High Levels of Physical Activity in Later Life Are Associated With Enhanced Markers of Mitochondrial Metabolism

Abstract

The age-associated reduction in muscle mass is well characterized; however, less is known regarding the mechanisms responsible for the decline in oxidative capacity also observed with advancing age. The purpose of the current study was therefore to compare mitochondrial gene expression and protein content between young and old recreationally active, and older highly active individuals. Muscle biopsies were obtained from the vastus lateralis of young males (YG: 22 ± 3 years) and older (OG: 67 ± 2 years) males not previously engaged in formal exercise and older male master cyclists (OT: 65 ± 5 years) who had undertaken cycling exercise for 32 ± 17 years. Comparison of gene expression between YG, OG, and OT groups revealed greater expression of mitochondrial-related genes, namely, electron transport chain (ETC) complexes II, III, and IV (p < .05) in OT compared with YG and OG. Gene expression of mitofusion (MFN)-1/2, mitochondrial fusion genes, was greater in OT compared with OG (p < .05). Similarly, protein content of ETC complexes I, II, and IV was significantly greater in OT compared with both YG and OG (p < .001). Protein content of peroxisome proliferator-activated receptor gamma, coactivator 1 α (PGC-1α), was greater in OT compared with YG and OG (p < .001). Our results suggest that the aging process per se is not associated with a decline in gene expression and protein content of ETC complexes. Mitochondrial-related gene expression and protein content are substantially greater in OT, suggesting that exercise-mediated increases in mitochondrial content can be maintained into later life.

Human, Athlete, Muscle, Mitochondria

Skeletal muscles of most older individuals are characterized by several adverse morphological changes (1–4), a phenotype directly influenced by physical activity (5–7). Some groups report an age-associated decline in fiber cross-sectional area in sprint-trained older individuals (8), whereas this association is not observed in highly trained master cyclists (6). This observation therefore questions the extent to which these adverse changes to skeletal muscle health are a function of the aging process or the interaction with age and physical inactivity (8).

Considerable attention has been given to determining the effects of aging on mitochondrial function in skeletal muscle (9). In addition to being associated with a decline in muscle fatigue resistance, aberrant mitochondrial function is proposed as a major contributor to sarcopenia (10). In support of this motion, previous research has reported an age-associated decline in mtDNA (11), in addition to a reduction in gene expression of mitochondrial transport chain subunits (12). The reduction in mitochondrial-related gene expression translates to the decline in mitochondrial-related protein content observed with aging (11), although this apparent reduction is not observed in middle-aged adults where protein content of electron transport chain (ETC) complex I is greater when compared with young (13). In addition, aging is associated with an increase in mtDNA mutation-deletions (14), an increase in oxidative damage (12), and altered protein content of factors involved in mitochondrial dynamics (15). These findings, however, are not universal, as other groups have reported that aging had no effect on mitochondrial associated gene networks (16), mitochondrial respiration and protein content of ETC subunits (17), and regulators of mitochondrial dynamics (18). Contrasting results may be due to a number of factors, one of which may be the physical activity status of old individuals included in these studies. For example, Disteffano and colleagues recently demonstrated that cardiorespiratory fitness has a greater influence on mitochondrial respiration than age (18). Although sedentary aging is associated with a variety of metabolic perturbations, it is suggested that these may be related to the low levels of physical activity rather than to the aging process per se (6). Or alternatively, physical activity remains beneficial even in advancing age. This latter notion is supported by studies demonstrating that older athletes, when compared with sedentary counterparts, have greater mitochondrial content, function (19), gene expression of ETC subunits (17), and protein content related to mitochondrial dynamics (18). In older individuals, mitochondrial volume density is correlated with exercise capacity, suggesting a role for physical activity to maintain mitochondrial content in this population (20). Importantly, older individuals retain the ability to positively respond to aerobic exercise training (20,21), placing regular physical exercise as an effective measure for improving mitochondrial content and function throughout the lifespan.

Several studies have sought to characterize the differences in gene and protein expression between either young and sedentary older adults or older sedentary and older athletes. However, few have examined the differences between young and old recreationally active and old highly active master athletes, thereby allowing the combined comparison of age and physical activity levels. Therefore, the purpose of the current study was to compare mitochondrial-related gene expression and protein abundance in the quadriceps muscle between recreationally active young (YG), old individuals who were healthy, but did not undertake regular exercise (OG) and older individuals who had been highly active through cycling exercise for many years (OT). We determined whether genes and proteins related to whole muscle metabolism, mitochondrial biogenesis, dynamics, and mitophagy were differentially expressed between groups to further characterize the aging muscle phenotype and the impact of physical activity.

