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Mitochondrial Enzyme Defects in Normal and Low-Frequency-Stimulated Muscles of Young and Aging Rats

Dejan korjanc^a, Georg Dünstl^a and Dirk Pette^a

^a Department of Biology, University of Konstanz, Germany

Dirk Pette, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany E-mail: dirk.pette{at}uni-konstanz.de.

Decision Editor: John A. Faulkner, PhD

	Abstract

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The age-related increase in cytochrome c-oxidase-deficient (COX^-) muscle fibers has been suggested to be positively correlated with mitochondrial content (Müller-Höcker, Brain Pathol. 1992; 2:149–158). As a way to test this relationship, tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway rats were exposed to chronic low-frequency stimulation (CLFS) for 50 days, an experimental protocol known to induce marked increases in mitochondrial content. CLFS produced elevated activity levels of COX and succinate dehydrogenase (SDH) in most fibers of young and aging rats. Some fibers low or deficient in COX and a few fibers low or deficient both in COX and SDH (COX^-/SDH^-) were detected in unstimulated muscles of young and, more frequently, aging rats. According to their myosin complement, these fibers were immunohistochemically identified as type I fibers. CLFS increased their number in young muscles, but reduced it in aging muscles. Stimulated aging muscles contained some very small, most likely newly formed COX⁺ and SDH⁺ type I fibers. Thus, the fraction of COX^- fibers was reduced in aging muscle by enhanced contractile activity.

ALTERATIONS of the mitochondrial genome are thought to substantially contribute to age-associated degenerative changes of muscle tissue. This assumption is based on the observation that mitochondrial DNA (mtDNA) of aging muscle contains increasing amounts of point and/or deletion mutations (see, e.g., ^{(1)(2)(3)(4)(5)(6)(7)(8)}). Mitochondrial DNA mutations most likely result from the action of free radicals generated as by-products in the respiratory chain. As a result of increasing amounts of mutated mtDNA, aging cardiac and skeletal muscles contain considerable amounts of muscle fibers deficient in mitochondrially encoded proteins. A well-established example is the age-related increase in cardiomyocytes and muscle fibers deficient in cytochrome c-oxidase (COX^-). Interestingly, COX^- fibers and cells are more numerous in muscles abundant in mitochondria (e.g., extraocular and cardiac muscles) than in muscles with low mitochondrial content ⁽⁷⁾⁽⁹⁾.

The observation that muscles of the so-called oxidative type contain more COX^- fibers than muscles low in mitochondria prompted us to test the suggested relationship between mitochondrial content and frequency of COX^- fibers in a direct experimental approach. Assuming that an experimentally induced increase in mitochondrial content would augment the frequency of COX^- fibers, we exposed fast-twitch muscles of young and aging Brown Norway (BN) rats to chronic low-frequency stimulation (CLFS). We were interested whether or not, and to what extent, this treatment affected the number of COX^- fibers. In view of our hypothesis that myosin isoform expression is under control of the overall cellular energy potential ^(10)(11), we were interested in investigating the myosin complement of COX-deficient fibers. According to this hypothesis, a lowered energy potential by an impairment of mitochondrial respiration might induce fast-to-slow transitions in myosin isoform expression.

The CLFS of fast-twitch muscle was chosen as an experimental model, because this treatment markedly increases the mitochondrial content ^(12)(13)(14) and has also been shown to produce similar increases in key enzymes of mitochondrial energy metabolism in fast-twitch muscles of young and aging BN rats ⁽¹⁵⁾. Therefore, we studied the distribution of COX^- fibers in chronically stimulated muscles by using the same experimental conditions as in our previous study ⁽¹⁵⁾. Fast-twitch tibialis anterior (TA) muscles of 15-week-old (young) and 101-week-old (aging) BN rats were exposed to CLFS for 50 days. Cross sections of stimulated and contralateral (unstimulated) TA muscles were analyzed for mitochondrial enzyme deficiencies by histochemical assays for COX and succinate dehydrogenase (SDH) activity. The fiber-type distribution of enzyme deficiencies was assessed immunohistochemically in serial sections by using monoclonal antibodies against specific myosin heavy chain (MHC) isoforms. Computer-assisted image analysis was applied to quantitatively evaluate the percentages of COX-deficient and SDH-deficient fibers, as well as changes in fiber size and fiber-type distribution.

