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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:350-359 (2008)
© 2008 The Gerontological Society of America

Age-Induced Morphological, Biochemical, and Functional Alterations in Isolated Mitochondria From Murine Skeletal Muscle

Pedro A. Figueiredo, Rita M. Ferreira, Hans Joachim Appell and José Alberto Duarte

1 CIAFEL, Faculty of Sports, University of Porto, Portugal.
2 Department of Physiology and Anatomy, German Sport University, Cologne, Germany.

Address correspondence to Pedro Alexandre Figueiredo, MSc, CIAFEL, University of Porto, Rua Dr. Plácido Costa, 91, 4200-450 Porto, Portugal. E-mail: pfigueiredo{at}ismai.pt


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Several in vitro studies about age-associated skeletal muscle mitochondrial dysfunction are somewhat conflicting, and this might be related to different normalization procedures. The objective of this study was to normalize the functional and biochemical data per number of mitochondria present in a mitochondrial suspension. Functional and biochemical parameters were obtained in mitochondrial suspensions from murine skeletal muscle of different ages. Mitochondrial respiratory function was polarographically measured using a Clark-type oxygen electrode. Biochemical analyses included determination of citrate synthase (CS) activity and total protein content in the mitochondrial suspension. Electron microscopy analysis of the suspensions allowed calculation of the number of mitochondria per milligram of protein. Our results conclude that advanced age is associated with mitochondrial dysfunction; moreover, from the correlation between morphological and biochemical data, it is evident that CS activity in the mitochondrial suspensions is a more accurate marker of mitochondrial mass than is total protein content.

Key Words: Aging • Citrate synthase • Respiratory function

AGING is characterized by a diminished functionality of organs, systems, and tissues of an individual, with a reduced capacity to withstand internal and/or external stimuli and therefore with an increased susceptibility to disease and death (1–3). At the cellular level, aging is also associated with an overall increased dysfunction, ranging from energetic deficits (2,4) to impaired stress tolerance (5,6). This statement suggests that the phenomenon of aging occurs at various levels of organization, from cells to tissues or complete organs. In this context, and regarding skeletal muscle, morphological degenerative alterations of mitochondria have been strongly associated with advancing age (7,8). These age-related alterations could be interpreted as a consequence or, as suggested by others (9,10), as the main cause for the loss of the overall cellular functionality, with an increased susceptibility to damage and death, that parallels chronological age (11–13). The supporters of the mitochondrial theory of aging state that age-related oxidative damage to mitochondria will result in a progressive reduction of mitochondrial bioenergetic capacity, leading to cellular energy deficits and compromising overall cellular functionality (7,10,13,14). However, despite the existence of morphological alterations, data in the literature about age-related mitochondrial dysfunction are somewhat uncertain, not only considering the maximal adenosine 5'-triphosphate (ATP) production rate but also when taking into account the activity of the complexes of the respiratory chain; whereas several authors have reported age-related decreases in mitochondrial function, both in humans (15) and animals (16), others (17,18) have failed to demonstrate that. In fact, some studies argue that mitochondrial capabilities in skeletal muscle are not affected by chronological age and exclude the possibility of a substantial or even moderate decrease in mitochondrial activities concerning the central bioenergetic reactions (18).

In this context, differences in (a) experimental design, (b) number of groups and age comparisons, (c) physical activity levels, and (d) mitochondrial populations are the main factors usually reported in the literature (15,19) to explain these inconclusive results among studies. Nevertheless, it is important to highlight that the methodological errors inherent to the process of tissue preparation and mitochondrial extraction for in vitro studies (18) might also bias the results. In fact, isolation of intact mitochondria from skeletal muscle presents several limitations, because the mitochondria are scarce and surrounded by the myofibrillar structures (20). The homogenization procedure, one of the most important steps in the process of isolation of mitochondria from other components (17), may leave some nonmitochondrial proteins in the mitochondrial suspension. Indeed, a major reason for this lack of consensus regarding the age-related mitochondrial dysfunction could be the assumption of some authors that the mitochondrial suspensions obtained from young and aged muscles contained equivalent amounts of nonmitochondrial protein and thereby similar degrees of contamination. For instance, in aged skeletal muscle, collagen is a likely candidate for excess contamination, as collagen is known to be more abundant in old muscles (21). Taking into account that the mitochondrial biochemical and functional status is frequently reported per total protein content in suspension, different degrees of contamination with nonmitochondrial proteins will bias the specific activities of isolated mitochondria and compromise the comparisons between various age groups (17).

