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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:715-723 (2005)
© 2005 The Gerontological Society of America

Age-Related Changes of Mitochondrial DNA Content and Mitochondrial Genotypic and Phenotypic Alterations in Rat Hind-Limb Skeletal Muscles

Vito Pesce1, Antonella Cormio1, Flavio Fracasso1, Angela M. S. Lezza1, Palmiro Cantatore1,3 and Maria Nicola Gadaleta1,2,3,

1 Department of Biochemistry and Molecular Biology
2 Centro di Eccellenza di Genomica in Campo Biomedico ed Agrario, University of Bari, Italy.
3 Institute of Biomembranes and Bioenergetics, National Council of Researchers, Bari, Italy.

Address correspondence to Professor Maria Nicola Gadaleta, Department of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4, 70125, Bari, Italy. E-mail: m.n.gadaleta{at}biologia.uniba.it


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Mitochondrial DNA (mtDNA) content relative to nuclear DNA content as well as mitochondrial transcription factor A (TFAM) content was measured in four hind-limb skeletal muscles, namely soleus (S), tibialis anterior (TA), gastrocnemius (G), and extensor digitorum longus (EDL) of adult rats. Content of mtDNA in 6-month-old rats is in the rank order of S > TA > G > EDL, and TFAM content is higher in S than in the other studied muscles. After the rat is 6 months of age, the mtDNA content decreases only in S and TA, whereas the TFAM content increases only in S. Deletions in mtDNA appear quite early in life in S and later on in the other muscles. Fibers defective for mitochondrial respiratory enzymes appear in rats at 15 months of age. In the oldest animals, the highest frequencies of occurrence of mtDNA deletions as well as of mitochondrial phenotypic alterations are found in S according to its highest mtDNA content and oxidative potential.

THE deep involvement of mitochondria in aging and age-related phenomena is a widely accepted idea (1–3) since the "mitochondrial theory of aging" (4–6) has gained progressively larger support among the aging theories. Mitochondria are known today as receiver and/or integrator organelles of signaling pathways (7,8) as well as potential loci of diseases (9). However, the involvement of mitochondria in aging relates particularly to their function as "powerhouses of the cell" which has been shown to be decreased by different extents by aging (1). This reduced bioenergetic capability is due to an age-related decrease of some respiratory enzyme activities which has been reported in various tissues and organisms by many authors (5,10), although a few others (11–13) suggested that such changes are more due to lifestyle or diet than to aging. The age-related decrease of the mitochondrial bioenergetic capability becomes dramatic for those postmitotic cells such as neurons and skeletal muscle fibers that heavily depend on oxidative metabolism for their power supply (10,14). In the literature, several examples of the mitochondrial dysfunction occurring in the aging process of skeletal muscle fibers in human (15,16), mouse (17), and rat (18) have been reported. Such mitochondrial bioenergetic decline has been demonstrated by means of various experimental approaches including histochemical techniques (16,19,20), which unveil at the cellular level the functionality of some mitochondrial respiratory chain enzymes and lead to the identification of different cellular phenotypes. The reactions for cytochrome c oxidase (COX) (21) and succinate dehydrogenase (SDH) (22) activities have allowed the detection of COX normal (COX-positive) and COX deficient (COX-negative) fibers as well as SDH normal (SDHn) and SDH hyperreactive (SDH++) fibers. The last type of fibers corresponds to the ragged red fibers (RRF), revealed by the Gomori modified trichrome staining (23), that exhibit a subsarcolemmal mitochondrial proliferation. The age-related mitochondrial dysfunction has been studied also by means of molecular biology techniques, used for the detection of mitochondrial DNA (mtDNA) modifications (24), point mutations (25,26) and/or deletions both at whole tissue (5,27) and at single fiber levels (25,28). In particular, a large deletion (nt 8103-12936), encompassing 4834 bp (29,30) from a genomic region of rat mtDNA similar to that of the 4977 bp human mtDNA "common deletion" (31,32), and other large deletions have been detected and demonstrated to accumulate in various tissues of aging rat (5,33). As for the origin of such mtDNA deletions, it has been suggested that the very fast rate of mitochondrial metabolism in rat might continuously produce a high level of reactive oxygen species (ROS) (34) which might cause extensive oxidative damage to mtDNA as well as to other mitochondrial molecules. Because of the physical closeness to the respiratory complexes, the exposure to a high reducing environment in the mitochondrial matrix, and the lack of a histone-like protein coverage, mtDNA is particularly prone to oxidative damage. The age-related increase of ROS production, demonstrated also in rodent mitochondria (35,36), might originate the accumulation of the oxidized adduct 8-oxo-7,8-dihydro-2'-deoxyguanosine, OH8dG, and other modified bases, with aging, in mtDNA. Oxidized adducts might then induce the quite precocious formation of the "common deletion" (34,37,38) and/or of other mtDNA deleted species through one of the different hypothesized mechanisms (5). It has been postulated that the age-related accumulation of mtDNA mutations in the protein-coding genes might damage the mitochondrial bioenergetic capability leading to a severe dysfunction. The age-related increased oxidative damage to mtDNA might also affect the replicative machinery of the mitochondrial genome and induce changes in the mtDNA content. In mtDNA maintenance, a very relevant role is assigned to the mitochondrial transcription factor A (TFAM). TFAM expression appears to be affected by different kinds of stimuli influencing mitochondrial proliferation (39,40). Others (41) and some of us reported increased amounts of mtDNA (19) and TFAM (42) in aged human skeletal muscle samples, as well as increased amounts of mtDNA and TFAM in several rat tissues (43).

