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Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice Background: Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. Results: Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. Conclusions: Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present 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, Senescence Background evidence actually suggests that mtDNA replication er- The universality of the aging phenomenon has evoked rors may be the more important culprit [2]. The demon- great interest in unveiling regenerative remedies and re- stration that multiple aspects of aging are accelerated in juvenation medicine designed to evade molecular insti- mutator mice harboring error-prone mitochondrial poly- gators of mammalian aging. Molecular investigations of merase gamma provides support for the causal role of age-related pathologies implicate mitochondrial DNA mtDNA replication errors in instigating mammalian (mtDNA) mutations as one of the primary instigators aging [3, 4]. Similar phenotypes have also been reported driving multisystem degeneration, stress intolerance, and in telomerase-deficient mice [5], where telomere dys- energy deficits [1]. It is intuitive to assume that the de function is associated with impaired mitochondrial bio- novo mtDNA mutations observed during aging are due genesis and metabolic failure resulting in progressive to accumulated, unrepaired oxidative damage, but some tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses as seen with aging * Correspondence: tarnopol@mcmaster.ca [5]. Indeed, epidemiological studies have correlated de- Department of Pediatrics, McMaster University, Hamilton, ON L8N 3Z5, creased telomere length in peripheral blood leukocytes, Canada 3 with higher mortality rates in individuals more than Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada 60 years old [6]. Furthermore, a recent study in Full 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 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the 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 Page 2 of 17 centenarians and their offspring found a positive link be- Endurance exercise protocol tween telomere length and longevity; in particular, those Endurance exercise protocol and tissue harvesting was with longer telomeres had an overall improved health carried out, as previously described, using an independ- profile, with decreased incidence of age-associated dis- ent cohort of mice [11]. Briefly, at 3 months of age, mice eases, better cognitive function, and improved lipid pro- were housed individually in micro-isolator cages in a files relative to controls [7]. temperature- and humidity-controlled room and main- The epidemic emergence of modern chronic diseases tained on a 12-h light–dark cycle with food and water largely stems from the adoption of a sedentary lifestyle ad libitum [13]. PolG mice and PolG-p53 MKO mice and excess energy intake [8]. There is incontrovertible were randomly assigned to sedentary (PolG-SED or evidence that endurance exercise extends life expectancy PolG-p53 MKO-SED) or forced-endurance (PolG-END and reduces the risk of chronic diseases in both rodents or PolG-p53 MKO-END) exercise groups (n =5–20/ and humans [9, 10]. We have previously shown that en- group; ♀ = ♂). None of the mice had been previously durance exercise effectively rescued progeroid aging in subjected to a structured exercise regiment. One week mutator mice concomitant with a reduction in mtDNA of pre-training was allowed to acclimatize mice in en- mutations, despite an inherent defect in mitochondrial durance exercise groups to the treadmill. Mice in endur- polymerase gamma (POLG1) proofreading function [11]. ance exercise groups were subjected to forced treadmill Exercise has also been shown to increase telomerase ac- exercise (Eco 3/6 treadmill; Columbus Instruments, tivity and reduce senescence markers [12]. These find- Columbus, Ohio) three times per week at 15 m/min for ings suggest a link between exercise-mediated metabolic 45 min for 6 months. A 5-min warm-up and cool-down adaptations and genomic (nuclear and mitochondrial) at 8 m/min were also included. PolG mice were age- and stability; however, the identity of this metabolic link re- sex-matched with sedentary littermate WT mice (n = 20; mains unknown. In this study, we have utilized PolG ♀ = ♂), which served as controls for the study to assess mice to investigate the mitochondrial-telomere dysfunc- if endurance exercise intervention can molecularly bring tion axis in the context of progeroid aging, and to PolG mice to normalcy. At 8 months of age, animals elucidate how exercise counteracts mitochondrial were euthanized and tissues were collected for molecular dysfunction and mtDNA mutation burden through analyses. The study was approved by the McMaster mitochondrial localization of the tumor suppressor University Animal Research and Ethics Board under the protein p53. global Animal Utilization Protocol # 12-03-09, and the experimental protocol strictly followed guidelines put Methods forth by the Canadian Council of Animal Care. Mice breeding +/D257A Heterozygous mice (C57Bl/6J, PolgA )for the Endurance stress test mitochondrial polymerase gamma knock-in mutation The mice were subjected to four separate endurance were a kind gift from Dr. Tomas A. Prolla, University stress tests over to indirectly assess improvements in of Wisconsin-Madison, USA [4]. We generated homo- aerobic capacity with exercise as previously described zygous knock-in mtDNA mutator mice (PolG; Pol- [11]. Briefly, animals from all groups were placed in indi- D257A/D257A gA ) and littermate wild-type (WT; PolgA vidual lanes on the treadmill and allowed to acclimatize +/+ ) from heterozygous mice-derived colony main- for 30 min to eliminate any confounding effects due to tained at the McMaster University Central Animal Fa- stress or anxiety related to a new environment. The test cility as previously described [11]. Muscle-specific p53 began with a 5-min warm-up session at 8 m/min, knock-out mice (p53 MKO) were bred by crossing followed by +1 m/min increase in speed every 2 min tm1Brn p53 flox mice (Trp53 /J) with muscle-creatine until the mouse reached exhaustion. A low-intensity kinase Cre recombinase mice (Tg(Ckmm-cre)5Khn/J) electrically stimulus was provided to ensure compliance. purchased from Jackson Laboratories. We generated Time to exhaustion (min) was recorded when the mouse genetically modified homozygous knock-in mtDNA sat at the lower end of the treadmill, near a shock bar, mutator mice with muscle-specific p53 knockout for >10 s and was unresponsive to further stimulation to (PolG-p53 MKO), by crossing heterozygous mice continue running. +/D257A (PolgA ) with p53 MKO mice. During breeding, all animals were housed three to five per cage in a 12-h light/dark cycle and were fed ad libitum Survival analysis (Harlan-Teklad 8640 22/5 rodent diet) after weaning. An independent cohort of animals from all groups was The presence of the polymerase gamma homozygous used to carry out survival analyses as previously de- knock-in mutation was confirmed as previously scribed [11], and Kaplan–Meier survival curves were cal- described [4]. culated using GraphPad Prism 4.0. Safdar et al. Skeletal Muscle (2016) 6:7 Page 3 of 17 Tissue harvesting Mouse embryonic fibroblast isolation and reporter assay Tissues were collected at the time of euthanasia as previ- Mouse embryonic fibroblasts (MEFs) were generated +/+ ously described [11]. Immediately following cervical dis- using standard techniques from WT (p53 ) and p53 −/− location, the chest cavity was exposed and the heart was knockout (KO) mice (p53 ). Cell used in the experi- removed rapidly, followed by the skeletal muscle (quad- ments were from passages 4–5. Promoter sequence for riceps femoris). The skeletal muscle (quadriceps femoris, PGC-1α was amplified by PCR from mouse muscle gen- tibialis anterior, and soleus) and heart were either (i) col- omic DNA and cloned into the pGL4 luciferase reporter lected in RNase-free cryovials, immediately immersed in vector (Promega, Madison, WI). The pG13-luc plasmid liquid nitrogen, and stored at −80 °C for later analysis of containing 13 copies of a synthetic p53 DNA binding DNA, RNA, protein, and enzyme activity or (ii) immedi- site was used as a positive control (which has been com- ately rinsed with phosphate buffer saline (PBS) and used prehensively characterized in Jackson et al., 2001 and for skeletal muscle and heart mitochondrial and nuclear Kern et al., 1991). A GFP expressing plasmid was used +/+ −/− fractionations. to 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- Hematopoietic stem and progenitor cell isolation scriptional activity was measured using Bright-Glo™ lu- Mouse hematopoietic stem and progenitor cells (HSC) ciferase reporter assay system (Promega, Madison, WI). were isolated according to the method of Ema et al. with minor modifications [14]. Marrow was flushed from the Total RNA isolation from skeletal muscle and heart femur and tibia using a 25-g needle, passed through a 50- Total RNA was isolated from ~25 mg of the skeletal μm sieve and counted with a hemocytometer. Cells were muscle (quadriceps femoris)and heartusing the incubated with primary antibodies for 90 min at 4 °C Qiagen total RNA isolation kit (Qiagen, Mississauga, followed by 20 min incubation in the appropriate second- ON) [11, 13]. RNA samples were treated with ary antibody at 4 °C. Lineage negative, and Sca-1 and c- RNase-free DNase on Qiagen spin-columns (Qiagen, Kit positive (LSK) population enriched for stem cells Mississauga, ON) to remove DNA contamination. were sorted using the EPICS ALTRA™ fluorescence- RNA integrity and concentration were assessed using activated cell sorter (Beckman Coulter, Mississauga, the Agilent 2100 Bioanalyzer (Agilent Technologies, ON) with gating strategies established using single- Palo Alto, CA) [13]. The average RIN (RNA integrity stained controls. The following antibodies were used: number) value for all samples was 9.64 ± 0.20 (scale lineage panel (BD Pharmingen™,Mississauga,ON), 1–10), ensuring a high quality of isolated RNA. anti-mouse Sca-1 Clone: E13-161.7 (BD Pharmingen™, Mississauga, ON), anti-mouse c-Kit Clone: 2B8 RNA, DNA, and protein isolation from HSC and SC (eBioscience, San Diego, CA), and streptavidin (Bio- Total RNA, DNA, and protein were isolated from HSC Source, Burlington, ON). and SC using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Mississauga, ON) according to the manufac- Satellite cell isolation turer’s instructions. Primary skeletal muscle satellite cells (SC) were isolated from WT, PolG-SED, and PolG-END mice using the Microarray analysis methods described previously [15] and subsequently Total RNA was extracted from skeletal muscle (quadri- purified by fluorescence-activated cell sorting. Briefly, ceps femoris) using the Qiagen RNeasy Micro kit the hind limb skeletal muscles were carefully dissected, (Qiagen, Mississauga, ON) and processed on Qiagen’s cleaned of fat and washed in cold PBS. Cells were re- QIAcube (Qiagen, Mississauga, ON) using the standard leased by mulching the tissue with scissors and incuba- manufacturer’s protocol. The samples were then checked tion in a collagenase/dispase solution three times, for quality using Nanodrop 2000 (Thermo Scientific, 12 min each, at 37 °C with further mechanical disruption Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent using a pipette between incubations. Following passage Technologies, Palo Alto, CA). TransPlex Whole Tran- through 70 and 30 μm filters, cells were stained using scriptome Amplification kit (Sigma-Aldrich , Oakville, primary antibody to c-met conjugated to PE (1:200, ON) was used to amplify complementary (cDNA) from eBioscience, San Diego, CA) and subjected to FACS the muscle RNA samples according to the manufac- sorting (EPICS ALTRA™, Beckman Coulter, Mississauga, turer’s instructions. Samples were amplified for 25 cycles ON). SC were pelleted in RNase-free cryovials, immedi- using the recommended cycling parameters. All samples ately immersed in liquid nitrogen, and stored at −80 °C were subsequently purified using Qiagen’s QIAquick for later analyses. PCR Purification kit (Qiagen, Mississauga, ON) and Safdar et al. Skeletal Muscle (2016) 6:7 Page 4 of 17 processed on Qiagen’s QIAcube (Qiagen, Mississauga, (Applied Biosystems Inc., Foster City, CA) [11]. Forward ON) using the standard “Cleanup QIAquick PCR for and reverse primers for the aforementioned genes amplification reactions” (Version 4) protocol. Samples (Additional file 1: Table S2) were designed based on se- were purified and examined using Nanodrop 2000 quences available in GenBank using the online MIT (Thermo Scientific, Wilmington, DE) and Agilent 2100 Primer 3 designer software (developed at Whitehead In- Bioanalyzer DNA 7500 chip (Agilent Technologies, Palo stitute and Howard Hughes Medical Institute by Steve Alto, CA) to ensure proper yield and quality of amplifi- Rozen and Helen Skaletsky) and were confirmed for spe- cation. To perform the microarray hybridization, 2 μgof cificity using the basic local alignment search tool. β-2 cDNA from each sample was labeled using NimbleGen’s microglobulin was used as a control house-keeping gene, One Color Labeling kit (Cat.# 05223555001; Roche as its expression was not affected with the experimental NimbleGen Inc., Madison, WI) according to the manu- intervention (data not shown). All samples were run in facturer’s protocol. Five micrograms of Cy3 labeled duplicate simultaneously with a negative control which samples were hybridized to Mus musculus 12x135k, contained no cDNA. Melting point dissociation curves NimbleGen Gene Expression Arrays (Cat.