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J Gerontol A Biol Sci Med Sci. 2009 September; 64A(9): 927–939.
Published online 2009 May 22. doi:  10.1093/gerona/glp066
PMCID: PMC2981452

Impact of Lifelong Sedentary Behavior on Mitochondrial Function of Mice Skeletal Muscle

Abstract

This study investigated the impact of lifelong sedentariness on skeletal muscle mass and mitochondrial function. Thirty C57BL/6 strain mice (2 months) were randomly divided into three groups (young-Y; old sedentary-OS; old active-OA). Young animals were sacrificed after 1 week of quarantine, and OS and OA groups were individually placed into standard cages and in cages with running wheels, respectively, until sacrifice (25 months). Body weights and hind-limb skeletal muscle wet weights were obtained from all groups. Mitochondrial respiratory functional measures (i.e., state 3 and 4 respiration, respiratory control ratio, and ratio of nanomoles of ADP phosphorylated by nanomoles of O2 consumed [ADP/O]) and biochemical markers of oxidative damage (aconitase activity, protein carbonyl derivatives, sulfhydryl groups) were measured in isolated mitochondrial suspensions. Our results reveal that lifelong sedentary behavior has a negative impact on the age-related loss of skeletal muscle mass and on the isolated mitochondrial function of mixed skeletal muscle of mice, which is associated with an increased oxidative damage to mitochondrial biomolecules.

Keywords: Voluntary activity, Muscle aging, Sarcopenia, Running wheel, Oxidative stress

AGING diminishes both cellular number and function in all organ systems. One of the most adversely affected tissues with increasing age is skeletal muscle (15). Indeed, one of the hallmarks of aging is a decrease in both skeletal muscle fiber number and fiber cross sectional area, known as sarcopenia. Moreover, advanced age is associated with a progressive impairment in muscle fiber structure and contractile function (6). This age-related skeletal muscle weakness leads to a loss of independence, decreased quality of life, and an increased risk of morbidity due to falls (4,7). Intrinsic mechanisms that contribute to aging-induced sarcopenia include a depressed capacity for cellular repair, impaired gene expression, reduced protein synthesis, and compromised mitochondrial function (79). Extrinsic mechanisms to muscle fibers, such as the impairment of neuromuscular function and alterations in the endocrine milieu, may also contribute to sarcopenia (4,7).

Beyond the aforementioned intrinsic and extrinsic mechanisms, it is feasible that several behavioral factors influence sarcopenia and the age-related damage to skeletal muscle mitochondria (2,3,10,11). Of these factors, a sedentary lifestyle is potentially one of the most deleterious contributors to sarcopenia and skeletal muscle dysfunction (3,12). Because physical exercise provides numerous positive adaptations to skeletal muscle including mitochondrial biogenesis (1316), a lifelong program of regular exercise is widely recommended (1618). Although physical exercise may provide potential benefits to retard the age-related skeletal muscle dysfunction, the existing literature does not provide a clear picture of the physiological impact of a sedentary lifestyle versus a lifelong active lifestyle on skeletal muscle mass and mitochondrial function. Indeed, most published reports using animal models to investigate this important issue suffer from one or more shortcomings. For example, some studies have incorporated only short durations of exercise programs (1921), and other studies used forced exercise modalities such as treadmill running or swimming (19,20,2224). A major disadvantage of a forced exercise program is that this training paradigm imposes significant psychological stress on the animals that may promote deleterious adaptations (25,26). Clearly, a program of forced exercise does not mimic the normal intermittent exercise routine of rodents in the wilderness.

Indeed, free access to a running wheel is a more appropriate approach in a rodent model of lifelong exercise to satisfy the animals’ physiological requirements for voluntary activity. With a voluntary model of exercise, physical activity is not externally imposed and the animals can run intermittently and freely. This type of methodological approach has been associated with positive physiological adaptations in adult and aged animal models, namely augmented VO2 max. (25), cardiac hypertrophy (27), increases in skeletal muscle mitochondrial enzyme expression (27,28), decreases in the basal level of H2O2 production by isolated mitochondria from cardiac muscle (29), upregulation of telomere-stabilizing proteins, reduced cellular senescence and apoptotic cell death in cardiomyocytes (30), and a shift from IIb to IIa myosin heavy chain expression (27,28). In addition, this type of lifelong voluntary physical activity (LVPA) has been associated with increases in mean lifespan (3133). However, although several studies have examined the role of LVPA on survival rates, little is known about the effects of LVPA on aging of skeletal muscle; this void forms the basis for the current experiments.

Therefore, this study investigated the impact of a lifelong sedentary lifestyle on both skeletal muscle mass and mitochondrial function. To achieve this objective, two markedly different models of physical activity were compared in mice; the physical activity in one group of animals was restricted to the space of each cage whereas the second group of animals had free access to a running wheel for voluntary exercise. We tested the hypothesis that compared with voluntary active behavior; a sedentary lifestyle would have negative effects on skeletal muscle mass and mitochondrial function.

