval, QC) as previouslydescribed [11]. Briefly, wells were coated with a monoclo-nal anti-histone antibody and incubated with homoge-nates. Nucleosomes were centrifuged at 100,000g followedby binding to the anti-histone antibody followed by theaddition of anti-DNA-peroxidase antibody that binds tothe DNA associated with the histones. The amount of per-oxidase retained in the immunocomplex was determinedspectrophotometrically with ABTS (2,2′-azino-bis[3-ethyl-benzthiazoline-6-sulphonic acid]) as a substrate. Resultswere expressed as arbitrary OD units normalized to mi-crograms of cytosolic protein.Quantification of mtDNA mutationsmtDNA mutations were quantified by the error-resistantsingle molecule approach [26]. Briefly, skeletal muscle(quadriceps femoris) DNA was subjected to limiting dilu-tion long-range PCR, where each positive PCR reactionwas initiated by a single mtDNA molecule. PCR was de-signed to amplify essentially the entire mitochondrialgenome using high-fidelity Phusion DNA polymerase,Safdar et al. Skeletal Muscle  (2016) 6:7 Page 7 of 17(New England Biolabs). Three to 9 amplified moleculeswere obtained per animal. Each amplified molecule wassequenced in its entirety using barcoded Illumina nextgeneration sequencing approach at a local core facility.Mutations were identified by comparing each molecule’ssequence to the standard C57Bl/6J mtDNA sequence(GenBank EF108336). Only 100 % mutations were con-sidered, which guaranteed the exclusion of artifacts [26].Mutant fractions were calculated by dividing the totalnumber of mutations by the number of nucleotides se-quenced per animal.p53 base excision repair activity assayAn in vitro fluorescence-based DNA primer p53 repairactivity assay was employed as previously described [27],with minor modifications. This assay utilized a double-stranded deoxyoligomers containing sequences identicalto the first 40 nucleotides of the mtDNA replication ori-gin as the primer-template substrate, with the 3′ end ofthe primer contained self-designed mismatch point mu-tation in the last three nucleotides (Additional file 1:Table S2). The 5′ and 3′ ends of the primer were chem-ically linked to a Black Hole Quencher®-1 and 6-carboxyfluorescein (FAM-1™) flourophore, respectively(Integrated DNA Technology®, Toronto, ON). The prem-ise of this assay is that, in the absence of proofreadingcapacity of mitochondrial polymerase gamma, primerextension requires the excision of the unpaired nucleo-tides by the 3′→5′ exonuclease activity which in turnwill be detected as an increase in fluorescence over time.The 20 μL reaction mixtures containing 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 1 mM DTT, 100 μg/mL ofBSA, 3′-end-FAM1™ primer-template substrate, 50 μMeach of dATP, dCTP, dGTP, and dTTP, and 40 μg ofWT, PolG-SED, and PolG-END skeletal muscle (quadri-ceps femoris) mitochondrial extracts were incubated at37 °C for 40 min with data collection at the end usingiCycler IQ™ real-time PCR detection system (BioRad,Mississauga, ON). To assess the requirement of p53 asan accessory mtDNA mismatch point mutation repairprotein, p53 repair activity assay was also carried out in(i) PolG-END skeletal muscle mitochondrial extract afterp53 immunodepletion and (ii) PolG-SED skeletal musclemitochondrial extract with addition of recombinant hu-man p53 (BD Biosciences, Mississauga, ON).StatisticsAll molecular indices between the groups (WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53MKO-END mice) were analyzed using two-tailed Stu-dent’s t test. The log-rank test was used to test for signifi-cant differences in life span distribution between groups.Statistical significance was established at a P ≤ 0.05. Dataare presented as mean ± standard error of the mean(SEM).Results and discussionEndurance exercise confers phenotypic protection,reduces mtDNA mutations, and attenuates oxidativedamage in PolG miceAged tissues display stochastic accumulation of mtDNAmutations that likely perpetuate respiratory chain defi-ciency and greater