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(The FASEB Journal. 2001;15:322-332.)
© 2001 FASEB

Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia

JONATHAN WANAGAT*,{dagger}, ZHENGJIN CAO{dagger}, PRANALI PATHARE{dagger} and JUDD M. AIKEN{dagger}1

* Medical Scientist Training Program, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA; and
{dagger} Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA

1Correspondence: Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Dr., Madison, WI 53706, USA. E-mail: jma{at}ahabs.wisc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The in vivo cellular impact of age-associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age-related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 x Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid-belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 ± 8 ETS abnormal fibers in the entire 5-month-old rectus femoris to 1094 ± 126 in the 38-month-old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady-state levels of oxidative nucleic damage. These data suggest a causal role for age-associated mitochondrial DNA deletion mutations in sarcopenia.—Wanagat, J., Cao, Z., Pathare, P., Aiken, J. M. Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia.


Key Words: aging • skeletal muscle • laser capture microdissection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SARCOPENIA IS DEFINED as the age-related loss of muscle mass and function and is observed in diverse mammalian species including primates and rodents. In humans, muscle mass declines ~40% between 20 and 80 years of age (1) and negatively affects mobility, energy intake, nutrition, independent living, and respiratory function (reviewed in ref 2 ). This age-associated muscle mass decrease is attributed to fiber atrophy and fiber loss. The principle molecular causes of fiber loss and atrophy are unknown, but proposed mechanisms include 1) contraction-induced injury; 2) deficient satellite cell recruitment, 3) denervation/renervation, 4) endocrine changes, and 5) oxidative stress (reviewed in refs 3 , 4 ).

Oxidative damage, secondary to endogenous or exogenous free radicals, is hypothesized to accumulate throughout the lifetime of an organism, eventually giving rise to physiological and structural changes that are recognized as aging (5) . This is reflected in skeletal muscle by the age-associated increases in oxidative damage to proteins, lipids, and nucleic acids (reviewed in ref 3 ). Approximately 90% of cellular oxygen is metabolized in mitochondria with 1–5% of this being converted to reactive oxygen species (ROS) as a normal by-product of electron transport system (ETS) complexes (6) . Mitochondria may, therefore, be the primary cellular source and target of endogenous reactive oxygen species. Protein carbonyls, a marker of protein oxidative damage, increase ~150% between 12 and 29 months of age in isolated mitochondria from mouse skeletal muscle, whereas the concentration of thiobarbituric acid-reactive substances, a marker of lipid oxidative damage, increases ~267% between 7 and 24 months in the same mitochondrial isolates (7) .

The mitochondrial genome may be particularly susceptible to oxidative damage during aging. This 16 kb closed circular DNA molecule is located in close proximity to the source of ROS and lacks cognates of the nuclear histone proteins that confer protection from ROS (8 , 9) . Of the three principle DNA repair activities detected in the nucleus (i.e., base excision, nucleotide excision, and recombinational repair), nucleotide excision repair activity has not yet been detected in mammalian mitochondria (reviewed in ref 10 ). Oxidative mitochondrial DNA (mtDNA) damage increases with age in human brain (11) , diaphragm (12) , heart (13 , 14) , lung (15) , and mouse liver (16) and may directly disrupt mitochondrial gene expression or cause mtDNA mutations. For example, a specific oxidative DNA lesion, 8-hydroxy deoxyguanosine (8-OHdG), causes point mutations in vitro (17) .

Oxidative damage to, or mutation of, the mitochondrial genome may affect the activity of those ETS complexes to which this genome makes its largest polypeptide contributions, namely, complexes I and IV (i.e., NADH dehydrogenase and cytochrome c oxidase, COX, respectively). Biochemical analyses of tissue homogenates demonstrates decreased complex I and IV activities with age in postmitotic tissues that rely heavily on the ETS and oxidative phosphorylation (OXPHOS) such as brain, heart, and skeletal muscle (18 19 20) . In situ histochemical studies demonstrate increased abundance of ETS abnormal cells with age in a variety of tissues including heart, extraocular muscles, limb muscles, parathyroid, and diaphragm (21 22 23 24 25 26 27 28 29 30 31 32 33 34 35) . An absence of the partially mitochondrial-encoded cytochrome c oxidase activity (COX-) is the most commonly reported abnormal ETS phenotype and is sometimes observed with an increase in the entirely nuclear-encoded succinate dehydrogenase activity (SDH++). The SDH hyperreactivity is likely due to compensatory nuclear up-regulation of mitochondrial biogenesis. The combination of COX- and SDH++ phenotypes within a skeletal muscle fiber represents the ragged red fiber (RRF) phenotype observed in Gomori trichrome staining of skeletal muscle from aged individuals or those with mitochondrial myopathies. Focal, segmental ETS abnormalities are associated with high levels of mitochondrial DNA deletion mutations in aging (21 , 34) and mitochondrial myopathies (36 37 38 39 40) . Cybrid cell studies of myopathic mitochondrial deletions demonstrate decreases in cell growth, ETS/OXPHOS activity, mitochondrial membrane potential, and rate of ATP synthesis (41 , 42) suggesting that similar in vivo deficits may occur in ETS abnormal cells or segmental portions of muscle fibers.