Bearing this in mind, reference enzymatic activities have been used to normalize enzymatic and functional activities of mitochondria (22,23). Citrate synthase (CS) activity has been widely used as a marker of mitochondrial mass based on the supposition that, being located in the matrix, this enzyme activity would be little affected by the homogenization procedure in intact mitochondria (17). However, despite the fact that the data in the literature assume that changes in mitochondrial content parallel changes in enzyme maximum velocity (Vmax) (24), there is to our knowledge no morphological study that has validated the utilization of a matrix enzyme, such as CS, as a marker for the morphologically preserved mitochondrial content assessed by transmission electron microscopy (TEM) examination.

Following the above-mentioned considerations, the aim of this study was to estimate the amount of mitochondria in mitochondrial suspensions obtained from young and mature animals. Moreover, expecting different mitochondrial concentrations, we intended as well to correlate the protein content, the concentration of mitochondria, and CS activity to verify which one of the two biochemical parameters (protein content vs CS activity) is the more representative value of the total mitochondrial content in the suspension. A further objective of this study was to perform a functional characterization of skeletal muscle mitochondria normalizing the data to the calculated number of mitochondria used to perform the functional assessment. We hypothesized that, when compared to protein content, CS activity should constitute a better marker of mitochondrial mass in the suspension, thus permitting an augmented accuracy of the results among different groups. Moreover, we also expected that skeletal muscle mitochondrial function was diminished with increased age when data is expressed per number of mitochondria.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Experimental Design
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 (ILAR, 1996). Sixteen male C57BL/6 mice were divided into two groups according to their ages: young group (YG, 3 months old; n = 8) and mature group (MG, 18 months old; n = 8). All animals were kept at constant temperature (21°C–25°C) on a 12-hour light/dark cycle with normal activity until death. Mice were provided with food and water ad libitum and were killed after 1 week of quarantine. The local Ethics Committee had approved the study.

Skeletal Muscle Extraction and Mitochondria Isolation
The animals were killed by cervical dislocation, and the hind-limb muscles (soleus, gastrocnemius, tibialis anterior, and quadriceps) were excised for preparation of isolated mitochondria. Skeletal muscle mitochondria were prepared by conventional methods of differential centrifugation, as previously described by Tonkonogi and Sahlin (25). Briefly, muscles were immediately minced in ice-cold isolation medium containing 100 mM sucrose, 0.1 mM EGTA, 50 mM Tris/HCl, 100 mM KCl, 1 mM KH2PO4, and 0.2% bovine serum albumin, pH 7.4. Minced blood-free tissue was rinsed and suspended in 10 mL of fresh medium containing bacterial proteinase (Nagarse E.C.3.4.21.62, type XXVII; Sigma, St. Louis, MO) at 0.2 mg/mL and stirred for 2 minutes. The sample was then carefully homogenized with a tightly fitted Potter–Elvehjem 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 CS activity and total protein content in skeletal muscle, the remaining homogenate was fractionated by centrifugation at 700 g for 10 minutes. The resulting pellet was removed, and the supernatant suspension was centrifuged at 10,000 g for 10 minutes. The supernatant was decanted, and the pellet was gently resuspended in isolation medium (1.3 mL/100 mg initial tissue) and centrifuged at 7000 g for 3 minutes. The supernatant was discarded, and the pellet, containing the mitochondrial fraction, was carefully resuspended (0.4 µL/mg initial tissue) in a medium containing 225 mM mannitol, 75 mM sucrose, 10 mM Tris, and 0.1 mM EDTA, pH 7.4. All mitochondrial isolation procedures were performed at 0°C–4°C. The mitochondrial suspensions were used within 2 hours after the excision of the muscles and were maintained on ice (0°C–4°C) throughout this period.