reactive oxygen species (ROS)-medi-ated damage [28]. To evaluate the underlying protectivemechanism of exercise on mitochondrial redox statusand mtDNA integrity, we profiled “terminally differenti-ated” (skeletal muscle and heart) and “proliferative” (Lin− Sca-1+ c-Kit + population enriched for hematopoieticstem and progenitor cells, “HSC’ and c-met+, satellitecells, “SC”) compartments of littermate wild-type (WT),sedentary PolG (PolG-SED), and forced-endurance exer-cised PolG (PolG-END) mice. As shown previously [11],and now confirmed in an independent cohort of miceutilized in this study, exercise rescued progeroid aging(Additional file 1: Figure S1A), increased life span(Additional file 1: Figure S1B), and reduced mtDNA mu-tations (Fig. 1a) in PolG mice.Initial characterization of PolG mice showed absenceof increased oxidative damage despite significant accu-mulation of mtDNA point mutations [4, 29]. We evalu-ated the presence of oxidative modifications and foundno difference in protein carbonyls (PC) and 4-hydroxy-2-nonenal (4-HNE) content in the muscle, heart, and SChomogenates of PolG-SED vs. WT (Additional file 1:Figure S1C). We surmised that since the absence of oxi-dative damage in the PolG tissues is due to cell-to-cellvariability, and that any one modification would be lowerthan the detectable limit in whole tissue homogenates[4, 29], we measured oxidative damage in mitochondrialfractions—the primary source of cellular ROS. Indeed,mitochondria from these tissues demonstrated a sub-stantial increase in H2O2 production, along with elevatedPC and 4-HNE content (Fig. 1b, c, and Additional file 1:Figure S1C, D, and F). These observations are consistentwith recent studies reporting higher PC levels in heartmitochondria of PolG mice [30] and increased mito-chondrial H2O2 production in vivo using mitochondria-targeted mass spectrometry probe MitoB [31]. Thishigher oxidative damage is also congruent with reducedsuperoxide dismutase 2 (SOD2) and catalase contentand activity in PolG-SED vs. WT (Fig. 1d and Additionalfile 1: Figure S1E). We hypothesize that the combinationof lower antioxidant capacity, coupled with elevatedROS production in PolG-SED mitochondria exacerbatesthe accumulation of mtDNA mutations. Consistent withthis notion, Vermulst et al. reported a significant reduc-tion in the frequency of mtDNA mutations in the heartSafdar et al. Skeletal Muscle  (2016) 6:7 Page 8 of 17Fig. 1 (See legend on next page.)Safdar et al. Skeletal Muscle  (2016) 6:7 Page 9 of 17tissue of transgenic animals that over-expressed humancatalase (CAT), a ROS scavenger, to mitochondria vs.age-matched (28 months old) wild-type mice [32]. Weobserved that exercise normalized mitochondrial H2O2production (Fig. 1b and Additional file 1: Figure S1D),and markers of oxidative damage (Fig. 1c and Additionalfile 1: Figure S1F) in PolG-END to WT levels and in-creased SOD2 and catalase content and activity (Fig. 1dand Additional file 1: Figure S1E). Together, our datasuggest that exercise reduces mtDNA point mutations,at least in part, via the up-regulation of cellular antioxi-dant capacity that subsequently serves to attenuate ROSlevels.Endurance exercise diminishes telomere erosion anddown-regulates aberrant p53 signaling and pathologicallevels of apoptosis in PolG miceSustained intrinsic accumulation of oxidative damagehas been implicated in telomere erosion that drivesage-related tissue degeneration [1]. In agreement withthis, we observed shorter telomeres in the heart,HSC, and SC from PolG-SED vs. WT (Fig. 1e).Genomic instability due to telomere shortening acti-vates tumor suppressor protein p53-mediated senes-cence/apoptotic signaling cascades [1]. Accordingly,nuclear p53 abundance in the muscle, heart, and SCof PolG-SED was enhanced (Fig. 1f ), concomitantlywith higher expression levels of the p53-responsivesenescence genes: p21WAF1, p16INK4A, and GADD45B(Additional file 1: Figure S1G) vs. WT. Mitochondrialdysfunction in PolG mice is associated with patho-logical systemic