# 05543797001; generated by the instrument were used to confirm the Roche NimbleGen Inc., Madison, WI), washed, and specificity of the amplified product. scanned according to manufacturer’s protocol. NimbleGen gene expression arrays were scanned using an Axon Tissue total DNA isolation GenePix 4200A scanner (Molecular Devices Inc., Down- Total DNA (genomic and mtDNA) was isolated from ingtown, PA) with settings of 100 POW and 300–350 ~15 mg of the skeletal muscle (soleus) and heart using photomultiplier (PMT). Pair files were generated for each the QIAamp DNA Mini kit (Qiagen, Mississauga, ON) array using NimbleScan software (Roche NimbleGen Inc., [11, 16]. DNA samples were treated with RNase Madison, WI). Resulting array data was analyzed with Bio- (Fermentas, Mississauga, ON) to remove RNA contam- conductor software (Bioconductor, Seattle, WA) in which ination. DNA concentration and quality were assessed the data were normalized and tested for significantly dif- using Nanodrop 2000 (Thermo Scientific, Wilmington, ferentially expressed genes which were assessed based DE). upon a 5 % false discovery rate (FDR). The gene array data reported here is deposited in Gene Expression Omnibus mtDNA copy number analysis (Accession Number: GSE75869) public functional genom- Mitochondrial DNA copy number, relative to the diploid ics data repository. The resulting data were input into In- chromosomal DNA content, was quantitatively analyzed genuity Pathway Analysis (Ingenuity Systems, Redwood from the skeletal muscle (soleus), heart, primary City, CA) to determine the over-represented gene categor- hematopoietic stem cells, primary satellite cells, and pri- ies using strict association. The normalized expression in mary fibroblasts using ABI 7300 real-time PCR (Applied these categories was plotted in a heat map using R script Biosystems, CA) [11, 16]. Primers were designed around and Bioconductor software (Bioconductor, Seattle, WA). COX-II region of the mitochondrial genome (Additional file 1: Table S2). Nuclear β-globin gene was used as a Real-time quantitative PCR housekeeping gene (Additional file 1: Table S2). The messenger RNA (mRNA) expression of peroxisome proliferator-activated receptor gamma co-activator 1 Average telomere length alpha (PGC-1α), mitochondrial transcription factor A Average telomere length was measured in heart, primary (TFAM), estrogen-related receptor alpha (ERRα), 5- hematopoietic stem cells, and primary satellite cell gen- aminolevulinate synthase (ALAS), cytochrome c oxidase omic DNA using a real-time quantitative PCR method subunit-I (COX-I), cytochrome c oxidase subunit-IV as previously described [17]. The premise of this assay is (COX-IV), complex I NADH dehydrogenase subunit 1 to measure an average telomere length ratio by quantify- (ND1), complex V subunit ATPase 6 (ATPase 6), cyclin- ing telomeric DNA with specially designed primer se- WAF1 dependent kinase inhibitor 1A (p21 ), cyclin- quences and dividing that amount by the quantity of a INK4A dependent kinase inhibitor 2A (p16 ), and growth single-copy gene [17]. All samples were run using a 7300 arrest and DNA-damage-inducible beta (GADD45B) Real-time PCR System (Applied Biosystems Inc., Foster ® ® were quantified using 7300 Real-time PCR System (Ap- City, CA) and SYBR Green chemistry (PerfeC a SYBR plied Biosystems Inc., Foster City, CA) and SYBR Green Green Supermix, ROX, Quanta BioSciences, Gaithers- chemistry (PerfeC a SYBR Green Supermix, ROX, burg, MD). A single-copy gene, 36B4, which encodes for Quanta BioSciences, Gaithersburg, MD) as previously the acidic ribosomal phosphoprotein PO, was used as a described [11, 13]. First-strand cDNA synthesis from control for amplification for every sample performed 1 μg of total RNA was performed with random primers [17, 18]. Each PCR reaction for the telomere and 36B4 using a high-capacity cDNA reverse transcription kit included 12.5 μL of 1x SYBR Green master mix Safdar et al. Skeletal Muscle (2016) 6:7 Page 5 of 17 (PerfeC a SYBR Green Supermix, ROX, Quanta BioSci- Chromatin immunoprecipitation assay ences, Gaithersburg, MD), 300 nM each of the forward Chromatin immunoprecipitation (ChIP) assay was per- and reverse telomere or 36B4 primers (Additional file 1: formed using an EZ-ChIP™ kit (Millipore, Billerica, MA) Table S2), 20 ng genomic DNA, and enough DNase/ as previously described [11]. Twenty-milligram piece of RNase-free H O (Applied Biosystems Inc., Foster City, the quadriceps femoris muscle was cross-linked in 5 mL of CA) to yield a 25-μL reaction. Cycling conditions for phosphate-buffered saline containing 1 % formaldehyde telomere are as follows: 95 °C for 10 min followed by for 10 min at room temperature. One milliliter of 10X gly- 30 cycles of data collection at 95 °C for 15 s and a 56 °C cine was added to stop fixation. Muscles were then ho- anneal-extend step for 1 min. Cycling conditions for mogenized in 1 mL of SDS lysis buffer supplemented with 36B4 are as follows: 95 °C for 10 min followed by 35 cy- protease inhibitor cocktail Complete, ETDA-free (Roche cles of data collection at 95 °C for 15 s, with 52 °C an- Applied Science, Mannheim, Germany). Chromatin was nealing for 20 s, followed by extension at 72 °C for 30. sheared by sonicating each sample on ice using a Branson Each sample was analyzed in duplicate, and the ratio of Digital Sonifier S-450D (output 20 %, 4 times for 20 s, telomere:36B4 was calculated. The average of these ra- with a 20-s pause each time; Branson Ultrasonics Corpor- tios was reported as the average telomere length ratio ation, Danbury, CT). Following centrifugation at 10,000×g (ATLR). at 4 °C for 10 min, the supernatant containing 1 mg of protein was diluted to 1 mL with dilution buffer. Ten mi- crograms of anti-p53 (FL-393) antibody (Santa Cruz Bio- Whole tissue lysate technology Inc., Santa Cruz, CA) was added per sample Total protein was extracted from tissue samples as pre- and incubated overnight at 4 °C. Anti-IgG antibody was viously described [11]. Briefly, ~30 mg of the skeletal used as a non-specific control. Sixty microliters of protein muscle (quadriceps femoris) and heart were homoge- G-agarose was added, and the sample was mixed for 1 h nized on ice in a 2-mL Wheaton glass homogenizer at 4 °C with rotation. Precipitated complexes were eluted (Fisher Scientific, Ottawa, ON) with 25 volumes of phos- in 100 μL of elution buffer, and cross-linking was reversed phate homogenization buffer [50 mM KPi, 5 mM EDTA, by the addition of 8 μL of 5 M NaCl per sample followed 0.5 mM DTT, 1.15 % KCl supplemented with a by incubation at 65 °C for 10 h. Co-immunoprecipitated Complete Mini, ETDA-free protease inhibitor cocktail DNA was purified according to the manufacturer’sin- tablet and a PhosSTOP, phosphatase inhibitor cocktail structions. Primers were designed to amplify the p53 bind- tablet (Roche Applied Science, Mannhein, Germany) per ing regions (−564 and −954) of the PGC-1α promoter 10 mL of buffer]. The lysate was centrifuged at 600g for (Additional file 1: Table S2). The amount of PGC-1α pro- 15 min at 4 °C to pellet cellular debris. The supernatant moter immunoprecipitated with p53 was quantified using was aliquoted, snap frozen in liquid nitrogen, and stored the 7300 Real-time PCR System (Applied Biosystems Inc., at −80 °C until further analysis. Foster City, CA) and SYBR Green chemistry (PerfeC a SYBR Green Supermix, ROX, Quanta BioSciences, Nuclear fractionation Gaithersburg, MD). Purified DNA from the input sample Nuclear fractions were prepared from 40 mg of the that did not undergo immunoprecipitation was PCR- freshly obtained skeletal muscle (quadriceps femoris), amplified using of β-globin primers (Additional file 1: heart, primary satellite cells, and primary fibroblasts Table S2) and was used to normalize signals from ChIP using a commercially available nuclear extraction kit assays. (Pierce NE-PER , Rockford, IL) as previously described [11, 16]. Briefly, samples were homogenized in CER-I Mitochondrial fractionation buffer containing protease inhibitor cocktail Complete, Mitochondrial fractions were isolated using differential ETDA-free (Roche Applied Science, Mannheim, centrifugation as previously outlined [11]. Briefly, the Germany) using an electronic homogenizer (Pro 250, skeletal muscle (quadriceps femoris and tibialis anterior), Pro Scientific, Oxford, CT, USA). Pellets containing nu- heart, primary satellite cells, and primary fibroblasts clei were obtained by centrifugation at 16,000g for were finely minced and homogenized on ice in 1:10 (wt/ 10 min at 4 °C and were subsequently washed four times vol) ice-cold isolation buffer A (10 mM sucrose, 10 mM in PBS to remove cytosolic contaminating proteins. Nu- Tris/HCl, 50 mM KCl, and 1 mM EDTA, and 0.2 % fatty clear proteins were extracted in NER buffer supple- acid-free BSA, pH 7.4, supplemented with protease in- mented with protease inhibitors [11]. Enrichment and hibitor cocktail Complete, ETDA-free [Roche Applied purity of nuclear fractions were confirmed by the abun- Science, Mannheim, Germany]) using a Potter-Elvehjem dance of nuclear histone H2B and absence of the cyto- glass homogenizer. The resulting homogenates were solic protein lactate dehydrogenase in Western blot centrifuged for 15 min at 700g, and the subsequent su- analyses as previously shown by our group [16]. pernatants were centrifuged for 20 min at 12,000g.The Safdar et al. Skeletal Muscle (2016) 6:7 Page 6 of 17 mitochondrial pellets from 12,000g spin were washed antibodies were used as a non-specific control. The and then re-suspended in a small volume of ice-cold iso- matrix was centrifuged at 16,000g for 30 s, and the pellet lation buffer B (10 mM sucrose, 0.1 mM EGTA/Tris, matrix-immune complex precipitate was washed four and 10 M Tris/HCl, pH 7.4, supplemented with protease times under stringent conditions (50 mM Tris-HCl, pH inhibitor cocktail Complete, ETDA-free [Roche Applied 7.4, 500 mM NaCl, 2 mM EDTA) and incubated over- Science, Mannheim, Germany]). All centrifugation steps night at 65 °C in the presence of 1 % SDS for cross- were carried out at 4 °C. The mitochondrial pellets were linking reversion. DNA was extracted from supernatants immediately frozen at −80 °C for further biochemical using the QIAamp DNA Mini kit (Qiagen, Mississauga, analyses. Enrichment and purity of mitochondrial frac- ON) according to the manufacturer’s instructions. tions were confirmed by the abundance of mitochondrial mtDNA COX-II and cytochrome b regions (Additional cytochrome c oxidase subunit IV protein and absence of file 1: Table S2) were quantified using 7300 Real-time the nuclear histone H2B and the cytosolic protein lactate PCR System (Applied Biosystems Inc., Foster City, CA) ® ® dehydrogenase in Western blot analyses as previously and SYBR Green chemistry (PerfeC a SYBR Green shown by our group [16]. Supermix, ROX, Quanta BioSciences, Gaithersburg, MD), as previously described [16]. Mitochondrial co-immunoprecipitation assay Mitochondrial co-immunoprecipitation assay was per- formed on isolated mitochondrial fractions using Pierce Western blotting and markers of oxidative damage Co-Immunoprecipitation Kit (Pierce, Rockford, IL) as Protein concentrations of whole tissue lysates, and mito- previously described [16]. Briefly, mitochondrial frac- chondrial and nuclear fractions were determined using a tions were homogenized in lysis buffer (0.025 M Tris, commercial assay (BCA Protein Assay, Pierce, Rockford, 0.15 M NaCl, 0.001 M EDTA, 1 % NP-40, 5 % glycerol, IL). Proteins were resolved on 10 or 12.5 % SDS-PAGE pH 7.4) supplemented with protease inhibitor cocktail gels depending on the molecular weight of the protein of Complete, ETDA-free (Roche Applied Science, interest. The gels were transferred onto Hybond ECL Mannheim, Germany). Two milligrams of mitochondrial nitrocellulose membranes (Amersham, Piscataway, NJ) fraction was pre-cleared by incubation with 100 μLof and immunoblotted using the following commercially control agarose resin to minimize non-specific binding. available primary antibodies: MitoProfile Total OXPHOS Forty micrograms of anti-p53 (FL-393) antibody (Santa Rodent cocktail (MS604) antibody (MitoSciences, Eugene, Cruz Biotechnology Inc., Santa Cruz, CA) was covalently OR); anti-PGC-1α (2178), anti-VDAC (4866), and anti-α/ coupled onto an amine-reactive resin. The pre-cleared β-tubulin (2148) antibodies (Cell Signaling Technology, lysates were subsequently incubated with antibody- Denver, MA); anti-p53 (MABE283-PAb421) antibody coupled beads overnight at 4 °C. Co-immunoprecipitates (EMD Millipore); anti-Tfam (sc-23588) and anti-NRF-1 were collected by centrifugation, boiled in 50 μLof (sc-33771) antibodies (Santa Cruz Biotechnology Inc., Laemmli sample buffer, and used for immunoblot ana- Santa Cruz, CA); anti-POLG1 antibody (a kind gift of Dr. lysis for POLG1 (a kind gift of Dr. William C. Copeland, William C. Copeland, National Institutes of Health, USA); National Institutes of Health) or anti-Tfam (sc23588, A- anti-citrate synthase antibody (a kind gift of Dr. Brian H. 17; Santa Cruz Biotechnology Inc., Santa Cruz, CA) anti- Robinson, The Hospital for Sick Children, Canada); anti- body. Anti-IgG antibodies were used as a non-specific 4-HNE (ab48506), anti-SOD2 (ab13533), anti-catalase WAF1 control. (ab1877), and anti-p21 (ab7960) antibodies (Abcam, Cambridge, MA); anti-Pax7 (Developmental Studies mtDNA immunoprecipitation assay Hybridoma Bank, University of Iowa, Iowa City, IO); and mtDNA immunoprecipitation was performed on skeletal anti-ERRα (EPR46Y) and anti-actin (NB600-535) anti- muscle mitochondrial fraction that was cross-linked and bodies (Novus Biologicals, Littleton, CO) [11, 16]. The sonicated as previously described [16, 19]. One milli- carbonylated protein content in whole tissue lysates and gram of mitochondrial fraction was pre-cleared in 25 % mitochondrial fractions was quantified by Western blot v/v pre-clearing matrix F (Santa Cruz Biotechnology, using OxyBlot Protein Detection kit (S7150; Millipore, Santa Cruz, CA) overnight at 4 °C. The supernatant was Bedford, MA) as per manufacturer’sinstructions. Allanti- then incubated with 20 μg of anti-p53 (FL-393) antibody bodies were used at 1:1000 dilution, except for anti-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and (1:10,000). Membranes were then incubated with the ExactaCruz™ F matrix (Santa Cruz Biotechnology, Santa appropriate anti-mouse or anti-rabbit horse radish Cruz, CA) [20] with mixing by end-over-end inversion peroxidase-linked secondary antibody (1:10,000) and visu- overnight at 4 °C in the presence of 5 μg of shared sal- alized by enhanced chemiluminescence detection reagent mon sperm DNA (Sigma-Aldrich , Oakville, ON) to re- (Amersham, Piscataway, NJ). Relative intensities of the duce non-specific DNA-bead interactions. Anti-IgG protein bands were digitally quantified by using NIH Safdar et al. Skeletal Muscle (2016) 6:7 Page 7 of 17 Image J, version 1.37, analysis software (Scion Image, combining 960 μLof K HPO buffer (50 mM with 2 4 NIH). 50 mM EDTA and 0.01 % Triton X-100, pH 7.2–7.4) with 30 μL of muscle homogenate. Ten microliters of H O 2 2 ROS assay (1 M) was added to the cuvette and mixed by inversion to Mitochondrial H O production was measured using the initiate the reaction. Absorbance was measured at 240 nm 2 2 Amplex Red Hydrogen Peroxide assay (A22188; Invitro- every 15 s for 2 min. Catalase activity was calculated and −1 −1 gen, Burlington, ON) as per manufacturer’s instructions. reported in μmol · min ·mg protein . All samples were Briefly, 40 μg of mitochondrial fraction was diluted in analyzed in duplicates on the Cary UV-vis spectrophotom- 50 μL reaction buffer (125 mM KCl, 10 mM HEPES, eter (Varion, Inc., Palo Alto, CA). 