Rat Hindlimb Unloading: Soleus and Extensor Digitorum. Longus Histochemistry, Mitochondrial DNA Content and Mitochondrial DNA Deletions. V. Pesce,1 A.
Bioscience Reports, Vol. 22, No. 1, February 2002 ( 2002)
MINI REVIEW
Rat Hindlimb Unloading: Soleus and Extensor Digitorum Longus Histochemistry, Mitochondrial DNA Content and Mitochondrial DNA Deletions V. Pesce,1 A. Cormio, F. Fracasso,1 A. M. S. Lezza,1, P. Cantatore1,2 and M. N. Gadaleta1,2,3 Receiûed Noûember 8, 2001 Mitochondrial phenotypic alterations, mitochondrial DNA content and mitochondrial DNA deletions in a slow, Soleus, and a fast, Extensor Digitorum Longus, skeletal muscle of 3- and 15-month-old hindlimb suspended rats have been studied. Cytochrome c oxidasenegative fibers appeared after unloading in all examined animals and their percentage increased with increasing unloading time. After 14 days of suspension the mitochondrial DNA content did not change in 3-month-old but decreased significantly in 15-month-old rats. Soleus was much more affected by unloading than Extensor Digitorum Longus. The mitochondrial DNA deletion of 4834 bp as well as other mtDNA deletions, researched with Long Distance-PCR, were absent in both studied muscles before and after unloading. KEY WORDS: Mitochondrial DNA; COX-negative fibers; skeletal muscles; hindlimb unloading.
INTRODUCTION It has been reported that, in both human and rodent, deficits of motor capacity, strength and endurance properties of skeletal muscle are induced by space flight (SF). The reduced muscle strength is associated, in part, with a reduction in muscle mass as reflected in smaller cross-sectional areas of both fast- and slow-twitch fibers. It seems that slow-twitch fibers exposed to microgravity are more sensitive to the atrophying process than fast-twitch ones. Accompanying the atrophy is a transformation of the fibers phenotype from slow to fast involving myosin heavy chain and sarcoplasmic reticulum protein isoforms [1]. It is known that slow fibers are characterized by a higher number and more differentiated mitochondria than fast fibers [2, 3]. Mitochondria are the major source of energy in the cell because they contain the oxidative phosphorylation system (OXPHOS) responsible for the maximum output of ATP. Mitochondria contain their own DNA whose genes encode components 1
Department of Biochemistry and Molecular Biology, University of Bari, via Orabona, 4, 70125, Bari, Italy. 2 Center for the Study of Mitochondria and Energetic Metabolism, Bari, Italy. 3 To whom correspondence should be addressed. Fax: 39-080-5443403; email: m.n.gadaletea@biologia. uniba.it 115 0144-8463兾02兾0200-0115兾0 2002 Plenum Publishing Corporation
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of the OXPHOS system. The expression of mitochondrial DNA (mtDNA) is therefore absolutely critical for the function of those tissues that are highly dependent on aerobic metabolism, such as skeletal muscle and heart [4]. Morphological and functional data were reported on rat and human skeletal muscle mitochondria in microgravity [3]. Oxidative enzymes were reported to be unaltered or slightly elevated both in the slow antigravity muscle, Soleus, as in the fast muscle, Extensor Digitorum Longus (EDL) [2, 3]. However, a loss of oxidative enzymes and of mitochondria from the subsarcolemmal and intermyofibrillar regions of the fiber with SF in the Soleus was reported [5]. Microgravity significantly reduced (41%) cytochrome c oxidase (COX) activity in skeletal muscle triceps brachii although mRNA levels of nuclear and mitochondrial encoded subunits remained unaltered [6]. A loss of mitochondrial proteins with SF has been reported in rats, although at a lower degree than that observed for the contractile proteins and for cell atrophy [3]. No data have been reported until now on mitochondrial biogenesis and on mtDNA content in microgravity. In this paper we used histochemical and PCR techniques to follow phenotypic and genotypic alterations of mitochondria as well as mitochondrial DNA content in a slow, Soleus, and a fast, EDL, skeletal muscle of 3- and 15-month-old hindlimb rats suspended up to 14 days. METHODS Animal Groups Rats were suspended by tail harnessing and housed separately in plastic box cages. The temporal changes during suspension were studied by killing the animals after 3, 11 and 14 days of suspension. Suspended rats were fed Purina chow and water ad libitum, and the control animals were pair fed. Experimental rats were suspended by their tails using a tail harness that consisted of a triangular shaped wire (18 gauge) sandwiched between two layers of vinyl cloth glued with Dural contact cement to the dorsal proximal four-fifths of the tail. The tail was washed, dried, and coated with cement