The present study characterizes age-associated ETS abnormalities in rat rectus femoris skeletal muscle fibers. The rat rectus femoris is a mixed fiber-type muscle composed of both type I and type II fibers as differentiated by conduction velocity and oxidative capacity. Sarcopenic changes occur in the rectus femoris muscle of aging Fischer 344 x Brown Norway F1 hybrid rats including mass loss, fiber loss, and fiber type switching. Exhaustive serial sectioning (i.e., through 1000 microns) and histological analysis of the rectus femoris muscle demonstrated an age-related increase in ETS abnormal fibers as calculated from volume density, an association of segmental abnormalities with fiber atrophy, fiber splitting, and a predilection for the involvement of type II fibers. Laser capture microdissection (LCM) and whole mitochondrial genome amplification revealed mtDNA deletion mutations in all of the fibers displaying the RRF phenotype. These observations suggest a model of mitochondrial deletion involvement in senescence of postmitotic cells and in the progression of mammalian sarcopenia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and tissue collection
Male Fisher 344 x Brown Norway F1 hybrid rats were purchased from the National Institute on Aging colony maintained by Harlan Sprague Dawley (Indianapolis, Ind.). The average life span of males in this strain is ~31 months on an ad libitum diet (43) . Rats were transferred 1 month before study (i.e., 4, 17, and 35 or 37 months of age) to the Shared Aging Rodent Facility at the Geriatric Research, Education, and Clinical Center (Wm. S. Middleton VA Hospital, Madison, Wis.). Rats were given free access to water and food. Rats were anesthetized with sodium pentobarbital (30 mg kg-1 to effect) and killed by exsanguination. The rectus femoris muscles were dissected from each animal. The muscle was transected at the mid-belly, embedded in OCT mounting media (Miles Inc., Elkhart, Ind.), and flash frozen in liquid nitrogen-cooled isopentane. Samples were stored at -80°C until analyzed.

Histochemistry
Rectus femoris muscle samples were raised to the sectioning temperature of -20°C. Ten micron-thick serial sections were obtained and placed on Probe-on Plus Slides (Fisher Scientific, Pittsburgh, Pa.). Sections were stored at -80°C until needed. Histochemical staining for COX and SDH activities were performed according to Seligman et al. (44) and Dubowitz (45) . Hematoxylin and eosin staining was performed according to the rapid H&E technique for cryostat sections (46) . Slides were examined on an Olympus BH2 microscope and digital images were collected using a Hitachi 3-chip CCD camera (Hitachi Inc, Japan) and ImagePro Plus software (Media Cybernetics, Atlanta, Ga.).

Determination of fiber type
Fiber type was determined as described in Aspnes et al. (24) . Briefly, immunohistochemistry was performed using an antibody specific for the heavy chain of fast myosin found in all type II muscle fiber subtypes (clone MY-32, Sigma, St. Louis, Mo.). Sections were air dried and then blocked for 45 min in 5% Bacto skim milk (Difco Laboratories, Detroit, Mich.) in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4). Sections were then incubated for 2 h at room temperature with the anti-myosin antibody (1:400) in PBS, followed by alkaline phosphatase-conjugated Affinipure donkey anti-mouse IgG with minimal rat cross-reactivity (1:2000) in PBS. Composite digital images were made encompassing the entire muscle cross section and the number of type I fibers (i.e., those that showed no staining) from each rectus femoris muscle was determined. Control sections incubated without primary antibody exhibited little nonspecific staining (data not shown).