Method

Ethical Approval

Prior to participation written informed consent was obtained from all subjects. Procedures were approved by the National Health Service Wandsworth Research Ethics Committee (reference number 12/LO/0457) and the University of Nottingham Faculty of Medicine and Health Science Research Ethics Committee (B/10/2010), with all procedures conforming to the Declaration of Helsinki. All human tissue collected, stored, and analyzed was done so in accordance with the Human Tissue Act.

Participants

The OT group is a subset from a large cross-sectional study previously described by Pollock and colleagues in which the physiological function of a group of amateur nonelite cyclists aged 55–79 years was evaluated (22). The inclusion criteria of males was the ability to cycle 100 km in under 6.5 hours and subjects were required to have undertaken this task twice in the 3 weeks prior to testing (22). Subjects included in the OG group (67 ± 2 years; 27 ± 2.8 kg/m²; n = 8, for subsequent gene expression and protein content analysis) were selected to have similar ages and body mass index (BMI) to those of the OT group (65 ± 5 years; 25.9 ± 2.1 kg/m²; n = 7, for subsequent gene expression analysis, n = 8 for subsequent protein content analysis). Subjects included in the YG group (21 ± 3 years; 23.9 ± 2.5 kg/m^2;n = 6, for subsequent gene expression analysis, n = 9 for subsequent protein content analysis) were selected to best match BMI from the older groups. Both YG and OG were recreationally active and performed activities of daily living but none were involved in a formal exercise training programme nor did they participate in more than two sessions of purposeful exercise each week. No differences between BMI are observed between groups and no differences between ages are observed between the OT and OG groups. Due to lack of tissue, the sample size was smaller for gene expression analysis as stated above.

Muscle Biopsy Sampling

Following administration of local anesthetic (1% lidocaine for the YG and OG; 2% lidocaine for the OT), a muscle sample was obtained from the vastus lateralis of all participants using the conchotome technique (23) for the YG and OG groups and the Bergstrom needle technique with applied suction for the OT group (6). All participants in the YG and OG groups were instructed not to exercise for 72 hours prior to their biopsy which was taken after an overnight fast (~12 hours) with water ad libitum. Participants in the OT group were asked to maintain habitual levels of physical activity the day prior to their biopsy.

RNA Isolation and Reverse Transcription

RNA was isolated from 15 to 20 mg of powdered muscle tissue using TRI Reagent/ReliaPrep spin columns. Briefly, all samples were homogenized in 1 mL of TRI Reagent (Sigma–Aldrich, Gillingham, United Kingdom) with the FastPrep 24 5G (MP Biomedicals, Santa Ana, CA) at a speed of 6 m/s for 40 seconds. Two hundred microliters of chloroform were added to each sample and mixed vigorously for 15 seconds, then incubated at room temperature for 5 minutes, and then centrifuged at 12,000g for 10 minutes at 4°C. The RNA (aqueous phase) was purified using Reliaprep spin columns (Promega, Madison, WI) as per manufacturer’s instructions. RNA concentrations were determined using the LVis function of the FLUOstar Omega microplate reader. RNA was diluted to 20 μg/μL and reverse transcribed to cDNA using the RT² First Strand kit (Qiagen, Manchester, United Kingdom).

Quantitative RT–PCR

Quantitative analysis of 84 genes was completed using custom designed 384-well RT² PCR Profiler Array (Qiagen) and RT² SYBR Green Mastermix (Qiagen) on a CFX384 Real-Time PCR Detection System following manufacturer’s instructions (Bio-Rad). Briefly, 2.8 ng of cDNA was added to each well. The absence of genomic DNA, the efficiency of reverse-transcription, and the efficiency of the PCR assay were assessed on each plate and conformed to the manufacturer’s limits in each case. The C(t) values for housekeeper genes beta actin (Refseq# NM_001101), heat shock protein 90 (Refseq# NM_007355), and beta-2-microglobulin (Refseq# NM_004048) showed no statistical differences between groups. Therefore, the geometric mean C(t) of all three housekeeper genes was used as an internal control (24). Statistical analysis was carried out on the ΔΔC(t) (ΔC(t) gene of interest − ΔC(t) mean of gene of interest of the YG). Data are presented as a fold change from YG as determined using the 2^−ΔΔC(t) method (25).