METHODS

Animals and Lifelong Interventions

Thirty C57BL/6 strain mice aged 2 months were randomly divided into three groups (young-Y; old sedentary-OS; old active-OA). One week after arrival in the laboratory, all Y animals (n = 10) were sacrificed whereas animals assigned to the OS (n = 10) and OA (n = 10) groups were individually placed into standard cages 355 × 235 × 190 mm (Ref. 2150E, Tecniplast, Italy) and in cages equipped with running wheels with 364 × 258 × 350 mm (Ref. 1284L0106, Tecniplast, Italy), respectively. Both OS and OA animals were sacrificed at an age of 25 months. The running wheels (25 cm in diameter) in the cages of the OA animals were equipped with a magnetic switch and a revolution counter to monitor the running distance for each animal throughout the duration of the experimental protocol.

Body weights of all mice were recorded weekly. Animals from both groups were maintained at constant environmental temperatures (21–25 °C) on a daily light schedule of 12 h of light versus dark until sacrifice. Mice were provided with food and water ad libitum. Housing and experimental treatment of animals were in accordance with the Guide for the Care and Use of Laboratory Animals from the Institute for Laboratory Animal Research. The local Ethics Committee had approved the study and the experiments were complied with the current national laws.

Animals Sacrifice, Skeletal Muscle Extraction, and Mitochondria Isolation

At the time of sacrifice (25 months), there were 8 and 10 animals in OS and OA group, respectively. Note that two animals from the OS group died during the experimental protocol (with 20 and 22 months of age, respectively) resulting in an n = 8 in this experimental group. The animals were sacrificed by cervical dislocation, and the hind-limb muscles (soleus, gastrocnemius, tibialis anterior, and quadriceps) were excised and weighed prior to mitochondria isolation. The muscles were cleared of fat and connective tissue, and wet muscle weight was determined as the difference between the weight of a given volume of cold isolation medium without the muscle sample and the weight of the same volume of cold isolation medium with the muscle sample. Afterwards, skeletal muscle mitochondria were isolated and prepared by conventional methods of differential centrifugation, as previously described by Tonkonogi and Sahlin (34). Briefly, muscles were immediately minced in ice-cold isolation medium containing 100 mM sucrose, 0.1 mM ethylene glycol tetraacetic acid, 50 mM Tris–HCl, 100 mM KCl, 1 mM KH2PO4, and 0.2% bovine serum albumin (BSA), pH 7.4. Minced blood-free tissue was rinsed and suspended in 10 ml of fresh medium containing 0.2 mg/mL bacterial proteinase (Nagarse E.C.3.4.21.62, type XXVII; Sigma, St Louis, MO) and stirred for 2 min. The sample was then carefully homogenized with a tightly fitted Potter-Elvehjen homogenizer and a Teflon pestle. After homogenization, three volumes of Nagarse-free isolation medium were added to the homogenate. After the extraction of 1 mL of this homogenate for biochemical assessment of citrate synthase (CS) activity and total protein content in skeletal muscle, the remaining homogenate was fractionated by centrifugation at 700 g for 10 min. The resulting pellet was removed, and the supernatant suspension was centrifuged at 10,000g during 10 min. The supernatant was decanted, and the pellet was gently resuspended in isolation medium (1.3 mL/100 mg initial tissue) and centrifuged at 7,000g for 3 min. The supernatant was discarded, and the final pellet, containing the mitochondrial fraction, was gently resuspended (0.4 μL/mg initial tissue) in a medium containing 225 mM mannitol, 75 mM sucrose, 10 mM Tris, and 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4. Total protein concentration in the mitochondrial suspension was estimated spectrophotometrically with the biuret method using BSA as standard. All mitochondrial isolation procedures were performed at 0–4 °C. The mitochondrial suspensions were used within 2 hours after the excision of the muscles and were maintained on ice (0–4 °C) throughout this period.

One aliquot from the final mitochondrial suspension was used for mitochondrial biochemical analysis at nonstimulated conditions. All the biochemical parameters were assessed in the whole mitochondrial suspension after treatment with 0.1% Triton X-100. Another aliquot was processed for morphological analysis. The remaining mitochondrial suspension was used for mitochondrial in vitro stimulation using traditional methods for measurements of the mitochondrial respiratory activity and a simulated exercise test with one, three, and six adenosine diphosphate (ADP) pulses interspersed by 1:30 minute each in which functional and biochemical data were obtained. The utilization of this in vitro test was an attempt to assess the capacity of mitochondria to reestablish their homeostatic balance between consecutive ADP stimulations as a function of time.

Biochemical Analysis in Skeletal Muscle Homogenate and Mitochondrial Fraction

Total protein determination.—

Total protein concentrations in skeletal muscle homogenate and in the mitochondrial suspension were determined using the protocol of Lowry and colleagues (35) and with the biuret method, respectively. In both assays, BSA served as the standard.