Determination of fiber number and fiber cross sectional areas
Total fiber number was determined from composite digital images of the entire muscle cross section at the middle belly. Digital images at a 200x magnification were taken of the entire muscle cross section and reconstructed. Every fiber in the muscle cross section was then counted by marking each fiber in the composite digital image with a digital pen and tablet (Wacom, Japan). This method avoided the need for estimations of fiber profiles per cross section. Fibers that were analyzed for cross sectional area along their length were digitally imaged at 200x magnification every 70 microns through a distance of 1000 microns. Calibration was performed using a stage micrometer. The outlines of the fibers were manually traced and fiber cross sectional area (CSA) in square microns measured digitally using Image Pro Plus software (Media Cybernetics). The CSA ratio is defined as the ratio of the smallest CSA within the ETS abnormal region divided by the average CSA ratio of the ETS normal region within the 1000 microns examined.

The volume density of the ETS abnormal regions was calculated by first counting the number of such regions that occurred within the 1000 microns of muscle examined. The cross sectional area of the entire muscle was measured. The volume of examined muscle was then calculated by multiplying the cross sectional area of the entire muscle (in square millimeters as determined from a digital image of an H&E stained muscle section) by 1 mm. The total volume of each rectus femoris muscle was determined by dividing its mass by 1.06 g ml-1, the specific gravity of muscle tissue (47) . The total number of ETS abnormal regions within the entire muscle was calculated by multiplying the volume density of abnormal regions by the volume of the muscle.

Immunohistochemical detection of oxidative nucleic acid damage
Frozen sections were fixed for 10 min in 10% buffered formalin. Formalin alters the antigenic epitopes by forming cross-links that involve calcium ions. To alleviate the cross-links and to retrieve the antigens, the slides were placed in citrate buffer (100 mM, pH 6.0) and autoclaved at 121°C for 8 min. Slides were blocked in Superblock (Pierce, Rockford, Ill.) for 30 min at room temperature, washed, and incubated with anti-8-hydroxy guanosine antibody (QED Biosciences, San Diego, Calif.) overnight at 4°C. This monoclonal antibody recognizes markers of DNA and RNA oxidative damage including 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanine, and 8-hydroxy guanosine. After overnight incubation with primary antibody, the slides were rinsed in distilled water, incubated with biotinylated goat anti-mouse IgG (1:200) for 1 h at room temperature, rinsed with distilled water and incubated with the avidin-biotin-peroxidase complex (Vector, Burlingame, Calif.) for 2 h at room temperature. Color development was performed with the Metal Enhanced DAB Substrate Kit (Pierce) for 10 min. Negative controls (i.e., incubation without the primary antibody) were included with each section and demonstrated little background staining.

Laser capture microdissection and whole mitochondrial genome amplification
Frozen sections of rat rectus femoris muscle were histochemically stained for SDH activity as described above. Sections were then dehydrated through an ethanol series and xylene. Slides were air dried and laser capture microdissection was performed the same day. Individual skeletal muscle fibers were microdissected using a PixCell II LCM (Arcturus, Calif.). Settings for LCM of skeletal muscle fiber were a laser spot size of 30 microns, a pulse power of 30 mW, and a pulse width of 50 ms. These settings worked equally well with sections on charged or uncharged glass slides. A single fiber section was captured per cap. Any extraneous noncaptured material was removed using the Cap-Sure pad (Arcturus). Total DNA was extracted using a modification of the protocol from Khrapko et al. (48) . Briefly, 1 µl of a digestion solution containing 2 mg/ml proteinase K, 0.5% sodium dodecyl sulfate and 10 mM EDTA was added to each section of skeletal muscle fiber and incubated at 37°C for 30 min in a humidified chamber. After digestion, 10 µl of water was added to the original 1 µl of digestion solution and the sample stored at -20°C until needed. One microliter of the DNA solution was subjected to whole mitochondrial genome amplification using ExpandTM Long Template PCR System (Roche, Nutley, N.J.). Polymerase chain reaction (PCR) amplification was conducted according to manufacturer’s protocol (93°C 3 min for hot start and 25 cycles of 93°C for 15 s, 68°C for 15 min). After primary PCR amplification, 1 µl of the primary PCR was used for 25 cycles of nested PCR. The outer primer pairs for primary PCR were F15671 (5'-CCATCCTCCGTGAAATCAACAACCCG-3') and R15377 (5'-CTTTGGGTGTTGATGGTGGGGAGGTAG-3'), which amplify a 16007 bp fragment of the rat mitochondrial genome. The inner primer pairs for nested PCR are F15826 (5'-AAGACATCTCGATGGTAACGGGTC-3') and R15233 (5'-CCAGAGATTGGTATGAGAATGAGG-3'), which amplify a 15708 bp fragment. Amplification products were subcloned into the pGEM®-T Easy vector (Promega) and sequenced using the ABI PRISM® BigDye Terminator Cycle Sequencing Kit at the University of Wisconsin-Madison DNA Sequencing Center.