apoptosis [4, 11], and consistent withthese observations, we found higher DNA fragmenta-tion (Fig. 2a) and caspase-3/9 activity (Additional file1: Figure S2A and B) in PolG-SED mice. Interestingly,exercise abrogated telomere shortening (Fig. 1e), re-duced p53 nuclear accumulation (Fig. 1f ), normalizedthe expression of p53-responsive senescence genes(Additional file 1: Figure S1G), and reduced patho-logical levels of apoptosis in PolG-END (Fig. 2a, andAdditional file 1: Figure S2A and B).Endurance exercise mitigates mitochondrial dysfunctionvia reduction in nuclear p53 that represses PGC-1αA causal role of mitochondrial-induced oxidative stressand telomere erosion, secondary to mtDNA mutations,suggests a direct link between p53 activation and mito-chondrial dysfunction [5]. Increasing the expression ofPGC-1α, a potent regulator of mitochondrial biogenesis,positively regulates the expression of antioxidants [33]and has been touted to attenuate aging-associated sarco-penia and metabolic dysfunction [34]. This prompted usto investigate whether activation of p53-mediated senes-cence signaling attenuates PGC-1α-triggered gene pro-gramming. We conducted in silico promoter analysisthat identified putative p53 binding elements in thePGC-1α promoter. These promoter regions were thencloned into a pGL4 luciferase reporter vector and trans-fected into p53+/+ and p53−/− mouse embryonic fibro-blasts (MEFs). A significant repression of PGC-1α-pGL4reporter activity was observed in the p53+/+ relative top53−/− MEFs (Additional file 1: Figure S2C). These re-sults are consistent with a recent study showing that nu-clear p53 can directly repress PGC-1α expression andpromote mitochondrial dysfunction [5]. To further testour hypothesis, we next performed an anti-p53 chroma-tin immunoprecipitation assay that showed physical en-richment of nuclear p53 at the PGC-1α promoter ofPolG-SED vs. WT mice (Fig. 2b). Together, these datasuggest that ROS-induced cellular damage prompts thenuclear accumulation of p53, which in turn activatesp53-responsive senescence genes while simultaneouslyrepressing the pro-metabolic activity of PGC-1α.qPCR analyses of the PolG-SED muscle, heart, HSCand SC confirmed lower expression of PGC-1α andstrong repression of its metabolic networks, includingoxidative phosphorylation, mitochondrial function, glu-coneogenesis, and fatty acid metabolism vs. WT (Fig. 2c,and Additional file 1: Figure S2D–G, and Table S1).Additionally, PolG-SED mice tissues and stem cells havereduced mtDNA copy number (Fig. 2D and Additionalfile 1: Figure S3A), lower mitochondrial complex I andcomplex IV enzyme activity (Additional file 1: Figure(See figure on previous page.)Fig. 1 Endurance exercise reduces random mtDNA somatic mutations, attenuates mitochondrial ROS-mediated oxidative damage, mitigatestelomere shortening, and reduces nuclear accumulation of p53 in mtDNA mutator mice. a Random mtDNA somatic mutation rate (per 1000nucleotides of mtDNA) in muscle (quadriceps femoris) WT, PolG-SED, and PolG-END mice (n = 4–5/group). b H2O2 production rate in musclemitochondrial fractions of WT, PolG-SED, and PolG-END (n = 5–7/group). Complex I and II substrates: P/M, pyruvate/malate and SUC, succinate(5 mM each), respectively. Complex I and III inhibitors: ROT, rotenone, and AA, antimycin A (0.5 μM each), respectively. c Protein carbonyls (PC)content in muscle (tibialis anterior) and heart mitochondrial fractions of WT, PolG-SED, and PolG-END (n = 5–7/group). d SOD2 and catalaseenzyme activity in the muscle (quadriceps femoris) and heart of WT, PolG-SED, and PolG-END (n = 7/group). e Average telomere length ratios inthe heart, hematopoietic stem and progenitor cells (HSC), and satellite cells (SC) of WT, PolG-SED, and PolG-END (n = 6–8/group). f Representativeblots of nuclear p53 content (~53 kDa) in the muscle (quadriceps femoris) and heart of WT, PolG-SED, and PolG-END (n = 5–8/group). Histone H2B(~14 kDa) was used as a nuclear loading control. (PolG-SED vs. both WT and PolG-END) = *P < 0.05, **P < 0.01; (PolG-END vs. WT) = †P < 0.05. Errorbars represent SEM. AU arbitrary unitsSafdar et al. Skeletal Muscle  (2016) 6:7 Page 10 of 17Fig. 2 (See legend on next page.)Safdar et al. Skeletal Muscle  (2016) 6:7 Page 11 of 17S3B), reduced mitochondrial electron transport chainsubunits protein content (Additional file 1: Figure S3C–I), and accumulation of swollen, pleomorphic, oversizedmitochondria (Fig. 2e). Endurance exercise decreasedbinding of p53 to the PGC-1α promoter (Fig. 2b), andthis effect was accompanied by the maintenance ofmtDNA copy number, increased expression of PGC-1αand its downstream metabolic network, enhanced mito-chondrial oxidative capacity, and restoration of mito-chondrial structural integrity in PolG-END (Fig. 2c–e,and Additional file 1: Figures S2D–G, Figure S3A–I, andTable S1). These observations collectively imply that ac-cumulating mtDNA mutations in PolG-SED mice leadto an increase in ROS generation that (i) promotes mito-chondrial dysfunction and telomere damage and (ii) sub-sequently triggers p53-regulated senescence pathways,thereby potentiating the loss of somatic and stem cellsvia apoptosis. In contrast, exercise reduced mtDNA mu-tations and maintained the cellular energy and redoxhomeostasis thereby circumventing telomere erosionculminating in the inhibition of accelerated systemicaging characteristic of PolG mice [3, 4].Endurance exercise-mediated repair of mtDNA mutationsis p53-dependentPOLG1 is the sole mitochondrial polymerase essentialfor mtDNA replication and repair via its 3′→5′ exo-nuclease activity [35]. Since exercise reduced mtDNAmutations in PolG mice, which lack proofreading cap-acity of POLG1, this raised an intriguing possibility thatexercise recruited a POLG1-independent mtDNA repairpathway(s) [11]. We found that despite elevated p53 nu-clear abundance in PolG-SED, the total p53 content inthe muscle, heart, and SC homogenates of all groupswas unaltered (Additional file 1: Figure S3J). This indi-cated that a basal pool of p53 is maintained intra-cellularly, with the distribution of p53 between thedifferent subcellular compartments dependant on thecellular stress milieu [27]. In vitro studies show that inresponse to intra- and extra-cellular insults such asROS, p53 translocates into the mitochondria where it in-teracts with the mtDNA and POLG1 [27]. Biochemicalanalysis of p53 has revealed an inherent 3′→5′exonuclease activity that helps p53 promote and main-tain mitochondrial genomic stability by executing baseexcision repair on damaged mtDNA [36]. The role ofmitochondrial p53 in the context of aging remains hith-erto unknown. Collectively, these observations led us tohypothesize that in the presence of an error-pronePOLG1, mitochondrial p53 will function as an accessoryfidelity-enhancing component of the mtDNA replicationmachinery in PolG mice.To test our hypothesis, we first assessed the submito-chondrial localization of p53 in skeletal muscle of WTmice. Subfractionation of skeletal muscle mitochondria in-dicated that mitochondrial p53 was primarily localized inthe mitochondrial matrix (Fig. 3a). Next, we measured themitochondrial abundance of p53 in our experimentalgroups. Unlike PolG-SED, in PolG-END mice, p53 prefer-entially resided in the mitochondria vs. nuclei of muscleand heart (Figs. 1f and 3b). To ascertain whether mito-chondrial ROS levels regulated p53 compartmentalization,we treated primary fibroblasts with rotenone, a complex Iinhibitor known to increase mitochondrial ROS, and ob-served a rapid increase in mitochondrial p53 content atlower dosages without a concomitant increase in nuclearp53 (Additional file 1: Figure S4A). Intriguingly, with in-creasing rotenone concentrations, we measured an in-crease in nuclear p53 abundance (Additional file 1: FigureS4A) and expression of its downstream targets (p16INK4Aand p21WAF1; Additional file 1: Figure S4B), along with aconcomitant reduction in mitochondrial p53 content(Additional file 1: Figure S4A), and mtDNA copy number(Additional