CS determination.—

CS activity was measured according to Coore and colleagues (36). In brief, the CoASH released from the reaction of acetyl-CoA with oxaloacetate was measured by its reaction with 5, 5¢-dithiobis-(2-nitrobenzoic acid) (DTNB) at 412 nm (molar extinction coefficient [ϵ] = 13.6 mM−1 × cm−1). CS activity was assessed in skeletal muscle homogenate and in the whole mitochondrial suspension after treatment with 0.1% Triton X-100.

Mitochondrial density (milligram/gram muscle wet weight) was estimated according to Kerner and colleagues (37). Briefly, the total CS activity in the whole skeletal muscle homogenate was initially calculated multiplying the CS activity per milligram of protein by the total amount of protein in the whole skeletal muscle homogenate, being further normalized to muscle wet weight (g). In order to estimate the mitochondrial density, the obtained value was divided by the CS specific activity assessed in isolated mitochondrial suspension. The recovery of mitochondria was measured as CS activity in the mitochondrial suspension relative to that in the skeletal muscle tissue homogenate.

Mitochondrial Preparation for Transmission Electron Microscopy

Mitochondrial preparation for transmission electron microscopy (TEM) analyses and further morphometric characterization has been previously described (38). Briefly, 100 μL of the mitochondrial suspension was centrifuged at 7,000g during 10 minutes, and the resulting pellet was fixed with 2.5% glutaraldehyde, post-fixed with 2% osmiumtetroxide, deyhdrated in graded alcohol, and embedded in LR White (Sigma, St Louis, MO). Ultra-thin sections mounted on copper grids (300 Mesh) were contrasted with uranyl acetate and lead citrate for transmission electron microscopy (Zeiss EM 10A) analysis. In order to obtain a global characterization of the pellet, several grids were prepared (five to eight grids per animal each containing three to four sections) from different zones ranging through the whole pellet.

Morphometric Analysis of the Mitochondrial Pellet

Morphometric analysis was performed as described in detail elsewhere (38) in at least 50 photos per mitochondrial pellet using a morphometric processing program (ImageJ, National Institutes of Health, Bethesda, MD). For each pellet, the mean number of mitochondria per micrometer and micrometer square was calculated from all analyzed micrographs, and their product was used to calculate the mitochondrial concentration in the pellet (number per cubic micrometer with further adjustment to the number per micro liter).

The mitochondrial concentration in each mitochondrial suspension was assessed as previously described (38) and used to normalize all functional and biochemical data.

Measurement of Mitochondrial Respiratory Activity

Traditional tests.—

After the determination of the total protein concentration in the mitochondrial suspension (estimated spectrophotometrically with the biuret method using BSA as standard), the mitochondrial respiratory function was polarographically measured using a Clark-type oxygen electrode (Hansatech DW 1, Norfork, UK). All assays were conducted in a 0.75-mL closed, magnetically stirred, and temperature controlled (25 °C) glass chamber. Mitochondria were added to the chamber (0.5 mg of protein) in a reaction buffer of 225 mM mannitol, 75 mM sucrose, 10 mM Tris, 10 mM KCl, 10 mM K2HPO4, and 0.1 mM EDTA, pH 7.5 (39). After a 1-minute equilibration period, mitochondrial respiration was initiated by adding pyruvate (5 mM) plus malate (2 mM) for Complex I–linked substrate assay or succinate (10 mM) plus rotenone (4 μM) for Complex II–linked substrate assay. State 3 respiration was determined after adding ADP to a final concentration of 200 μM; state 4 respiration was measured as the rate of oxygen consumption in the absence of ADP phosphorylation. The respiratory control ratio (RCR), that is, the ratio between state 3 and state 4 respiration, and ADP/O were calculated according to Estabrook (40), using 235 nmol O2/mL as the value for the solubility of oxygen at 25 °C. To quantify mitochondrial inner membrane permeability and the maximal rate of uncoupled oxidative phosphorylation, oligomycin (final concentration of 1.5 μg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; 2 μM), respectively, were sequentially added during ADP-stimulated (final concentration of 1 mM) state 3 respiration. The utilization of oligomycin avoids the permeability to protons through the ATP synthase creating an oligomycin-inhibited state 3 respiration; the comparison of oligomycin-inhibited state 3 respiration with a normal state 4 allows establishing the degree of mitochondrial inner membrane integrity. After obtaining the oligomycin-inhibited state 3 respiration, the addition of CCCP induces an uncoupling of the respiratory chain enabling to assess its maximal activity because the permeability in the presence of CCCP is always maximal (41).