Statistics
Comparisons between the three age groups were made using one way ANOVA where data were normally distributed with equal variances. The Tukey test was used for multiple comparisons. The strength of the association of ETS abnormality length and CSA ratio was performed using Spearman rank order correlation. Kernel density estimates of fiber CSA ratios were generated using S software. Comparisons of fiber type proportions of ETS abnormal fibers were determined using {chi}2 analysis. Statistical significance for all tests was set at P<0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Age-related sarcopenic changes in rat rectus femoris muscle
Body mass, in the Fischer 344 x Brown Norway F1 hybrid rats, increased ~40% between 5 and 18 months but then did not change significantly between 18 and 36–38 months of age (Table 1 ). Age had a significant biphasic effect on rectus femoris mass, which increased 22% between 5 and 18 months, followed by a 45% decrease from 18 to 38 months.


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  		Table 1. Age-related sarcopenic changes in the F344BNF1 hybrid rat

Age had a significant effect on total fiber number (P=0.011). Total fiber number at the mid-belly decreased 7% from 5 to 18 months (Table 1) , a decrease that was not statistically significant. From 18 to 38 months, total fiber number significantly decreased 25% (Table 1) . Type II fibers, the predominant fiber type in the rat rectus femoris, significantly decreased in proportion between 18 and 38 months, whereas type I fibers increased in proportion with age (Table 1) . The absolute number of type I fibers in a single mid-belly section increased with age from 243 ± 56 at 5 months to 981 ± 338 at 38 months, indicating that the increase in percentage of type I fibers was not due solely to loss of type II fibers. The change in fiber type proportions was accompanied by extensive fiber type grouping in histological sections of the 38-month-old rectus femoris.

Age-related increases in fibers displaying ETS abnormalities
Mitochondrial histochemical abnormalities in the rat rectus femoris were detected using a combination of COX and SDH staining of adjacent, serial sections. Serial, 10 micron-thick sections were examined at 70 micron intervals through a total length of 1000 microns along the muscle’s longitudinal axis and the fibers harboring an ETS abnormality were counted. A loss of COX activity was associated with one of three SDH phenotypes. SDH activity within a fiber that was higher than other fibers within a section was scored as SDH-hyperreactive (SDH++; Fig. 1A , B , C ). SDH activity within a fiber that was similar to the other fibers within a section was scored as SDH-normal (SDHnl; Fig. 1D , E , F ). SDH activity within a fiber that was lower than the other fibers within a section was scored as SDH-low (SDHlow; Fig. 1G , H , I ). The proportion of ETS abnormal fibers that displayed the RRF phenotype (i.e., COX-/SDH++) increased with age (P<0.001). There were no ETS abnormal fibers with the RRF phenotype observed in the 5-month-old muscles, one RRF in an 18-month-old muscle, whereas 42% of the total ETS abnormal fibers in the 38-month-old animals displayed the RRF phenotype.



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  		Figure 1. Three categories of observed ETS abnormalities in the rat rectus femoris muscle. ETS abnormal fiber is denoted by an X. H&E staining (A, D, G: white spots in fiber cytoplasm are artifacts of H&E staining of frozen sections), COX staining (B, E, H), and SDH staining (C, F, I). Micrographs in each column are consecutive serial sections. Each column of micrographs represents an individual fiber. Micrographs A–C demonstrate a ragged red fiber with COX negative (COX-) and SDH hyperreactive (SDH++) staining. Micrographs D–F show a fiber displaying an absence of COX activity with normal (i.e., similar to other fibers in the section) SDH activity (SDHnl). Micrographs G–I show a fiber lacking COX activity with decreased (i.e., lower than other fibers in the section) SDH activity. Black bar represents 50 microns.

Volume density of ETS abnormal fibers (i.e., number of ETS abnormal segments per cubic millimeter of tissue) increased with age (P<0.001). The number of ETS abnormal segments per cubic millimeter of rectus femoris muscle was 0.291 ± 0.027 in the 5-month-old muscles, 0.182 ± 0.024 in the 18-month-old muscles, and 1.039 ± 0.225 in the 38-month-old muscles (5 and 18<38, P<0.05). Using the volume density of ETS abnormal fibers and the volume of each muscle (i.e., calculated from muscle mass and specific gravity), the total number of ETS abnormal regions in an entire muscle was calculated and demonstrated an age-related increase (P<0.05). The calculated numbers of ETS abnormal segments in an entire muscle was 289 ± 8 from the 5-month-old rats, 231 ± 49 in the 18-month-old rats, and 1094 ± 126 in the 38-month-old rats. No fibers were observed that harbored more than one ETS abnormal segment within the 1000 microns examined.