The goals of this study were the measurement of the mtDNA and TFAM contents in four different hind-limb skeletal muscles of adult rats (6 months old)—namely, soleus (S), tibialis anterior (TA), gastrocnemius (G), and extensor digitorum longus (EDL), the determination of their age-related changes, as well as the analysis of the lifetime evolution of some mitochondrial genotypic and phenotypic alterations. We report here that the content of mtDNA in the four hind-limb skeletal muscles of adult rat is in the rank order of S > TA > G > EDL and that the TFAM content is significantly higher in S than in the other muscles. As far as age-related changes, the mtDNA content decreases only in S and TA, whereas the TFAM content increases only in S. Quite early in life mtDNA deletions appear, and later on (at 15 months of age), fibers defective for some mitochondrial respiratory enzymes appear. In the oldest animals the highest frequencies of occurrence of mtDNA deletions as well as of fibers altered for some mitochondrial respiratory enzymes are found in S according to its highest mtDNA content and oxidative potential.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Muscle Samples
Male Wistar rats were maintained two per cage on a 12-hour light/dark cycle at 25°C, and had access to standard laboratory chow and water ad libitum. All procedures were in accordance with the guiding principles in the care and use of laboratory animals of Bari University. Thirty rats of different ages (3, 6, 12, 15, 18, and 28 months) were killed, and four hind-limb muscles (S, G, TA, and EDL) were quickly excised and immediately frozen in isopentane cooled by liquid nitrogen and stored in liquid nitrogen until analysis.

Determination of mtDNA Content
Total DNA was prepared from about 40–50 mg of skeletal muscle samples as described by Arnaudo and colleagues (44). Total DNA (5 µg) was digested with Pvu II (MBI-Fermentas, Vilnius, Lithuania) and run on a 0.35% agarose gel (Seakem Gold; FMC BioProducts, Rockland, ME). The gel was blotted onto a Hybond-N membrane (Amersham-Pharmacia, Little Chalfont, U.K.) and simultaneously hybridized with a mitochondrial and a nuclear probe. The mitochondrial probe was a 658-bp fragment obtained by polymerase chain reaction (PCR) using the primers DN (L 15758-15777) and DR (H 117-98) described by Dinardo and colleagues (43). Rat mtDNA nucleotide positions are according to Gadaleta and colleagues (45). The nuclear probe was a 413-bp fragment containing part of the 18S rRNA gene and subcloned in the TA vector (Clontech, Palo Alto, CA). Both probes were labeled by random priming (Random Primed DNA Labeling Kit; Roche, Basel, Switzerland) and used in a 10:1 ratio of the nuclear DNA (nDNA) probe to the mtDNA probe. Blotting, prehybridization, hybridization, and washings were carried out as described by Sambrook and colleagues (46). The filter was exposed to an X-ray film at –70°C with an intensifying screen, and the hybridization signals were quantified by densitometry with an LKB-Pharmacia Ultrascan-XL Laser densitometer equipped with GelScan-LX-Evaluation software (LKB-Pharmacia Biotechnology, Uppsala, Sweden).

Determination of TFAM Content
Rabbit anti-rat TFAM antiserum was a gift from Dr. H. Inagaki (Department of Chemistry, National Industrial Research Institute of Nagoya, Japan). Goat anti-rabbit immunoglobulin G–horseradish peroxidase (hrp) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-mouse actin immunoglobulin M ascites and goat anti-mouse immunoglobulin M–hrp were purchased from Oncogene Research Products (Boston, MA). Total proteins were extracted with sodium dodecyl sulfate (SDS) gel-loading buffer (50 mM Tris–HCl [pH 6.8], 100 mM dithiothreitol, 2% SDS, 10% glycerol) and their amounts were quantified using the Bradford method (Bio-Rad Laboratories, Hercules, CA) according to the supplier's instructions. Total proteins (2.5 µg) were separated into 12% SDS-polyacrylamide slab minigels and electroblotted onto polyvinylidene difluoride membrane (Amersham-Pharmacia). The membrane was subjected to incubation with primary antiserum, washings, incubation with secondary antibodies, and final washings according to Frigeri and colleagues (47). Secondary antibodies were labeled with hrp and detection (ECL-Plus; Amersham-Pharmacia) was performed according to the supplier's instructions. Antibodies were used at the following dilutions: the anti-TFAM primary antibody (1:2500), the antirabbit secondary antibody (1:5000), the anti-actin primary antibody (1:2000), the antimouse secondary antibody (1:5000). Autoradiographs were analyzed by laser densitometry with the Chemi Doc System and Quantity One software (Bio-Rad Laboratories). Preliminary titration experiments allowed us to establish the amounts of protein extracts (2.5–25 µg) which gave a signal in the linear range of the relation: densitometric value/blotted proteins. Different exposures of each western blot, in the time range between 1 second and 5 minutes, were taken to ensure the linearity of the response for both assayed proteins.

Detection of the 4834-bp-Long mtDNA Deletion
About 50 mg of skeletal muscle was used to extract nucleic acids (48). DNA (100 ng) was amplified by using the following primers: 7825-For (L 7825-7844) and 13117-Rev (H 13117-13099) for the 4834-bp-long mtDNA deletion (mtDNA4834; 459-bp product, annealing temperature 60°C), as reported in Pesce and colleagues (49). A secondary seminested PCR round, using one of the primers of the first amplification (13117-Rev) and the new internal primer 7978-For (L 7978–7997) with a 1-µl aliquot of the first amplification, at an annealing temperature of 57°C, was performed to obtain a 306-bp product. PCR products were size-fractionated on a 1.5% agarose gel in 1 x Tris-borate-EDTA buffer and visualized by ethidium bromide staining. Only those animals not showing the expected amplification products in two successive PCR rounds were considered to be not harboring the searched mtDNA deletion. The identity of the amplification products was confirmed by direct DNA sequencing as described elsewhere (50).