5 mM MgCl , 2 mM K HPO , pH 7.44) to determine 2 2 4 mitochondrial respiratory chain complex I (5 mM pyru- Caspase-3 and caspase-9 enzyme activity vate/malate) or complex II (5 mM succinate) driven Caspase-3 and caspase-9 enzyme activity was measured H O production with and without inhibitors (0.5 μM using fluorometric protease assays caspase-3/CPP32 and 2 2 rotenone, complex I inhibitor, and 0.5 μM antimycin A, caspase-9/Mch6, respectively (Biovision, Mountain View, complex III inhibitor). Mitochondrial H O production CA) according to manufacturer’s instructions. Briefly, 2 2 was measured after the addition of 50 μL of reaction the assays are based on the detection of cleavage of the buffer containing horseradish peroxidase and Amplex substrate DEVD-AFC (AFC: 7-amino-4-trifluoromethyl Red. Fluorescence was followed at an excitation wave- coumarin) by caspase-3 and LEHD-AFC (AFC: 7-amino- length of 545 nm and an emission wavelength of 590 nm 4-trifluoromethyl coumarin) by caspase-9. Uncleaved for 5 min using fluorescence microplate reader (Tecan DEVD-AFC and LEHD-AFC fluoresce at λ = 400 nm, max Safire, MTX Lab Systems, Inc., Vienna, VA). The slope upon cleavage of the respective substrate by caspase-3 of the increase in fluorescence is converted to the rate of or caspase-9, free AFC emits a yellow-green fluorescence H O production with a standard curve. All of the assays (λ = 505 nm), which was quantified using a fluores- 2 2 max were performed at 25 °C. The results are expressed as cence microplate reader (Tecan Safire, MTX Lab Sys- −1 −1 pmoles.min .mg protein . tems, Inc., Vienna, VA). Results were expressed as raw fluorescence units per milligram of cytosolic protein. Mitochondrial respiratory chain complex I and IV enzyme activity Apoptosis cell death detection ELISA Mitochondrial ETC complex I and complex IV activities Apoptotic DNA fragmentation was quantified in the skel- were determined in tissue lysates following established etal muscle (quadriceps femoris), heart, primary protocols [11, 21–23]. All samples were analyzed in du- hematopoietic stem cells, and primary satellite cells by plicates on the Cary UV-vis spectrophotometer (Varion, measuring the amount of cytosolic mono- and oligo- PLUS Inc., Palo Alto, CA). nucleosomes using a Cell Death detection ELISA assay (Roche Applied Science, Laval, QC) as previously Superoxide dismutase and catalase enzyme activity described [11]. Briefly, wells were coated with a monoclo- Muscle total superoxide dismutase (Mn-SOD and Cu/Zn- nal anti-histone antibody and incubated with homoge- SOD) activity was determined in muscle lysates by meas- nates. Nucleosomes were centrifuged at 100,000g followed uring the kinetic consumption of superoxide radical (O ) by binding to the anti-histone antibody followed by the by SOD in a competitive reaction with cytochrome c,as addition of anti-DNA-peroxidase antibody that binds to previously described [20]. Absorption was recorded at the DNA associated with the histones. The amount of per- 550 nm and was observed every 15 s for 2 min at 37 °C. oxidase retained in the immunocomplex was determined One unit (U) of SOD activity was defined as the amount spectrophotometrically with ABTS (2,2′-azino-bis[3-ethyl- of enzyme that caused a 50 % inhibition of the reduction benzthiazoline-6-sulphonic acid]) as a substrate. Results of cytochrome c. Total SOD activity was expressed in were expressed as arbitrary OD units normalized to mi- −1 U.mg of protein . In a separate cuvette, the same sample crograms of cytosolic protein. was analyzed under identical conditions in the presence of 0.2 M KCN (pH 8.5–9.5), a potent inhibitor of cytosolic Quantification of mtDNA mutations Cu/Zn-SOD [24], for determination of mitochondrial Mn- mtDNA mutations were quantified by the error-resistant SOD activity. Cu/Zn-SOD activity was approximated by single molecule approach [26]. Briefly, skeletal muscle subtracting Mn-SOD activity from total SOD activity. (quadriceps femoris) DNA was subjected to limiting dilu- Both Mn-SOD and Cu/Zn-SOD activity were expressed in tion long-range PCR, where each positive PCR reaction −1 U.mg protein . Catalase activity was determined by was initiated by a single mtDNA molecule. PCR was de- measuring the kinetic decomposition of H O as previ- signed to amplify essentially the entire mitochondrial 2 2 ously described [25]. Catalase activity was measured by genome using high-fidelity Phusion DNA polymerase, Safdar et al. Skeletal Muscle (2016) 6:7 Page 8 of 17 (New England Biolabs). Three to 9 amplified molecules are presented as mean ± standard error of the mean were obtained per animal. Each amplified molecule was (SEM). sequenced in its entirety using barcoded Illumina next generation sequencing approach at a local core facility. Results and discussion Mutations were identified by comparing each molecule’s Endurance exercise confers phenotypic protection, sequence to the standard C57Bl/6J mtDNA sequence reduces mtDNA mutations, and attenuates oxidative (GenBank EF108336). Only 100 % mutations were con- damage in PolG mice sidered, which guaranteed the exclusion of artifacts [26]. Aged tissues display stochastic accumulation of mtDNA Mutant fractions were calculated by dividing the total mutations that likely perpetuate respiratory chain defi- number of mutations by the number of nucleotides se- ciency and greater reactive oxygen species (ROS)-medi- quenced per animal. ated damage [28]. To evaluate the underlying protective mechanism of exercise on mitochondrial redox status and mtDNA integrity, we profiled “terminally differenti- p53 base excision repair activity assay ated” (skeletal muscle and heart) and “proliferative” (Lin An in vitro fluorescence-based DNA primer p53 repair − Sca-1+ c-Kit + population enriched for hematopoietic activity assay was employed as previously described [27], stem and progenitor cells, “HSC’ and c-met+, satellite with minor modifications. This assay utilized a double- cells, “SC”) compartments of littermate wild-type (WT), stranded deoxyoligomers containing sequences identical sedentary PolG (PolG-SED), and forced-endurance exer- to the first 40 nucleotides of the mtDNA replication ori- cised PolG (PolG-END) mice. As shown previously [11], gin as the primer-template substrate, with the 3′ end of and now confirmed in an independent cohort of mice the primer contained self-designed mismatch point mu- utilized in this study, exercise rescued progeroid aging tation in the last three nucleotides (Additional file 1: (Additional file 1: Figure S1A), increased life span Table S2). The 5′ and 3′ ends of the primer were chem- (Additional file 1: Figure S1B), and reduced mtDNA mu- ically linked to a Black Hole Quencher -1 and 6- tations (Fig. 1a) in PolG mice. carboxyfluorescein (FAM-1™) flourophore, respectively Initial characterization of PolG mice showed absence (Integrated DNA Technology , Toronto, ON). The prem- of increased oxidative damage despite significant accu- ise of this assay is that, in the absence of proofreading mulation of mtDNA point mutations [4, 29]. We evalu- capacity of mitochondrial polymerase gamma, primer ated the presence of oxidative modifications and found extension requires the excision of the unpaired nucleo- no difference in protein carbonyls (PC) and 4-hydroxy- tides by the 3′→5′ exonuclease activity which in turn 2-nonenal (4-HNE) content in the muscle, heart, and SC will be detected as an increase in fluorescence over time. homogenates of PolG-SED vs. WT (Additional file 1: The 20 μL reaction mixtures containing 50 mM Tris– Figure S1C). We surmised that since the absence of oxi- HCl (pH 7.5), 5 mM MgCl , 1 mM DTT, 100 μg/mL of dative damage in the PolG tissues is due to cell-to-cell BSA, 3′-end-FAM1™ primer-template substrate, 50 μM variability, and that any one modification would be lower each of dATP, dCTP, dGTP, and dTTP, and 40 μgof than the detectable limit in whole tissue homogenates WT, PolG-SED, and PolG-END skeletal muscle (quadri- [4, 29], we measured oxidative damage in mitochondrial ceps femoris) mitochondrial extracts were incubated at fractions—the primary source of cellular ROS. Indeed, 37 °C for 40 min with data collection at the end using mitochondria from these tissues demonstrated a sub- iCycler IQ™ real-time PCR detection system (BioRad, stantial increase in H O production, along with elevated 2 2 Mississauga, ON). To assess the requirement of p53 as PC and 4-HNE content (Fig. 1b, c, and Additional file 1: an accessory mtDNA mismatch point mutation repair Figure S1C, D, and F). These observations are consistent protein, p53 repair activity assay was also carried out in with recent studies reporting higher PC levels in heart (i) PolG-END skeletal muscle mitochondrial extract after mitochondria of PolG mice [30] and increased mito- p53 immunodepletion and (ii) PolG-SED skeletal muscle chondrial H O production in vivo using mitochondria- 2 2 mitochondrial extract with addition of recombinant hu- targeted mass spectrometry probe MitoB [31]. This man p53 (BD Biosciences, Mississauga, ON). higher oxidative damage is also congruent with reduced superoxide dismutase 2 (SOD2) and catalase content Statistics and activity in PolG-SED vs. WT (Fig. 1d and Additional All molecular indices between the groups (WT, PolG- file 1: Figure S1E). We hypothesize that the combination SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 of lower antioxidant capacity, coupled with elevated MKO-END mice) were analyzed using two-tailed Stu- ROS production in PolG-SED mitochondria exacerbates dent’s t test. The log-rank test was used to test for signifi- the accumulation of mtDNA mutations. Consistent with cant differences in life span distribution between groups. this notion, Vermulst et al. reported a significant reduc- Statistical significance was established at a P ≤ 0.05. Data tion in the frequency of mtDNA mutations in the heart Safdar et al. Skeletal Muscle (2016) 6:7 Page 9 of 17 Fig. 1 (See legend on next page.) Safdar et al. Skeletal Muscle (2016) 6:7 Page 10 of 17 (See figure on previous page.) Fig. 1 Endurance exercise reduces random mtDNA somatic mutations, attenuates mitochondrial ROS-mediated oxidative damage, mitigates telomere shortening, and reduces nuclear accumulation of p53 in mtDNA mutator mice. a Random mtDNA somatic mutation rate (per 1000 nucleotides of mtDNA) in muscle (quadriceps femoris) WT, PolG-SED, and PolG-END mice (n =4–5/group). b H O production rate in muscle 2 2 mitochondrial 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 catalase enzyme activity in the muscle (quadriceps femoris) and heart of WT, PolG-SED, and PolG-END (n = 7/group). e Average telomere length ratios in the heart, hematopoietic stem and progenitor cells (HSC), and satellite cells (SC) of WT, PolG-SED, and PolG-END (n =6–8/group). f Representative blots 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. Error bars represent SEM. AU arbitrary units tissue of transgenic animals that over-expressed human Endurance exercise mitigates mitochondrial dysfunction catalase (CAT), a ROS scavenger, to mitochondria vs. via reduction in nuclear p53 that represses PGC-1α age-matched (28 months old) wild-type mice [32]. We A causal role of mitochondrial-induced oxidative stress observed that exercise normalized mitochondrial H O and telomere erosion, secondary to mtDNA mutations, 2 2 production (Fig. 1b and Additional file 1: Figure S1D), suggests a direct link between p53 activation and mito- and markers of oxidative damage (Fig. 1c and Additional chondrial dysfunction [5]. Increasing the expression of file 1: Figure S1F) in PolG-END to WT levels and in- PGC-1α, a potent regulator of mitochondrial biogenesis, creased SOD2 and catalase content and activity (Fig. 1d positively regulates the expression of antioxidants [33] and Additional file 1: Figure S1E). Together, our data and has been touted to attenuate aging-associated sarco- suggest that exercise reduces mtDNA point mutations, penia and metabolic dysfunction [34]. This prompted us at least in part, via the up-regulation of cellular antioxi- to investigate whether activation of p53-mediated senes- dant capacity that subsequently serves to attenuate ROS cence signaling attenuates PGC-1α-triggered gene pro- levels. gramming. We conducted in silico promoter analysis that identified putative p53 binding elements in the Endurance exercise diminishes telomere erosion and PGC-1α promoter. These promoter regions were then down-regulates aberrant p53 signaling and pathological cloned into a pGL4 luciferase reporter vector and trans- +/+ −/− levels of apoptosis in PolG mice fected into p53 and p53 mouse embryonic fibro- Sustained intrinsic accumulation of oxidative damage blasts (MEFs). A significant repression of PGC-1α-pGL4 +/+ has been implicated in telomere erosion that drives reporter activity was observed in the p53 relative to −/− age-related tissue degeneration [1]. In agreement with p53 MEFs (Additional file 1: Figure S2C). These re- this, we observed shorter telomeres in the heart, sults are consistent with a recent study showing that nu- HSC, and SC from PolG-SED vs. WT (Fig. 1e). clear p53 can directly repress PGC-1α expression and Genomic instability due to telomere shortening acti- promote mitochondrial dysfunction [5]. To further test vates tumor suppressor protein p53-mediated senes- our hypothesis, we next performed an anti-p53 chroma- cence/apoptotic signaling cascades [1]. Accordingly, tin immunoprecipitation assay that showed physical en- nuclear p53 abundance in the muscle, heart, and SC richment of nuclear p53 at the PGC-1α promoter of of PolG-SED was enhanced (Fig. 1f), concomitantly PolG-SED vs. WT mice (Fig. 2b). Together, these data with higher expression levels of the p53-responsive suggest that ROS-induced cellular damage prompts the WAF1 INK4A senescence genes: p21 ,p16 , and GADD45B nuclear accumulation of p53, which in turn activates (Additional file 1: Figure S1G) vs. WT. Mitochondrial p53-responsive senescence genes while simultaneously dysfunction in PolG mice is associated with patho- repressing the pro-metabolic activity of PGC-1α. logical systemic apoptosis [4, 11], and consistent with qPCR analyses of the PolG-SED muscle, heart, HSC these observations, we found higher DNA fragmenta- and SC confirmed lower expression of PGC-1α and tion (Fig. 2a) and caspase-3/9 activity (Additional file strong repression of its metabolic networks, including 1: Figure S2A and B) in PolG-SED mice. Interestingly, oxidative phosphorylation, mitochondrial function, glu- exercise abrogated telomere shortening (Fig. 1e), re- coneogenesis, and fatty acid metabolism vs. WT (Fig. 2c, duced p53 nuclear accumulation (Fig. 1f), normalized and Additional file 1: Figure S2D–G, and Table S1). the expression of p53-responsive senescence genes Additionally, PolG-SED mice tissues and stem cells have (Additional file 1: Figure S1G), and reduced patho- reduced mtDNA copy number (Fig. 2D and Additional logical levels of apoptosis in PolG-END (Fig. 2a, and file 1: Figure S3A), lower mitochondrial complex I and Additional file 1: Figure S2A and B). complex IV enzyme activity (Additional file 1: Figure Safdar et al. Skeletal Muscle (2016) 6:7 Page 11 of 17 Fig. 2 (See legend on next page.) Safdar et al. Skeletal Muscle (2016) 6:7 Page 12 of 17 (See figure on previous page.) Fig. 2 Endurance exercise prevents dysregulated mitochondrial-induced apoptosis, reduces nuclear p53-mediated repression of PGC-1α, induces PGC-1α regulated gene networks, restores mtDNA copy number, and normalizes mitochondrial morphology in mtDNA mutator mice. a Nuclear DNA fragmentation (apoptotic index) in the heart, HSC, and SC of WT, PolG-SED, and PolG-END (n =7–10/group). b ChIP assay showing reduced p53 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-globin gene 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 bars represent SEM. AU arbitrary units S3B), reduced mitochondrial electron transport chain exonuclease activity that helps p53 promote and main- subunits protein content (Additional file 1: Figure S3C– tain mitochondrial genomic stability by executing base I), and accumulation of swollen, pleomorphic, oversized excision repair on damaged mtDNA [36]. The role of mitochondria (Fig. 2e). Endurance exercise decreased mitochondrial p53 in the context of aging remains hith- binding of p53 to the PGC-1α promoter (Fig. 2b), and erto unknown. Collectively, these observations led us to this effect was accompanied by the maintenance of hypothesize that in the presence of an error-prone mtDNA copy number, increased expression of PGC-1α POLG1, mitochondrial p53 will function as an accessory and its downstream metabolic network, enhanced mito- fidelity-enhancing component of the mtDNA replication chondrial oxidative capacity, and restoration of mito- machinery in PolG mice. chondrial structural integrity in PolG-END (Fig. 2c–e, To test our hypothesis, we first assessed the submito- and Additional file 1: Figures S2D–G, Figure S3A–I, and