file 1: Figure S4C). The increase in nuclearp53 paralleled the decrease in PGC-1α mRNA expression(Additional file 1: Figure S5A), further supporting the in-hibitory effects of p53 on PGC-1α. Furthermore, the up-regulation of rotenone-evoked nuclear p53 content wasattenuated in fibroblasts pre-treated with a ROS scavenger,N-acetylcysteine (Additional file 1: Figure S5B), in tandemwith higher PGC-1α mRNA expression (Additional file 1:Figure S5C). Thus, p53 preferentially shuttles intomitochondria in response to physiological ROS levels,which abrogates the negative regulation of PGC-1α asexerted by p53 residing in the nucleus. Next, wesought to elucidate if mitochondrial p53 interacted(See figure on previous page.)Fig. 2 Endurance exercise prevents dysregulated mitochondrial-induced apoptosis, reduces nuclear p53-mediated repression of PGC-1α, inducesPGC-1α regulated gene networks, restores mtDNA copy number, and normalizes mitochondrial morphology in mtDNA mutator mice. a NuclearDNA fragmentation (apoptotic index) in the heart, HSC, and SC of WT, PolG-SED, and PolG-END (n = 7–10/group). b ChIP assay showing reducedp53 enrichment of PGC-1α promoter (positions −954/−564) with exercise in the muscle and heart of WT, PolG-SED, and PolG-END (n = 6/group).c Gene expression of PGC-1α and its downstream targets in muscle (quadriceps femoris), d mtDNA copy number normalized to nuclear β-globingene in the muscle (soleus) and heart, and e representative electron micrographs of myofibers (quadriceps femoris) and cardiomyocytes (heart)from WT, PolG-SED, and PolG-END mice (n = 4–7/group). Asterisk (PolG-SED vs. both WT and PolG-END): *P < 0.05, **P < 0.01, ***P < 0.001. Error barsrepresent SEM. AU arbitrary unitsSafdar et al. Skeletal Muscle  (2016) 6:7 Page 12 of 17Fig. 3 (See legend on next page.)Safdar et al. Skeletal Muscle  (2016) 6:7 Page 13 of 17with POLG1 and Tfam in the mitochondrial matrix.We performed co-immunoprecipitation reactions andfound that mitochondrial p53 formed a complex withPOLG1 and Tfam complexed at mtDNA (Fig. 3c–e).The p53-POLG1-Tfam complex at mtDNA was higherin PolG-END vs. PolG-SED and WT (Fig. 3c–e).These observations are consistent with a recent studyreporting p53 translocation to of p53 to the mito-chondria and subsequent formation of p53-Tfam-mtDNA complex in skeletal muscle of WT mice inresponse to an acute bout of endurance exercise [37].These results led us to conclude that the preferentialsubcellular localization of p53 to mitochondria vs.nuclear compartment is a “universal” exercise-inducedphenomenon and likely plays a role in mediatingbeneficial effects of endurance exercise on improvingmitochondrial content/function and amelioratingdysfunction.Since both PolG-SED and PolG-END mice have defect-ive POLG1 proofreading capacity, we believe that the re-duction in total mtDNA mutational burden in PolG-ENDmice (Fig. 1a) is mediated by mitochondrial p53 levels inresponse to endurance exercise. Hence, we sought toevaluate whether mitochondrial p53 can repair mtDNAmutations, independent of the proofreading capacity ofPOLG1. A fluorescence-based in vitro DNA primerextension-mutation repair assay displayed an efficient re-pair of double-stranded oligonucleotides, with artificiallyadded mismatch point mutations, incubated with ex vivomuscle mitochondrial extract of PolG-END vs. PolG-SED(Fig. 4a). PolG-END mitochondria failed to repair thesemutations upon p53 immunodepletion (Fig. 4b), whileaddition of recombinant p53 increased PolG-SED mito-chondrial mutation repair efficiency (Additional file 1:Figure S6A). Clearly, mitochondrial p53 plays a vital rolein the maintenance of mtDNA integrity in the presence ofdefective POLG1 in mutator mice.However, to conclusively attribute causality to mito-chondrial p53 in mediating mtDNA repair in vivo in re-sponse to endurance exercise, a “double genetically alteredmutator mouse model” was needed where changes inmtDNA mutation burden can be assessed in a backgroundof p53 over-expression or knockdown. It