Detection of Multiple mtDNA Deletions by Long Distance-PCR
Used primers were 5881-For (L 5881-5905) and 15141-Rev (H 15141-15118) (48), and the PCR mixture contained 100 ng of DNA, 0.1 µM of each primer, 200 µM dNTPs, 1X reaction buffer (Takara Bio Inc., Shiga, Japan) and 2.5 U of Long Amplification Taq Polymerase (Takara) in a 50-µl reaction volume. The PCR conditions consisted of denaturation at 94°C for 30 seconds, annealing at 60°C for 1 minute, and extension at 68°C for 1.5 minute for 15 cycles, followed by denaturation at 94°C for 30 seconds, annealing at 60°C for 1 minute, and extension at 68°C for 1.5 minutes plus 10 s/cycle for 15 cycles and a final extension of 7 minutes at 68°C in a Peltier Thermal Cycler (MJ Research, Watertown, MA). PCR products were separated on a 1.5% agarose gel (Amresco, Solon, OH) in 1X TBE buffer and visualized by ethidium bromide staining. To increase the sensibility of the technique and to exclude the possibility that the low-molecular-weight bands were due to PCR artifacts, a secondary seminested long-distance PCR (LD-PCR) round, very similar to the first, was performed on an aliquot of the first reaction mixture using the 15141-Rev primer and the new internal primer 5974-For (L 5974–5996).

Histochemistry
Serial 8-µm-thick transverse sections from frozen muscles were cut with the Cryotome (HM 505 E-Microm, Walldorf, Germany), mounted on polylysine-coated glass slides, and stained for COX activity (21), SDH activity (22), and both activities. Fibers with abnormal accumulation of mitochondria, the so-called "ragged red fibers" (RRF) for their red staining in the subsarcolemmal region with the modified Gomori trichrome stain, appeared hyperreactive with the modified SDH stain (SDH++). The modified SDH stain was chosen to count the number of SDH++ fibers (RRF) because it is more sensitive than the modified Gomori trichrome staining (23). Sections were viewed on a Zeiss transmitted light microscope (Carl Zeiss, Jena, Germany) and photographed using a 10x objective. In each specimen from 400 to 700 fibers of two to three representative fields were analyzed on a photomicrograph, and the percentage of those having no detectable COX activity and normal SDH activity (COXneg/SDHn) and no detectable COX activity and hyperreactive SDH activity (COXneg/SDH++) was calculated.

Statistics
Statistical analysis was carried out by using SPSS version 11.0 software (SPSS Inc., Chicago, IL). The comparison among the four hind-limb skeletal muscles was made using one-way analysis of variance (where data were normally distributed with equal variance). The Tukey test was used for multiple comparisons. Results in adult (6-month-old) and old (28-month-old) rats were compared using Student's t test for unpaired data. Statistical significance for all tests was set at p <.05.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
mtDNA Content
The mtDNA content relative to nDNA content (mtDNA/nDNA) was measured in the four hind-limb skeletal muscles to verify its eventual differences among S, TA, G, and EDL in adult rats (6-month-old rats) as well as its muscle-specific changes with age.

From 23 to 25 samples of the four hind-limb muscles (S, TA, G, and EDL), derived from rats ranging from 3 to 28 months of age, were analyzed. The analysis was performed by hybridizing Pvu II-digested total DNA with a mitochondrial and a nuclear probe. The mitochondrial probe detected a band of 16.3 Kbp corresponding to linearized mtDNA, whereas the nuclear probe detected a 12-Kbp band, corresponding to a Pvu II fragment of the nuclear 18S rDNA gene. Figure 1A shows an example of Southern blot hybridization carried out on S samples from rats 6, 15, and 28 months of age. The ratio of the intensities of the two bands was used to estimate the amount of mtDNA relative to nDNA in each sample. Comparisons among the different muscles were made, setting equal to 1 the mtDNA/nDNA value of EDL from 6-month-old rats, as reported in Figure 1B. The mtDNA content increases in all examined muscles passing from the 3-month-old animals to the 6-month-old ones confirming that the postnatal development and/or differentiation of such skeletal muscles is completed at 6 months of age (51). At this age, comparing the mtDNA/nDNA values of the analyzed hind-limb muscles with that of S (3.0), TA has a value of 2.0 (33% less mtDNA than S), G has a value of 1.5 (50% less mtDNA than S), and the glycolytic muscle EDL has a value of 1.0 (66% less mtDNA than S) (Figure 1B, # = statistically significant differences, p <.05). After 6 months of age, the mtDNA content shows in S a gradual decline reaching an overall statistically significant reduction of 52% of the initial value in the 28-month-old rats. The decrease of mtDNA content begins later in life in TA, after 15 months of age, so that the mtDNA content of the 28-month-old rat is reduced by 38% with respect to the 6-month-old rat. The mtDNA content in G shows a peculiar trend because it decreases from 6 months through 15 months of age and, after such age, it increases again reaching approximately the value of the 6-month-old adult animal; however, such age-related changes in this muscle never reach statistical significance. In EDL, there is no age-related change of the mtDNA content.