chondrial localization of p53 in skeletal muscle of WT Table S1). These observations collectively imply that ac- mice. Subfractionation of skeletal muscle mitochondria in- cumulating mtDNA mutations in PolG-SED mice lead dicated that mitochondrial p53 was primarily localized in to an increase in ROS generation that (i) promotes mito- the mitochondrial matrix (Fig. 3a). Next, we measured the chondrial dysfunction and telomere damage and (ii) sub- mitochondrial abundance of p53 in our experimental sequently triggers p53-regulated senescence pathways, groups. Unlike PolG-SED, in PolG-END mice, p53 prefer- thereby potentiating the loss of somatic and stem cells entially resided in the mitochondria vs. nuclei of muscle via apoptosis. In contrast, exercise reduced mtDNA mu- and heart (Figs. 1f and 3b). To ascertain whether mito- tations and maintained the cellular energy and redox chondrial ROS levels regulated p53 compartmentalization, homeostasis thereby circumventing telomere erosion we treated primary fibroblasts with rotenone, a complex I culminating in the inhibition of accelerated systemic inhibitor known to increase mitochondrial ROS, and ob- aging characteristic of PolG mice [3, 4]. served a rapid increase in mitochondrial p53 content at lower dosages without a concomitant increase in nuclear Endurance exercise-mediated repair of mtDNA mutations p53 (Additional file 1: Figure S4A). Intriguingly, with in- is p53-dependent creasing rotenone concentrations, we measured an in- POLG1 is the sole mitochondrial polymerase essential crease in nuclear p53 abundance (Additional file 1: Figure INK4A for mtDNA replication and repair via its 3′→5′ exo- S4A) and expression of its downstream targets (p16 WAF1 nuclease activity [35]. Since exercise reduced mtDNA and p21 ; Additional file 1: Figure S4B), along with a mutations in PolG mice, which lack proofreading cap- concomitant reduction in mitochondrial p53 content acity of POLG1, this raised an intriguing possibility that (Additional file 1: Figure S4A), and mtDNA copy number exercise recruited a POLG1-independent mtDNA repair (Additional file 1: Figure S4C). The increase in nuclear pathway(s) [11]. We found that despite elevated p53 nu- p53 paralleled the decrease in PGC-1α mRNA expression clear abundance in PolG-SED, the total p53 content in (Additional file 1: Figure S5A), further supporting the in- the muscle, heart, and SC homogenates of all groups hibitory effects of p53 on PGC-1α. Furthermore, the up- was unaltered (Additional file 1: Figure S3J). This indi- regulation of rotenone-evoked nuclear p53 content was cated that a basal pool of p53 is maintained intra- attenuated in fibroblasts pre-treated with a ROS scavenger, cellularly, with the distribution of p53 between the N-acetylcysteine (Additional file 1: Figure S5B), in tandem different subcellular compartments dependant on the with higher PGC-1α mRNA expression (Additional file 1: cellular stress milieu [27]. In vitro studies show that in Figure S5C). Thus, p53 preferentially shuttles into response to intra- and extra-cellular insults such as mitochondria in response to physiological ROS levels, ROS, p53 translocates into the mitochondria where it in- which abrogates the negative regulation of PGC-1α as teracts with the mtDNA and POLG1 [27]. Biochemical exerted by p53 residing in the nucleus. Next, we analysis of p53 has revealed an inherent 3′→5′ sought to elucidate if mitochondrial p53 interacted Safdar et al. Skeletal Muscle (2016) 6:7 Page 13 of 17 Fig. 3 (See legend on next page.) Safdar et al. Skeletal Muscle (2016) 6:7 Page 14 of 17 (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 Tfam in mtDNA mutator mice. a Mitochondrial p53 is primarily localized in the matrix. Muscle mitochondria were subfractionated into outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), and matrix (Mx) fractions, and these fractions were immunoblotted 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), and d 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 to mtDNA (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 units with POLG1 and Tfam in the mitochondrial matrix. mutator mouse model” was needed where changes in We performed co-immunoprecipitation reactions and mtDNA mutation burden can be assessed in a background found that mitochondrial p53 formed a complex with of p53 over-expression or knockdown. It was unfeasible to POLG1 and Tfam complexed at mtDNA (Fig. 3c–e). generate PolG mice with over-expression of p53, as previ- The p53-POLG1-Tfam complex at mtDNA was higher ous work has shown that mice engineered with hyper- in PolG-END vs. PolG-SED and WT (Fig. 3c–e). active p53 alleles display show stem cell depletion and These observations are consistent with a recent study premature aging phenotype themselves [38]. On the other reporting p53 translocation to of p53 to the mito- hand, whole-body p53 knockout mice die prematurely of chondria and subsequent formation of p53-Tfam- cancer [39] and do not breed efficiently with heterozygous +/D257A mtDNA complex in skeletal muscle of WT mice in PolgA mice, and thus could not be bred with PolG response to an acute bout of endurance exercise [37]. mice to efficiently study the effects of exercise. Hence, we These results led us to conclude that the preferential created a new genetically modified mutator mouse with subcellular localization of p53 to mitochondria vs. muscle-specific p53 deletion (PolG-p53 MKO). At basal nuclear compartment is a “universal” exercise-induced levels, PolG-p53 MKO-SED mice demonstrated an accel- phenomenon and likely plays a role in mediating erated progeriod phenotype and significant accumulation beneficial effects of endurance exercise on improving of random mtDNA mutations in muscle compared to mitochondrial content/function and ameliorating PolG-SED mice (Fig. 4e and Additional file 1: Figure S6B). dysfunction. To our surprise, endurance exercise not only failed to Since both PolG-SED and PolG-END mice have defect- reduce mtDNA point mutations but also did not rescue ive POLG1 proofreading capacity, we believe that the re- progeroid aging, sarcopenia, exercise intolerance, mito- duction in total mtDNA mutational burden in PolG-END chondrial morphology anomalies, and deficits in mito- mice (Fig. 1a) is mediated by mitochondrial p53 levels in chondrial content and function such as mtDNA copy response to endurance exercise. Hence, we sought to number, mitochondrial electron transport chain protein evaluate whether mitochondrial p53 can repair mtDNA content, and COX activity in skeletal muscle of PolG-p53 mutations, independent of the proofreading capacity of MKO mice (Fig. 4c–g and Additional file 1: Figure S6B– POLG1. A fluorescence-based in vitro DNA primer E). This suggests that the exercise-mediated repair of extension-mutation repair assay displayed an efficient re- mtDNA mutations in vivo is dependent on mitochondrial pair of double-stranded oligonucleotides, with artificially p53 adjuvant repair capacity. Furthermore, unlike PolG- added mismatch point mutations, incubated with ex vivo END, mitochondrial extracts from PolG-p53 MKO failed muscle mitochondrial extract of PolG-END vs. PolG-SED to repair mutations in vitro in the primer extension- (Fig. 4a). PolG-END mitochondria failed to repair these mutation repair assay (Additional file 1: Figure S6F). Thus, mutations upon p53 immunodepletion (Fig. 4b), while exercise-induced maintenance of mtDNA stability is con- addition of recombinant p53 increased PolG-SED mito- tingent on mitochondrially localized p53 and represents a chondrial mutation repair efficiency (Additional file 1: viable therapy for pre-symptomatic patients carrying Figure S6A). Clearly, mitochondrial p53 plays a vital role POLG1 exonuclease domain mutations known to cause in the maintenance of mtDNA integrity in the presence of pathology [35]. defective POLG1 in mutator mice. However, to conclusively attribute causality to mito- Conclusions chondrial p53 in mediating mtDNA repair in vivo in re- Here, we show that exercise promotes mitochondrial sponse to endurance exercise, a “double genetically altered oxidative capacity and cellular redox dynamics via PGC- Safdar et al. Skeletal Muscle (2016) 6:7 Page 15 of 17 Fig. 4 Endurance exercise-mediated repair of mtDNA mutations is mitochondrial p53-dependent. a A fluorescence-based in vitro DNA primer extension-mutation repair assay in muscle mitochondrial extracts from WT, PolG-SED, and PolG-END (n= 6–8/group) to assess the excision of the unpaired 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 trials in WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice (n= 5–6/group). d Representative electron micrographs of myofibers (quadriceps femoris) from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END (n= 4/group). e Random mtDNA somatic 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-p53 MKO-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 units 1α-mediated expression networks, thus preventing the Intriguingly, stress-mediated subcellular localization of accumulation of oxidative damage, abrogating genotoxic the tumor suppressor protein p53 determines its pro- or damage, and repressing apoptosis in mutator mice. anti-survival function and seems indispensible for the Safdar et al. Skeletal Muscle (2016) 6:7 Page 16 of 17 exercise-mediated mtDNA repair and mitochondrial bio- Cu/Zn-SOD); SOD2: superoxide dismutase 2 (mitochondrial; Mn-SOD); TFAM: mitochondrial transcription factor A; VDAC: voltage-dependent anion genesis. The work summarized here opens up viable ave- channel; WT: wild-type mice. nues of research in cancer biology where mitochondrial dysfunction and genomic instability have been impli- Competing interests The authors declare that they have no competing interests. cated [1]. It will be of potential clinical interest to see if exercise-induced mitochondrial-targeted p53 might rep- Authors’ contributions resent a therapeutic intervention for aging-associated AS and MAT designed the research; AS, KK, JMF, AS, MDL, APWJ, YK, IAS, YK, pathologies such as insulin resistance, diabetes, and car- 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 BPH diovascular diseases, which manifest telomere shortening analyzed the data; and AS wrote the manuscript. All authors have been in conjunction with mitochondrial dysfunction [40, 41]. involved in drafting and revising the manuscript and have approved the final Indeed, while exercise and an active lifestyle are the manuscript. most prominent therapies to reduce the incidence and Acknowledgements pathogenicity of diabetes, insulin resistance, and cardio- We like to acknowledge the late Mrs. Suzanne Southward (McMaster vascular diseases [42, 43], therapeutic modalities that University) for assisting in enzyme assays and Dr. William C. Copeland (NIH) for his kind donation of POLG1 antibody. This work was supported by the promise to recapitulate some of the effects of exercise Canadian Institutes of Health Research (CIHR) grant and a kind donation warrant further attention. The telomere–p53–PGC-1α from Mr. Warren Lammert and family to M.A.T. A. Safdar was funded by axis provides a molecular basis of how telomere erosion Banting Fellowship (CIHR) and American Federation for Aging Research and Ellison Medical Foundation (EMF) Fellowship. A. Saleem is funded by Natural and mitochondrial dysfunction can modulate systemic Sciences and Engineering Research Council of Canada postdoctoral aging of tissues and stem cell compartments. Under- fellowship. K.K. was supported by the United Mitochondrial Disease standing the upstream signaling cascades and posttrans- Foundation, the NIH (AG019787) and the EMF Senior Scholarship. The authors declare no competing and financial interests. T.A.P. was awarded a lational modifications that promote mitochondrial D257A US patent 7,126,040 for the Polg mouse model. T.A.P. is a partial owner localization of p53 may allow for the generation of of LifeGen Technologies, specializing in nutrigenomics, as well as a Scientific pharmaceutical analogs, novel therapeutic strategies to Advisory Board member for Nu Skin Enterprises. M.A.T. is the founder, president, and CEO of Exerkine Corporation and a member of its scientific antagonize mitochondrial genomic decay, and cellular advisory board. senescence in age-associated pathologies. Author details Department of Kinesiology, McMaster University, Hamilton, ON L8N 3Z5, Additional file 2 Canada. Department of Pediatrics, McMaster University, Hamilton, ON L8N 3Z5, Canada. Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada. Department of Medical Sciences, McMaster University, Additional file 1: Figure S1. Endurance exercise confers complete Hamilton, ON L8N 3Z5, Canada. Department of Medical Physics & Applied phenotype protection, suppresses early mortality, mitigates mitochondrial Radiation Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada. ROS-mediated oxidative damage, increases cellular antioxidant capacity, 6 7 Northeastern University, Boston, MA 02115, USA. Buck Institute for Research and 4 prevents cellular senescencmutator mice. Figure S2. Endurance on Aging, Novato, CA 94945, USA. School of Health and Exercise Sciences, exercise prevents dysregulated mitochondrial-induced apoptosis and University of British Columbia Okanagan, Kelowna, BC V1V 1V7, Canada. reduces nuclear p53-mediated repression of PGC-1α and promotes Division of Cardiovascular Disease, University of Alabama, Birmingham, AL mitochondrial biogenesis in mutator mice. Figure S3. Endurance exercise 35294, USA. Perelman School of Medicine, University of Pennsylvania, promotes systemic mitochondrial biogenesis in mtDNA mutator mice. Philadelphia, PA 19104, USA. Departments of Genetics, University of Figure S4. Magnitude of mitochondrial ROS (physiological vs. Wisconsin, Madison, WI 53706, USA. Departments of Medical Genetics, pathological) regulates p53 subcellular localization. Figure S5. University of Wisconsin, Madison, WI 53706, USA. Pre-treatment with exogenous antioxidant preferentially shuttles p53 to mitochondria in response to stress. Figure S6. Endurance exercise- Received: 11 October 2015 Accepted: 5 January 2016 mediated attenuation of sarcopenia, increase in endurance capacity, skeletal muscle mitochondrial biogenesis, and repair of muscle mtDNA mutations is p53 dependent. Table S1. WT, PolG-SED, and PolG-END Skeletal Muscle Microarray IPA-GO Analysis. Table S2. Real-time PCR References primer sequences. (PDF 1601 kb) 1. Sahin E, Depinho RA. Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature. 2011;464(7288):520–8. 2. Larsson NG. Somatic mitochondrial DNA mutations in mammalian aging. 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Telomere DNA; ND1: complex I NADH dehydrogenase subunit 1; NRF1: nuclear dysfunction induces metabolic and mitochondrial compromise. Nature. INK4A respiratory factor 1; p16 : cyclin-dependent kinase inhibitor 2A; 2011;470(7334):359–65. WAF1 p21 : cyclin-dependent kinase inhibitor 1A; p53: tumor suppressor protein 6. Cawthon RM, Smith KR, O'Brien E, Sivatchenko A, Kerber RA. Association 53; PC: protein carbonyls; PGC-1α: peroxisome proliferator-activated receptor between telomere length in blood and mortality in people aged 60 years gamma co-activator 1 alpha; PolG: polymerase gamma mutator mice; or older. Lancet. 2003;361(9355):393–5. POLG1: mitochondrial polymerase gamma; ROS: reactive oxygen species; 7. Atzmon G, Cho M, Cawthon RM, Budagov T, Katz M, Yang X, et al. Evolution SC: satellite cells; SED: sedentary; SOD1: superoxide dismutase 1 (cytosolic; in health and medicine Sackler colloquium: genetic variation in human Safdar et al. 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Trifunovic A, Hansson A, Wredenberg A, Rovio AT, Dufour E, Khvorostov I, et � Convenient online submission al. Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production. Proc Natl Acad Sci U S A. 2005;102(50): � Thorough peer review 17993–8. � Inclusion in PubMed and all major indexing services 30. Dai DF, Chen T, Wanagat J, Laflamme M, Marcinek DJ, Emond MJ, et al. � Maximum visibility for your research Age-dependent cardiomyopathy in mitochondrial mutator mice is attenuated by overexpression of catalase targeted to mitochondria. Aging Submit your manuscript at Cell. 2010;9(4):536–44. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