Age-related COX and SDH histochemical abnormalities are associated with fiber atrophy
Muscle fibers harboring mitochondrial abnormalities frequently displayed a striking decrease in cross sectional area (CSA) concurrent with the abnormalities. For example, a fiber from the rectus femoris muscle of a 38-month-old hybrid rat initially demonstrated a typical CSA and the COX and SDH activities appeared normal (i.e., no different from the majority of fibers in the section) (Fig. 2 , column 1). Within the region displaying the ETS abnormality, the CSA decreased (Fig. 2 , columns 2–4). The same fiber then became both COX and SDH normal regaining a normal CSA (Fig. 2 , column 5). Of 131 ETS abnormal fibers examined, 9 fibers (7%) were found to be interrupted within the ETS abnormal segment. Kernel density distributions of CSA ratios in phenotypically normal, control fibers and ETS abnormal fibers at each of the three ages showed that none of the control fibers had a CSA ratio smaller than 0.5, whereas 33 of 131 abnormal fibers (25.2%) had a ratio below 0.5 (Fig. 3 ).



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  		Figure 2. Atrophy concomitant with an ETS abnormality in a fiber from a 38-month-old hybrid rat rectus femoris. Row A, COX staining. Row B, SDH staining; row C, CSA silhouettes of ETS abnormal fiber denoted by arrowhead in row B (number is CSA in square microns). Row D, reconstruction of CSA silhouettes: black silhouettes are from micrographs shown in columns 1–5, gray CSA silhouettes measured but not shown columns 1–5, white silhouettes extrapolations from measured CSA silhouettes. Distance (in microns) between micrographs in columns 1 and 2, 280; 2 and 3, 490; 3 and 4, 420; 4 and 5, 420. Black bar in micrograph A1 denotes 50 microns in rows A and B.



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  		Figure 3. Association of ETS abnormal region and fiber atrophy. Histograms (gray bars) and kernel density estimates (black line) of ETS abnormal fiber CSA ratio (i.e., as defined in Materials and Methods). Fibers from three animals were examined for each age group. Gray vertical bar on left indicates lowest CSA ratio observed in the corresponding control fibers.

Length of ETS abnormal region is associated with fiber atrophy
Length was determined for each ETS abnormal segment censoring those segments that extended beyond the 1000 microns examined. There was not a significant relationship between ETS abnormality length and age (P=0.439). The average length of the ETS abnormal regions (in microns) was 239 ± 17 at 5 months, 266 ± 33 at 18 months, and 267 ± 14 at 38 months of age. ETS abnormality length was negatively correlated to CSA ratio (P<0.001; r=0.523), demonstrating that fibers with longer ETS abnormal regions were more likely to have smaller CSA ratios.

ETS abnormalities are associated with fiber splitting
ETS abnormal fibers in the 38-month-old rat rectus femoris frequently split into two or more distinct skeletal muscle fibers (Fig. 4 ). There was a significant positive relationship between the presence of an ETS abnormality and splitting within the 1000 microns examined compared to randomly selected, ETS normal control fibers (P<0.001). Of the 197 ETS abnormal fibers analyzed, 68 fibers displayed fiber splitting, in contrast to 4 of 86 ETS normal, control fibers. Fiber splitting was also age associated with one split ETS abnormal fiber in an 18-month-old animal whereas all other split fibers occurred in the 38-month-old rectus femoris muscles.



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  		Figure 4. Fiber splitting in an ETS abnormal fiber from a 38-month-old hybrid rat rectus femoris. H&E (A, D, G), COX staining (B, E, H), SDH staining (C, F, I). ‘X’ denotes ETS abnormal fiber. Micrographs in each column (A–C, D–F, G–I) are consecutive serial sections. Micrographs A–C show an ETS abnormal fiber displaying the RRF phenotype (COX-/SDH2+). Further along its length, the ETS abnormal fiber exists as seven separate muscle fibers (D) with normal ETS phenotype (E, F); arrowheads indicate invaginations of the plasma membrane. Micrographs G–I display same fiber as one large and one small fiber with normal ETS phenotype. Distance between A–C and D–F, 770 microns; between D–F and G–I, 210 microns. A) Black bar denotes 50 microns.