One aliquot from the mitochondrial suspension was used for biochemical analysis. The remaining mitochondrial suspension was processed for measurement of mitochondrial respiratory activity and for morphological analysis.

Biochemical Analysis in Skeletal Muscle Homogenate and Mitochondrial Fraction
Total protein determination.-- Total protein concentration in skeletal muscle homogenate and in the mitochondrial suspension was spectrophotometrically estimated according to Lowry and colleagues (26) and with the biuret method, respectively, using bovine serum albumin as standard.

CS determination.-- CS activity was measured according to Coore and colleagues (27) by spectrophotometric (412 nm) measurement of the amount of 5,5-dithiobis (2-nitrobenzoate) that reacted with acetyl-coenzyme A (CoA) upon release from the reaction of acetyl-CoA with oxaloacetate. CS activity was assessed in skeletal muscle homogenate and in the whole mitochondrial suspension after treatment with 0.1% Triton X-100.

Mitochondrial density (mg/g muscle wet wt) was estimated according to Kerner and colleagues (28) with the division of CS activity of skeletal muscle homogenate (U/g wet wt) by the CS specific activity in isolated mitochondrial suspension (U/mg mitochondrial protein). The recovery of mitochondria was measured as CS activity in the mitochondrial suspension relative to that in the skeletal muscle tissue homogenate.

Measurement of Mitochondrial Respiratory Activity
Mitochondrial respiratory function was polarographically measured using a Clark-type oxygen electrode (Hansatech DW 1; Norfork, U.K.). All assays were conducted in a 0.75-mL closed thermostated (25°C) and magnetically stirred glass chamber containing 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, in accordance with Tonkonogi and colleagues (29). After a 1-minute equilibration period, mitochondrial respiration was initiated by adding pyruvate (5 mM) plus malate (2 mM) or succinate (10 mM) plus rotenone (4 µM). State 3 respiration was determined after adding adenosine diphosphate (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 RCR, i.e., the ratio between state 3 and state 4 respiration, and ADP/O were calculated according to Estabrook (30), 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 added during state 3 respiration with saturated amounts of ADP (final concentration of 1 mM).

Mitochondrial Preparation for TEM
For morphological and morphometric characterization, 100 µL of the mitochondrial suspension was centrifuged at 7000 g for 10 minutes, and the resulting pellet was fixed with 2.5% glutaraldehyde, postfixed with 2% osmium tetroxide, dehydrated in graded alcohol, and embedded in LR White. Ultrathin sections mounted on copper grids (300 mesh) were contrasted with uranyl acetate and lead citrate for TEM (Zeiss EM 10A; Carl Zeiss, Oberkochen, Germany) analysis. To obtain a global characterization of the pellet, several grids were prepared (5–8 grids per animal, each one having 3–4 sectioning cuts) from different zones ranging the whole pellet.

Morphometric Analysis of the Mitochondrial Pellet
Morphometric analysis was performed in at least 50 photos per mitochondrial pellet using a morphometric processing program (ImageJ; NIH Image). In each photograph, the areas of mitochondria, their number per micrometer and square micrometer were determined. Taking into account the magnification of each micrograph, the number of mitochondria per micrometer was assessed by counting the mitochondria that overlie four lines that crossed the center of the micrograph, traced horizontally (n = 1), vertically (n = 1), and obliquely (n = 2) (Figure 1A), being the final number of mitochondria per micrometer established as the mean value of the four counts; the number of mitochondria per square micrometer was evaluated by counting the total mitochondria present in each micrograph. For each pellet, the mean number of mitochondria per micrometer and square micrometer 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 milliliter).