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Abstract

Background: Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. Results: Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. Conclusions: Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present 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, Senescence Background evidence actually suggests that mtDNA replication er- The universality of the aging phenomenon has evoked rors may be the more important culprit [2]. The demon- great interest in unveiling regenerative remedies and re- stration that multiple aspects of aging are accelerated in juvenation medicine designed to evade molecular insti- mutator mice harboring error-prone mitochondrial poly- gators of mammalian aging. Molecular investigations of merase gamma provides support for the causal role of age-related pathologies implicate mitochondrial DNA mtDNA replication errors in instigating mammalian (mtDNA) mutations as one of the primary instigators aging [3, 4]. Similar phenotypes have also been reported driving multisystem degeneration, stress intolerance, and in telomerase-deficient mice [5], where telomere dys- energy deficits [1]. It is intuitive to assume that the de function is associated with impaired mitochondrial bio- novo mtDNA mutations observed during aging are due genesis and metabolic failure resulting in progressive to accumulated, unrepaired oxidative damage, but some tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses as seen with aging * Correspondence: tarnopol@mcmaster.ca [5]. Indeed, epidemiological studies have correlated de- Department of Pediatrics, McMaster University, Hamilton, ON L8N 3Z5, creased telomere length in peripheral blood leukocytes, Canada 3 with higher mortality rates in individuals more than Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada 60 years old [6]. Furthermore, a recent study in Full 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 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the 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 Page 2 of 17 centenarians and their offspring found a positive link be- Endurance exercise protocol tween telomere length and longevity; in particular, those Endurance exercise protocol and tissue harvesting was with longer telomeres had an overall improved health carried out, as previously described, using an independ- profile, with decreased incidence of age-associated dis- ent cohort of mice [11]. Briefly, at 3 months of age, mice eases, better cognitive function, and improved lipid pro- were housed individually in micro-isolator cages in a files relative to controls [7]. temperature- and humidity-controlled room and main- The epidemic emergence of modern chronic diseases tained on a 12-h light–dark cycle with food and water largely stems from the adoption of a sedentary lifestyle ad libitum [13]. PolG mice and PolG-p53 MKO mice and excess energy intake [8]. There is incontrovertible were randomly assigned to sedentary (PolG-SED or evidence that endurance exercise extends life expectancy PolG-p53 MKO-SED) or forced-endurance (PolG-END and reduces the risk of chronic diseases in both rodents or PolG-p53 MKO-END) exercise groups (n =5–20/ and humans [9, 10]. We have previously shown that en- group; ♀ = ♂). None of the mice had been previously durance exercise effectively rescued progeroid aging in subjected to a structured exercise regiment. One week mutator mice concomitant with a reduction in mtDNA of pre-training was allowed to acclimatize mice in en- mutations, despite an inherent defect in mitochondrial durance exercise groups to the treadmill. Mice in endur- polymerase gamma (POLG1) proofreading function [11]. ance exercise groups were subjected to forced treadmill Exercise has also been shown to increase telomerase ac- exercise (Eco 3/6 treadmill; Columbus Instruments, tivity and reduce senescence markers [12]. These find- Columbus, Ohio) three times per week at 15 m/min for ings suggest a link between exercise-mediated metabolic 45 min for 6 months. A 5-min warm-up and cool-down adaptations and genomic (nuclear and mitochondrial) at 8 m/min were also included. PolG mice were age- and stability; however, the identity of this metabolic link re- sex-matched with sedentary littermate WT mice (n = 20; mains unknown. In this study, we have utilized PolG ♀ = ♂), which served as controls for the study to assess mice to investigate the mitochondrial-telomere dysfunc- if endurance exercise intervention can molecularly bring tion axis in the context of progeroid aging, and to PolG mice to normalcy. At 8 months of age, animals elucidate how exercise counteracts mitochondrial were euthanized and tissues were collected for molecular dysfunction and mtDNA mutation burden through analyses. The study was approved by the McMaster mitochondrial localization of the tumor suppressor University Animal Research and Ethics Board under the protein p53. global Animal Utilization Protocol # 12-03-09, and the experimental protocol strictly followed guidelines put Methods forth by the Canadian Council of Animal Care. Mice breeding +/D257A Heterozygous mice (C57Bl/6J, PolgA )for the Endurance stress test mitochondrial polymerase gamma knock-in mutation The mice were subjected to four separate endurance were a kind gift from Dr. Tomas A. Prolla, University stress tests over to indirectly assess improvements in of Wisconsin-Madison, USA [4]. We generated homo- aerobic capacity with exercise as previously described zygous knock-in mtDNA mutator mice (PolG; Pol- [11]. Briefly, animals from all groups were placed in indi- D257A/D257A gA ) and littermate wild-type (WT; PolgA vidual lanes on the treadmill and allowed to acclimatize +/+ ) from heterozygous mice-derived colony main- for 30 min to eliminate any confounding effects due to tained at the McMaster University Central Animal Fa- stress or anxiety related to a new environment. The test cility as previously described [11]. Muscle-specific p53 began with a 5-min warm-up session at 8 m/min, knock-out mice (p53 MKO) were bred by crossing followed by +1 m/min increase in speed every 2 min tm1Brn p53 flox mice (Trp53 /J) with muscle-creatine until the mouse reached exhaustion. A low-intensity kinase Cre recombinase mice (Tg(Ckmm-cre)5Khn/J) electrically stimulus was provided to ensure compliance. purchased from Jackson Laboratories. We generated Time to exhaustion (min) was recorded when the mouse genetically modified homozygous knock-in mtDNA sat at the lower end of the treadmill, near a shock bar, mutator mice with muscle-specific p53 knockout for >10 s and was unresponsive to further stimulation to (PolG-p53 MKO), by crossing heterozygous mice continue running. +/D257A (PolgA ) with p53 MKO mice. During breeding, all animals were housed three to five per cage in a 12-h light/dark cycle and were fed ad libitum Survival analysis (Harlan-Teklad 8640 22/5 rodent diet) after weaning. An independent cohort of animals from all groups was The presence of the polymerase gamma homozygous used to carry out survival analyses as previously de- knock-in mutation was confirmed as previously scribed [11], and Kaplan–Meier survival curves were cal- described [4]. culated using GraphPad Prism 4.0. Safdar et al. Skeletal Muscle (2016) 6:7 Page 3 of 17 Tissue harvesting Mouse embryonic fibroblast isolation and reporter assay Tissues were collected at the time of euthanasia as previ- Mouse embryonic fibroblasts (MEFs) were generated +/+ ously described [11]. Immediately following cervical dis- using standard techniques from WT (p53 ) and p53 −/− location, the chest cavity was exposed and the heart was knockout (KO) mice (p53 ). Cell used in the experi- removed rapidly, followed by the skeletal muscle (quad- ments were from passages 4–5. Promoter sequence for riceps femoris). The skeletal muscle (quadriceps femoris, PGC-1α was amplified by PCR from mouse muscle gen- tibialis anterior, and soleus) and heart were either (i) col- omic DNA and cloned into the pGL4 luciferase reporter lected in RNase-free cryovials, immediately immersed in vector (Promega, Madison, WI). The pG13-luc plasmid liquid nitrogen, and stored at −80 °C for later analysis of containing 13 copies of a synthetic p53 DNA binding DNA, RNA, protein, and enzyme activity or (ii) immedi- site was used as a positive control (which has been com- ately rinsed with phosphate buffer saline (PBS) and used prehensively characterized in Jackson et al., 2001 and for skeletal muscle and heart mitochondrial and nuclear Kern et al., 1991). A GFP expressing plasmid was used +/+ −/− fractionations. to 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- Hematopoietic stem and progenitor cell isolation scriptional activity was measured using Bright-Glo™ lu- Mouse hematopoietic stem and progenitor cells (HSC) ciferase reporter assay system (Promega, Madison, WI). were isolated according to the method of Ema et al. with minor modifications [14]. Marrow was flushed from the Total RNA isolation from skeletal muscle and heart femur and tibia using a 25-g needle, passed through a 50- Total RNA was isolated from ~25 mg of the skeletal μm sieve and counted with a hemocytometer. Cells were muscle (quadriceps femoris)and heartusing the incubated with primary antibodies for 90 min at 4 °C Qiagen total RNA isolation kit (Qiagen, Mississauga, followed by 20 min incubation in the appropriate second- ON) [11, 13]. RNA samples were treated with ary antibody at 4 °C. Lineage negative, and Sca-1 and c- RNase-free DNase on Qiagen spin-columns (Qiagen, Kit positive (LSK) population enriched for stem cells Mississauga, ON) to remove DNA contamination. were sorted using the EPICS ALTRA™ fluorescence- RNA integrity and concentration were assessed using activated cell sorter (Beckman Coulter, Mississauga, the Agilent 2100 Bioanalyzer (Agilent Technologies, ON) with gating strategies established using single- Palo Alto, CA) [13]. The average RIN (RNA integrity stained controls. The following antibodies were used: number) value for all samples was 9.64 ± 0.20 (scale lineage panel (BD Pharmingen™,Mississauga,ON), 1–10), ensuring a high quality of isolated RNA. anti-mouse Sca-1 Clone: E13-161.7 (BD Pharmingen™, Mississauga, ON), anti-mouse c-Kit Clone: 2B8 RNA, DNA, and protein isolation from HSC and SC (eBioscience, San Diego, CA), and streptavidin (Bio- Total RNA, DNA, and protein were isolated from HSC Source, Burlington, ON). and SC using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Mississauga, ON) according to the manufac- Satellite cell isolation turer’s instructions. Primary skeletal muscle satellite cells (SC) were isolated from WT, PolG-SED, and PolG-END mice using the Microarray analysis methods described previously [15] and subsequently Total RNA was extracted from skeletal muscle (quadri- purified by fluorescence-activated cell sorting. Briefly, ceps femoris) using the Qiagen RNeasy Micro kit the hind limb skeletal muscles were carefully dissected, (Qiagen, Mississauga, ON) and processed on Qiagen’s cleaned of fat and washed in cold PBS. Cells were re- QIAcube (Qiagen, Mississauga, ON) using the standard leased by mulching the tissue with scissors and incuba- manufacturer’s protocol. The samples were then checked tion in a collagenase/dispase solution three times, for quality using Nanodrop 2000 (Thermo Scientific, 12 min each, at 37 °C with further mechanical disruption Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent using a pipette between incubations. Following passage Technologies, Palo Alto, CA). TransPlex Whole Tran- through 70 and 30 μm filters, cells were stained using scriptome Amplification kit (Sigma-Aldrich , Oakville, primary antibody to c-met conjugated to PE (1:200, ON) was used to amplify complementary (cDNA) from eBioscience, San Diego, CA) and subjected to FACS the muscle RNA samples according to the manufac- sorting (EPICS ALTRA™, Beckman Coulter, Mississauga, turer’s instructions. Samples were amplified for 25 cycles ON). SC were pelleted in RNase-free cryovials, immedi- using the recommended cycling parameters. All samples ately immersed in liquid nitrogen, and stored at −80 °C were subsequently purified using Qiagen’s QIAquick for later analyses. PCR Purification kit (Qiagen, Mississauga, ON) and Safdar et al. Skeletal Muscle (2016) 6:7 Page 4 of 17 processed on Qiagen’s QIAcube (Qiagen, Mississauga, (Applied Biosystems Inc., Foster City, CA) [11]. Forward ON) using the standard “Cleanup QIAquick PCR for and reverse primers for the aforementioned genes amplification reactions” (Version 4) protocol. Samples (Additional file 1: Table S2) were designed based on se- were purified and examined using Nanodrop 2000 quences available in GenBank using the online MIT (Thermo Scientific, Wilmington, DE) and Agilent 2100 Primer 3 designer software (developed at Whitehead In- Bioanalyzer DNA 7500 chip (Agilent Technologies, Palo stitute and Howard Hughes Medical Institute by Steve Alto, CA) to ensure proper yield and quality of amplifi- Rozen and Helen Skaletsky) and were confirmed for spe- cation. To perform the microarray hybridization, 2 μgof cificity using the basic local alignment search tool. β-2 cDNA from each sample was labeled using NimbleGen’s microglobulin was used as a control house-keeping gene, One Color Labeling kit (Cat.# 05223555001; Roche as its expression was not affected with the experimental NimbleGen Inc., Madison, WI) according to the manu- intervention (data not shown). All samples were run in facturer’s protocol. Five micrograms of Cy3 labeled duplicate simultaneously with a negative control which samples were hybridized to Mus musculus 12x135k, contained no cDNA. Melting point dissociation curves NimbleGen Gene Expression Arrays (Cat.