was unfeasible togenerate PolG mice with over-expression of p53, as previ-ous work has shown that mice engineered with hyper-active p53 alleles display show stem cell depletion andpremature aging phenotype themselves [38]. On the otherhand, whole-body p53 knockout mice die prematurely ofcancer [39] and do not breed efficiently with heterozygousPolgA+/D257A mice, and thus could not be bred with PolGmice to efficiently study the effects of exercise. Hence, wecreated a new genetically modified mutator mouse withmuscle-specific p53 deletion (PolG-p53 MKO). At basallevels, PolG-p53 MKO-SED mice demonstrated an accel-erated progeriod phenotype and significant accumulationof random mtDNA mutations in muscle compared toPolG-SED mice (Fig. 4e and Additional file 1: Figure S6B).To our surprise, endurance exercise not only failed toreduce mtDNA point mutations but also did not rescueprogeroid aging, sarcopenia, exercise intolerance, mito-chondrial morphology anomalies, and deficits in mito-chondrial content and function such as mtDNA copynumber, mitochondrial electron transport chain proteincontent, and COX activity in skeletal muscle of PolG-p53MKO mice (Fig. 4c–g and Additional file 1: Figure S6B–E). This suggests that the exercise-mediated repair ofmtDNA mutations in vivo is dependent on mitochondrialp53 adjuvant repair capacity. Furthermore, unlike PolG-END, mitochondrial extracts from PolG-p53 MKO failedto repair mutations in vitro in the primer extension-mutation repair assay (Additional file 1: Figure S6F). Thus,exercise-induced maintenance of mtDNA stability is con-tingent on mitochondrially localized p53 and represents aviable therapy for pre-symptomatic patients carryingPOLG1 exonuclease domain mutations known to causepathology [35].ConclusionsHere, we show that exercise promotes mitochondrialoxidative capacity and cellular redox dynamics via PGC-(See figure on previous page.)Fig. 3 Endurance exercise increases the abundance of p53 in mitochondrial matrix where it interacts with mtDNA in a complex with POLG1 and Tfamin mtDNA mutator mice. a Mitochondrial p53 is primarily localized in the matrix. Muscle mitochondria were subfractionated into outer mitochondrialmembrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), and matrix (Mx) fractions, and these fractions wereimmunoblotted for the compartment-specific proteins TOMM22 (~16 kDa), cytochrome c (~14 kDa), COX-IV (~17 kDa), and CS (~45 kDa), respectively,and also for p53 (~53 kDa) and Tfam (~24 kDa). Representative blots of b mitochondrial p53 content (~53 kDa) in the muscle and heart of WT, PolG-SED, PolG-END (n = 6–8/group), c p53 co-immunoprecipitation (IP) followed by immunoblotting (IB) for mitochondrial transcription factor A (Tfam;~24 kDa) to assess mitochondrial p53-Tfam complex content in muscle and heart mitochondria from WT, PolG-SED, PolG-END (n = 4–5/group), andd p53 co-IP followed by IB for POLG1 (~140 kDa) to assess mitochondrial p53-POLG1 complex content in muscle and heart mitochondria from WT,PolG-SED, and PolG-END (n = 6–8/group). VDAC (~32 kDa) was used as a mitochondrial loading control. e p53-POLG1-Tfam complex is bound tomtDNA (quantified using two independent mtDNA regions: COX-II and cytochrome b) in muscle mitochondrial fractions of WT, PolG-SED, and PolG-END mice (n = 4–6/group). A non-specific IgG antibody was used as negative control antibody. Asterisk (PolG-SED vs. both WT and PolG-END):*P < 0.05, **P < 0.01; dagger (PolG-END vs. WT): †P < 0.05. Error bars represent SEM. AU arbitrary unitsSafdar et al. Skeletal Muscle  (2016) 6:7 Page 14 of 171α-mediated expression networks, thus preventing theaccumulation of oxidative damage, abrogating genotoxicdamage, and repressing apoptosis in mutator mice.Intriguingly, stress-mediated subcellular localization ofthe tumor suppressor protein p53 determines its pro- oranti-survival function and seems indispensible for theFig. 