	Methods

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Animals, Low-Frequency Stimulation, and Muscles
The experiments were performed on 5 young (15 weeks) and 5 aging (101 weeks) male rats of the BN strain (supplied by the TNO Prevention and Health Center for Ageing Research, Leiden, the Netherlands). CLFS was performed with electrodes implanted laterally to the peroneal nerve of the left hind limb, using a portable stimulator fixed to the animal's shoulder. Stimulation at a frequency of 10 Hz was applied 10 hours per day ⁽¹⁶⁾. After 50 days, the animals were euthanized and TA muscles from stimulated (left) and contralateral (right) hind limbs were excised, blotted, and weighed (Table 1 ). The slightly stretched muscles were immersed in melting isopentane (-159°C) and kept at -80°C until they were analyzed.

As a way to delineate type I fibers expressing slow myosin, two monoclonal antibodies were used—the NOQ7.5.4D ⁽¹⁹⁾ and the 7HCs15 ⁽²⁰⁾. Type IIA fibers were identified by a monoclonal antibody (7HCS11) that recognizes myosin heavy chains MHCIIa and MHCI (S. Düsterhöft, I. Ruhdel, and D. Pette, unpublished data, 1992). For immunohistochemistry, 9-µm-thick frozen sections were air dried, washed three times in phosphate-buffered saline (PBS), and incubated for 30 minutes in 3% H₂O₂ in methanol. The sections were washed again (10 minutes, three times) and incubated for 1 hour in a blocking solution (1% bovine serum albumin, 10% horse serum in PBS; pH 7.4). Excess blocking solution was removed and the primary monoclonal antibody overlaid and incubated for 60 minutes at room temperature in a moist chamber. Primary antimouse IgG monoclonal antibodies were diluted in blocking solution as follows: NOQ7.5.4D, 1:400; 7HCS15, 1:40; and SC-71, 1:1000. Sections were washed once for 10 minutes in blocking solution and reacted for 30 minutes with biotinylated horse-antimouse IgG (1:200 diluted) as secondary antibody. After they were washed in PBS, sections were incubated with VECTASTAIN ABC reagent (Vector Laboratories, Burlingame, CA) for 30 minutes, washed three times for 5 minutes in PBS, and reacted for 6 minutes with a peroxidase substrate solution (VECTOR, DAB substrate kit for peroxidase, Vector Laboratories). The reaction was stopped by several washes with distilled water. Sections were dehydrated, cleared, and mounted in Entellan (Merck, Darmstadt, Germany).

Microphotometric Image Analysis
Qualitative enzyme histochemistry and immunohistochemistry was performed on serial cross sections to determine fiber-type distribution of COX and/or SDH deficiencies as well as changes in fiber type and fiber size. A previously described computer-assisted image analysis system was used for quantitative evaluation of the staining reactions ⁽²¹⁾. Reactions for COX and SDH, and immunohistochemical stainings for MHCI and MHCIIa were analyzed in 20 TA muscles from 10 rats. Fibers were examined at 400-fold magnification on the computer screen (10-fold primary magnification). In each section, 6–10 fields with approximately 60 fibers were evaluated. A total of 9254 fibers were analyzed, namely 1844 in contralateral and 1824 in stimulated TA muscles of young rats, and 2688 in contralateral and 2898 fibers in aging rats. On the average, 509 ± 157 (SD) fibers were evaluated per muscle.

As judged by visual inspection, only fibers unambiguously stained or unstained were included in our study. According to the histochemically assessed activities of the two enzymes, the following fiber types were distinguished: (a) COX positive, SDH positive (COX⁺/SDH⁺); (b) COX negative, SDH positive (COX^-/SDH⁺); and (c) COX negative, SDH negative (COX^-/SDH^-). The data were expressed as number of fibers per square millimeter or as percentage of investigated fibers.

Statistical Analysis
All results are given as means ± SE. A Student's t test was used to determine if differences existed between contralateral and stimulated muscles in young rats and aging rats, as well as between contralateral muscles from young and aging rats or stimulated muscles from young and aging rats. The level of significance was set at p < .05.

	Results

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Effects of CLFS on Body and Muscle Weights
On the average, body weight of the young animals increased by 23% during the stimulation period. Conversely, body weight of the aging rats decreased by 7% during CLFS. A similar decrease in body weight of aging rats exposed to the same stimulation protocol was observed in our previous study ⁽¹⁵⁾. CLFS led to similar reductions in muscle weight in young (-11%) and aging (-8%) rats.