# 05543797001; generated by the instrument were used to confirm the Roche NimbleGen Inc., Madison, WI), washed, and specificity of the amplified product. scanned according to manufacturer’s protocol. NimbleGen gene expression arrays were scanned using an Axon Tissue total DNA isolation GenePix 4200A scanner (Molecular Devices Inc., Down- Total DNA (genomic and mtDNA) was isolated from ingtown, PA) with settings of 100 POW and 300–350 ~15 mg of the skeletal muscle (soleus) and heart using photomultiplier (PMT). Pair files were generated for each the QIAamp DNA Mini kit (Qiagen, Mississauga, ON) array using NimbleScan software (Roche NimbleGen Inc., [11, 16]. DNA samples were treated with RNase Madison, WI). Resulting array data was analyzed with Bio- (Fermentas, Mississauga, ON) to remove RNA contam- conductor software (Bioconductor, Seattle, WA) in which ination. DNA concentration and quality were assessed the data were normalized and tested for significantly dif- using Nanodrop 2000 (Thermo Scientific, Wilmington, ferentially expressed genes which were assessed based DE). upon a 5 % false discovery rate (FDR). The gene array data reported here is deposited in Gene Expression Omnibus mtDNA copy number analysis (Accession Number: GSE75869) public functional genom- Mitochondrial DNA copy number, relative to the diploid ics data repository. The resulting data were input into In- chromosomal DNA content, was quantitatively analyzed genuity Pathway Analysis (Ingenuity Systems, Redwood from the skeletal muscle (soleus), heart, primary City, CA) to determine the over-represented gene categor- hematopoietic stem cells, primary satellite cells, and pri- ies using strict association. The normalized expression in mary fibroblasts using ABI 7300 real-time PCR (Applied these categories was plotted in a heat map using R script Biosystems, CA) [11, 16]. Primers were designed around and Bioconductor software (Bioconductor, Seattle, WA). COX-II region of the mitochondrial genome (Additional file 1: Table S2). Nuclear β-globin gene was used as a Real-time quantitative PCR housekeeping gene (Additional file 1: Table S2). The messenger RNA (mRNA) expression of peroxisome proliferator-activated receptor gamma co-activator 1 Average telomere length alpha (PGC-1α), mitochondrial transcription factor A Average telomere length was measured in heart, primary (TFAM), estrogen-related receptor alpha (ERRα), 5- hematopoietic stem cells, and primary satellite cell gen- aminolevulinate synthase (ALAS), cytochrome c oxidase omic DNA using a real-time quantitative PCR method subunit-I (COX-I), cytochrome c oxidase subunit-IV as previously described [17]. The premise of this assay is (COX-IV), complex I NADH dehydrogenase subunit 1 to measure an average telomere length ratio by quantify- (ND1), complex V subunit ATPase 6 (ATPase 6), cyclin- ing telomeric DNA with specially designed primer se- WAF1 dependent kinase inhibitor 1A (p21 ), cyclin- quences and dividing that amount by the quantity of a INK4A dependent kinase inhibitor 2A (p16 ), and growth single-copy gene [17]. All samples were run using a 7300 arrest and DNA-damage-inducible beta (GADD45B) Real-time PCR System (Applied Biosystems Inc., Foster ® ® were quantified using 7300 Real-time PCR System (Ap- City, CA) and SYBR Green chemistry (PerfeC a SYBR plied Biosystems Inc., Foster City, CA) and SYBR Green Green Supermix, ROX, Quanta BioSciences, Gaithers- chemistry (PerfeC a SYBR Green Supermix, ROX, burg, MD). A single-copy gene, 36B4, which encodes for Quanta BioSciences, Gaithersburg, MD) as previously the acidic ribosomal phosphoprotein PO, was used as a described [11, 13]. First-strand cDNA synthesis from control for amplification for every sample performed 1 μg of total RNA was performed with random primers [17, 18]. Each PCR reaction for the telomere and 36B4 using a high-capacity cDNA reverse transcription kit included 12.5 μL of 1x SYBR Green master mix Safdar et al. Skeletal Muscle (2016) 6:7 Page 5 of 17 (PerfeC a SYBR Green Supermix, ROX, Quanta BioSci- Chromatin immunoprecipitation assay ences, Gaithersburg, MD), 300 nM each of the forward Chromatin immunoprecipitation (ChIP) assay was per- and reverse telomere or 36B4 primers (Additional file 1: formed using an EZ-ChIP™ kit (Millipore, Billerica, MA) Table S2), 20 ng genomic DNA, and enough DNase/ as previously described [11]. Twenty-milligram piece of RNase-free H O (Applied Biosystems Inc., Foster City, the quadriceps femoris muscle was cross-linked in 5 mL of CA) to yield a 25-μL reaction. Cycling conditions for phosphate-buffered saline containing 1 % formaldehyde telomere are as follows: 95 °C for 10 min followed by for 10 min at room temperature. One milliliter of 10X gly- 30 cycles of data collection at 95 °C for 15 s and a 56 °C cine was added to stop fixation. Muscles were then ho- anneal-extend step for 1 min. Cycling conditions for mogenized in 1 mL of SDS lysis buffer supplemented with 36B4 are as follows: 95 °C for 10 min followed by 35 cy- protease inhibitor cocktail Complete, ETDA-free (Roche cles of data collection at 95 °C for 15 s, with 52 °C an- Applied Science, Mannheim, Germany). Chromatin was nealing for 20 s, followed by extension at 72 °C for 30. sheared by sonicating each sample on ice using a Branson Each sample was analyzed in duplicate, and the ratio of Digital Sonifier S-450D (output 20 %, 4 times for 20 s, telomere:36B4 was calculated. The average of these ra- with a 20-s pause each time; Branson Ultrasonics Corpor- tios was reported as the average telomere length ratio ation, Danbury, CT). Following centrifugation at 10,000×g (ATLR). at 4 °C for 10 min, the supernatant containing 1 mg of protein was diluted to 1 mL with dilution buffer. Ten mi- crograms of anti-p53 (FL-393) antibody (Santa Cruz Bio- Whole tissue lysate technology Inc., Santa Cruz, CA) was added per sample Total protein was extracted from tissue samples as pre- and incubated overnight at 4 °C. Anti-IgG antibody was viously described [11]. Briefly, ~30 mg of the skeletal used as a non-specific control. Sixty microliters of protein muscle (quadriceps femoris) and heart were homoge- G-agarose was added, and the sample was mixed for 1 h nized on ice in a 2-mL Wheaton glass homogenizer at 4 °C with rotation. Precipitated complexes were eluted (Fisher Scientific, Ottawa, ON) with 25 volumes of phos- in 100 μL of elution buffer, and cross-linking was reversed phate homogenization buffer [50 mM KPi, 5 mM EDTA, by the addition of 8 μL of 5 M NaCl per sample followed 0.5 mM DTT, 1.15 % KCl supplemented with a by incubation at 65 °C for 10 h. Co-immunoprecipitated Complete Mini, ETDA-free protease inhibitor cocktail DNA was purified according to the manufacturer’sin- tablet and a PhosSTOP, phosphatase inhibitor cocktail structions. Primers were designed to amplify the p53 bind- tablet (Roche Applied Science, Mannhein, Germany) per ing regions (−564 and −954) of the PGC-1α promoter 10 mL of buffer]. The lysate was centrifuged at 600g for (Additional file 1: Table S2). The amount of PGC-1α pro- 15 min at 4 °C to pellet cellular debris. The supernatant moter immunoprecipitated with p53 was quantified using was aliquoted, snap frozen in liquid nitrogen, and stored the 7300 Real-time PCR System (Applied Biosystems Inc., at −80 °C until further analysis. Foster City, CA) and SYBR Green chemistry (PerfeC a SYBR Green Supermix, ROX, Quanta BioSciences, Nuclear fractionation Gaithersburg, MD). Purified DNA from the input sample Nuclear fractions were prepared from 40 mg of the that did not undergo immunoprecipitation was PCR- freshly obtained skeletal muscle (quadriceps femoris), amplified using of β-globin primers (Additional file 1: heart, primary satellite cells, and primary fibroblasts Table S2) and was used to normalize signals from ChIP using a commercially available nuclear extraction kit assays. (Pierce NE-PER , Rockford, IL) as previously described [11, 16]. Briefly, samples were homogenized in CER-I Mitochondrial fractionation buffer containing protease inhibitor cocktail Complete, Mitochondrial fractions were isolated using differential ETDA-free (Roche Applied Science, Mannheim, centrifugation as previously outlined [11]. Briefly, the Germany) using an electronic homogenizer (Pro 250, skeletal muscle (quadriceps femoris and tibialis anterior), Pro Scientific, Oxford, CT, USA). Pellets containing nu- heart, primary satellite cells, and primary fibroblasts clei were obtained by centrifugation at 16,000g for were finely minced and homogenized on ice in 1:10 (wt/ 10 min at 4 °C and were subsequently washed four times vol) ice-cold isolation buffer A (10 mM sucrose, 10 mM in PBS to remove cytosolic contaminating proteins. Nu- Tris/HCl, 50 mM KCl, and 1 mM EDTA, and 0.2 % fatty clear proteins were extracted in NER buffer supple- acid-free BSA, pH 7.4, supplemented with protease in- mented with protease inhibitors [11]. Enrichment and hibitor cocktail Complete, ETDA-free [Roche Applied purity of nuclear fractions were confirmed by the abun- Science, Mannheim, Germany]) using a Potter-Elvehjem dance of nuclear histone H2B and absence of the cyto- glass homogenizer. The resulting homogenates were solic protein lactate dehydrogenase in Western blot centrifuged for 15 min at 700g, and the subsequent su- analyses as previously shown by our group [16]. pernatants were centrifuged for 20 min at 12,000g.The Safdar et al. Skeletal Muscle (2016) 6:7 Page 6 of 17 mitochondrial pellets from 12,000g spin were washed antibodies were used as a non-specific control. The and then re-suspended in a small volume of ice-cold iso- matrix was centrifuged at 16,000g for 30 s, and the pellet lation buffer B (10 mM sucrose, 0.1 mM EGTA/Tris, matrix-immune complex precipitate was washed four and 10 M Tris/HCl, pH 7.4, supplemented with protease times under stringent conditions (50 mM Tris-HCl, pH inhibitor cocktail Complete, ETDA-free [Roche Applied 7.4, 500 mM NaCl, 2 mM EDTA) and incubated over- Science, Mannheim, Germany]). All centrifugation steps night at 65 °C in the presence of 1 % SDS for cross- were carried out at 4 °C. The mitochondrial pellets were linking reversion. DNA was extracted from supernatants immediately frozen at −80 °C for further biochemical using the QIAamp DNA Mini kit (Qiagen, Mississauga, analyses. Enrichment and purity of mitochondrial frac- ON) according to the manufacturer’s instructions. tions were confirmed by the abundance of mitochondrial mtDNA COX-II and cytochrome b regions (Additional cytochrome c oxidase subunit IV protein and absence of file 1: Table S2) were quantified using 7300 Real-time the nuclear histone H2B and the cytosolic protein lactate PCR System (Applied Biosystems Inc., Foster City, CA) ® ® dehydrogenase in Western blot analyses as previously and SYBR Green chemistry (PerfeC a SYBR Green shown by our group [16]. Supermix, ROX, Quanta BioSciences, Gaithersburg, MD), as previously described [16]. Mitochondrial co-immunoprecipitation assay Mitochondrial co-immunoprecipitation assay was per- formed on isolated mitochondrial fractions using Pierce Western blotting and markers of oxidative damage Co-Immunoprecipitation Kit (Pierce, Rockford, IL) as Protein concentrations of whole tissue lysates, and mito- previously described [16]. Briefly, mitochondrial frac- chondrial and nuclear fractions were determined using a tions were homogenized in lysis buffer (0.025 M Tris, commercial assay (BCA Protein Assay, Pierce, Rockford, 0.15 M NaCl, 0.001 M EDTA, 1 % NP-40, 5 % glycerol, IL). Proteins were resolved on 10 or 12.5 % SDS-PAGE pH 7.4) supplemented with protease inhibitor cocktail gels depending on the molecular weight of the protein of Complete, ETDA-free (Roche Applied Science, interest. The gels were transferred onto Hybond ECL Mannheim, Germany). Two milligrams of mitochondrial nitrocellulose membranes (Amersham, Piscataway, NJ) fraction was pre-cleared by incubation with 100 μLof and immunoblotted using the following commercially control agarose resin to minimize non-specific binding. available primary antibodies: MitoProfile Total OXPHOS Forty micrograms of anti-p53 (FL-393) antibody (Santa Rodent cocktail (MS604) antibody (MitoSciences, Eugene, Cruz Biotechnology Inc., Santa Cruz, CA) was covalently OR); anti-PGC-1α (2178), anti-VDAC (4866), and anti-α/ coupled onto an amine-reactive resin. The pre-cleared β-tubulin (2148) antibodies (Cell Signaling Technology, lysates were subsequently incubated with antibody- Denver, MA); anti-p53 (MABE283-PAb421) antibody coupled beads overnight at 4 °C. Co-immunoprecipitates (EMD Millipore); anti-Tfam (sc-23588) and anti-NRF-1 were collected by centrifugation, boiled in 50 μLof (sc-33771) antibodies (Santa Cruz Biotechnology Inc., Laemmli sample buffer, and used for immunoblot ana- Santa Cruz, CA); anti-POLG1 antibody (a kind gift of Dr. lysis for POLG1 (a kind gift of Dr. William C. Copeland, William C. Copeland, National Institutes of Health, USA); National Institutes of Health) or anti-Tfam (sc23588, A- anti-citrate synthase antibody (a kind gift of Dr. Brian H. 17; Santa Cruz Biotechnology Inc., Santa Cruz, CA) anti- Robinson, The Hospital for Sick Children, Canada); anti- body. Anti-IgG antibodies were used as a non-specific 4-HNE (ab48506), anti-SOD2 (ab13533), anti-catalase WAF1 control. (ab1877), and anti-p21 (ab7960) antibodies (Abcam, Cambridge, MA); anti-Pax7 (Developmental Studies mtDNA immunoprecipitation assay Hybridoma Bank, University of Iowa, Iowa City, IO); and mtDNA immunoprecipitation was performed on skeletal anti-ERRα (EPR46Y) and anti-actin (NB600-535) anti- muscle mitochondrial fraction that was cross-linked and bodies (Novus Biologicals, Littleton, CO) [11, 16]. The sonicated as previously described [16, 19]. One milli- carbonylated protein content in whole tissue lysates and gram of mitochondrial fraction was pre-cleared in 25 % mitochondrial fractions was quantified by Western blot v/v pre-clearing matrix F (Santa Cruz Biotechnology, using OxyBlot Protein Detection kit (S7150; Millipore, Santa Cruz, CA) overnight at 4 °C. The supernatant was Bedford, MA) as per manufacturer’sinstructions. Allanti- then incubated with 20 μg of anti-p53 (FL-393) antibody bodies were used at 1:1000 dilution, except for anti-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and (1:10,000). Membranes were then incubated with the ExactaCruz™ F matrix (Santa Cruz Biotechnology, Santa appropriate anti-mouse or anti-rabbit horse radish Cruz, CA) [20] with mixing by end-over-end inversion peroxidase-linked secondary antibody (1:10,000) and visu- overnight at 4 °C in the presence of 5 μg of shared sal- alized by enhanced chemiluminescence detection reagent mon sperm DNA (Sigma-Aldrich , Oakville, ON) to re- (Amersham, Piscataway, NJ). Relative intensities of the duce non-specific DNA-bead interactions. Anti-IgG protein bands were digitally quantified by using NIH Safdar et al. Skeletal Muscle (2016) 6:7 Page 7 of 17 Image J, version 1.37, analysis software (Scion Image, combining 960 μLof K HPO buffer (50 mM with 2 4 NIH). 50 mM EDTA and 0.01 % Triton X-100, pH 7.2–7.4) with 30 μL of muscle homogenate. Ten microliters of H O 2 2 ROS assay (1 M) was added to the cuvette and mixed by inversion to Mitochondrial H O production was measured using the initiate the reaction. Absorbance was measured at 240 nm 2 2 Amplex Red Hydrogen Peroxide assay (A22188; Invitro- every 15 s for 2 min. Catalase activity was calculated and −1 −1 gen, Burlington, ON) as per manufacturer’s instructions. reported in μmol · min ·mg protein . All samples were Briefly, 40 μg of mitochondrial fraction was diluted in analyzed in duplicates on the Cary UV-vis spectrophotom- 50 μL reaction buffer (125 mM KCl, 10 mM HEPES, eter (Varion, Inc., Palo Alto, CA). 