Immunoblotting

Approximately 25 mg of powdered muscle tissue was homogenized in 300 μL of ice-cold sucrose lysis buffer (50 mM Tris, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na₄P₂O₇-10H₂O, 270 mM sucrose, 1 M Triton-X, 25 mM β-glycerophosphate, 1 μM Trichostatin A, 10 mM Nicatinamide, 1 mM 1,4-Dithiothreitol, 1% Phosphatase Inhibitor Cocktail 2; Sigma, 1% Sigma Phosphatase Inhibitor Cocktail 2; Sigma, 4.8% cOmplete Mini Protease Inhibitor Cocktail; Roche) using the FastPrep 24 5G (MP Biomedicals, Santa Ana, CA) at a speed of 6 m/s for 40 seconds and repeated three times. Samples were then centrifuged at 4°C at a speed of 8,000g for 10 minutes to remove insoluble material. Protein content was determined from the DC protein assay (Bio-Rad, Hercules, CA) using FLUOstar Omega at an absorbance of 750 nm. Laemmli samples buffer was added and samples were boiled for 5 minutes, equal amounts of protein (20–50 μg) was separated by SDS-PAGE on 8%–12.5% gels at a constant current of 23 mA per gel. Proteins were transferred to BioTrace NT nitrocellulose membranes (Pall Life Sciences, Pensacola, FL) via wet transfer at 100 V for 1 hour on ice. Membranes were stained with Ponceau S (Sigma–Aldrich, Gillingham, United Kingdom) and imaged to assure even loading and for future normalization. Membranes were blocked in 3% dry-milk in tris-buffered saline with tween (TBST) for 1 hour prior to an overnight primary antibody incubation at 4°C. Membranes were washed in TBST three times prior to incubation in appropriate horse radish peroxidase-conjugated secondary antibody at room temperature for one hour. Membranes were then washed in TBST three times prior to antibody detection via enhanced chemiluminescence horseradish peroxidase substrate detection kit (Millipore, Watford, United Kingdom). Imaging and band quantification were undertaken using a G:Box Chemi-XR5 (Syngene, Cambridge, United Kingdom). Mean pixel intensity was determined for each band. All bands were normalized to a gel control in addition to the corresponding ponceau image. Statistical analysis was carried out on normalized values for each protein of interest.

Antibodies

All primary antibodies were prepared in TBST. Antibodies used were as follows: total OXPHOS human WB antibody cocktail (ab11041; abcam, 1:1000 in unboiled samples), PGC-1Antibody (AB3242; Merck, 1:1000), pyruvate dehydrogenase antibody (PDH; #2784; Cell signaling technology, 1:1000), Citrate synthase (SAB2701077; MitoSciences, 1:1000), and Sirtuin3 (SirT3 D22A3; Cell signaling technology, 1:1000). Very long-chain acyl CoA dehydrogenase (VLCAD; 1:5000), long-chain acyl CoA dehydrogenase (LCAD; 1:5000), and medium-chain acyl-CoA dehydrogenase (MCAD; 1:5000) were kind gifts from Prof Jerry Vockley, University of Pittsburgh, PA. Appropriate secondary antibodies either antirabbit (7,074) or antimouse (7,076) from Cell Signaling Technology were used at a concentration of 1:10000 in TBST.

Statistical Analysis

Statistical analysis was performed using Prism 7 (GraphPad Software Incorporated). To assess differences between YG, OG, and OT groups, a one-way analysis of variance (ANOVA) was completed on individual genes and proteins independently, and multiple comparisons were assessed using Tukey’s test. The normal distribution of data was verified using the Shapiro–Wilk normality test. Values are presented as mean ± standard deviation.

Results

Mitochondrial-Related Genes Expression Profile Is Altered in Master Athletes

The mRNA expression of 84 target genes was assessed in YG (n = 6), OG (n = 8), and OT (n = 7) groups, and gene expression is presented as a fold change from the YG group (Supplementary Table 1). Of the 84 genes assessed, 23 were differentially expressed (p < .05) (Supplementary Table 1). Differentially expressed genes of mitochondrial-related proteins and transcriptional regulators are presented in Figure 1. Differentially expressed genes related to skeletal muscle structural remodeling are presented in Figure 2. Differentially expressed genes related to substrate metabolism are presented in Figure 3.