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  		Figure 1. Age-related changes of the mitochondrial DNA content relative to nuclear DNA content (mtDNA/nDNA) in the four analyzed hind-limb rat skeletal muscles. A, Representative Southern blot hybridization carried out on soleus muscle (S) samples from rats of 6, 15, and 28 months of age. Top and bottom bands show signals, respectively, from the mtDNA (16.3 Kb) and the nDNA fragment containing the 18S rRNA gene (12.0 Kb). B, Quantitative estimate of the mtDNA content relative to nDNA in different hind-limb skeletal muscles. The histograms show for each age group of animals the mtDNA content (mtDNA/nDNA) of the four hind-limb skeletal muscles normalized to that of the extensor digitorum longus muscle (EDL) samples from 6-month-old rats (indicated by the broken line). Bars represent the average ± standard error of values obtained from three preparations of each sample analyzed in triplicate. #p <.05 in tibialis anterior (TA), gastrocnemius (G), and EDL versus S of 6-month-old rats using analysis of variance (ANOVA); §p <.05 in EDL versus S of 28-month-old rats using ANOVA for the four muscles; *p <.05, obtained comparing (in the same muscle) animals of different age groups versus the 6-month age group using ANOVA; n = number of analyzed animals

 
A comparison of the mtDNA relative content among the four hind-limb muscles in the 28-month-old rats shows that no more differences exist among TA and G versus S, whereas the mtDNA content is still significantly lower in EDL than in S (–41%) (Figure 1B, § = statistically significant differences, p <.05).

TFAM Content
Because TFAM content was recently reported by some of us (43) to increase with age in several rat tissues together with the mtDNA content, we measured the TFAM content in the four hind-limb muscles from 6- and 28-month-old rats. Total proteins from the same skeletal muscle samples used for the mtDNA content determination were analyzed by SDS-polyacrylamide gel electrophoresis, blotted onto a polyvinylidene difluoride membrane, and incubated with antibodies against TFAM and {alpha}-actin. Figure 2A shows a representative western blot carried out on S from rats of 6 and 28 months of age. The ratio of the intensities of the two bands was used to estimate the amount of TFAM in each sample (Figure 2B). The TFAM content of the four skeletal muscles in both the 6-month-old and the 28-month-old rats reaches the highest value in S, showing, in the 6-month-old rats, a rank order of TA > G > EDL for the respective values (Figure 2B, # = statistically significant differences; p <.05, S versus G and EDL). In the oldest rats, the TFAM contents from G, TA, and EDL are significantly lower than those from S (Figure 2B, § = statistically significant differences, p <.05). TFAM content, when the values for the same muscle from 6- and 28-month-old rats are compared, significantly changes only in S, where, in the oldest rats with respect to the adult ones, the TFAM content increases from 0.5 to 0.72 (44%) (Figure 2B, *p <.05).



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  		Figure 2. Age-related changes in mitochondrial transcription factor A (TFAM) content in the four hind-limb muscles from young and old rats. A, Representative western blot carried out on soleus muscle (S) samples from rats 6 and 28 months of age. Top and bottom bands show, respectively, signals from rat {alpha}-actin protein and from rat TFAM protein. B, Quantitative estimate of the TFAM content relative to {alpha}-actin in different hind-limb skeletal muscles. The histograms show the TFAM content relative to the {alpha} actin content (TFAM/{alpha} actin) of the four hind-limb skeletal muscles in 6- and 28-month-old rats. Bars represent the average ± standard error of values obtained, respectively, from analysis in triplicate of the four protein extracts obtained independently from each young and each old animal. #p <.05, gastrocnemius muscle (G) and extensor digitorum longus muscle (EDL) versus S in 6-month-old rats using analysis of variance; §p <.05 in TA, G, and EDL versus S of 28-month-old rats using analysis of variance; *p <.05, soleus muscle (S) for the adult-old comparison using Student's t test; n = number of analyzed animals

 
mtDNA Deletions
We detected in four hind-limb muscles the presence of the mtDNA4834 deletion, by using two-rounds of PCR with a nested primer. Figure 3A shows a representative gel for the detection of the 4834-bp deletion in three different skeletal muscles (S, EDL, and TA) from the same 28-month-old rat. The ethidium bromide-stained gel shows the expected PCR products (459 bp), deriving from the primary amplification of mtDNA harboring the "common deletion" only in S and TA. The seminested PCR on the same samples was necessary to reveal the presence of such deletion as a nested PCR product of 306 bp also in EDL (Figure 3B).



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  		Figure 3. Detection of the 4834-bp deletion and of multiple deletions by primary and seminested polymerase chain reaction (PCR). Gels are representative of PCR-amplified products. A, Ethidium bromide staining of a 1.5% agarose gel shows the 459-bp PCR products deriving from the primary amplification of the 4834-bp deletion in soleus (S) and tibialis anterior (TA) skeletal muscles of a 28-month-old rat. M1 = DNA size standard (pBR DNA x Hinf I: 1631, 517-506, 394, 344, 298). B, Ethidium bromide staining of a 1.5% agarose gel shows the 306-bp products deriving from the seminested PCR on aliquots from the same reactions shown in gel A. M2 = DNA size standard (3000, 2000, 1500, 1200, 1031, 900, 800, 700, 600, 500, 400, 300, 200 bp). C, Ethidium bromide staining of a 1.5% agarose gel shows the presence of multiple mitochondrial DNA deleted species detected by primary and seminested long-distance-PCR (LD-PCR) in S of a 28-month-old rat. Lane 1, primary LD-PCR products; lane 2, seminested LD-PCR products deriving from the primary LD-PCR. M3 = DNA size standard (10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 750 bp)

 
The number of analyzed animals really harboring the mtDNA4834 deletion (expressed as % of occurrence) in each of the four muscles from rats belonging to different age groups is reported in the Figure 4 insets. Such percentage of occurrence increases with age in the four analyzed muscles although with muscle-specific differences. In fact, mtDNA4834 appears in one of the five analyzed S samples from the 3-month-old rats, then the percentage of occurrence gradually increases up to 28 months of age when 100% of S samples harbor mtDNA4834.