Consecutive ADP-stimulation test.—

In order to mimic the in vivo metabolic stress imposed to mitochondria during muscular exercise, we performed a series of repeated and consecutive bouts of ADP-stimulated mitochondrial respiration using in a Clark-type oxygen electrode to determine oxygen consumption (Hansatech DW 1) following a previously described protocol (42). Briefly, this in vitro test consisted of four assays performed on each of the mitochondrial suspensions (Figure 1); assay I: after a 60-second equilibration period, mitochondrial respiration was initiated by adding pyruvate (5 mM) plus malate (2 mM) followed by 30-second stabilization; assay II: after a 60-second equilibration period, mitochondrial respiration was initiated by adding pyruvate (5 mM) plus malate (2 mM) followed by 30-second stabilization and the addition of one pulse of ADP followed by 90 seconds; assay III: after a 60-second equilibration period, mitochondrial respiration was initiated by adding pyruvate (5 mM) plus malate (2 mM) followed by 30-second stabilization and the addition of three consecutive pulses of ADP interspersed by 90 seconds each; assay IV: after a 60-second equilibration period, mitochondrial respiration was initiated by adding pyruvate (5 mM) plus malate (2 mM) followed by 30-second stabilization and the addition of six consecutive pulses of ADP interspersed by 90 seconds each.

Figure 1.
Consecutive ADP stimulation test. See text for detailed description.

In assays II, III, and IV, mitochondrial respiratory function was assessed by analyzing several functional parameters (i.e., state 3 and state 4 respiratory rates, the RCR, and ADP/O) in each of the ADP cycles. At the end of each of the four assays, the content of the oxygen chamber was collected and used for biochemical analysis (Figure 1). The content of the oxygen chamber at the end of assay I represent the mitochondrial basal condition (prestimulation); the content of the oxygen chamber after assays II, III, and IV correspond to different degrees of ADP stimulation. In all assays, a certain quantity of the oxygen chamber (500 μL) was used for the determination of protein carbonyl derivatives and total mitochondrial sulfhydryl (SH) groups. Furthermore, a portion of the oxygen chamber (250 μL) was centrifuged for 15 minutes at 10,000g in order to determine the content of extramitochondrial cytochrome c. Aconitase (ACON) activity was also quantified in assay I in order to indirectly assess the age-related superoxide radical generation at basal conditions.

ACON activity.—

The activity of ACON was assayed because ACON activity is redox sensitive, and diminished ACON activity has been interpreted as an index of superoxide radical generation and mitochondrial oxidative damage (43). ACON activity was measured spectrophotometrically by monitoring the formation of cis-aconitate at 240 nm after the addition of 20 mM isocitrate at 25 °C, according to Krebs and Holzach (44). One unit of activity was defined as the amount of enzyme necessary to produce 1 μM cis-aconitate/minute (ϵ at 240nm [ϵ240] = 3.6 mM−1 × cm−1].

Protein carbonyl derivatives.—

For the protein carbonyl derivatives assay, a certain volume of the oxygen chamber containing 20 μg of protein was derivatized with dinitrophenylhydrazine. Briefly, the sample was mixed with 1 volume of 12% sodium dodecyl sulfate plus 2 volume of 20 mM dinitrophenylhydrazine prepared in 10% trifluoroacetic acid, followed by 30 minutes of dark incubation, after which 1.5 volume of 2 M Tris/18.3% β-mercaptoethanol was added. A negative control was simultaneously prepared for each sample. After the derivatized proteins were diluted in Tris-buffered saline (TBS) to obtain a final concentration of 0.001 μg/μL, a 100-μL volume was slot blotted into a Hybond-polyvinylidene difluoride membrane. Immunodetection of carbonyls was then performed using rabbit polyclonal antidinitrophenyl (1:2000; V0401 DakoCytomation, Germany) as the primary antibody and anti-rabbit IgG peroxidase (Amersham Pharmacia Biotech, Buckinghamshire, UK) as the second antibody (1:2,000 dilution). The bands were visualized by treating the immunoblots with enhanced chemiluminescence (ECL) chemiluminescence reagents (Amersham Pharmacia Biotech), according to the supplier's instructions, followed by exposure to X-ray films (Sigma, Kodak Biomax Light Film, St. Louis, MO). The films were analyzed with QuantityOne Software version 4.3.1 (Bio-Rad, Hercules, CA).

Total mitochondrial SH groups.—

The oxidatively modified SH groups, including GSH and other SH-containing proteins, were quantified by spectrophotometric measurement according to the method proposed by Hu (45). Briefly, after 1-minute centrifugation at 1,000g, 50 μL of supernatant was added to a medium containing 150 μL of 0.25 M Tris, 790 μL of methanol, and 10 μL of 10 mM 5,5’-dithio-bis (2-nitrobenzoic acid). The colorimetric assay was performed at 414 nm against a blank test. Total SH content was calculated using the following molar extinction coefficient: ϵ414 = 13.6 mM−1 × cm−1.

Extramitochondrial cytochrome c.—

The content of extramitochondrial cytochrome c was determined in the supernatant after centrifugation and isolation of the mitochondrial pellet. Equal amounts of supernatant proteins were electrophoresed on a 15% SDS–polyacrylamide gel electrophoresis gel, followed by blotting on a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech). After blotting, nonspecific binding was blocked with 5% nonfat dry milk in TBS with Tween 20 and the membrane was incubated with anticytochrome c (1:1000; cat. number 556433; Pharmingen, San Diego, CA) antibody for 2 hours at room temperature, washed, and incubated with secondary horseradish peroxidase–conjugated anti-mouse (Amersham Pharmacia Biotech) for 2 hours. The membrane was then washed and developed with Western blotting chemiluminescence reagents (Amersham Pharmacia Biotech) according to manufacturer's instructions, followed by exposure to X-ray films (Sigma). The films were analyzed with QuantityOne Software (Bio-Rad).