Association of ETS abnormalities and fiber type
Segmental ETS abnormalities occurred in both type I and type II fibers in the rat rectus femoris. There was no significant relationship between fiber type and presence of ETS abnormality in the 5-month-old rat rectus femoris (P=0.640). ETS abnormalities were, however, more likely to occur in type II fibers than in type I fibers in the 18-month-old (P=0.003) and 38-month-old rats (P=0.009).

Increased steady-state levels of oxidative nucleic acid damage in rat RRFs
Immunohistochemical staining with an antibody specific for DNA and RNA oxidative damage demonstrated the presence of increased steady-state levels of oxidative nucleic acid damage in rat RRFs. Of 67 fibers with the RRF phenotype, 29 fibers displayed positive staining that was localized to the ETS abnormal segment (Fig. 5 ). From a total of 74,398 fibers (i.e., from all ages) examined through 1000 microns, 37 displayed positive staining for oxidative nucleic acid damage: 29 fibers displayed the RRF phenotype (COX-/SDH++), 3 fibers displayed the COX-/SDHnl phenotype, and 5 fibers were ETS normal.



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  		Figure 5. Immunohistochemical staining for oxidative nucleic acid damage in rat RRF. X denotes RRF from a 38-month-old rat rectus femoris and is same RRF shown in Fig. 1A , B , C . A) Section taken when fiber displayed RRF phenotype. B) Fiber when ETS phenotype was normal. Black bar in panel A denotes 50 microns.

MtDNA deletions concomitant with the RRF phenotype
Laser capture microdissection was used to isolate sections of 29 individual ragged-red skeletal muscle fibers from the rectus femoris of a 38-month-old hybrid rat (Fig. 6A , B , C ). Total DNA was isolated and whole genome amplification was used to detect rearrangements of the mitochondrial genome. The entire 16 kb mitochondrial genome was amplified from a 10 micron-thick section of an ETS normal fiber (Fig. 6D , lane 1). Using this technique, we detected shorter than wild-type genomes in all of the RRFs. Amplification products varied in length between 11,000 bp and 7000 bp (e.g., Fig. 6D , lanes 2–4). MtDNA deletion mutations were not detected in ETS normal fibers from the same sections.



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  		Figure 6. LCM of skeletal muscle fiber and detection of mtDNA deletions by whole mitochondrial genome PCR. A—C) LCM of a single rat skeletal muscle fiber. X denotes fiber of interest. A) Section before capture. B) Section after capture showing removal of target fiber. C) Captured fiber section. Black bar in panel C denotes 50 microns. D) Size fractionation of whole mitochondrial genome amplification products from a single ETS normal fiber (lane 1) and three rat RRFs (lanes 2–4). E) Sequence analysis of breakpoints from four different RRFs. Letters in parentheses indicate deleted bases that could be from either end of the breakpoint. Boldface letters indicate a direct repeat.

Identification of specific deletion breakpoints associated with the RRF phenotype
Deletion products from 16 RRFs were cloned and sequenced. All of the RRFs harbored unique deletion breakpoint and one breakpoint was flanked by a direct repeat sequence (Fig. 6E ). The deletion size varied from 6410 bp to 8851 bp, eliminating 8–11 of the 13 polypeptides encoded by the mitochondrial genome as well as up to 9 mitochondrial tRNA genes. Multiple microdissections along the same RRF amplified identically sized products, supporting the clonal nature of the mtDNA deletion (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The biological impact of age-associated mitochondrial abnormalities is unknown. In situ analyses of age-associated mitochondrial biochemical abnormalities suggest that these abnormalities may have their greatest and initial impact at the level of individual cells in postmitotic tissues. Previous studies rely on a single or an undefined number of serial sections to measure the abundance of ETS abnormal fibers (21 , 22 , 24 , 28 29 30 31 , 34) ; however, these studies provide scant information regarding sarcopenic changes (i.e., mass or fiber loss) in the muscles being examined. The abundance of mitochondrial histochemical abnormalities is commonly reported as the proportion of total fibers or number per area of tissue section and the proportions are typically very low, e.g., 0.07% in rat vastus lateralis (24) , 0.9% in rhesus monkey vastus lateralis (34) , 0.37% in human limb muscle (22) . Area densities of ETS abnormal fibers are, however, difficult to interpret since both the segmental abnormality and the tissue being studied are three dimensional. Since such objects are sectioned in a reference volume in proportion to their height normal to the section, their volume densities must be determined (49) . The use of serial sectioning through a known volume of hybrid rat rectus femoris allowed us to measure the volume densities of segmental ETS abnormalities and estimate the number of abnormalities in the entire muscle.