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  		Figure 4. Analysis of the presence of the age-related mitochondrial DNA (mtDNA) deletions in the four rat hind-limb skeletal muscles. Insets show, for each muscle, the percentage of animals bearing the mtDNA4834 (% occurrence) in each different age group. Bars of the histograms show the number of mtDNA deleted species, detected by long-distance primary and seminested polymerase chain reaction, in soleus, tibialis anterior, gastrocnemius, and extensor digitorum longus muscles of each analyzed animal. Abscissa = ages (months) of all analyzed rats. No deletions have been found where no bars are reported

 
In the other hind-limb muscles analyzed, mtDNA4834 appears for the first time at 6 months of age; thereafter, its occurrence increases gradually with age. In the oldest animals, a different percentage of occurrence is found in the various muscles: in fact, about 75% of analyzed animals bear such deletion in G and TA, whereas only 50% of the animals bear the deletion in EDL. G and TA show, at different ages, an intermediate percentage of occurrence between the values of S and EDL.

To obtain a more comprehensive representation of the presence of mtDNA deletions in each analyzed animal, the DNA extracted from the four hind-limb muscles was analyzed by LD-PCR (48). Figure 3C shows results of a typical LD-PCR experiment on DNA extracted from the S muscle of a 28-month-old rat. The ethidium bromide-stained gel shows multiple bands deriving from the amplification of deleted mtDNA species (Lane 1). Lane 2 shows the products deriving from the seminested LD-PCR carried out with an aliquot of the primary LD-PCR. Analyzing the gel containing 22–28 samples of muscles derived from rats of different ages, we found that each specimen has a distinctive pattern of mtDNA deletions. By counting the different bands obtained in all analyzed samples, we could detect at least 20 different mtDNA deleted species. This number is likely an underestimate, due to factors such as the resolution power of the gel, which prevented discrimination between bands having similar molecular weights. Figure 4 shows the number of mtDNA deleted species found in each analyzed specimen. All samples from rats younger than 28 months of age carried 0–2 deletions, whereas samples from 28-month-old rats had 0–11 deletions. More than one deletion was found earlier (6 months of age) in S than in the other muscles. Also, the percentage of samples harboring more than one deletion among the 28-month-old rats is muscle-specific: all samples from S, four from TA, three from G, and two from EDL of six analyzed samples for each muscle harbor more than one deletion.

Histochemical Analysis
To verify whether the genotypic alterations reported above were associated with the alteration of all fibers in a chosen muscle or a mosaic of fibers with different respiratory activities was originated in the analyzed muscles, as already reported (52), a histochemical analysis of the samples was performed. Samples of the four skeletal muscles from rats of 3, 6, 15, and 28 months of age were analyzed for COX and SDH activities by staining serial 8-µm-thick transverse sections of frozen muscles. The number of animals, among all those assayed, showing COX-negative/SDHn or COX-negative/SDH++ (RRF) fibers in the four studied muscles is expressed as percentage of occurrence for each age group in Figure 5. Among the samples from rats younger than 15 months of age, none bears COX-negative/SDHn fibers or COX-negative/RRF. A muscle-specific increase with age of the percentage of occurrence of the COX-negative/SDHn fibers was found. At 15 months of age, S, TA, and EDL showed COX-negative/SDHn fibers with different frequencies, whereas no skeletal muscle sample but G carried COX-negative/RRF. In the 28-month-old rats, COX-negative/SDHn fibers were present in all four examined muscles with a percentage of occurrence that ranges from about 30% in EDL and in TA to 70% in G and to 80% in S, whereas COX-negative/RRF fibers were present in 80% of S and G examined samples and in 60% of TA, and were absent in EDL samples.



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  		Figure 5. Age-related percentage of occurrence of cytochrome c oxidase (COX)-negative/succinate dehydrogenase normal (SDHn) and COX-negative/SDH++ fibers in the four hind-limb rat skeletal muscles. Histograms show for each muscle the percentage of animals bearing phenotypically altered fibers (% occurrence) in each age group of animals. 400–700 fibers in 2–3 representative fields for each specimen were analyzed. n = number of analyzed animals

 
No COX-positive/RRF fibers were found in any of the examined muscles from each age group of animals. The overall percentage of COX-negative/RRF fibers in S, TA, and G, that is, the number of COX-negative/RRF counted in each specific sample, was around 0.5% in the oldest animals, whereas the overall percentage of COX-negative/SDHn fibers is 0.6% (not reported data).


   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In this study we report for the first time, to our knowledge, the mtDNA and the TFAM contents of four hind-limb skeletal muscles, namely S, TA, G, and EDL from adult Wistar rats as well as their age-related changes. Furthermore, to better characterize the aging process at mitochondrial level in these four hind-limb skeletal muscles, are reported also the age-related changes involving the percentage of occurrence of mtDNA4834, other deleted species, and some altered mitochondrial phenotypes.

It has been reported for a long time that the concentration of mtDNA is directly proportional to the oxidative capacity of mammalian striated muscles (53). More recently, He and colleagues (54) reported that the absolute copy number of mtDNA in single type I oxidative fibers of human skeletal muscle is double that in single type II glycolytic fibers. The rank order of mtDNA contents for the four hind-limb muscles in 6-month-old rats (from highest to lowest) is S > TA > G > EDL. Such order corresponds to the respective degrees of dependence on oxidative metabolism already reported for these muscles in adult Wistar and other rat strains (55–58). In particular, S has the highest mtDNA content relative to nDNA among the other age-matched muscles, in agreement with its highest mitochondrial content (56) and its fiber type composition (about 90% type I plus type IIA), whereas EDL has the lowest mtDNA content and a fiber type composition mostly glycolytic (about 70% type IIB) (55–58). TA and G have intermediate mtDNA content between S and EDL and a mixed fiber type composition (55–58).