4 Endurance exercise-mediated repair of mtDNA mutations is mitochondrial p53-dependent. a A fluorescence-based in vitro DNA primerextension-mutation repair assay in muscle mitochondrial extracts from WT, PolG-SED, and PolG-END (n = 6–8/group) to assess the excision of theunpaired artificial point mutations. b p53 immunodepletion prevents mutation repair in muscle mitochondrial extracts from PolG-END (n = 5/group). A non-specific IgG antibody was used as negative control antibody. c Endurance stress test time to exhaustion in four independent trialsin WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice (n = 5–6/group). d Representative electron micrographs ofmyofibers (quadriceps femoris) from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END (n = 4/group). e Random mtDNAsomatic mutation rate (per 1000 nucleotides of mtDNA) in muscle (quadriceps femoris) WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice (n = 3–4/group). f mtDNA copy number in muscle mitochondria from PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53MKO-END mice (n = 4–5/group) relative to WT mice (horizontal line). g Cytochrome c oxidase (COX) activity in muscle from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice (n = 4–5/group). Asterisk (PolG-SED vs. both WT and PolG-END): *P < 0.05, **P < 0.01, ***P< 0.001; dagger (PolG-p53 MKO-SED vs. PolG-SED OR PolG-p53 MKO-END vs. PolG-END): †P < 0.05; closed circle (PolG-END vs. PolG-SED): ●P < 0.05,●●P < 0.01, ●●●P < 0.001. Error bars represent SEM. AU arbitrary unitsSafdar et al. Skeletal Muscle  (2016) 6:7 Page 15 of 17exercise-mediated mtDNA repair and mitochondrial bio-genesis. The work summarized here opens up viable ave-nues of research in cancer biology where mitochondrialdysfunction and genomic instability have been impli-cated [1]. It will be of potential clinical interest to see ifexercise-induced mitochondrial-targeted p53 might rep-resent a therapeutic intervention for aging-associatedpathologies such as insulin resistance, diabetes, and car-diovascular diseases, which manifest telomere shorteningin conjunction with mitochondrial dysfunction [40, 41].Indeed, while exercise and an active lifestyle are themost prominent therapies to reduce the incidence andpathogenicity of diabetes, insulin resistance, and cardio-vascular diseases [42, 43], therapeutic modalities thatpromise to recapitulate some of the effects of exercisewarrant further attention. The telomere–p53–PGC-1αaxis provides a molecular basis of how telomere erosionand mitochondrial dysfunction can modulate systemicaging of tissues and stem cell compartments. Under-standing the upstream signaling cascades and posttrans-lational modifications that promote mitochondriallocalization of p53 may allow for the generation ofpharmaceutical analogs, novel therapeutic strategies toantagonize mitochondrial genomic decay, and cellularsenescence in age-associated pathologies.Additional fileAdditional file 1: Figure S1. Endurance exercise confers completephenotype protection, suppresses early mortality, mitigates mitochondrialROS-mediated oxidative damage, increases cellular antioxidant capacity,and 4 prevents cellular senescencmutator mice. Figure S2. Enduranceexercise prevents dysregulated mitochondrial-induced apoptosis andreduces nuclear p53-mediated repression of PGC-1α and promotesmitochondrial biogenesis in mutator mice. Figure S3. Endurance exercisepromotes systemic mitochondrial biogenesis in mtDNA mutator mice.Figure S4. Magnitude of mitochondrial ROS (physiological vs.pathological) regulates p53 subcellular localization. Figure S5.Pre-treatment with exogenous antioxidant preferentially shuttles p53 tomitochondria in response to stress. Figure S6. Endurance exercise-mediated attenuation of sarcopenia, increase in endurance capacity,skeletal muscle mitochondrial biogenesis, and repair of muscle mtDNAmutations is p53 dependent. Table S1. WT, PolG-SED, and PolG-ENDSkeletal Muscle Microarray IPA-GO Analysis. Table S2. Real-time PCRprimer sequences. (PDF 1601 kb)Abbreviations4-HNE: 4-hydroxy-2-nonenal; ALAS: 5-aminolevulinate synthase; ATPase6: complex