Statistical Analysis

For all dependent variables, a one-way analysis of variance was used to analyze the differences between the three groups. When appropriate post hoc comparisons were determined with Scheffe test. The Statistical Package for the Social Sciences (SPSS version 10.0) was used for all analyses. Significance was taken as p <.05.

RESULTS

LVPA of OA animals are shown in Figure 2. Voluntary physical activity levels of the OA animals peaked at the fourth and fifth week of the experiment. Note that activity declined during the subsequent 10 weeks and reached a new plateau of activity at approximately week 16 with animals running approximately 5 km per day until the end of the experiment (Figure 2).

Figure 2.
Lifelong voluntary physical activity. Data points represent the daily average distance per day (km) (mean ± SD).

Animal body weights and skeletal muscle CS activity are reported in Table 1. As indicated from the skeletal muscle wet weight and muscle weight/body weight ratio, aging and inactivity were associated with a decline in skeletal muscle mass in OS animals. This age-related decline in skeletal muscle mass was attenuated by physical activity. Compared with both, the young and sedentary old animals, skeletal muscle CS activity was significantly higher in the old active animals. Interestingly, this CS activity in the old sedentary animals did not differ from the younger animals.

Table 1.
Body Weights, Skeletal Muscle Wet Weights, and CS Activity in Muscle Homogenate and in Isolated Mitochondria From Young, Old Sedentary, and Old Active Animals

Quantification of the number of mitochondria revealed a significantly higher number of mitochondria in the old sedentary animals compared with both the young and old active animals (Table 2). This finding is corroborated by the measurement of CS activity within the mitochondrial suspension (Table 1); however, the protein content in the mitochondrial suspensions was not significantly different among groups. Previously we have reported (38) that varying degrees of contamination exist in skeletal muscle mitochondrial isolation across animals of differing age groups. Therefore, to avoid this important confounding variable, we determined the number of mitochondria in the mitochondrial suspension of each animal in order to normalize all functional and biochemical data to the number of mitochondria in the assay sample.

Table 2.
Characterization of the Mitochondrial Suspensions Obtained From Young, Old Sedentary, and Old Active Animals

Lifelong Inactivity and Mitochondrial Functional Alterations

Traditional measurements of mitochondrial respiration.—

Skeletal muscle mitochondria were isolated with high integrity in all groups under study as documented by the RCR values (Table 3) and by their morphological appearance in the TEM analysis (data not shown).

Table 3.
Functional Data Obtained From Skeletal Muscle Mitochondria Isolated From Young (Y), Old Sedentary (OS), and Old Active (OA) Animals With Complex I–Linked Substrates

Functional data obtained from skeletal muscle mitochondria of all groups is shown in Tables 3 and and4.4. Compared with young animals, OA and OS animals evidenced a significant decline in state 3 respiration and RCR with both complex I– and II–linked substrates, being this reduction more pronounced in OS animals. To assess the mitochondrial phosphorylation efficiency, we calculated the ADP/O ratio normalized to the number of mitochondria (Table 3 and and4).4). Our results reveal that when expressed per mitochondria, ADP/O values are significantly diminished in the old sedentary animals compared with both the young and old active animals. Interestingly, compared with both the young and old sedentary animals, ADP/O ratios (complex I substrates) were significantly higher in the old active animals; this finding suggests that aging per se did not influence on mitochondrial phosphorylation efficiency.

Table 4.
Functional Data Obtained From Skeletal Muscle Mitochondria Isolated From Young (Y), Old Sedentary (OS), and Old Active (OA) Animals With Complex II–Linked Substrates (Succinate [10 mM] and Rotenone [4 μM])

Our results collectively indicate that the age-related functional impairment of skeletal muscle mitochondria is targeted at the state 3 respiratory rates and that a lifelong sedentary behavior exacerbates this age-related mitochondrial dysfunction. In order to confirm this data, we have performed an additional assay where we analyzed the oligomycin-inhibited state 3 respiration and CCCP-induced uncoupled respiration (Table 5).

Table 5.
Effect of Age and Lifelong Physical Activity on Oligomycin-Inhibited State 3 Respiration and CCCP-Induced Uncoupled Respiration in Skeletal Muscle Mitochondria Isolated From Young, Old Sedentary, and Old Active Animals

No significant differences existed between the experimental groups in the oligomycin-inhibitied state 3 respiration. In contrast, uncoupled respiration with CCCP was significantly diminished with age although the decrease in state CCCP respiration was greater in the old sedentary animals.