Figure 1.

Mitochondrial-related gene expression is greater in highly active older individuals. mRNA of genes related to mitochondrial content in recreationally active young (YG, n = 6; black bars), old individuals who were healthy, but did not undertake regular exercise (OG, n = 8; gray bars), older individuals who had been highly active through cycling exercise for many years (OT, n = 7; open bars). Data are presented as a fold change from YG. (a) sirtuin 3 (SIRT3), (b) peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), (c) citrate synthase (CS), (d) ubiquinol-cytochrome c reductase core protein I (UQCRC1), (e) ubiquinol-cytochrome c reductase core protein II (UQCRC2), (f) cytochrome c oxidase subunit IV isoform 1 (COX4I2), (g) cytochrome c oxidase assembly homolog 10 (COX10), (h) succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), (i) succinate dehydrogenase complex, subunit B, iron sulfur (SDHB), (j) translocase of inner mitochondrial membrane 8 homolog A (TIMM8A). ^aSignificantly different from YG, ^bsignificantly different from OG.

Figure 2.

Skeletal muscle-related gene expression is greater in highly active older individuals. mRNA expression of genes related to skeletal muscle remodeling in recreationally active young (YG, n = 6; black bars), old individuals who were healthy, but did not undertake regular exercise (OG, n = 8; gray bars), older individuals who had been highly active through cycling exercise for many years (OT, n = 7; open bars). Data are presented as a fold change from YG. (a) vascular endothelial growth factor A (VEGFA), (b) vascular endothelial growth factor B (VEGFB), (c) myogenic differentiation 1 (MyoD1), (d) BTG family, member 2 (BTG2), (e) mitofusin 1 (MFN1), (f) mitofusin 2 (MFN2), (g) sestrin 2 (SESN2), (h) phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), and (i) zinc finger, matrin-type 3 (ZMAT3). ^aSignificantly different from YG, ^bsignificantly different from OG.

Figure 3.

Substrate metabolism-related gene expression is greater in highly active older individuals. mRNA expression of genes related to substrate metabolism in recreationally active young (YG, n = 6; black bars), old individuals who were healthy, but did not undertake regular exercise (OG, n = 8; gray bars), older individuals who had been highly active through cycling exercise for many years (OT, n = 7; open bars). Data are presented as a fold change from YG. (a) isocitrate dehydrogenase 2 (NADP+) (IDH2), (b) malate dehydrogenase 1, NAD (MDH1), (c) pyruvate dehydrogenase kinase, isozyme 4 (PDK4), and (d) nuclear receptor subfamily 1, group D, member 2 (NR1D2). ^aSignificantly different from YG, ^bsignificantly different from OG.

Mitochondrial-Related Protein Content

Protein content was assessed in YG (n = 9), OG (n = 8), and OT (n = 8). Protein content of each complex of the electron transport chain was assessed via immunoblotting. There was no difference between YG and OG for any protein assessed. Protein content of complexes I, II, and IV was 7.1-, 1.9-, and 1.3-fold greater in OT compared with OG and 5.6-, 2.1-, and 1.2-fold greater compared with YG (p < .05) (Figure 4a). There are no differences in protein content of complexes III and V across groups (p > .05). Protein content of PGC-1α was 2.9-fold greater in OT compared with OG (p < .05) and 5.4-fold greater compared with YG (p < .001) (Figure 4c). Protein content of CS was 1.9-fold greater in the OT compared with both the OG (p < .001) and 1.3-fold greater in the OT compared with the YG (p < .001) (Figure 4d). Additionally, protein content of SIRT3 was 1.8-fold greater in OT compared with both OG (p < .001) and YG (p ≤ .001) (Figure 4e).

Figure 4.

Mitochondrial-related protein content is greater in highly active older individuals. Protein content in recreationally active young (YG, n = 9; black bars), old individuals who were healthy, but did not undertake regular exercise (OG, n = 8; gray bars), older individuals who had been highly active through cycling exercise for many years (OT, n = 8; open bars). (a) Protein content of mitochondrial enzymes (complexes I-V), (b) Representative images of protein content of mitochondrial enzymes (complexes I–V). (c) Protein content of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), (d) citrate synthase (CS), (e) sirtuin 3 (SIRT3). (f) Representative images of protein content of PGC-1α,CS and SIRT3 in YG, OG, and OT. All values were normalized to their respective ponceau stain. Data are presented as mean ± standard deviation. ^bSignificantly different from OG and YG.