before applying the cloth strips. The harness was further strengthened by loosely wrapping the tail with vinyl strips and elastic tape (Medipore-3M). This method of harnessing distributed the load along the length of the tail, avoiding excessive tension on a small area. Rats were suspended according to the non-invasive procedure of Morey-Holton [7]. Daily health checks confirmed that the exposed tip of the tail remained pink, indicating adequate blood flow. A fish swivel was attached to the exposed apex of the wire triangle, and fishing line was tied to the swivel to elevate the rat’s hindquarters, unloading the hindlimbs. The height of suspension was adjusted so that the hind feed just cleared the grid floor. The forelimbs maintained contact with the floor, thus allowing the animals access to food and water. Histochemistry Serial eight-microns-thick transverse sections from frozen muscle biopsies were cut with the cryotome (HM 505 E-Microm), mounted on polylysine-coated glass
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slides and stained for COX activity [8], for SDH activity [9] and for both activities. 250 to 500 fibers for each muscle sample were analyzed. 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. The modified SDH reaction was chosen to count the number of RRF because it was more sensitive than the modified Gomori trichrome staining [10]. Sections were viewed on a Zeiss transmitted light microscope and photographed using a 10× objective. In each specimen the total number of fibers in a representative selected field was counted on a photomicrograph and the percentage of those having no detectable COX activity (COXnegative), normal COX activity (COX-positive), normal SDH activity (SDHn) and hyperreactive SDH activity (RRF) was calculated.
Determination of mtDNA Content in Skeletal Muscle Biopsies Total DNA was prepared from about 40–50 mg of skeletal muscle as described by Arnaudo et al. [11] and suspended in 30 µl of water. 5 µg of total DNA were digested with Pvu II (MBI-Fermentas) and run on a 0.35% agarose gel (Seakem Gold, FMC). The gel was blotted onto a Hybond-N membrane (Amersham-Pharmacia) and simultaneously hybridized with a mitochondrial and a nuclear probe. The mitochondrial probe was a 657 bp fragment obtained by PCR using the primers DN (L 15758–15777) and DR (G 117–98). PCR reaction contained 0.1 µM of each primer, 100 ng DNA, 200 µM dNTPs, 2.5 U Taq Polymerase (Roche Mol. Biochem., Germany), 1.5 mM MgCl2 in a 100 µl volume of 1× reaction buffer. PCR conditions were: denaturation of 94°C for 1 min, annealing at 51°C for 1 min and extension at 72°C for 1 min for 25 cycles. The nuclear probe was a 413 bp fragment containing part of the 18S rRNA gene and subcloned in the TA vector (Clontech). Both probes were labeled by random priming (Random Primed DNA Labeling Kit, Roche Mol. Biochem., Germany) 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 et al. [12]. The filter was exposed to a X-ray film at −70°C with an intensifying screen and the hybridization signals were quantified by densitometry with a LKB-Pharmacia Ultrascan-XL Laser densitometer equipped with a GelScan-LX-Evaluation software. Detection of Mitochondrial DNA Deletion of 4834 bp (mtDNA4834) About 50 mg of skeletal muscle were used to extract nucleic acids enriched for mtDNA [13]. The DNA pellet was suspended in 50 µl of water and stored at 4°C. 100 ng of DNA were amplified as described elsewhere [14] by using the following primers: 7825–For (L 7825-7844) and 13117 Rev (H 13117-13099) for mtDNA4834 (460 bp product, annealing temperature 60°C). The initial PCR reaction contained 0.1 µM of each primer, 200 µM dNTPs, 100 ng DNA and 2.5 U Taq polymerase (Roche Mol. Biochem., Germany) in a 100 µl volume of 1Breaction buffer. The PCR conditions consisted of denaturation at 94°C for 1 min, annealing at 60°C for
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1 min and extension at 72°C for 1 min in a Peltier Thermal Cycler (MJ Research) for 35 cycles. A secondary seminested PCR round, using one of the primers of the first amplification (13117 Rev) and a new internal primer 7978 For (L 7978–7997) with a 1 µl aliquot of the first amplification mixture diluted 1:100 at an annealing temperature of 60°C, was performed to obtain a 309 bp product. PCR products were size-fractionated on a 1.5% agarose gel (Amresco) 1BTBE buffer and visualized by ethidium bromide staining. Only those subjects not showing the expected amplification products in two successive PCR rounds were considered not containing the searched mtDNA deletions. Rat mtDNA nucleotide positions are according to Gadaleta et al. [15]. The identity of the amplification products was confirmed by direct DNA sequencing as described elsewhere [16].