Figure 01
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  		Figure 1. Methodological procedures to quantify the mitochondrial concentration in each suspension. A, Number of mitochondria per micrometer was assessed by counting the mitochondria that underlie the four lines crossing the center of the micrograph; the final number of mitochondria per micrometer was established as the mean value of the four counts. The number of mitochondria per square micrometer was evaluated by counting the total mitochondria present in each micrograph. B, Assessment of the correction factor for centrifugation-induced compaction, to adjust the data drawn from the microscopic evaluation of the pellet to the real volume of mitochondrial suspension

 
Afterward, to estimate the mitochondrial concentration in the mitochondrial suspension from the data obtained in the pellet, we assessed the degree of compaction induced by the centrifugation procedure using the following method: From each suspension, a small quantity (25 µL) was introduced in a glass capillary tube the tip of which was then closed. Subsequently, the sample was centrifuged at 7000 g for 10 minutes and the pellet length was measured (mm, with a 0.1 mm-graded ruler) and divided by the length of the glass capillary tube occupied by the 25 µL of mitochondrial suspension (Figure 1B). This ratio, representing the correction factor necessary to expand each pellet to the length occupied by 25 µL of mitochondrial suspension, was multiplied by the respective mitochondrial concentration calculated from the TEM analysis, to estimate the real number of mitochondria per volume of suspension.

Statistical Analysis
Means and standard deviations were calculated for all variables in both groups. Independent samples t test was used to analyze the differences in the variables between groups. The Spearmen correlation coefficient was used to analyze the correlations between total protein determination, CS activity, and concentration of mitochondria using simultaneously the data from all animals. The Statistical Package for the Social Sciences (SPSS version 10.0; Chicago, IL) was used for all analyses. Significance was taken as p ≤.05.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Body weights and skeletal muscle CS activity are shown in Table 1. A reduction in skeletal muscle CS activity is apparent in the mature animals, suggesting an impaired skeletal muscle oxidative capacity and mitochondrial mass in the older animals. Likewise, mitochondrial density (mg mitochondrial protein/g skeletal muscle wet wt) was significantly lower in the mature animals.


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

 
Skeletal muscle mitochondria were isolated with high levels of structural integrity, both in the young and mature animals as documented by the RCR values (Table 3) and by their morphological appearance in the electron microscopy analysis. When analyzing our mitochondrial suspensions, we found that the morphometric results shown in Table 2 clearly indicate a higher number of these organelles in the suspension of the mature animals, which is in agreement with the CS activity of isolated mitochondria and with the mitochondrial recovery data in both groups (Table 1). Despite the differences in mitochondrial concentration, we did not find significant differences in the mitochondrial areas between the young and the mature animals (YG = 0.16 ± 0.12 µm2 and MG = 0.11 ± 0.09 µm2). The qualitative analysis of the electron micrographs revealed the existence of nonmitochondrial material in both groups (Figure 2) suggesting some degree of contamination in the mitochondrial fractions, which was more pronounced in the younger animals. Despite the differences observed in TEM analysis, the total protein content in the mitochondrial suspension was not significantly different between the groups (Table 2).


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  		Table 3. Functional Data Obtained From Skeletal Muscle Mitochondria Isolated From Young and Mature Animals With Complex I–Linked Substrates, Pyruvate (5 mM), and Malate (2 mM).

 

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  		Table 2. Characterization of the Mitochondrial Suspensions Obtained From Both Age Groups.

 

Figure 02
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  		Figure 2. Electron micrographs of mitochondrial pellets obtained from skeletal muscle of young (A and C) and mature animals (B and D). A and C, Note that mitochondria from young animals present a normal structure with high variability in their shape and dimensions. Their cristae are numerous, clearly visible, and well defined. In the intermitochondrial space, several membrane-like cellular debris as well as various kinds of grouped filamentous structures are visible, suggestive for myofibril-like structures. B and D, Mitochondria from mature animals also present a high variability of dimensions; however, their shape appears more constant and regular, but their cristae are less apparent and not well defined. The intermitochondrial space also reveals the existence of some membranous and filamentlike cellular debris

 
Concerning the correlation between the protein content in the mitochondrial fraction, CS activity of isolated mitochondria, and the morphometric data, strong correlations were found between CS activity and the number of mitochondria (r = 0.815; p <.05) and a somewhat lower, yet significant correlation was obvious between protein content and CS activity (r = 0.608; p <.05) as well as between protein content and the number of mitochondria (r = 0.324; p <.05).