TFAM content in the four muscles from adult (6-month-old) rats seems to correspond well to the respective mtDNA content. In fact, the TFAM amount shows the highest value in S, and progressively decreases in the age-matched muscles from TA to G to EDL. This further confirms the tight relationship already reported between the mtDNA content and the level of the transacting factor TFAM (39,59,60).

With aging, a decrease of mtDNA content begins earlier in S (from 6 months on) to progressively reach the lowest value in the 28-month-old samples. The results reported here for S muscle from Wistar rats are consistent with the age-related decrease of the relative mtDNA content (from 6 months to 27 months) demonstrated in the same S muscle from Fisher rats by Barazzoni and colleagues (61). Furthermore, a more recent study reported the age-related changes of molecular and fiber composition in rat S (62). In that study, consistent with the results reported here, a loss of full-size mtDNA and an increase of deleted species as well as a decrease of COX activity in S of 28-month-old rat were found. Furthermore, the same authors reported a high variability in old rat S of the fiber type composition and atrophic changes accompanied by the slow–to-fast fiber type transition. The fiber type transition can explain the loss of mtDNA in S reported here. The mixed fiber type muscle TA also shows an age-related progressive decrease of its mtDNA content. However, mtDNA loss begins later in life (from 15 months on) in this muscle and proceeds faster than in the slow-twitch predominant S. In the above-cited article by Barazzoni and colleagues (61), the reported decrease of mtDNA content with aging in both separated portions of G was smaller than that in S and was explained as a common trend of the skeletal muscle group. Our data about the mtDNA content in G come from the joined portions of muscle isolated from a rat strain different from that used in (61); furthermore, our data were determined at six distinct ages, unveiling a more complex trend in mtDNA content changes during the life span that, however, never reached statistical significance.

As far as TFAM content is concerned in aging rats, an increase of both mtDNA and TFAM contents in rat cerebellum, kidney, and liver (but not in heart) has been found by some of us (43), whereas in the muscles studied here the muscle-specific differences prevail over a common trend (Figure 2). In fact, in the 28-month-old samples from S, there is an age-related increase of the protein that apparently is in contrast with the contemporaneous decrease of the mtDNA content. Although TFAM content was measured in the homogenate of the four muscles and the COX-negative/RRF fibers were reported as percentage of occurrence in the examined samples, one possible explanation of the level of TFAM in 28-month-old rat could be that the gradual decrease of mtDNA with age in S induces TFAM expression to compensate for the loss of mtDNA with mitochondrial proliferation. However, because such proliferating mitochondria should contain mainly mutated mtDNA molecules (33), all examined samples of S from 28-month-old rats show COX-negative/RRF. A similar hypothesis might explain the age-related increase of TFAM in G from the 28-month-old animals and the decrease of the TFAM amount in age-matched TA, only a few samples of which harbor COX-negative/RRF fibers. As for EDL, again, there is consistency between the lack of any age-related change both in the mtDNA content and in the TFAM amount and the total absence of COX-negative/RRF fibers.

Conclusion
A good consistency between the mtDNA content and the respective dependence on oxidative metabolism, according to the literature (55–58), in different hind-limb muscles from adult rats has been found as well as between mtDNA content and TFAM content. An age-related trend common in the analyzed muscles includes (a) the increase in the occurrence of mtDNA4834 and of multiple mtDNA deletions that might be responsible for the decreased activity of COX shown by the histochemical results and (b) the increase in the occurrence of COX-negative/SDHn and COX-negative/RRF phenotypes. Muscle-specific differences with aging seem to be linked essentially to the mtDNA and TFAM contents of the examined skeletal muscles. In agreement with the mitochondrial theory of aging, the S muscle, which contains more mtDNA than the other three examined muscles do, seems to be more exposed to oxidative stress and ROS production, whereas EDL seems to be almost unaffected by the age-related oxidative stress, and TA and G have an intermediate behavior. On the whole, it should be noted that the occurrence of mitochondrial genotypic and phenotypic alterations is very low in our oldest rats (28-month-old), whereas the loss of mtDNA seems to be the major age-linked change. If this is the final outcome of a remote process that has caused the life-long continuous loss of damaged fibers leading to sarcopenia or the real picture of a small entity process in aging rat skeletal muscle still needs to be conclusively discovered through further research.


   Acknowledgments
 
This work has been supported by funds from Sigma Tau–Industrie Farmaceutiche Riunite S.p.A. (Contract D.S./2000/C.R./no. 20), from the Italian Space Agency (ASI; Contract I/R/322/02), from Ministero dell'Istruzione, dell'Università e della Ricerca-Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale 2002 (to M.N.G.) and from the University of Bari–Progetti di Ateneo 2003 (to A.M.S. L.), -Progetti di Ateneo 2005 (to V.P.). We thank R. Longo for word processing.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received October 5, 2004