Long Distance-PCR Primers used were 5881-For (L 5881–5906) and 15141-Rev (H 15141–15118). Gene Amp carry-over prevention kit (Perkin Elmer–Norwalk) was used. The PCR mixture contained 100 ng of DNA, 0.1 µM of each primer, 200 µM dNTP (with dUTP instead of dTTP), 2.5 mM MgCl2 and 2.5 U of Long Amplification Taq Polymerase (Takara—Japan) in a 100 µl reaction. PCR products were separated on a 1.5% agarose gel (Amresco—OH, USA) in 1BTBE buffer and visualized by ethidium bromide staining. To exclude that the low molecular weight bands were due to PCR artefacts, a secondary seminested LD-PCR round, very similar to the first, was performed on an aliquot of the first reaction mixture added after a 2 min at 50°C and 10 min at 94°C step, employing the 15118-Rev primer and a new internal primer 5974-For (L 5974–5996). In all analyzed cases we obtained a pattern consistent with that observed in the first PCR round (data not shown).
Statistics Statistical analysis was carried out by using StatView SECversion 1.03 software (Abacus Concepts, Berkeley, CA).
RESULTS Atrophy Suspension caused a progressive loss of mass of the two studied muscles (wet wt). In Fig. 1A the loss of mass of the Soleus and EDL in 15-month-old animals is reported. The loss of mass was 39% in Soleus and 27% in EDL. In Fig. 1B the ratio of muscle weight (mg) to total body weight (g) is reported. The values of this ratio were for the Soleus and the EDL muscles of 15-month-old animals, respectively, 0.45J0.02 (mean JSD) and 0.51J0.02 (mean JSD) in control rats and 0.34J0.2 (mean JSD) and 0.45J0.02 (mean JSD) after 14 days of unloading. Therefore, the Soleus lost 24% and the EDL 12% of their mass after 14 days of unloading.
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Fig. 1. (A): Relationship between mean Soleus and EDL mass (mg) and duration of hindlimb rat suspension. (B) Relationship between the ratio of mean Soleus and EDL mass (mg) and mean body weight (g) and the duration of hindlimb suspension. The number of 15-month-old used animals was: 11 controls, 5 hindlimb suspended for 3 days, 3 for 11 days and 3 for 14 days. Each point represents the mean JSD. *G Significantly different from control ( pF0.05).
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Histochemistry Staining for COX and SDH activities to identify COX-negative fibers and RRF as well as double staining to distinguish two subtypes of RRF, COX-positive RRF and COX-negative RRF, in 3- and 15-month-old rats were performed on Soleus and EDL skeletal muscles. COX-negative fibers were absent in 3-month-old and present in 50% of 15-month-old control rats whereas, after unloading, they appeared in all examined animals. In Fig. 2 the mean percentage of COX-negative fibers, respectively, in Soleus and EDL skeletal muscles of 3-month-old rats, unloaded for 14 days, is reported. After 14 days hindlimb suspension of 3-month-old rats, 0.4% COXnegative fibers in Soleus muscle and 0.2% in EDL, respectively, have been found. In Fig. 3 the mean percentage of COX-negative fibers, respectively, in Soleus and EDL skeletal muscles of 15-month-old rats, unloaded for 3, 11 and 14 days, is reported. COX-negative fibers appeared 3 days after suspension in both muscles of all analyzed rats and their percentage increased after 11 days of suspension. After 14 days of suspension the Soleus muscle showed fibers disorganization whereas a three-fold increase of the COX-negative fibers percentage was found in EDL muscle. RRF were not found in 3- and 15-month-old control and unloaded rats. Mitochondrial DNA Content In order to investigate whether a variation of mtDNA copy number occurred in the two rat skeletal muscles after unloading, we measured the mtDNA兾nDNA ratio in 3-month-old and 15-month-old rats, by hybridizing Pvu II-digested total DNA extracted from Soleus and from EDL muscles with a mitochondrial and nuclear probe. The mitochondrial probe detected a band of 16.2 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 (data not shown). The ratio of the intensities of the two bands was used to estimate the relative amount of mtDNA in each subject. No change of the mtDNA兾nDNA ratio was found in 3
Fig. 2. Percentage of COX-negative fibers in the skeletal muscle of 3-month-old rats after 14 days of unloading. Three controls and 3 unloaded rats were used. Values represent the mean JSE of the percentages of COX-negative fibers.