Protein Content Related to Substrate Metabolism

Protein content of pyruvate dehydrogenase (PDH), LCAD, and MCAD was 3-, 1.8-, and 1.6-fold greater, respectively, in OT compared with OG and 2.8-, 1.9-, and 2.1-fold greater, respectively, compared with YG (p < .05) (Figure 5a–c). Protein content of VLCAD was 1.7-fold greater in OT compared with OG (p < .05) (Figure 5d).

Figure 5.

Substrate metabolism-related protein content is greater in highly active older individuals. Protein content in recreationally active young (YG, n = 9; black bars), old individuals who were healthy, but did not undertake regular exercise (OG, n = 8; gray bars), older individuals who had been highly active through cycling exercise for many years (OT, n = 8; open bars). (a) protein content of pyruvate dehydrogenase (PDH), (b) long-chain acyl CoA dehydrogenase (LCAD), (c) medium-chain acyl-CoA dehydrogenase (MCAD), and (d) very long-chain acyl CoA dehydrogenase (VLCAD). (e) Representative images of protein content of PDH, LCAD, MCAD, and VLCAD. All values were normalized to their respective ponceau stain. Data are presented as mean ± standard deviation. ^aSignificantly different from OG, ^bsignificantly different from OG and YG.

Discussion

Many studies have sought to determine the effect of aging on mitochondrial content and function (11–15,17,18,26); however, few have accounted for the divergent levels of physical activity in the older population. Here, we have directly compared young and old recreationally active adults in addition to old highly aerobically trained (master cyclists) adults. The inclusion of these three distinct groups allows the effects of activity to be established and therefore what extent declines in function can be attributed to chronological aging (8). The primary finding of this study is that highly active older adults have greater mitochondrial-related protein content and gene expression in comparison to recreationally active untrained young and older adults. Importantly, no observable age-associated reduction in mitochondrial-related protein content and gene expression (except for CS mRNA expression) was observed in the recreationally active older group in which the negative effects of inactivity are eliminated. In addition, we report no change in OXPHOS, PGC-1α, or mitochondrial fusion and fission-related gene expression with age.

Previous work has described a reduction in mtDNA, mitochondrial protein content, and ATP production in older adults who had similar physical activity levels to young counterparts (<30 minutes, <2 d/wk) (11). However, more recent work suggests that mitochondrial-related protein content may not be affected by aging (18). Our results are in line with the latter in which chronological age does not affect mitochondrial-related protein content (Figure 4a). A lower expression in OG compared with YG is only observed with respect to CS (Figure 1c) and UQCRC1 (Figure 1d) gene expression. However, this was not observed at the protein level (Figure 4c). Although chronological aging per se did not affect mitochondrial-related protein content and gene expression in our hands, the impact of high levels of physical activity in advanced age was substantial. We report greater expression of genes related to the electron transport chain (SDHA, SDHB, UQCRC1, UQCRC2, and COX4I2) in the OT compared with both the YG and OG groups (Figure 1). These results are supported by greater protein content of complexes I, II, and IV of the electron transport chain in the OT compared with YG and OG groups (Figure 4a). Larsen and coworkers have reported an association between protein content of complexes II and V and total mitochondrial content measured via mitochondrial volume by transmission electron microscopy (27). Although not a direct measure of mitochondrial content, our results are in accordance with previous work demonstrating a greater mitochondrial volume density and content of ETC proteins in old active compared with sedentary adults (19,20). Given that endurance exercise training is well established as a potent activator of mitochondrial biogenesis (28), the greater mitochondrial-related gene expression and protein content in the OT group are likely due to their high physical activity levels and the metabolic demands placed on skeletal muscle.