Consecutive ADP-stimulation test.—

Similar to the age-related changes in mitochondrial function observed with the traditional tests, the age and sedentary-related alterations in mitochondrial function were also present in the consecutive ADP-stimulation tests (Figure 3). Specifically, state 3 respiratory rate and RCR were acutely diminished in all groups with increasing number of trials. In contrast, state 4 respiration rate was significantly higher in the sixth ADP stimulation, when compared with data from the first and second ADP challenge, in all groups under investigation. As to the ADP/O ratio normalized to the number of mitochondria, no change occurred across the successive ADP stimulations.

Figure 3.
Functional data obtained from skeletal muscle mitochondria isolated from young, old sedentary, and old active animals with complex I–linked substrates, pyruvate (5 mM), and malate (2 mM), in the consecutive ADP stimulation test. Data are mean ...

A schematic representation of the mitochondrial oxygen consumption in the consecutive ADP-stimulation test (assay IV) is shown in Figure 4 for young, old sedentary, and old active animals. Note that isolated mitochondria from old sedentary animals were unable to reestablish baseline state 4 conditions after the fourth, fifth, and sixth ADP stimulation. In contrast, this functional impairment seems to be not that much apparent in young and in old active animals.

Figure 4.
Schematic representation of the assay IV (six ADP “cycles”) from the consecutive ADP-stimulation test in examples of one young, one old sedentary, and one old active animal.

Consecutive ADP-stimulation test.—

When stimulated with consecutive ADP additions, mitochondria from all groups exhibited a significant increase in protein carbonyls and extramitochondrial cytochrome c content. In contrast, consecutive ADP additions resulted in a decrease of the SH group levels in all groups under study (Figure 5). Note that these oxidative modifications of mitochondria were not present after a single ADP stimulation (assay II) in all groups under study (data not shown). This observation suggests that the repeated ADP stimulations resulted in increased reactive oxygen species (ROS) production as the test proceeded.

Figure 5.
Acute biochemical alterations induced by the consecutive ADP stimulation test in skeletal muscle mitochondria isolated from young, old sedentary, and old active animals. Prestimulation (pre-) represents basal conditions; post- (3 ADP) and post- (6 ADP) ...

Impact of Age and Physical Inactivity on Mitochondrial Oxidative Damage

To evaluate the impact of lifelong inactivity on the level of mitochondrial oxidative damage, we determined the activity and content of several oxidative stress biomarkers in mitochondria of young, old sedentary, and old active animals in basal conditions (assay I) (Table 6 and Figure 5 “prestimulation”).

Table 6.
Biochemical Data Obtained From Skeletal Muscle Mitochondria Isolated From Young, Old Sedentary, and Old Active Animals

Compared with both young and old active animals, ACON activity was significantly lower in the old sedentary animals. Moreover, compared with both young and old sedentary animals, mitochondrial from old active animals contained significantly higher ACON activity. We also observed an age-related increase in mitochondrial protein oxidation as indicated by the increased content of protein carbonyls; note that the age-related increase in protein oxidation was exacerbated in the old sedentary group. Similarly, mitochondria from sedentary animals contained a diminished level of SH groups when compared with both young and active old animals.

DISCUSSION

Overview of the Principal Findings

The main message of this study is that lifelong sedentary behavior accelerates both age-related sarcopenia and skeletal muscle mitochondrial dysfunction in mice. Moreover, our findings reveal that lifelong sedentary behavior increases the level of oxidative damage in skeletal muscle mitochondria. A detailed discussion of these major findings and a discussion of the experimental model follow.

Rationale Behind the Experimental Model

The goal of these experiments was to determine whether a lifetime sedentary behavior would exacerbate aged-related sarcopenia and skeletal muscle mitochondrial dysfunction. To achieve this objective, we studied two groups of animals; one group was confined to a small cage for 23 months (i.e., old sedentary), and the second group was housed with free access to a running wheel (i.e., old active). We reason that the activity pattern of the old active animals resembles the normal activity of animals in the wild and therefore these animals served as our control group (i.e., normal activity). In contrast, our sedentary old animals were housed in an environment that fostered an abnormal activity pattern of sedentary behavior and therefore, we consider these animals to be the experimental group (i.e., forced sedentary activity). In this context, we believe that the findings obtained from the normal activity group represents the aging process per se, whereas the results obtained from the sedentary animals represent the impact of sedentary behavior on the aging process.

In addition, consistent with previous findings (38), we also observed varying levels of impurity among the mitochondrial pellets (data not shown) indicating contamination of the mitochondrial suspensions. Specifically, we performed morphological analyses of the isolated mitochondria (data not shown) and observed varying degrees of mitochondria-like membranous debris in samples with large variability between both experimental groups and among animals within the same group. Further, our mitochondrial suspensions reveal a significantly higher yield of intact mitochondria in the old sedentary animals (Table 2) that agrees with the CS activity measured within the mitochondrial fraction and with the mitochondrial recovery data (Table 1). Note, however, the protein content in the mitochondrial suspensions did not differ between groups. Therefore, normalizing our mitochondrial functional measures to total protein content, as typically done in the literature, could bias our experimental results. In this context, although CS activity is a better marker of mitochondrial mass than the total protein content (38), the normalization of our results to the CS activity also has limitations because the mitochondrial suspensions can be contaminated with extramitochondrial CS resulting from damage to mitochondria during the isolation procedure. Therefore, given these important methodological considerations, our discussion will focus on the mitochondrial respiratory data normalized to the number of mitochondria.