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Fig. 3. Percentage of COX-negative fibers in the skeletal muscle of 15-month-old control rats and rats after 3, 11 and 14 days of unloading. The number of animals used was the same as in Fig. 1. #G Morphological disorganization of the skeletal muscle. Values represent the mean JSE of the percentages of COX-negative fibers, calculated by excluding, in the controls, animals without COX-negative fibers. Significant differences in the percentage of COX-negative fibers (Student’s t-test) were found for the comparisons between the values of control rats and rats after 11 days of unloading for Soleus and between those of control rats and rats after 14 days of unloading for EDL ( pF0.05).
months old rats after 14 days of suspension (data not reported). However, the mtDNA content increased after 3 days of unloading in the Soleus muscle of 15month-old rats and then significantly decreased reaching 40% of the control value after 14 days of suspension (Fig. 4A). The mtDNA content of the EDL muscle was also reduced after 14 days of unloading although at a lower extent (Fig. 4B). MtDNA Deletions Soleus and EDL muscle samples were screened by two PCR rounds with nested primers for the presence of the mtDNA4834 in 3- and 15-month-old rats. Although it was found in 28-month-old rats (A. Cormio, personal communication), such deletion was absent both in 3- and 15-month-old control and unloaded animals here studied (results not shown). In order to obtain a more comprehensive representation of mtDNA deletions in each animal we used LD-PCR. With this technique, which revealed in 28-month-old rats 4 to 8 bands on the gel, corresponding to subgenomic mtDNA particles (A. Cormio, personal communication), no mtDNA deletions at all were found here in 3- and 15-month-old rats, both before and after unloading (results not shown). DISCUSSION Mitochondria play a central role in processes such as stress response, metabolic remodeling, apoptosis and aging [17]. After SF some endocrine axes seem to undergo changes resembling those generally described during aging [18, 19]. We reported recently the results of an extensive analysis of age-related mitochondrial phenotypic and genotypic alterations in a large number of skeletal muscle biopsies from healthy
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Fig. 4. Relative mitochondrial DNA content in Soleus and EDL skeletal muscles of 15-month-old control rats and rats after 3, 11 and 14 days of hindlimb suspension. The relative mtDNA content was obtained from the ratio of signals intensities corresponding to mtDNA and nDNA bands and determined in 11 control rats, 5 rats after 3 days, 3 after 11 days and 3 after 14 days of hindlimb suspension. The relative mtDNA content was normalized with respect to the control rats value. Data are the mean JSD of two experiments. *GStatistically significant decrease of mtDNA content in Soleus muscle after 14 days of hindlimb suspension.
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human individuals. We were able to show that, in individuals older than 50 years of age, the appearance of COX-positive RRF and, thereafter, of COX-negative RRF, the increase of the mtDNA4977 and the increase of mtDNA deleted species were accompanied by the increase of mtDNA content as an attempt of the cell to counteract the age-related mitochondrial dysfunction [14]. Consistent with such a compensatory response was, after 50 years of age, the increased level of nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (Tfam) [20]. These results prompted us to verify in two rat skeletal muscles, known to have a different fibers composition, namely Soleus and EDL, and in microgravity conditions, known to induce atrophy of slow fibers and兾or transformation of slow to fast-twitch fibers [1–3], the involvement of mitochondria and of their genetic system. Here we report, for the first time, that microgravity induces the appearance of COX-negative fibers in Soleus and EDL skeletal muscles of young and adult rats and a depletion of mtDNA in adult rats. Moreover, no RRF and no mtDNA deletions in both groups of rats, before and after unloading, were found. A 41% reduction of COX activity was already reported in the skeletal muscle triceps brachii after 6 days of microgravity with no change of mRNA for some mtDNA and nDNA-encoded COX subunits. The Authors suggested that protein degradation was responsible for the reduced COX activity measured [6]. These results were obtained by measuring COX activity in tissue homogenate so that if the COX deficiency was equally distributed in all fibers was not clear. The histochemical results here reported show that the deficiency of COX activity affects in a mosaiclike fashion both the Soleus and EDL muscles of all young and adult examined rats after 3, 11 and 14 days of unloading. These data, together with the high degree of mtDNA depletion after 14 days of unloading in