5 mM MgCl , 2 mM K HPO , pH 7.44) to determine 2 2 4 mitochondrial respiratory chain complex I (5 mM pyru- Caspase-3 and caspase-9 enzyme activity vate/malate) or complex II (5 mM succinate) driven Caspase-3 and caspase-9 enzyme activity was measured H O production with and without inhibitors (0.5 μM using fluorometric protease assays caspase-3/CPP32 and 2 2 rotenone, complex I inhibitor, and 0.5 μM antimycin A, caspase-9/Mch6, respectively (Biovision, Mountain View, complex III inhibitor). Mitochondrial H O production CA) according to manufacturer’s instructions. Briefly, 2 2 was measured after the addition of 50 μL of reaction the assays are based on the detection of cleavage of the buffer containing horseradish peroxidase and Amplex substrate DEVD-AFC (AFC: 7-amino-4-trifluoromethyl Red. Fluorescence was followed at an excitation wave- coumarin) by caspase-3 and LEHD-AFC (AFC: 7-amino- length of 545 nm and an emission wavelength of 590 nm 4-trifluoromethyl coumarin) by caspase-9. Uncleaved for 5 min using fluorescence microplate reader (Tecan DEVD-AFC and LEHD-AFC fluoresce at λ = 400 nm, max Safire, MTX Lab Systems, Inc., Vienna, VA). The slope upon cleavage of the respective substrate by caspase-3 of the increase in fluorescence is converted to the rate of or caspase-9, free AFC emits a yellow-green fluorescence H O production with a standard curve. All of the assays (λ = 505 nm), which was quantified using a fluores- 2 2 max were performed at 25 °C. The results are expressed as cence microplate reader (Tecan Safire, MTX Lab Sys- −1 −1 pmoles.min .mg protein . tems, Inc., Vienna, VA). Results were expressed as raw fluorescence units per milligram of cytosolic protein. Mitochondrial respiratory chain complex I and IV enzyme activity Apoptosis cell death detection ELISA Mitochondrial ETC complex I and complex IV activities Apoptotic DNA fragmentation was quantified in the skel- were determined in tissue lysates following established etal muscle (quadriceps femoris), heart, primary protocols [11, 21–23]. All samples were analyzed in du- hematopoietic stem cells, and primary satellite cells by plicates on the Cary UV-vis spectrophotometer (Varion, measuring the amount of cytosolic mono- and oligo- PLUS Inc., Palo Alto, CA). nucleosomes using a Cell Death detection ELISA assay (Roche Applied Science, Laval, QC) as previously Superoxide dismutase and catalase enzyme activity described [11]. Briefly, wells were coated with a monoclo- Muscle total superoxide dismutase (Mn-SOD and Cu/Zn- nal anti-histone antibody and incubated with homoge- SOD) activity was determined in muscle lysates by meas- nates. Nucleosomes were centrifuged at 100,000g followed uring the kinetic consumption of superoxide radical (O ) by binding to the anti-histone antibody followed by the by SOD in a competitive reaction with cytochrome c,as addition of anti-DNA-peroxidase antibody that binds to previously described [20]. Absorption was recorded at the DNA associated with the histones. The amount of per- 550 nm and was observed every 15 s for 2 min at 37 °C. oxidase retained in the immunocomplex was determined One unit (U) of SOD activity was defined as the amount spectrophotometrically with ABTS (2,2′-azino-bis[3-ethyl- of enzyme that caused a 50 % inhibition of the reduction benzthiazoline-6-sulphonic acid]) as a substrate. Results of cytochrome c. Total SOD activity was expressed in were expressed as arbitrary OD units normalized to mi- −1 U.mg of protein . In a separate cuvette, the same sample crograms of cytosolic protein. was analyzed under identical conditions in the presence of 0.2 M KCN (pH 8.5–9.5), a potent inhibitor of cytosolic Quantification of mtDNA mutations Cu/Zn-SOD [24], for determination of mitochondrial Mn- mtDNA mutations were quantified by the error-resistant SOD activity. Cu/Zn-SOD activity was approximated by single molecule approach [26]. Briefly, skeletal muscle subtracting Mn-SOD activity from total SOD activity. (quadriceps femoris) DNA was subjected to limiting dilu- Both Mn-SOD and Cu/Zn-SOD activity were expressed in tion long-range PCR, where each positive PCR reaction −1 U.mg protein . Catalase activity was determined by was initiated by a single mtDNA molecule. PCR was de- measuring the kinetic decomposition of H O as previ- signed to amplify essentially the entire mitochondrial 2 2 ously described [25]. Catalase activity was measured by genome using high-fidelity Phusion DNA polymerase, Safdar et al. Skeletal Muscle (2016) 6:7 Page 8 of 17 (New England Biolabs). Three to 9 amplified molecules are presented as mean ± standard error of the mean were obtained per animal. Each amplified molecule was (SEM). sequenced in its entirety using barcoded Illumina next generation sequencing approach at a local core facility. Results and discussion Mutations were identified by comparing each molecule’s Endurance exercise confers phenotypic protection, sequence to the standard C57Bl/6J mtDNA sequence reduces mtDNA mutations, and attenuates oxidative (GenBank EF108336). Only 100 % mutations were con- damage in PolG mice sidered, which guaranteed the exclusion of artifacts [26]. Aged tissues display stochastic accumulation of mtDNA Mutant fractions were calculated by dividing the total mutations that likely perpetuate respiratory chain defi- number of mutations by the number of nucleotides se- ciency and greater reactive oxygen species (ROS)-medi- quenced per animal. ated damage [28]. To evaluate the underlying protective mechanism of exercise on mitochondrial redox status and mtDNA integrity, we profiled “terminally differenti- p53 base excision repair activity assay ated” (skeletal muscle and heart) and “proliferative” (Lin An in vitro fluorescence-based DNA primer p53 repair − Sca-1+ c-Kit + population enriched for hematopoietic activity assay was employed as previously described [27], stem and progenitor cells, “HSC’ and c-met+, satellite with minor modifications. This assay utilized a double- cells, “SC”) compartments of littermate wild-type (WT), stranded deoxyoligomers containing sequences identical sedentary PolG (PolG-SED), and forced-endurance exer- to the first 40 nucleotides of the mtDNA replication ori- cised PolG (PolG-END) mice. As shown previously [11], gin as the primer-template substrate, with the 3′ end of and now confirmed in an independent cohort of mice the primer contained self-designed mismatch point mu- utilized in this study, exercise rescued progeroid aging tation in the last three nucleotides (Additional file 1: (Additional file 1: Figure S1A), increased life span Table S2). The 5′ and 3′ ends of the primer were chem- (Additional file 1: Figure S1B), and reduced mtDNA mu- ically linked to a Black Hole Quencher -1 and 6- tations (Fig. 1a) in PolG mice. carboxyfluorescein (FAM-1™) flourophore, respectively Initial characterization of PolG mice showed absence (Integrated DNA Technology , Toronto, ON). The prem- of increased oxidative damage despite significant accu- ise of this assay is that, in the absence of proofreading mulation of mtDNA point mutations [4, 29]. We evalu- capacity of mitochondrial polymerase gamma, primer ated the presence of oxidative modifications and found extension requires the excision of the unpaired nucleo- no difference in protein carbonyls (PC) and 4-hydroxy- tides by the 3′→5′ exonuclease activity which in turn 2-nonenal (4-HNE) content in the muscle, heart, and SC will be detected as an increase in fluorescence over time. homogenates of PolG-SED vs. WT (Additional file 1: The 20 μL reaction mixtures containing 50 mM Tris– Figure S1C). We surmised that since the absence of oxi- HCl (pH 7.5), 5 mM MgCl , 1 mM DTT, 100 μg/mL of dative damage in the PolG tissues is due to cell-to-cell BSA, 3′-end-FAM1™ primer-template substrate, 50 μM variability, and that any one modification would be lower each of dATP, dCTP, dGTP, and dTTP, and 40 μgof than the detectable limit in whole tissue homogenates WT, PolG-SED, and PolG-END skeletal muscle (quadri- [4, 29], we measured oxidative damage in mitochondrial ceps femoris) mitochondrial extracts were incubated at fractions—the primary source of cellular ROS. Indeed, 37 °C for 40 min with data collection at the end using mitochondria from these tissues demonstrated a sub- iCycler IQ™ real-time PCR detection system (BioRad, stantial increase in H O production, along with elevated 2 2 Mississauga, ON). To assess the requirement of p53 as PC and 4-HNE content (Fig. 1b, c, and Additional file 1: an accessory mtDNA mismatch point mutation repair Figure S1C, D, and F). These observations are consistent protein, p53 repair activity assay was also carried out in with recent studies reporting higher PC levels in heart (i) PolG-END skeletal muscle mitochondrial extract after mitochondria of PolG mice [30] and increased mito- p53 immunodepletion and (ii) PolG-SED skeletal muscle chondrial H O production in vivo using mitochondria- 2 2 mitochondrial extract with addition of recombinant hu- targeted mass spectrometry probe MitoB [31]. This man p53 (BD Biosciences, Mississauga, ON). higher oxidative damage is also congruent with reduced superoxide dismutase 2 (SOD2) and catalase content Statistics and activity in PolG-SED vs. WT (Fig. 1d and Additional All molecular indices between the groups (WT, PolG- file 1: Figure S1E). We hypothesize that the combination SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 of lower antioxidant capacity, coupled with elevated MKO-END mice) were analyzed using two-tailed Stu- ROS production in PolG-SED mitochondria exacerbates dent’s t test. The log-rank test was used to test for signifi- the accumulation of mtDNA mutations. Consistent with cant differences in life span distribution between groups. this notion, Vermulst et al. reported a significant reduc- Statistical significance was established at a P ≤ 0.05. Data tion in the frequency of mtDNA mutations in the heart Safdar et al. Skeletal Muscle (2016) 6:7 Page 9 of 17 Fig. 1 (See legend on next page.) Safdar et al. Skeletal Muscle (2016) 6:7 Page 10 of 17 (See figure on previous page.) Fig. 1 Endurance exercise reduces random mtDNA somatic mutations, attenuates mitochondrial ROS-mediated oxidative damage, mitigates telomere shortening, and reduces nuclear accumulation of p53 in mtDNA mutator mice. a Random mtDNA somatic mutation rate (per 1000 nucleotides of mtDNA) in muscle (quadriceps femoris) WT, PolG-SED, and PolG-END mice (n =4–5/group). b H O production rate in muscle 2 2 mitochondrial 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 catalase enzyme activity in the muscle (quadriceps femoris) and heart of WT, PolG-SED, and PolG-END (n = 7/group). e Average telomere length ratios in the heart, hematopoietic stem and progenitor cells (HSC), and satellite cells (SC) of WT, PolG-SED, and PolG-END (n =6–8/group). f Representative blots 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. Error bars represent SEM. AU arbitrary units tissue of transgenic animals that over-expressed human Endurance exercise mitigates mitochondrial dysfunction catalase (CAT), a ROS scavenger, to mitochondria vs. via reduction in nuclear p53 that represses PGC-1α age-matched (28 months old) wild-type mice [32]. We A causal role of mitochondrial-induced oxidative stress observed that exercise normalized mitochondrial H O and telomere erosion, secondary to mtDNA mutations, 2 2 production (Fig. 1b and Additional file 1: Figure S1D), suggests a direct link between p53 activation and mito- and markers of oxidative damage (Fig. 1c and Additional chondrial dysfunction [5]. Increasing the expression of file 1: Figure S1F) in PolG-END to WT levels and in- PGC-1α, a potent regulator of mitochondrial biogenesis, creased SOD2 and catalase content and activity (Fig. 1d positively regulates the expression of antioxidants [33] and Additional file 1: Figure S1E). Together, our data and has been touted to attenuate aging-associated sarco- suggest that exercise reduces mtDNA point mutations, penia and metabolic dysfunction [34]. This prompted us at least in part, via the up-regulation of cellular antioxi- to investigate whether activation of p53-mediated senes- dant capacity that subsequently serves to attenuate ROS cence signaling attenuates PGC-1α-triggered gene pro- levels. gramming. We conducted in silico promoter analysis that identified putative p53 binding elements in the Endurance exercise diminishes telomere erosion and PGC-1α promoter. These promoter regions were then down-regulates aberrant p53 signaling and pathological cloned into a pGL4 luciferase reporter vector and trans- +/+ −/− levels of apoptosis in PolG mice fected into p53 and p53 mouse embryonic fibro- Sustained intrinsic accumulation of oxidative damage blasts (MEFs). A significant repression of PGC-1α-pGL4 +/+ has been implicated in telomere erosion that drives reporter activity was observed in the p53 relative to −/− age-related tissue degeneration [1]. In agreement with p53 MEFs (Additional file 1: Figure S2C). These re- this, we observed shorter telomeres in the heart, sults are consistent with a recent study showing that nu- HSC, and SC from PolG-SED vs. WT (Fig. 1e). clear p53 can directly repress PGC-1α expression and Genomic instability due to telomere shortening acti- promote mitochondrial dysfunction [5]. To further test vates tumor suppressor protein p53-mediated senes- our hypothesis, we next performed an anti-p53 chroma- cence/apoptotic signaling cascades [1]. Accordingly, tin immunoprecipitation assay that showed physical en- nuclear p53 abundance in the muscle, heart, and SC richment of nuclear p53 at the PGC-1α promoter of of PolG-SED was enhanced (Fig. 1f), concomitantly PolG-SED vs. WT mice (Fig. 2b). Together, these data with higher expression levels of the p53-responsive suggest that ROS-induced cellular damage prompts the WAF1 INK4A senescence genes: p21 ,p16 , and GADD45B nuclear accumulation of p53, which in turn activates (Additional file 1: Figure S1G) vs. WT. Mitochondrial p53-responsive senescence genes while simultaneously dysfunction in PolG mice is associated with patho- repressing the pro-metabolic activity of PGC-1α. logical systemic apoptosis [4, 11], and consistent with qPCR analyses of the PolG-SED muscle, heart, HSC these observations, we found higher DNA fragmenta- and SC confirmed lower expression of PGC-1α and tion (Fig. 2a) and caspase-3/9 activity (Additional file strong repression of its metabolic networks, including 1: Figure S2A and B) in PolG-SED mice. Interestingly, oxidative phosphorylation, mitochondrial function, glu- exercise abrogated telomere shortening (Fig. 1e), re- coneogenesis, and fatty acid metabolism vs. WT (Fig. 2c, duced p53 nuclear accumulation (Fig. 1f), normalized and Additional file 1: Figure S2D–G, and Table S1). the expression of p53-responsive senescence genes Additionally, PolG-SED mice tissues and stem cells have (Additional file 1: Figure S1G), and reduced patho- reduced mtDNA copy number (Fig. 2D and Additional logical levels of apoptosis in PolG-END (Fig. 2a, and file 1: Figure S3A), lower mitochondrial complex I and Additional file 1: Figure S2A and B). complex IV enzyme activity (Additional file 1: Figure Safdar et al. Skeletal Muscle (2016) 6:7 Page 11 of 17 Fig. 2 (See legend on next page.) Safdar et al. Skeletal Muscle (2016) 6:7 Page 12 of 17 (See figure on previous page.) Fig. 2 Endurance exercise prevents dysregulated mitochondrial-induced apoptosis, reduces nuclear p53-mediated repression of PGC-1α, induces PGC-1α regulated gene networks, restores mtDNA copy number, and normalizes mitochondrial morphology in mtDNA mutator mice. a Nuclear DNA fragmentation (apoptotic index) in the heart, HSC, and SC of WT, PolG-SED, and PolG-END (n =7–10/group). b ChIP assay showing reduced p53 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-globin gene 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 bars represent SEM. AU arbitrary units S3B), reduced mitochondrial electron transport chain exonuclease activity that helps p53 promote and main- subunits protein content (Additional file 1: Figure S3C– tain mitochondrial genomic stability by executing base I), and accumulation of swollen, pleomorphic, oversized excision repair on