The rectus femoris muscle of an 38-month-old hybrid rat contains ~7000 fibers as measured by a single section through the muscle mid-belly (Table 1) . Assuming that ETS abnormalities occur once per fiber, our estimation of 1094 ETS abnormal regions in the 38-month-old rat rectus femoris suggests that ~15% of the fibers contain regions of severely compromised mitochondrial function. This number reflects the steady-state levels of these abnormalities in the 38-month-old rat rectus femoris since the number of fibers lost from the population due to an ETS abnormality cannot be measured using in situ histological techniques. Thus, these calculations are necessarily an underestimate of the total number of affected fibers occurring throughout the lifetime of the animal.

A large number of ETS abnormalities in the muscles of old rats is not sufficient to implicate them in age-related changes in these tissues. The connection between mitochondrial abnormalities and specific age-associated histological changes such as fiber atrophy and splitting suggests a cellular impact beyond the oxidative metabolic dysfunction. Fiber atrophy is a common feature of aging skeletal muscle in humans (50) and rats (51 52 53 54 55) . ETS abnormal segments in aging rat skeletal muscle correlate with a striking decrease in fiber CSA suggesting a causal role for mitochondrial abnormalities in fiber atrophy. The negative correlation between the length of the ETS abnormal region and change in CSA suggests that the abnormality reaches a threshold length where, in the center of the abnormality, the fiber contains predominantly dysfunctional mitochondria. In the ETS abnormal region, there is no longer support of cellular processes (e.g., ATP production) from adjacent areas containing functional mitochondria; nuclear gene transcription and translation are affected and fiber atrophy ensues. The small regions of fiber atrophy associated with segmental ETS abnormalities are unlikely to contribute directly to the muscle atrophy, but may represent a step in the progression from fiber dysfunction to fiber loss. The appearance of fibers that appeared to be ‘pinched off’ at the ETS abnormal region suggests that these regions may be prone to breakage and death of the remaining portions of the fiber. Therefore, ETS abnormal regions may contribute to a chronic loss of fibers, a salient feature of sarcopenia.

Our observations indicate that fibers harboring a segmental ETS abnormality are significantly more likely to demonstrate splitting within 1000 microns of the abnormal segment. Skeletal muscle fiber splitting is observed in various neuromuscular disorders (e.g., muscular dystrophy, familial amyloidotic polyneuropathy), increased physical stress (e.g., removal of synergist muscles and chronic overload), and aging (45 , 56 57 58 59 60) and has been attributed to regeneration, degeneration, splitting or fusion of mature fibers, and may involve satellite cells (61) . The involvement of physical stress seems unlikely in 38-month-old hybrid rats that are largely immobile (J. Wanagat, unpublished observations). Satellite cell numbers are decreased in aged skeletal muscle; thus, the splitting seen with aging likely is not due to regeneration (reviewed in ref 62 ). Since splitting typically occurred outside the ETS abnormal segment and some phenotypically normal, control fibers showed splitting, it would not appear to be the cause of the age-associated ETS abnormalities. The causal link between segmental ETS abnormalities and fiber splitting is unclear and the impact of splitting on fiber contractile function is unknown.

The three histochemical phenotypes observed (i.e., COX-/SDH++, COX-/SDHnl and COX-/SDHlow) suggest distinct processes in the formation of these abnormalities. ETS abnormalities in the young and middle aged animals are more likely to be of the SDHnl or SDHlow phenotypes. The SDHnl phenotype may be a precursor to the RRF phenotype, which is first seen in middle-aged rats and predominates in the older animals. The SDHlow phenotype observed at all ages may be the result of mtDNA depletion, but also may be due to the degeneration of muscle fibers that were damaged or denervated and are experiencing a loss of mitochondria. Conversely, they may represent regenerating fibers that have not yet achieved their full mitochondrial complement.

Fiber loss in rodents and humans involves primarily type II fibers (3) and the reason for the selective type II loss is not understood. The present study demonstrates that ETS abnormal fibers in the rat rectus femoris are more likely to occur within type II fibers, which agrees with studies showing a similar fiber type association in rhesus monkey quadriceps (34 , 35) . Type II fibers are subcategorized into fast oxidative glycolytic (IIa) with a fast conduction velocity and high oxidative capacity and fast glycolytic fibers (IIb) that have high conduction velocities and low oxidative capacities. Therefore, one might surmise that type IIb fibers are least likely to display age-associated mitochondrial abnormalities. There is, however, little information regarding in vivo mitochondrial biogenesis, turnover, or antioxidant defenses in specific fiber types. Our findings of increased type I fibers with age in the rectus femoris are similar to findings in the soleus, plantaris, and diaphragm muscles with age in rats (3) .