In addition, we report greater PGC-1α gene expression in the OT compared with the OG group (Figure 1b) and greater PGC-1α protein content in OT compared with both the YG and OG groups (Figure 4b). These results are in partial contrast with previous work reporting a reduction in PGC-1α protein content with aging in both “high” and “low” functioning sedentary older adults (29). As PGC-1α is an important regulatory signaling node for the initiation of mitochondrial biogenesis, a greater expression in the OT group may reflect a greater mitochondrial mass due to greater habitual physical activity levels in this cohort. Thus, our results suggest that mitochondrial biogenesis is primarily affected by activity status rather than aging. PGC-1 α also regulates the gene expression of SIRT3 (30), a NAD⁺-dependent deacetylase highly involved in metabolism (31). Accordingly, we report greater expression (Figure 1a) and protein content (Figure 4d) of SIRT3, in the OT group compared with both the YG and the OG groups. Again, this observation suggests that aging per se is not associated with a reduction in markers of mitochondrial metabolism; rather that exercise training has a positive effect on SIRT3 induction irrespective of age. Skeletal muscle mitochondrial function is believed, in part, to be regulated by the tumor suppressor p53 (32). Here, we demonstrate that gene expression of p53 targets, SESN2 and PMPAIP1, is increased in the OT group although not affected by aging (Figure 2). ZMAT3, another target of p53, has a greater expression in both OG and OT (Figure 2g and h). Interestingly, no change in p53 expression was observed between groups (p > .05) (Supplementary Table 1). With the design of the current study, we are unable to state whether the increase in p53 target genes is beneficial or detrimental in maintaining muscle health.

Information regarding content of proteins involved in mitochondrial dynamics in aging skeletal muscle is equivocal with some reports indicating either no change or a reduction (15,18,29). Recently, Tezze and colleagues have reported greater expression of OPA1, MFN1/2, and DRP1 in “older sportsmen” and young individuals compared with old inactive individuals (15). Balan and colleagues report greater mitochondrial protein content of MFN2 and OPA1 in active young and older adults in comparison to sedentary age-matched subjects; however, they do not observe an age-associated reduction in protein content (26). This is partially confirmed by our findings where expression of MFN1 and MFN2 was greater in OT compared with OG; however, in our hands aging did not result in a reduced expression (Figure 2e and f). This may relate to possible differences in the activity status of the recreational active older participants in the present study and the “sedentary seniors” in the study of Tezze and colleagues (15). Additionally, we did not observe differences in expression of other genes related to mitochondrial dynamics, such as OPA1 and FIS1 (Supplementary Table 1). Our results suggest that aging does not impair the dynamic remodeling of the mitochondrial reticulum but highly active older adults may require enhanced mitochondrial remodeling to support the stresses placed on the skeletal muscle as evidenced by greater gene expression of MFN1/2.

Although we report very few changes associated with chronological aging with respect to protein and gene expression, it is important to note that skeletal muscle of older individuals remains a highly plastic tissue and responds positively to exercise training (33–37). Although older individuals’ response to resistance exercise training may be attenuated in comparison to young adults, they are able to increase muscle fiber size (37). In line with this, endurance exercise training in older individuals results in improved maximal oxygen uptake, mitochondrial enzyme activities, and increase in muscle fiber size (38–40). Importantly, a recent analysis of cross-sectional data demonstrates that when older adults commence intense training and competition in later life (>50 years), no differences are observed with respect to performance and body composition (fat mass and leg lean mass) compared with adults having trained their entire adult life (21). Thus, further highlighting the ability of older individuals to positively respond to exercise.

One of the limitations of the present study was the lack of precise quantification of levels of physical activity in the groups that described themselves as being recreationally active. It is likely, however, that the OG did not reflect the majority of older people who are known to engage in reduced levels of physical activity (41). Thus, comparison with the OT group is, if anything, most likely to be an underestimate of the differences in phenotype between lifelong exercisers and most of the older population.

In summary, we observed greater mitochondrial-related gene expression and protein content in highly active older individuals compared with young and aged-matched recreationally active individuals. Chronological aging in recreationally active individuals did not affect mitochondrial-related gene and protein expression. Collectively these data suggest that high levels of physical activity, even in advancing age, result in greater mitochondrial-related gene expression and protein content compared with young- and age-matched untrained individuals. Furthermore, our results highlight that chronological aging does not result in a reduction in mitochondrial content and function and supports the notion that skeletal muscle retains the ability to positively respond to stimuli even in advancing age.

Acknowledgments

The authors would like to thank the research participants for their time and effort. All authors gave their final approval of the version of the article to be published. P.J.A., S.D.R., and A.P. designed the study. P.J.A., S.D.R., R.D.P., N.R.L., D.J.W., B.E.P., and K.S. organized and carried out sample collection. S.J., S.A., and K.A.O. performed experiments. S.J., S.A., and A.P. completed data analysis. S.J. and A.P. performed the statistical analysis of the data. S.J. and A.P. wrote the manuscript.

Conflict of Interest

None declared.