Lifelong Sedentariness has a Major Impact on Sarcopenia

To compare skeletal muscle mass among our three experimental groups that differed in body mass, we used the ratio of skeletal muscle mass to body mass. Our findings reveal that sedentary behavior exacerbates age-related sarcopenia in mice as indicated by the observation that the skeletal muscle/body mass ratio was significantly greater in the old active animals compared with that of old sedentary animals (Table 1). This conclusion is further supported by the finding that no differences existed in the skeletal muscle mass/body mass ratio between old active animals and the young animals. Collectively, these data support the concept that sedentary behavior plays a major role in the etiology of sarcopenia and that long-term prevention strategies to retard age-induced sarcopenia should include a lifelong program of physical activity (46).

Lifelong Sedentary Behavior Exacerbates Age-Related Functional Impairments of Mitochondria

Traditionally, mitochondrial function has been evaluated by the assessment of oxygen consumption during a single measurement of both state 3 and state 4 respiration. Using this approach, several studies have reported that aging is associated with diminished mitochondrial function (38,47,48). Similarly, compared with young animals, our results demonstrate an age-related impairment in mitochondrial function in both the old active and sedentary animals as documented by a decrease in state 3 respiratory rate with both complex I– and complex II–linked substrates. When comparing the state 3 respiratory rates between complex I– and complex II–linked substrate assays, we observed a similar functional impairment for both the OS and OA animals. These results indicate that both complex I– and complex II–supported respiration were similarly influenced by increasing age and further exacerbated by sedentary lifestyles, as documented by the higher mitochondrial functional impairment in OS animals in both complex I– and complex II–linked substrates. These results agree with previous findings suggesting that age-related mitochondrial functional impairment might be explained by decreased activities of complex I, III, and IV of mitochondrial electron transport chain, caused by oxidative lesions of mitochondrial DNA that encode these subunits (49). Our finding that the mitochondrial functional impairments are greatest in the old sedentary group (OS) compared with the old active animals (OA) suggests that lifelong inactivity has a negative impact on mitochondrial function. Notably, we postulate that the age-related decline in state 3 mitochondrial respiration is age and inactivity related and not due to confounding variables (e.g.,, membrane damage during isolation) because no differences in state 4 respiration existed between the OA and OS groups. This argument is also supported by the finding that the oligomycin-inhibited state 3 respiration does not differ between groups indicating that the inner mitochondrial membrane integrity was not affected by age, inactivity, or by the mitochondrial isolation procedures. In support of this position, Navarro and Boveris (50) have recently concluded that there is no clear evidence that inner mitochondrial membrane permeability is altered with age.

As an extension beyond traditional mitochondrial function testing, our experiments included a new and innovative approach for testing mitochondrial function using a series of consecutive ADP-stimulation tests. This in vitro test was an attempt to assess the capacity of mitochondria to reestablish their homeostatic balance between consecutive ADP stimulations (e.g., simulated bouts of exercise). We hypothesized that, compared with young animals, mitochondria from old animals would have a lower capacity to maintain homeostasis when challenged with repeated ADP stimulations. Moreover, we predicted that lifelong inactivity would exacerbate this mitochondrial functional impairment in old animals. Our results support this postulate as we observed an age-associated decline in the maximal mitochondrial respiratory capacity as documented by the lower state 3 respiratory rates and RCR in both the old sedentary group and old active group, when compared with the young animals. Moreover, old sedentary animals demonstrated a greater impairment of mitochondrial function when compared with old active animals. Although the pattern of functional decline across ADP stimulation trials was similar in all groups (Figure 3), mitochondria from old sedentary animals exhibited significant functional impairments during each ADP stimulation cycle (i.e., lower state 3 respiratory rate, RCR, and ADP/O ratios). Finally, state 4 respiratory rates were increased significantly in all experimental groups during the repetitive ADP trials, indicating an increase in mitochondrial respiratory uncoupling.

Our results suggest that mitochondria from younger and old active animals were able to phosphorylate all the ADP added in each of the repetitive ADP “cycles” (Figure 4). In contrast, mitochondria isolated from old sedentary animals were unable to respond similarly; in fact, it was evident in all the experiments (data not shown) that from the fourth ADP cycle onwards, mitochondria from the old sedentary animals were unable to phosphorylate all the ADP added, suggesting that ATP homeostasis could not be maintained in response to this energetic challenge. This finding could be due to a decreased ADP phosphorylation capacity at the level of electron transport and/or proton pumping, thereby diminishing the proton electrochemical gradient and compromising the generation of ATP. Ultimately, the ADP/O, normalized to the number of mitochondria was not altered between the ADP cycles in all groups studied, suggesting that the efficiency of ADP phosphorylation was not acutely changed.