Effects of CLFS on Fiber Types
Type IIB and IID fibers contain low COX and SDH activities. Deficiencies in COX or SDH activity would have been difficult to detect in these fibers by qualitative enzyme histochemistry. Therefore, type IIB and IID fibers were not included in the present study. The identification of fibers with enzyme defects was restricted to immunohistochemically identified type I and type IIA fibers. Unstimulated TA muscles from young and aging rats contained similar percentages of type I and type IIA fibers (Fig. 1 and Fig. 2). CLFS increased the percentage of type IIA fibers in young (from 40% to 51%) and aging (from 38% to 47%) rats (Fig. 1). Small increases in type I fibers were observed in young and aging rats, but these changes were not significant (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Figure 1. Percentage of type IIA fibers in the deep portion of control and 50-day stimulated tibialis anterior muscles of young (22 wk) and aging (108 wk) Brown Norway rats. Values are means ± SE. *p < .05; ***p < .001.

View larger version (24K): [in this window] [in a new window]   Figure 2. Percentage of type I fibers in the deep portion of control and 50-day stimulated tibialis anterior muscles of young (22 wk) and aging (108 wk) Brown Norway rats. Values are means ± SE.

View larger version (118K): [in this window] [in a new window]   Figure 3. Serial sections of the deep portion of a control tibialis anterior muscle from a young (22 wk) Brown Norway rat histochemically stained for cytochrome c-oxidase (COX) and succinate dehydrogenase (SDH) activities or immunohistochemically reacted with monoclonal antibodies specific to myosin heavy chain (MHC)I or MHCI + MHCIIa. * = type I fibers; = type IIA fibers. Bar is 100 µm.

View larger version (134K): [in this window] [in a new window]   Figure 4. Serial sections of the deep portion of a 50-day stimulated tibialis anterior muscle from a young (22 wk) Brown Norway rat stained for cytochrome c-oxidase (COX) and succinate dehydrogenase (SDH) activities or reacted with monoclonal antibodies specific for myosin heavy chain (MHC)I or MHCI + MHCIIa. * = type I fibers; = type IIA fibers. Bar is 100 µm.

View larger version (23K): [in this window] [in a new window]   Figure 5. Percentage distribution of COX+/SDH+, COX-/SDH+, and COX-/SDH- type I fibers in the deep portions of control and 50-day stimulated tibialis anterior muscles from young (22 wk) Brown Norway rats. Values are means ± SE (n = 5 for each condition). COX = cytochrome c-oxidase; SDH = succinate dehydrogenese. *p < .05; **p < .01.

View larger version (22K): [in this window] [in a new window]   Figure 6. Percentage distribution of COX+/SDH+, COX-/SDH+, and COX-/SDH- type I fibers in the deep portions of control and 50-day stimulated tibialis anterior muscles from aging (108 wk) Brown Norway rats. Values are means ± SE (n = 5 for each condition). COX = cytochrome c-oxidase; SDH = succinate dehydrogenase. *p < .05; ***p < .001.

The effects of CLFS in muscles of aging rats differed markedly from those in young muscles. Contrary to stimulated young muscles, CLFS led to a marked decrease of the COX^-/SDH⁺ type I fibers (from 4.0% to 0.9%) in muscles of aging rats (Table 2 and Table 3 ; Fig. 6). COX^-/SDH^- fibers, however, increased from 1.2% to 2.3%. As a result, the percentage of COX⁺/SDH⁺ type I fibers was approximately twofold higher in stimulated than in control muscles of aging rats.

An interesting finding was the appearance of small type I fibers in stimulated muscles, especially of aging rats. These small type I fibers were all COX⁺/SDH⁺ (Fig. 7). According to a morphometric analysis of the changes in fiber size, stimulated muscles displayed a shift of the cross-sectional areas toward smaller values. This was more pronounced in the stimulated muscles of aging rats than in those of young rats (Fig. 8). In addition, COX⁺/SDH⁺ type I fibers with cross-sectional areas <800 µm were more numerous (approximately twofold) in stimulated TA muscles of aging rats than in those of young rats.

View larger version (128K): [in this window] [in a new window]   Figure 7. Serial sections of the deep portion of a 50-day stimulated tibialis anterior muscle from an aging (108 wk) Brown Norway rat stained for cytochrome c-oxidase (COX) and succinate dehydrogenase (SDH) activities or reacted with monoclonal antibodies specific to myosin heavy chain (MHC)I or MHCI + MHCIIa. * = type I fibers low in or without detectable COX and SDH activities; = small COX+/SDH+ type I fibers; = type IIA fibers. Bar is 100 µm.

View larger version (28K): [in this window] [in a new window]   Figure 8. Histograms of type I fiber sizes in the deep portions of control and 50-day stimulated tibialis anterior muscles from young and aging Brown Norway rats.