Accepted January 30, 2005


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Cadenas E, Davies KJA. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med. 2000;29:222-230.[Medline]
  2. Chomyn A, Attardi G. MtDNA mutations in aging and apoptosis. Biochem Biophys Res Commun. 2003;304:519-529.[Medline]
  3. Dufour E, Larsson NG. Understanding aging: revealing order out of chaos. Biochim Biophys Acta. 2004;1658:122-132.[Medline]
  4. Harman D. The biologic clock: the mitochondria? J Am Geriatr Soc. 1972;20:145-147.[Medline]
  5. Gadaleta MN, Kadenbach B, Lezza AMS, et al. Age-linked changes in the genotype and phenotype of mitochondria. In: Papa S, Guerrieri F, Tager J, eds. Frontiers of Cellular Bioenergetics: Molecular Biology, Biochemistry, and Physiopathology. London: Plenum Publishing Company Ltd; 1999:693–728.
  6. Trifunovic A, Wredengerg A, Falkenberg M, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004;429:417-423.[Medline]
  7. Nisoli E, Clementi E, Moncada S, Carruba MO. Mitochondrial biogenesis as a cellular signaling framework. Biochem Pharmacol. 2004;67:1-15.[Medline]
  8. Goldenthal MJ, Marìn-Garcìa J. Mitochondrial signaling pathways: a receiver/integrator organelle. Mol Cell Biochem. 2004;262:1-16.[Medline]
  9. DiMauro S. Mitochondrial medicine. Biochim Biophys Acta 2004;1659:107-114.[Medline]
  10. DiMauro S, Tanji K, Bonilla E, Pallotti F, Schon A. Mitochondrial abnormalities in muscle and other aging cells: classification, causes, and effects. Muscle Nerve. 2002;26:597-607.[Medline]
  11. Barrientos A, Casademont J, Rotig A, et al. Absence of relationship between the level of electron transport chain activities and aging in human skeletal muscle. Biochem Biophys Res Commun. 1996;229:536-539.[Medline]
  12. Chretien D, Gallego J, Barrientos A, et al. Biochemical parameters for the diagnosis of mitochondrial respiratory chain deficiency in humans, and their lack of age-related changes. Biochem J. 1998;329:249-254.
  13. Brierley EJ, Johnson MA, James OF, Turnbull DM. Effects of physical activity and age on mitochondrial function. Q J Med. 1996;89:251-258.
  14. Lezza AMS, Boffoli D, Scacco S, Cantatore P, Gadaleta MN. Correlation between mitochondrial DNA 4977-bp deletion and respiratory chain enzyme activities in aging human skeletal muscles. Biochem Biophys Res Commun. 1994;205:772-779.[Medline]
  15. Brierley EJ, Hohnson MA, Lightowlers RN, James OFW, Turnbull DM. Role of mitochondrial DNA mutations in human aging: implications for the central nervous system and muscle. Ann Neurol. 1998;43:217-223.[Medline]
  16. Müller-Höcker J. Cytochrome c oxidase deficient fibers in the limb muscle and diaphragm of man without muscular disease: an age-related alteration. J Neurol Sci. 1990;100:14-19.[Medline]
  17. Kwong L, Sohal RS. Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse. Arch Biochem Biophys. 2000;373:16-22.[Medline]
  18. Paradies G, Ruggiero FM, Petrosillo G, Quagliariello E. Age-dependent decline in the cytochrome c oxidase activity in rat heart mitochondria: role of cardiolipin. FEBS Lett. 1997;406:136-138.[Medline]
  19. Pesce V, Cormio A, Fracasso F, et al. Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle. Free Radic Biol Med. 2001;30:1223-1233.[Medline]
  20. Wanagat J, Cao Z, Pathare P, Aiken JM. Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia. FASEB J. 2001;15:322-332.[Abstract/Free Full Text]
  21. Seligman AM, Karnovsky MJ, Wasserkrug HL, Hanker JSV. Non-droplet ultrastructural demonstration of cytochrome c oxidase activity with a polymerasing osmiophilic reagent diaminobenzidine (DAB). J Cell Biol. 1968;38:1-14.[Abstract/Free Full Text]
  22. Dubowitz UK, Brooke MH. Muscle Biopsy: A Modern Approach. Philadelphia, PA: W. B. Saunders; 1973.
  23. Rifai Z, Welle S, Kamp C, Thornton CA. Ragged red fibers in normal aging and inflammatory myopathy. Ann Neurol. 1995;37:24-29.[Medline]
  24. Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 2003;17:1195-1214.[Abstract/Free Full Text]
  25. Nekhaeva E, Bodyak ND, Kraytsberg Y, et al. Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues. Proc Natl Acad Sci U S A. 2002;99:5521-5526.[Abstract/Free Full Text]
  26. Zhang J, Asin-Cayuela J, Fish J, et al. Strikingly higher frequency in centenarians and twins of mtDNA mutation causing remodeling of replication origin in leukocytes. Proc Natl Acad Sci U S A. 2003;100:1116-1121.[Abstract/Free Full Text]
  27. Gadaleta MN, Lezza AMS, Cantatore P. Mitochondrial DNA deletions. In: Yu B, ed. Methods in Aging Research. Boca Raton, FL: CRC Press; 1999:475–511.
  28. Cormio A, Milella F, Vecchiet J, Felzani G, Gadaleta MN, Cantatore P. Mitochondrial DNA mutations in RRF of healthy subjects of different age. Neurobiol Aging. 2005;26:655-664.[Medline]
  29. Gadaleta MN, Rainaldi G, Lezza AMS, Milella F, Fracasso F, Cantatore P. Mitochondrial DNA copy number and mitochondrial DNA deletion in adult and senescent rat. Mutat Res. 1992;275:181-193.[Medline]
  30. Yowe DL, Ames BN. Quantitation of age-related mitochondrial DNA deletions in rat tissues shows that their pattern of accumulation differs from that of humans. Gene. 1998;209:23-30.[Medline]
  31. Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M, DiMauro S. A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA. Science. 1989;244:346-349.[Abstract/Free Full Text]