The oxidative damage colocalizing with RRF segments provides strong evidence that mtDNA deletions lead to increased in vivo steady-state levels of ROS. The increased ROS levels could be due to increased in vivo production of ROS or decreased antioxidant defenses within the ETS abnormal fiber segment. The loss of mitochondrially encoded subunits may disrupt efficient flow of electrons through the ETS, increasing in vivo ROS generation and subsequent oxidative damage (63 , 64) . The increased oxidative damage in ETS abnormal segments indicates yet another route of injury due to the mitochondrial dysfunction.

MtDNA deletion mutations appear to be the fundamental molecular basis for age-associated ETS abnormalities and their subsequent negative cellular effects. The systematic LCM and whole genome amplification of all RRFs within a defined amount of muscle tissue, in the present study, reveals the strict correlation of the RRF phenotype with deletions involving the mitochondrial major arc (i.e., the larger mtDNA segment situated between the origins of replication). MtDNA deletions are detected in all fibers displaying the RRF phenotype and, in those fibers, only within the ETS abnormal segment. Wild-type mtDNA is not detectable within the RRF segments (Fig. 6 , lanes 2–4) as previously demonstrated in our in situ hybridization studies of RRFs in rhesus monkey skeletal muscle (34 , 35) . The precise identity of the deletion breakpoints demonstrates the specific mitochondrial genotype that is responsible for the ETS abnormality and variability in the breakpoint. Direct repeat sequences are not required in the mechanism of mtDNA deletion formation, as they were not present in the majority of deletion products. We found no evidence of mtDNA duplication mutations.

The present study suggests the following sequence of events linking mtDNA deletions to sarcopenia (Fig. 7 ). Unknown mechanisms (e.g., errors of replication, oxidative damage) cause the aberrant removal of a portion of a mitochondrial genome (i.e., a mtDNA deletion mutation). This deletion begins to accumulate, perhaps because of a replicative advantage of the smaller genome. The mitochondria containing the deleted genomes accumulate within a segment of the muscle fiber and the resulting disruption of mitochondrial-encoded complexes of the ETS is sensed by the nucleus as a decrease in cellular energy metabolism. Compensatory action by the nucleus results in up-regulation of mitochondrial biogenesis and the expression of nuclear-encoded mitochondrial genes (e.g., SDH), resulting in the RRF phenotype. The accumulation of dysfunctional mitochondria continues along the length of the muscle fiber within the confines of the plasma membrane until it reaches a length where the affected portion can no longer maintain normal cellular homeostasis. The dysfunctional mitochondria produce increased amounts of ROS that cause oxidative damage and interfere with ROS-mediated cell signaling pathways. Disruption of nuclear gene expression decreases synthesis of myofibrillar proteins leading to decrease in sarcomere number resulting in fiber atrophy. The ETS abnormal region may also affect the production of neurogenic factors and could result in disruption of the neuromuscular junction and subsequent denervation. We have also observed an apparent decrease in nuclear number within the ETS abnormal regions (unpublished observations), suggesting the activation of cellular apoptotic pathways resulting in fiber death.



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  		Figure 7. Model of mtDNA deletion involvement in sarcopenia. Illustration of skeletal muscle fiber with neuromuscular junction (NMJ) and peripherally located nuclei. Red, COX- phenotype. Yellow, COX-/SDH2+ phenotype. Figure is not drawn to scale.

Our findings suggest the involvement of mitochondrial abnormalities in mammalian sarcopenia and indicate a progression from specific molecular events to cellular impact and eventual age-related organ damage. Understanding the genotypic and phenotypic changes within individual skeletal muscle fiber segments harboring mtDNA deletions will provide a major advance in elucidating the in vivo cellular effect of these mutations and present opportunities directed at determining the causal role of mtDNA mutations in aging and age-related diseases.


   ACKNOWLEDGMENTS
 
This work was supported by grants R01 AG11604, P01 AG11915, and T32 AG00213 from the National Institutes of Health. We thank Sue McKiernan and Dr. Debbie McKenzie for critical review of this manuscript and Dr. Tom Pugh for technical assistance with the immunohistochemical staining for oxidative nucleic acid damage.

Received for publication May 12, 2000. Revision received July 28, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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