adult rats, might suggest that the COX deficiency is due to a selective mtDNA depletion in affected fibers. However, the values of relative mtDNA content here reported have been obtained from whole tissues extracts so that it is impossible to specify if unloading affects the relative mtDNA content (a) of all fibers of the tissue, (b) of COX-negative fibers only, (c) of a higher number of fibers than that reported in (b) i.e. fibers with defective Complex I that, in our case, might appear as COX-positive. Complex I defects have been reported in unloading rats [21]. In fact, in the case of mtDNA depletion all complexes of the OXPHOS system, except Complex II, which has only nDNA-encoded polypeptides, should be affected. Therefore, the COX-negative fibers found in the skeletal muscles here analyzed might be fibers defective in Complex IV or fibers in which all the OXPHOS system is altered. Reduced mtDNA availability has been reported to be a limiting factor for mitochondrial gene expression in type II but not in type I fibers [22, 23]. On the other hand, an increased oxidative capacity, following exercise training or chronic electrical stimulation [22, 23], was primarily associated, at molecular level, with increased mitochondrial DNA content in glycolytic type II fibers and with increased mitochondrial transcripts in highly oxidative type I muscle fibers [24]. Unstimulated fibers, due to hindlimb suspension of rat, might lose signals inducing mtDNA replication and mitochondrial biogenesis and兾or receive apoptotic signals. No fibers with hyperproliferation of mitochondria (RRF) have been found in Soleus and EDL muscles before and after unloading. The absence of mtDNA
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deletions in the presence of COX-negative fibers reported in unloading rats suggests that mtDNA replication is impaired. This is in agreement with the decrease of mtDNA content after loading here reported and with the results already reported by Riley et al. [5] who found that in Soleus, after 14 days of unloading, SDH activity, measured as marker of the respiratory chain activity and presence of mitochondria, was reduced and type I and IIa fibers decreased in area of 63% and 43%, respectively; both subsarcolemmal and intermyofibrillar mitochondria were degraded, with the first ones being degraded more rapidly. The same Authors reported a much less severe atrophy in EDL [5], in good agreement with the data here reported. Reduced ATP availability, because of OXPHOS system dysfunction, can interfere with nuclear gene expression and cause a decreased synthesis of myofibrillar proteins leading to a decrease in sarcomers number resulting in fiber atrophy [19, 25, 26]. An increased degradation of myonuclei has been reported in Soleus after unloading suggesting the activation of cellular apoptotic pathways. Apoptosis has also been hypothesized as a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting [27]. More detailed analyzes at molecular level and shorter times treatments are necessary to understand which is the mitochondrial role in triggering the fiber type change and兾or the atrophy in skeletal muscles exposed to microgravity [28]. ACKNOWLEDGMENTS We are grateful to Dr. Anna De Marzo and Adriana Di Benedetto for technical assistance and Ms. R. Longo for word processing. This work was supported by Italian Space Agency (ASI) grants, Contract No. I兾R.168兾00 and Sigma Tay—Industrie Farmaceutiche Riunite S.p.A. REFERENCES 1. Baldwin, K. M. (1996) Effect of spaceflight on the functional, biochemical, and metabolic properties of skeletal muscle. Med. Sci. Sports Exerc. 28:983–987. 2. Edgerton, V. R. and Roy, R. R. (1996) Neuromuscular adaptations to actual and simulated spaceflight. In: Handbook of Physiology, Enûironmental Physiology. Bethesda, MD: Am. Physiol. Soc., sec. 4, Vol. II, 32:721–763. 3. Fitts, R. H., Riley, D. R., and Widrick, J. J. (2000) Microgravity and skeletal muscle. J. Appl. Physiol. 89:823–839. 4. Wallace, D. C. (1999) Mitochondrial diseases in man and mouse. Science 283:1482–1488. 5. Riley, D. A., Slocum, G. R., Bain, J. L. W., Sedlak, F. R., Sowa, T. E., and Mellender, J. W. (1990) Rat hindlimb unloading: soleus histochemistry, ultrastructure, and electromyography. J. Appl. Physiol. 69:58–66. 6. Connor, M. K. and Hood, D. A. (1998) Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles. J. Appl. Physiol. 84:593–598. 7. Morey-Holton, E. R. and Globus, R. K. (1998) Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight. Bone 22:83S–88S. 8. Seligman, A. M., Karnovsky, M. J., Wasserkrug, H. L., and Hanker, J. W. V. (1968) Non-droplet ultrastructural demonstration of cytochrome c oxidase activity with a polymerising osmiophilic reagent diaminobenzidine (DAB). J. Cell. Biol. 38:1–14. 9. Dubowitz, U. K. and Brooke, M. H. eds. (1973) Muscle Biopsy: A Modern Approach. Saunders, W.B.: Philadelphia, PA, USA.
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