Characterization of Skeletal Muscle Mitochondria
Biochemical data concerning CS activity normalized to the number of mitochondria is presented in Figure 3. We found a significantly diminished CS activity in the mitochondria isolated from the mature animals when data was expressed per mitochondrium, suggesting that isolated mitochondria from mature animals present a reduced oxidative capacity when compared to their younger counterparts.


Figure 03
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  		Figure 3. Effect of age on citrate synthase activity when expressed per mitochondrium. Data are mean ± standard deviation and are expressed as nmoL/min/mitochondria. Filled bar: young animals; open bar: mature animals. *Significantly different from young animals (p <.05)

 
Functional data obtained from the isolated mitochondria of young and mature animals, namely the state 3 and state 4 respiratory rates as well as the RCR and the ADP/O values are shown in Tables 3 and 4.


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  		Table 4. Functional Data Obtained From Skeletal Muscle Mitochondria Isolated From Young and Mature Animals With Complex II–Linked Substrates, Succinate (10 mM), and Rotenone (4 µM).

 
It appears obvious that mature animals evidence a significant impairment in the respiratory rates of isolated skeletal muscle mitochondria energized with malate + pyruvate (M+P) and succinate + rotenone (S+R), irrespective of whether data are expressed per protein content, per CS activity, or per mitochondrium. This diminished state 3 respiration in the mature animals is somewhat evident with the complex I–linked substrates (–57.68% and –52.20% variation with complex I substrates and complex II substrates, respectively, with data normalized to CS activity). In contrast, state 4 respiration has not been significantly affected by increased age. The RCR values decreased significantly in the older animals, when using both M+P and S+R as respiratory substrates. On the contrary, the ratio of ADP to oxygen consumption showed no alterations with age. Moreover, the RCR data, as well as the state 3 and state 4 respiratory rates with M+P and S+R, suggest that the functional impairment is mainly targeted at the state 3 respiratory rates. These data are further supported by the results of the oligomycin-inhibited state 3 respiration and CCCP-induced uncoupled respiration, shown in Figure 4.


Figure 04
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  		Figure 4. Effect of age on oligomycin-inhibited state 3 respiration (A–C) and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced uncoupled respiration (D–F) in skeletal muscle mitochondria isolated from young and mature animals. Data are mean ± standard deviation and are expressed as nmol O2 consumed/min/mg of protein (A and D), as nmol O2 consumed/CS activity (B and E), and as nmol O2 consumed/min/mitochondria (C and F). Respiration was induced with pyruvate (5 mM) and malate (2 mM) as energizing substrates and saturated ADP concentration (1 mM) to initiate state 3 respiration. State 3 was inhibited after the addition of oligomycin (final concentration 1.5 µg/mL) and CCCP (final concentration 2 µM) to uncouple mitochondrial respiration. Filled bars: young animals; open bars: mature animals. *Significantly different from young animals (p <.05)

 
In fact, mitochondria from the mature animals energized with M+P and stimulated with excess ADP showed no significant alterations of the respiratory rate in the presence of oligomycin (state 4) when compared with their younger counterparts. In contrast, uncoupled mitochondrial respiration with CCCP was significantly diminished in the mature animals.