  32. Cortopassi GA, Arnheim N. Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic Acids Res. 1990;18:6927-6933.[Abstract/Free Full Text]
  33. Cao Z, Wanagat J, McKiernan SH, Aiken JM. Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. Nucleic Acids Res. 2001;29:4502-4508.[Abstract/Free Full Text]
  34. Barja G. Endogenous oxidative stress: relationship to aging, longevity and caloric restriction. Ageing Res Rev. 2002;1:397-411.[Medline]
  35. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273:59-63.[Abstract]
  36. Gredilla R, Barja G, Lopez-Torres M. Effect of short-term caloric restriction on H2O2 production and oxidative DNA damage in rat liver mitochondria and location of the free radical source. J Bioenerg Biomembr. 2001;33:279-287.[Medline]
  37. Ozawa T. Mitochondrial cardiomyopathy. Herz. 1994;19:105-118.[Medline]
  38. Lezza AMS, Mecocci P, Cormio A, et al. Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in old subjects but not in Alzheimer's disease patients brain. FASEB J. 1999;13:1083-1088.[Abstract/Free Full Text]
  39. Shadel GS, Clayton DA. Mitochondrial DNA maintenance in vertebrates. Annu Rev Biochem. 1997;66:409-435.[Medline]
  40. Hood DA. Invited review: contractile activity-induced mitochondrial biogenesis in skeletal muscle. J Appl Physiol. 2001;90:1137-1157.[Abstract/Free Full Text]
  41. Barrientos A, Casademont J, Cardellach F, et al. Qualitative and quantitative changes in skeletal muscle mtDNA and expression of mitochondrial-encoded genes in the human aging process. Biochem Mol Med. 1997;62:165-171.[Medline]
  42. Lezza AMS, Pesce V, Cormio A, et al. Increased expression of mitochondrial transcription factor A and nuclear respiratory factor-1 in skeletal muscle from aged human subjects. FEBS Lett. 2001;501:74-78.[Medline]
  43. Dinardo MM, Musicco C, Fracasso F, et al. Acetylation and level of mitochondrial transcription factor A in several organs of young and old rats. Biochem Biophys Res Commun. 2003;301:187-191.[Medline]
  44. Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 1991;337:508-510.[Medline]
  45. Gadaleta G, Pepe G, De Candia G, Quagliariello C, Sbisà E, Saccone C. The complete nucleotide sequence of the Rattus norvegicus mitochondrial genome: cryptic signals revealed by comparative analysis between vertebrates. J Mol Evol. 1989;28:497-516.[Medline]
  46. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  47. Frigeri A, Nicchia GP, Verbavatz JM, Valenti G, Svelto M. Expression of aquaporin-4 in fast-twitch fibers of mammalian skeletal muscle. J Clin Invest. 1998;102:695-703.[Medline]
  48. Aspnes L, Lee CM, Weindruch R, Chung S, Roecher E, Aiken J. Caloric restriction reduces fiber loss and mitochondrial abnormalities in aged rat muscle. FASEB J. 1997;11:573-581.[Abstract]
  49. Pesce V, Cormio A, Fracasso F, Lezza AMS, Cantatore P, Gadaleta MN. Rat hindlimb unloading: soleus and extensor digitorum longus histochemistry, mitochondrial DNA content and mitochondrial DNA deletions. Biosci Rep. 2002;22:115-125.[Medline]
  50. Lezza AMS, Cormio A, Gerardi P, et al. Mitochondrial DNA deletions in the oculopharyngeal muscular dystrophy. FEBS Lett. 1997;418:167-170.[Medline]
  51. Rubinstein NA, Kelly AM. Development of muscle fiber specialization in the rat hindlimb. J Cell Biol. 1981;90:128-144.[Abstract/Free Full Text]
  52. Bua EA, McKiernan SH, Wanagat J, McKenzie D, Aiken JM. Mitochondrial abnormalities are more frequent in muscles undergoing sarcopenia. J Appl Physiol. 2002;92:2617-2624.[Abstract/Free Full Text]
  53. Sanders Williams R. Mitochondrial gene expression in mammalian striated muscle. J Biol Chem. 1986;261:12390-12394.[Abstract/Free Full Text]
  54. He L, Chinnery PF, Durham SE, et al. Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR. Nucleic Acids Res. 2002;30:e68.[Abstract/Free Full Text]
  55. Armstrong RB, Phelps RO. Muscle fiber type composition of the rat hindlimb. Am J Anatomy. 1984;171:259-272.[Medline]
  56. Delp MD, Duan C. Composition and size of type I, IIA, IID/X, and IIB fibers and citrate synthase activity of rat muscle. J Appl Physiol. 1996;80:261-270.[Abstract/Free Full Text]
  57. Staron RS, Kraemer WJ, Hikida RS, Fry AC, Murray JD, Campos GER. Fiber type composition of four hindlimb muscles of adult Fisher 344 rats. Histochem Cell Biol. 1999;111:117-123.[Medline]
  58. Moura IMW, Dos Santos FF, Moura JAA, Curi R, Fernandes LC. Creatine supplementation induces alteration in cross-sectional area in skeletal muscle fibers of Wistar rats after swimming training. J Sports Sci Med. 2002;1:87-95.
  59. Poulton J, Morten K, Freeman-Emmerson C, et al. Deficiency of the human mitochondrial transcription factor h-mtTFA in infantile mitochondrial myopathy is associated with mtDNA depletion. Hum Mol Genet. 1994;3:1763-1769.[Abstract/Free Full Text]
  60. Ekstrand MI, Falkenberg M, Rantanen A, et al. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum Mol Genet. 2004;13:935-944.[Abstract/Free Full Text]
  61. Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem. 2000;275:3343-3347.[Abstract/Free Full Text]
  62. Yarovaya NO, Kramarova L, Borg J, Kovalenko SA, Caragounis A, Linnane AW. Age-related atrophy of rat soleus muscle is accompanied by changes in fibre type composition, bioenergy decline and mtDNA rearrangements. Biogerontology. 2002;3:25-27.[Medline]




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