	Discussion

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Confirming previous studies ⁽⁷⁾⁽⁹⁾, we show that TA muscles of young rats contain a minor fraction of COX^- fibers that increases with age. In addition, we show that young and aging muscles contain a few fibers low or deficient both in COX and SDH activity. To our knowledge, such fibers have yet to be described. Their appearance is not easy to explain because SDH is nuclear encoded, whereas COX is a product of both the nuclear and mitochondrial genomes. A combination of SDH and COX deficiency might, therefore, result from (a) an impaired function of the nuclear genome, (b) an impaired function of nuclear and mitochondrial genomes, and/or (c) a disruption of their coordinated functions. Another possible explanation could be an impaired mitochondrial import of extramitochondrially synthesized peptides of the two enzymes.

CLFS produces a conspicuous increase of COX^-, as well as of COX^-/SDH^- type I fibers in young muscle. Collectively, the percentage of these two fiber populations increases from 2.7% in the control to 5.4% in the stimulated muscles of young rats. This finding is in support of the assumption underlying this study, namely that an elevation in mitochondrial content leads to an increase in fibers with impaired respiratory chain function.

The data on COX^- and COX^-/SDH^- fibers in stimulated muscles of aging rats are opposite to the CLFS-induced changes in young muscle: CLFS decreases the number and percentage of COX^- fibers below control values (Table 2 and Table 3 ). Enhanced contractile activity, therefore, appears to be beneficial to aging muscle. In contrast, CLFS increases the number of COX^-/SDH^- fibers. When COX^- and COX^-/SDH^- fibers are combined, their overall percentage amounts to 3.2% in stimulated aging muscles as compared with 5.2% in the controls. At the level of absolute fiber numbers, the percentage of COX^- and COX^-/SDH^- fibers yields slightly higher values (Table 3 ). This difference may be due to the stimulation-induced decrease in fiber size, which causes an increase in total fiber per unit area.

Enhanced contractile activity by CLFS leads to the appearance of very small COX⁺ and SDH⁺ type I fibers, especially in aging muscle. We interpret these fibers as satellite cell-derived myotubes and newly formed fibers. CLFS thus seems to enhance fiber regeneration. As a consequence, the newly formed COX⁺/SDH⁺ type I fibers "dilute" the fraction of COX^- fibers. Some of the latter probably have converted into COX^-/SDH^- fibers, the more advanced stages of fiber lesion. Obviously, the pool of regenerating fibers is small in 50-day stimulated aging muscles. However, fiber regeneration does probably occur throughout the whole stimulation period. Regenerating fibers formed at earlier time points have matured by 50 days and may no longer be delineated by size from normal, surviving fibers. Thus, satellite cell proliferation and increases in satellite cell number have been observed in rat muscles exposed to CLFS for only 5 days ^(22)(23) and were also detected in the muscles under study ⁽²⁴⁾.

A remarkable finding is that regenerating fibers are more numerous in aging than in young muscles. A possible explanation is that CLFS produces more mechanical damage in aging than in young muscle. A higher vulnerability of aging muscle by increased contractile activity as compared with young muscle has been reported in several studies ^(25)(26)(27).

COX^- and COX^-/SDH^- fibers are obviously restricted to the population of type I fibers. A possible explanation of this finding is that type I fibers are more sensitive to free-radical-induced alterations than type II fibers. Another and more plausible explanation is that COX^- and COX^-/SDH^- fibers have undergone fast-to-slow fiber-type transition. This assumption is in agreement with our hypothesis that the expression of MHC isoforms in skeletal muscle is under control of the overall cellular energy potential. Thus, the (ATP)/(ADP_free) ratio (where ATP is adenosine triphosphate and ADP is adenosine diphosphate) is greatly depressed in rabbit muscle undergoing CLFS-induced fast-to-slow transformation ^(11)(28). As shown by single fiber studies, fast-to-slow fiber-type transitions occur in the direction of decreasing (ATP)/(ADP_free) ratios ⁽¹¹⁾. We, therefore, speculate that the observed mitochondrial enzyme defects cause local impairments of oxidative phosphorylation, and thus depressions of the cellular ATP phosphorylation potential, which in turn leads to an upregulation of slow myosin.

In summary, we show that CLFS-induced increases in mitochondrial content of rat TA muscle are accompanied in young animals by increases in COX^- and COX^-/SDH^- type I fibers. Although COX^- and COX^-/SDH^- fibers are more numerous in muscles of aging rats than in those of young rats, CLFS decreases their fraction in aging muscle, most likely by enhancing regeneration of COX⁺ and SDH⁺ fibers.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (Pe 62/25-1).

We thank Ms. Elmi Leisner for excellent technical assistance.

Received January 29, 2001

Accepted July 17, 2001

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