   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
The main findings of the present study clearly indicate that mitochondria isolated from skeletal muscle of mature mice present an altered morphological, biochemical, and functional status when compared with younger animals. The TEM analysis of the mitochondrial pellets revealed distinct degrees of impurity in both groups—the young and the mature animals—throughout the complete pellet (Figure 2). In fact, different degrees of nonmitochondrial material were evident in the mitochondrial pellets of both groups, which could lead to an overestimation of the presumed mitochondrial protein determination. Micrographs obtained from the mitochondrial pellets of the young animals (Figure 2) presented some myofibril-like structures and other nonmitochondrial material between the mitochondria. In contrast, the existence of nonmitochondrial material in the micrographs obtained from the older animals was not so evident. In this context, despite the fact that collagen fibers have been identified as potential candidates for an excess contamination of the skeletal muscle mitochondrial suspensions drawn from older animals (21), the analysis of the TEM micrographs did not substantiate this assumption. Moreover, our morphometric results showed clearly that, for the same amount of total protein, the mitochondrial suspensions originated from the mature animals had a higher number of mitochondria when compared with the mitochondrial suspensions from the younger animals (Table 2). These results are supported by the higher mitochondrial recovery data in the mature animals (19.65 ± 5.38% and 48.97 ± 9.05% for young and mature animals, respectively) when compared with their younger counterparts. In fact, it has been suggested that the abundance of myofibrillar proteins may complicate the extraction of mitochondria from skeletal muscle and thereby reduce the mitochondrial yield (31), which could explain the lower mitochondrial yields observed in the younger animals. In contrast, the higher number of mitochondria in the suspensions obtained from the mature animals could not be explained by differences in the sedimentation coefficients of the mitochondria, because it had been demonstrated that the ranges of particle size and density are essentially unchanged with age (18). Although it is claimed that mitochondria are somewhat bigger in aged tissues (32), our results failed to demonstrate such when considering the mitochondrial areas. Moreover, the determination of the protein content in the mitochondrial suspensions did not reveal any significant differences between the groups, which is in contrast with the morphometric results and the mitochondrial CS activity data, but is consistent with the idea of some degree of contamination in the suspensions. The higher content of mitochondria in the suspension obtained from the mature animals was confirmed by a correspondingly higher CS activity and supports the utilization of this enzyme as a marker of mitochondrial mass. In fact, it is suggested that CS activity, being located in the matrix, would be little affected by the homogenization procedure in intact mitochondria, and it can therefore be assumed as a good choice for a reference activity in mitochondrial assays (17). To our knowledge, this study is the first to demonstrate, with the utilization of TEM analysis, that when compared with the total protein content, CS activity is a better marker for the mitochondrial fraction in suspension obtained from skeletal muscle tissue. This issue could be particularly important when comparatively analyzing different groups (e.g., young vs old, trained vs untrained).

The correlation coefficients between the CS activity and the morphometric data indicate statistically significant and relevant correlation coefficients between the mitochondrial CS activity and the number of mitochondria, which reinforces the utilization of CS activity as a marker of mitochondrial content. Likewise, we have also found significant correlation coefficients, although lower, between protein content and CS activity, as well as between protein content and the morphometric data, which may be explained by the fact that, despite not statistically significant, the total protein content of the mitochondrial suspensions follows the same tendency as the CS activity and the morphometric results, suggesting that the majority of proteins in the mitochondrial suspensions certainly is of mitochondrial origin. Nevertheless, when expressed per mitochondrium, CS activity was significantly lower in the mature animals. In accordance with the well-described role of CS as an indicator of bioenergetic oxidative capacity, these data suggest that individual mitochondria from the mature animals have a diminished oxidative capacity. Considering this, CS activity could also present some limitations when studying different age groups; however, despite the above-mentioned limitation, our results strongly suggest that the utilization of CS as a marker of mitochondrial mass, to establish a reference activity, would be a more accurate approach than the simple utilization of the total protein content of a determined mitochondrial suspension.

Isolation of intact skeletal muscle mitochondria is a very delicate process in which any artificial damage will induce structural alterations and functional impairments, namely related with the activities associated with oxidative phosphorylation, specifically the state 3 respiration and the RCR values (33). The TEM qualitative analysis concerning the structural integrity of the mitochondria population isolated from the skeletal muscles of young and mature animals revealed that they were isolated with high integrity, and this was further supported by the RCR values (Table 3), indicating that the organelle damage induced by the isolation procedure was negligible. In fact, RCR values obtained both in the young and in the older animals were comparable with other data reported elsewhere on mitochondria isolated from skeletal muscle of different ages (34). However, the mitochondria isolated from skeletal muscle of mature animals, when compared with mitochondria from the younger animals, presented an altered morphological appearance, with regard to their shape and cristae ultrastructure. In this context, several age-related alterations in mitochondrial morphology have been reported, like matrix vacuolization and shortened cristae (7). In addition, when considering the mitochondrial areas, as previously mentioned, our results have failed to demonstrate differences between groups.