Inactivity Increases Mitochondrial Oxidative Damage in Skeletal Muscle

Another important finding of this study is that age-related increases in mitochondrial oxidative damage are exacerbated by a lifelong of sedentary behavior. Indeed, our data reveal increased levels of oxidative damage in skeletal muscle mitochondria isolated from old sedentary animals, documented by the diminished ACON activity, significantly lower SH groups, and higher levels of protein carbonyls. Mitochondria isolated from old active animals also contained increased levels of oxidative damage, but the levels were lower than the old sedentary animals. Collectively this data suggests that aging per se does contribute to increased mitochondrial oxidative damage and that sedentary behavior exacerbates this problem.

It is feasible that oxidative damage to mitochondria may account for, at least in part, the age- and inactivity-related decline in mitochondria function. For example, our findings indicate that lifelong inactivity results in diminished ACON activity, which is likely linked to an oxidative-induced downregulation of enzyme activity. Indeed, this enzyme is particularly sensitive to a reaction with superoxide that damages the iron–sulfur clusters [4Fe–4S] in its active site (51). This increased oxidative damage to this mitochondrial protein could diminish tricarboxylic acid cycle flux leading to decreased electron flow within the respiratory chain and therefore decreased oxidative phosphorylation (52).

Previous studies have reported an age-related decline in ACON activity in skeletal muscle mitochondria (52). Interestingly, our results reveal that this age-related decline in ACON activity is eliminated by a lifetime voluntary activity. Our data do not disclose the mechanism behind the ability of physical exercise to prevent an age-related decline in ACON activity. However, a reduction in mitochondrial H2O2 production after exercise training has been reported in skeletal muscle (53) and Judge and colleagues (29) found that H2O2 production in cardiac subsarcolemmal and interfibrillar mitochondria is reduced in rats subjected to lifelong physical activity. Therefore, the commonly observed age-related decline in ACON activity in skeletal muscle may not due to aging per se but may result from a lifetime of inactivity. A potential explanation for this postulate is that skeletal muscle inactivity is associated with higher levels of mitochondrial ROS production and therefore increased oxidative damage to mitochondrial proteins such as ACON (54).

When assessing the basal levels of mitochondrial oxidative damage, our results confirm the existence of an age-related increase in mitochondrial oxidative damage (Table 6), and these findings agree with published reports (51,52,5557). In fact, the present study clearly demonstrates an age-related increase in mitochondrial protein carbonyls, and this increase is exacerbated in the old sedentary group. Moreover, old sedentary animals exhibit a diminished content of SH groups when compared with both the younger and old active animals.

Note that the repetitive ADP-stimulation test (six ADP stimulations) resulted in a significant increase in the release of cytochrome c from the mitochondria in all groups. This finding suggests that this rigorous challenge increased mitochondrial outer membrane permeability. Importantly, the magnitude of cytochrome c release following our repetitive ADP challenges was consistently higher in mitochondria obtained from the old animals compared with the young. Furthermore, when comparing the old active animals versus the old sedentary at resting conditions, cytochrome c release was already significantly higher in mitochondria from the old sedentary animals. Also, note that an increase in mitochondrial protein oxidation occurred with the repetitive ADP-stimulations in all experimental groups. This increase in protein oxidation is particularly evident in the older animals and further exacerbated in the sedentary animals and could be due to a (i) higher production of ROS resulting from the increase in mitochondrial respiration or (ii) an impaired antioxidant capacity. Similar conclusions have been reached by McArdle and colleagues (58).

Summary and Future Directions

In summary, our study demonstrates that lifelong sedentary behavior has a major negative impact on the age-related changes of mice skeletal muscle mitochondrial respiration, namely at the level of the complex I and complex II and perhaps additional impairment in the function of the ATP synthase complex. We postulate that this mitochondrial dysfunction may be associated with an age-dependent and sedentary behavior–dependent increased oxidative damage to the mitochondrial biomolecules. Note, however, that our results only provide evidence of an age- and sedentary-related dysfunction of isolated mitochondria obtained from mixed skeletal muscle fiber types of mice, as our final mitochondrial suspensions were obtained from differing muscle phenotypes containing different mitochondrial characteristics (9,5961). Therefore, given the well described variability of age-related alterations among skeletal muscles and fiber types (5,62,63) the extrapolation of our results to a specific muscle or fiber type must be done with caution. Additionally, despite being potentially applied to other species, the extrapolation of our results to other type of animals should be done with prudence due to the variability of the aging process between species.

Future experiments should address the biochemical mechanisms responsible for the potential of regular physical exercise to prevent age-related impairments in skeletal muscle mitochondrial function. Finally, based upon our results, it can be argued that many of the age-related events that occur in skeletal muscle mitochondria of rodents housed in small cages without assess to running wheels may be due to inactivity and not to the aging process per se.

FUNDING

This work was supported by a grant by Fundação para a Ciência e Tecnologia (PTDC/10DES/70757/2006). P.A.F. is supported by a grant of Programa Operacional Ciência e Inovação 2010 and Fundo Social Europeu.

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