damaged mtDNA [36]. The role of mitochondria (Fig. 2e). Endurance exercise decreased mitochondrial p53 in the context of aging remains hith- binding of p53 to the PGC-1α promoter (Fig. 2b), and erto unknown. Collectively, these observations led us to this effect was accompanied by the maintenance of hypothesize that in the presence of an error-prone mtDNA copy number, increased expression of PGC-1α POLG1, mitochondrial p53 will function as an accessory and its downstream metabolic network, enhanced mito- fidelity-enhancing component of the mtDNA replication chondrial oxidative capacity, and restoration of mito- machinery in PolG mice. chondrial structural integrity in PolG-END (Fig. 2c–e, To test our hypothesis, we first assessed the submito- and Additional file 1: Figures S2D–G, Figure S3A–I, and chondrial localization of p53 in skeletal muscle of WT Table S1). These observations collectively imply that ac- mice. Subfractionation of skeletal muscle mitochondria in- cumulating mtDNA mutations in PolG-SED mice lead dicated that mitochondrial p53 was primarily localized in to an increase in ROS generation that (i) promotes mito- the mitochondrial matrix (Fig. 3a). Next, we measured the chondrial dysfunction and telomere damage and (ii) sub- mitochondrial abundance of p53 in our experimental sequently triggers p53-regulated senescence pathways, groups. Unlike PolG-SED, in PolG-END mice, p53 prefer- thereby potentiating the loss of somatic and stem cells entially resided in the mitochondria vs. nuclei of muscle via apoptosis. In contrast, exercise reduced mtDNA mu- and heart (Figs. 1f and 3b). To ascertain whether mito- tations and maintained the cellular energy and redox chondrial ROS levels regulated p53 compartmentalization, homeostasis thereby circumventing telomere erosion we treated primary fibroblasts with rotenone, a complex I culminating in the inhibition of accelerated systemic inhibitor known to increase mitochondrial ROS, and ob- aging characteristic of PolG mice [3, 4]. served a rapid increase in mitochondrial p53 content at lower dosages without a concomitant increase in nuclear Endurance exercise-mediated repair of mtDNA mutations p53 (Additional file 1: Figure S4A). Intriguingly, with in- is p53-dependent creasing rotenone concentrations, we measured an in- POLG1 is the sole mitochondrial polymerase essential crease in nuclear p53 abundance (Additional file 1: Figure INK4A for mtDNA replication and repair via its 3′→5′ exo- S4A) and expression of its downstream targets (p16 WAF1 nuclease activity [35]. Since exercise reduced mtDNA and p21 ; Additional file 1: Figure S4B), along with a mutations in PolG mice, which lack proofreading cap- concomitant reduction in mitochondrial p53 content acity of POLG1, this raised an intriguing possibility that (Additional file 1: Figure S4A), and mtDNA copy number exercise recruited a POLG1-independent mtDNA repair (Additional file 1: Figure S4C). The increase in nuclear pathway(s) [11]. We found that despite elevated p53 nu- p53 paralleled the decrease in PGC-1α mRNA expression clear abundance in PolG-SED, the total p53 content in (Additional file 1: Figure S5A), further supporting the in- the muscle, heart, and SC homogenates of all groups hibitory effects of p53 on PGC-1α. Furthermore, the up- was unaltered (Additional file 1: Figure S3J). This indi- regulation of rotenone-evoked nuclear p53 content was cated that a basal pool of p53 is maintained intra- attenuated in fibroblasts pre-treated with a ROS scavenger, cellularly, with the distribution of p53 between the N-acetylcysteine (Additional file 1: Figure S5B), in tandem different subcellular compartments dependant on the with higher PGC-1α mRNA expression (Additional file 1: cellular stress milieu [27]. In vitro studies show that in Figure S5C). Thus, p53 preferentially shuttles into response to intra- and extra-cellular insults such as mitochondria in response to physiological ROS levels, ROS, p53 translocates into the mitochondria where it in- which abrogates the negative regulation of PGC-1α as teracts with the mtDNA and POLG1 [27]. Biochemical exerted by p53 residing in the nucleus. Next, we analysis of p53 has revealed an inherent 3′→5′ sought to elucidate if mitochondrial p53 interacted Safdar et al. Skeletal Muscle (2016) 6:7 Page 13 of 17 Fig. 3 (See legend on next page.) Safdar et al. Skeletal Muscle (2016) 6:7 Page 14 of 17 (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 Tfam in mtDNA mutator mice. a Mitochondrial p53 is primarily localized in the matrix. Muscle mitochondria were subfractionated into outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), and matrix (Mx) fractions, and these fractions were immunoblotted 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), and d 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 to mtDNA (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 units with POLG1 and Tfam in the mitochondrial matrix. mutator mouse model” was needed where changes in We performed co-immunoprecipitation reactions and mtDNA mutation burden can be assessed in a background found that mitochondrial p53 formed a complex with of p53 over-expression or knockdown. It was unfeasible to POLG1 and Tfam complexed at mtDNA (Fig. 3c–e). generate PolG mice with over-expression of p53, as previ- The p53-POLG1-Tfam complex at mtDNA was higher ous work has shown that mice engineered with hyper- in PolG-END vs. PolG-SED and WT (Fig. 3c–e). active p53 alleles display show stem cell depletion and These observations are consistent with a recent study premature aging phenotype themselves [38]. On the other reporting p53 translocation to of p53 to the mito- hand, whole-body p53 knockout mice die prematurely of chondria and subsequent formation of p53-Tfam- cancer [39] and do not breed efficiently with heterozygous +/D257A mtDNA complex in skeletal muscle of WT mice in PolgA mice, and thus could not be bred with PolG response to an acute bout of endurance exercise [37]. mice to efficiently study the effects of exercise. Hence, we These results led us to conclude that the preferential created a new genetically modified mutator mouse with subcellular localization of p53 to mitochondria vs. muscle-specific p53 deletion (PolG-p53 MKO). At basal nuclear compartment is a “universal” exercise-induced levels, PolG-p53 MKO-SED mice demonstrated an accel- phenomenon and likely plays a role in mediating erated progeriod phenotype and significant accumulation beneficial effects of endurance exercise on improving of random mtDNA mutations in muscle compared to mitochondrial content/function and ameliorating PolG-SED mice (Fig. 4e and Additional file 1: Figure S6B). dysfunction. To our surprise, endurance exercise not only failed to Since both PolG-SED and PolG-END mice have defect- reduce mtDNA point mutations but also did not rescue ive POLG1 proofreading capacity, we believe that the re- progeroid aging, sarcopenia, exercise intolerance, mito- duction in total mtDNA mutational burden in PolG-END chondrial morphology anomalies, and deficits in mito- mice (Fig. 1a) is mediated by mitochondrial p53 levels in chondrial content and function such as mtDNA copy response to endurance exercise. Hence, we sought to number, mitochondrial electron transport chain protein evaluate whether mitochondrial p53 can repair mtDNA content, and COX activity in skeletal muscle of PolG-p53 mutations, independent of the proofreading capacity of MKO mice (Fig. 4c–g and Additional file 1: Figure S6B– POLG1. A fluorescence-based in vitro DNA primer E). This suggests that the exercise-mediated repair of extension-mutation repair assay displayed an efficient re- mtDNA mutations in vivo is dependent on mitochondrial pair of double-stranded oligonucleotides, with artificially p53 adjuvant repair capacity. Furthermore, unlike PolG- added mismatch point mutations, incubated with ex vivo END, mitochondrial extracts from PolG-p53 MKO failed muscle mitochondrial extract of PolG-END vs. PolG-SED to repair mutations in vitro in the primer extension- (Fig. 4a). PolG-END mitochondria failed to repair these mutation repair assay (Additional file 1: Figure S6F). Thus, mutations upon p53 immunodepletion (Fig. 4b), while exercise-induced maintenance of mtDNA stability is con- addition of recombinant p53 increased PolG-SED mito- tingent on mitochondrially localized p53 and represents a chondrial mutation repair efficiency (Additional file 1: viable therapy for pre-symptomatic patients carrying Figure S6A). Clearly, mitochondrial p53 plays a vital role POLG1 exonuclease domain mutations known to cause in the maintenance of mtDNA integrity in the presence of pathology [35]. defective POLG1 in mutator mice. However, to conclusively attribute causality to mito- Conclusions chondrial p53 in mediating mtDNA repair in vivo in re- Here, we show that exercise promotes mitochondrial sponse to endurance exercise, a “double genetically altered oxidative capacity and cellular redox dynamics via PGC- Safdar et al. Skeletal Muscle (2016) 6:7 Page 15 of 17 Fig. 4 Endurance exercise-mediated repair of mtDNA mutations is mitochondrial p53-dependent. a A fluorescence-based in vitro DNA primer extension-mutation repair assay in muscle mitochondrial extracts from WT, PolG-SED, and PolG-END (n= 6–8/group) to assess the excision of the unpaired 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 trials in WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice (n= 5–6/group). d Representative electron micrographs of myofibers (quadriceps femoris) from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END (n= 4/group). e Random mtDNA somatic 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-p53 MKO-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 units 1α-mediated expression networks, thus preventing the Intriguingly, stress-mediated subcellular localization of accumulation of oxidative damage, abrogating genotoxic the tumor suppressor protein p53 determines its pro- or damage, and repressing apoptosis in mutator mice. anti-survival function and seems indispensible for the Safdar et al. Skeletal Muscle (2016) 6:7 Page 16 of 17 exercise-mediated mtDNA repair and mitochondrial bio- Cu/Zn-SOD); SOD2: superoxide dismutase 2 (mitochondrial; Mn-SOD); TFAM: mitochondrial transcription factor A; VDAC: voltage-dependent anion genesis. The work summarized here opens up viable ave- channel; WT: wild-type mice. nues of research in cancer biology where mitochondrial dysfunction and genomic instability have been impli- Competing interests The authors declare that they have no competing interests. cated [1]. It will be of potential clinical interest to see if exercise-induced mitochondrial-targeted p53 might rep- Authors’ contributions resent a therapeutic intervention for aging-associated AS and MAT designed the research; AS, KK, JMF, AS, MDL, APWJ, YK, IAS, YK, pathologies such as insulin resistance, diabetes, and car- 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 BPH diovascular diseases, which manifest telomere shortening analyzed the data; and AS wrote the manuscript. All authors have been in conjunction with mitochondrial dysfunction [40, 41]. involved in drafting and revising the manuscript and have approved the final Indeed, while exercise and an active lifestyle are the manuscript. most prominent therapies to reduce the incidence and Acknowledgements pathogenicity of diabetes, insulin resistance, and cardio- We like to acknowledge the late Mrs. Suzanne Southward (McMaster vascular diseases [42, 43], therapeutic modalities that University) for assisting in enzyme assays and Dr. William C. Copeland (NIH) for his kind donation of POLG1 antibody. This work was supported by the promise to recapitulate some of the effects of exercise Canadian Institutes of Health Research (CIHR) grant and a kind donation warrant further attention. The telomere–p53–PGC-1α from Mr. Warren Lammert and family to M.A.T. A. Safdar was funded by axis provides a molecular basis of how telomere erosion Banting Fellowship (CIHR) and American Federation for Aging Research and Ellison Medical Foundation (EMF) Fellowship. A. Saleem is funded by Natural and mitochondrial dysfunction can modulate systemic Sciences and Engineering Research Council of Canada postdoctoral aging of tissues and stem cell compartments. Under- fellowship. K.K. was supported by the United Mitochondrial Disease standing the upstream signaling cascades and posttrans- Foundation, the NIH (AG019787) and the EMF Senior Scholarship. The authors declare no competing and financial interests. T.A.P. was awarded a lational modifications that promote mitochondrial D257A US patent 7,126,040 for the Polg mouse model. T.A.P. is a partial owner localization of p53 may allow for the generation of of LifeGen Technologies, specializing in nutrigenomics, as well as a Scientific pharmaceutical analogs, novel therapeutic strategies to Advisory Board member for Nu Skin Enterprises. M.A.T. is the founder, president, and CEO of Exerkine Corporation and a member of its scientific antagonize mitochondrial genomic decay, and cellular advisory board. senescence in age-associated pathologies. Author details Department of Kinesiology, McMaster University, Hamilton, ON L8N 3Z5, Additional file 2 Canada. Department of Pediatrics, McMaster University, Hamilton, ON L8N 3Z5, Canada. Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada. Department of Medical Sciences, McMaster University, Additional file 1: Figure S1. Endurance exercise confers complete Hamilton, ON L8N 3Z5, Canada. Department of Medical Physics & Applied phenotype protection, suppresses early mortality, mitigates mitochondrial Radiation Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada. ROS-mediated oxidative damage, increases cellular antioxidant capacity, 6 7 Northeastern University, Boston, MA 02115, USA. Buck Institute for Research and 4 prevents cellular senescencmutator mice. Figure S2. Endurance on Aging, Novato, CA 94945, USA. School of Health and Exercise Sciences, exercise prevents dysregulated mitochondrial-induced apoptosis and University of British Columbia Okanagan, Kelowna, BC V1V 1V7, Canada. reduces nuclear p53-mediated repression of PGC-1α and promotes Division of Cardiovascular Disease, University of Alabama, Birmingham, AL mitochondrial biogenesis in mutator mice. Figure S3. Endurance exercise 35294, USA. Perelman School of Medicine, University of Pennsylvania, promotes systemic mitochondrial biogenesis in mtDNA mutator mice. Philadelphia, PA 19104, USA. Departments of Genetics, University of Figure S4. Magnitude of mitochondrial ROS (physiological vs. Wisconsin, Madison, WI 53706, USA. Departments of Medical Genetics, pathological) regulates p53 subcellular localization. Figure S5. University of Wisconsin, Madison, WI 53706, USA. Pre-treatment with exogenous antioxidant preferentially shuttles p53 to mitochondria in response to stress. Figure S6. Endurance exercise- Received: 11 October 2015 Accepted: 5 January 2016 mediated attenuation of sarcopenia, increase in endurance capacity, skeletal muscle mitochondrial biogenesis, and repair of muscle mtDNA mutations is p53 dependent. Table S1. WT, PolG-SED, and PolG-END Skeletal Muscle Microarray IPA-GO Analysis. Table S2. Real-time PCR References primer sequences. (PDF 1601 kb) 1. Sahin E, Depinho RA. Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature. 2011;464(7288):520–8. 2. Larsson NG. Somatic mitochondrial DNA mutations in mammalian aging. 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