The functional assessment of skeletal muscle mitochondria in both groups revealed a general decline in the functionality of this organelle in the mature animals, which is in accordance with several studies demonstrating that mitochondrial function is compromised with advancing age, both in humans (15,34) and mice (16). In the present study, mitochondria from the older animals exhibited a significant decline in the state 3 respiratory rate, both with complex I- and complex II-linked substrates (Tables 3 and 4), which indicates that the maximal rate of mitochondrial oxygen consumption was affected by age. The functional impairment at the level of state 3 respiration was slightly higher in the complex I-linked substrate assay, which suggests that complex I-mediated respiration was more affected than succinate-supported respiration. In fact, the activities of mitochondrial electron transport chain (ETC) complexes—namely complex I, III, and IV—have been reported to decrease with age, whereas complex II appears to be unaffected (13). This could be related to the fact that many of the subunits of complex I, III, and IV of the mitochondrial ETC are encoded by mitochondrial DNA (mtDNA), whereas complex II is encoded by nuclear DNA (35). In this context, the age-related increases in oxidative stress and mitochondrial oxidative damage levels, supported by several studies (16,36), can induce significant oxidative damage to the mtDNA that could have serious implications in age-related mitochondrial heteroplasmy and dysfunction (7,13,37). In addition, complex I activity might be affected because of its particular vulnerability to oxidative attack, due to its higher content of Fe-S clusters (13). In contrast to the above-mentioned state 3 respiration data, we have failed to demonstrate significant differences in state 4 respiration between mitochondria isolated from both groups. In this respect, it appears that the permeability of the inner mitochondrial membrane was not affected by age.

To further confirm the above-mentioned results, we have performed an additional mitochondrial assay, where we used oligomycin and CCCP in mitochondria previously energized with M+P and stimulated with ADP. This assay avoids the possible interference of the permeability to protons through the ATP synthase on state 4 conditions, and ensures that the variations in membrane permeability do not interfere with the inhibition of the respiratory chain, because the permeability in the presence of CCCP is always maximal (38). In this context, the maximal rate of uncoupled respiration was significantly reduced in the mature animals confirming the state 3 respiration data and further supporting that increased age is associated with a decreased maximal functional capacity of the respiratory chain. In contrast, the respiration in the presence of oligomycin was not altered by age in these animals, suggesting that the integrity of the inner mitochondrial membrane was not affected, which is in accordance with the state 4 respiration results previously presented.

Concerning the RCR values, mitochondria from mature animals presented significantly decreased RCR when compared with their younger counterparts, which is consistent with the idea that advancing age might affect the coupling between the ETC and oxidative phosphorylation. Bearing in mind that RCR is a functional variable that represents the functional and structural integrity of respiring mitochondria (38), this result suggests that mitochondria from mature animals are less functional than those from the younger animals. This is mainly a consequence of the decrease in state 3 respiration in the mitochondria from the older animals, as state 4 was unchanged. In contrast, the mitochondrial phosphorylation efficiency (ADP/O ratio) did not appear to be affected by age, which indicates that oxidative phosphorylation was not altered in the mitochondria from the mature animals. Therefore, we might hypothesize that the functional impairments in the mitochondria from aged animals were mainly related to decreased activities of the complex I– and complex II-supported respiration, with little or no functional impairments at the level of the ATP synthase complex.

Summary
Our data suggest that increased age is associated with morphological, biochemical, and functional alterations in isolated skeletal muscle mitochondria. Moreover, the CS activity measured in the mitochondrial suspensions appears to be a more accurate marker of the mitochondrial mass when compared with the determination of the protein content.


   Acknowledgments
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 Abstract
 Methods
 Results
 Discussion
 References
 
This work was supported by a grant by Fundação para a Ciência e Tecnologia (PTDC/10DES/70757/2006). Pedro A. Figueiredo is supported by a grant of Programa Operacional Ciência e Inovação 2010 and Fundo Social Europeu (FSE).


   Footnotes
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 Discussion
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Decision Editor: Huber R. Warner, PhD

Received August 24, 2007

Accepted December 17, 2007


   References
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 Abstract
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 Discussion
 References
 

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