V subunit ATPase 6; COX-I: cytochrome c oxidase subunit-I; COX-II: cytochrome c oxidase subunit-II; COX-IV: cytochrome c oxidase subunit-IV;END: endurance exercise; ERRα: estrogen-related receptor alpha;GADD45B: growth arrest and DNA-damage-inducible, beta; H2O2: hydrogenperoxide; HSC: hematopoietic stem and progenitor cells; MEF: mouseembryonic fibroblasts; MKO: muscle-specific knockout; mtDNA: mitochondrialDNA; ND1: complex I NADH dehydrogenase subunit 1; NRF1: nuclearrespiratory factor 1; p16INK4A: cyclin-dependent kinase inhibitor 2A;p21WAF1: cyclin-dependent kinase inhibitor 1A; p53: tumor suppressor protein53; PC: protein carbonyls; PGC-1α: peroxisome proliferator-activated receptorgamma co-activator 1 alpha; PolG: polymerase gamma mutator mice;POLG1: mitochondrial polymerase gamma; ROS: reactive oxygen species;SC: satellite cells; SED: sedentary; SOD1: superoxide dismutase 1 (cytosolic;Cu/Zn-SOD); SOD2: superoxide dismutase 2 (mitochondrial; Mn-SOD);TFAM: mitochondrial transcription factor A; VDAC: voltage-dependent anionchannel; WT: wild-type mice.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAS and MAT designed the research; AS, KK, JMF, AS, MDL, APWJ, YK, IAS, YK,DIO, JPL, SR, GP, MA, BPH, and GCR performed the research; KK, GP, ZA, TAP,and MAT contributed the new reagents/analytic tools; AS, YK, and BPHanalyzed the data; and AS wrote the manuscript. All authors have beeninvolved in drafting and revising the manuscript and have approved the finalmanuscript.AcknowledgementsWe like to acknowledge the late Mrs. Suzanne Southward (McMasterUniversity) for assisting in enzyme assays and Dr. William C. Copeland (NIH)for his kind donation of POLG1 antibody. This work was supported by theCanadian Institutes of Health Research (CIHR) grant and a kind donationfrom Mr. Warren Lammert and family to M.A.T. A. Safdar was funded byBanting Fellowship (CIHR) and American Federation for Aging Research andEllison Medical Foundation (EMF) Fellowship. A. Saleem is funded by NaturalSciences and Engineering Research Council of Canada postdoctoralfellowship. K.K. was supported by the United Mitochondrial DiseaseFoundation, the NIH (AG019787) and the EMF Senior Scholarship. Theauthors declare no competing and financial interests. T.A.P. was awarded aUS patent 7,126,040 for the PolgD257A mouse model. T.A.P. is a partial ownerof LifeGen Technologies, specializing in nutrigenomics, as well as a ScientificAdvisory Board member for Nu Skin Enterprises. M.A.T. is the founder,president, and CEO of Exerkine Corporation and a member of its scientificadvisory board.Author details1Department of Kinesiology, McMaster University, Hamilton, ON L8N 3Z5,Canada. 2Department of Pediatrics, McMaster University, Hamilton, ON L8N3Z5, Canada. 3Department of Medicine, McMaster University, Hamilton, ONL8N 3Z5, Canada. 4Department of Medical Sciences, McMaster University,Hamilton, ON L8N 3Z5, Canada. 5Department of Medical Physics & AppliedRadiation Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada.6Northeastern University, Boston, MA 02115, USA. 7Buck Institute for Researchon Aging, Novato, CA 94945, USA. 8School of Health and Exercise Sciences,University of British Columbia Okanagan, Kelowna, BC V1V 1V7, Canada.9Division of Cardiovascular Disease, University of Alabama, Birmingham, AL35294, USA. 10Perelman School of Medicine, University of Pennsylvania,Philadelphia, PA 19104, USA. 11Departments of Genetics, University ofWisconsin, Madison, WI 53706, USA. 12Departments of Medical Genetics,University of Wisconsin, Madison, WI 53706, USA.Received: 11 October 2015 Accepted: 5 January 2016References1. 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Diabetes Metab. 2010;36(5):346–51.43. Buchner DM. Physical activity and prevention of cardiovascular disease inolder adults. Clin Geriatr Med. 2009;25(4):661–75. viii.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Safdar et al. Skeletal Muscle  (2016) 6:7 Page 17 of 17


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