Báo cáo Y học: Mitochondrial DNA deletion mutations A causal role in sarcopenia docx

MINIREVIEW Mitochondrial DNA deletion mutations A causal role in sarcopenia Debbie McKenzie, Entela Bua, Susan McKiernan, Zhengjin Cao, Jonathan Wanagat and Judd M. Aiken Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI, USA Mitochondrial DNA (mtDNA) deletion mutations accu- mulate with age in tissues of a variety of species. Although the relatively low calculated abundance of these deletion mutations in whole tissue homogenates led some investiga- tors to suggest that these mutations do not have any phy- siological impact, their focal and segmental accumulation suggests that they can, and do, accumulate to levels sufficient to affect the metabolism of a tissue. This phenomenon is most clearly demonstrated in skeletal muscle, where the accumulation of mtDNA deletion mutations remove critical subunits that encode for the electron transport system (ETS). In this review, we detail and provide evidence for a molecular basis of muscle fiber loss with age. Our data suggest that the mtDNA deletion mutations, which are generated in tissues with age, cause muscle fiber loss. Within a fiber, the process begins with a mtDNA replication error, an error that results in a loss of 25–80% of the mitochondrial genome. This smaller genome is replicated and, through a process not well understood, eventually comprises the majority of mtDNA within the small affected region of the muscle fiber. The preponderance of the smaller genomes results in a dysfunc- tional ETS in the affected area. As a consequence of both the decline in energy production and the increase in oxidative damage in the region, the fiber is no longer capable of self- maintenance, resulting in the observed intrafiber atrophy and fiber breakage. We are therefore proposing that a pro- cess contained within a very small region of a muscle fiber can result in breakage and loss of muscle fiber from the tissue. Keywords: muscle loss; intrafiber atrophy; aging. INTRODUCTION Adenosine triphosphate is the carrier of free energy in most living cells and is generated by the process of oxidative phosphorylation that occurs in the mitochondrion. This free energy is required for mechanical work, active transport of molecules and ions and the synthesis of biomolecules. Disturbances in energy production, due to mitochondrial DNA (mtDNA) mutations, have been shown, in mito- chondrial myopathies and cellular myopathy models, to have a negative impact on the function of cells, specific tissues and, ultimately, the whole animal. These mutations include missense mutations, protein synthesis mutations, copy number mutations and insertion-deletion mutations [1]. Mitochondrial deletion mutations present as human disease states in a number of mitochondrial myopathies. The evolving, progressive nature of mtDNA mutations has led researchers to focus on the contribution of mtDNA mutations to the aging process. Sarcopenia is a clinically recognized manifestation of the aging process that presents as muscle mass and function loss over time. We used young, middle-aged and old muscles from rodents and primates to test whether mtDNA deletion mutations are associated with the negative physiological impact of sarcopenia. SARCOPENIA An age-related loss of muscle mass and function occurs in skeletal muscle of a variety of mammalian species; this process is referred to as sarcopenia. In humans, specific skeletal muscles undergo a  40% decline in muscle mass between the ages of 20 and 80 years [2]. The public health ramifications of this large decline are evident in the clinical presentation, which includes decreased mobility, energy intake and respiratory function. These declines affect both the nutrition and the ability of elderly people to live independently. Progressive muscle wasting has also been demonstrated in rodents and nonhuman primates. These sarcopenic changes are evidenced by a significant reduction in muscle cross- sectional area, muscle mass loss and fiber number loss over time. In the Fischer 344 · Brown Norway (FBN) hybrid rat, the difference between the rectus femoris muscles of 18- and 38-month-old animals is striking. Muscle cross- sectional area is reduced by 30% in the older animals and the muscle composition is more heterogeneous including an increase in fibrotic tissue. A significant reduction in muscle mass (45%) is observed between 18- and 36/38-months of age as well as a significant (27%) loss of muscle fibers counted at the midbelly (Fig. 1). Although the molecular events responsible for sarcopenia are unknown, the muscle mass loss is due to fiber atrophy [2–4] and fiber loss [2,5,6]. A variety of mechanisms Correspondence to J. M. Aiken, 1656 Linden Drive, Madison, WI 53706, USA. Fax: + 1 608 262 7420, Tel.: + 1 608 262 7362, E-mail: aiken@ahabs.wisc.edu Abbreviations: mtDNA, mitochondrial DNA; ETS, electron transport system; FBN, Fischer 344 · Brown Norway rat; MERRF, myoclonic epilepsy and ragged red fiber; CPEO, chronic progressive external ophthalmoplegia; KSS, Kearns–Sayre syndrome; COX, cytochrome c oxidase; SDH, succinate dehydrogenase; LCM, laser capture micro- dissection; CSA, cross-sectional area. (Received 28 November 2001, accepted 15 February 2002) Eur. J. Biochem. 269, 2010–2015 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02867.x have been proposed for fiber loss. These include contrac- tion-induced injury, deficient satellite cell recruitment, denervation/renervation, endocrine changes, oxidative stress and mitochondrial DNA damage. We propose that the latter two mechanisms, in concert, contribute to the progressive age-related loss of muscle mass. Our working hypothesis is based on the idea that oxidative damage to the mitochondrial genome has the potential to trigger a deletion event. Accumulation of these mtDNA deletion mutations would cause a decline in the energy production of the affected cell(s), result in abnormal electron transport system (ETS) enzyme phenotypes, atro- phy and would, ultimately, lead to fiber loss. Working with an animal model known to undergo sarcopenia, we addressed the question of whether mtDNA deletion mutations initiate the events that lead to sarcope- nia. We examined a quadricep muscle, rectus femoris, from three different age groups of FBN rats (5-, 18-, and 36/38- month-old). Fiber number at the midbelly, individual fiber cross-sectional area, electron transport system abnormalities and mtDNA deletion mutations were analyzed. In the remainder of this review, we will present data that support the hypothesis that mitochondrial DNA deletion mutations play a critical role in sarcopenia. ACCUMULATION OF mtDNA DELETION MUTATIONS WITH AGE Mitochondria generate most of the energy in cells. They contain their own genomes (2–10 per mitochondria) that replicate independently of the nuclear genome [7]. The mitochondrial genome, in mammals, is  16 kb in length and encodes 22 tRNA, 13 subunits of the electron transport system and its own 16S and 26S ribosomal RNAs [8]. The mitochondrial transcripts are synthesized as long polycis- tronic messages and are transcribed from both strands. The mtDNA genome is thought to be a major target of oxidative damage for several reasons. First, the mtDNA genome is located adjacent to the primary source of reactive oxygen species, the electron transport system (reviewed in Cadenas & Davies [9]). The lack of histone cognates [10] and the minimal repair systems [11] in the mitochondria (as compared to the nucleus) increase the likelihood of oxidative damage occurring and being maintained in the mitochondrial genome. The levels of oxidatively damaged bases in mtDNA are 10- to 20-fold higher than that observed in nuclear DNA [12,13]. Also, the contiguous, compact nature of the mitochondrial coding region (all but the displacement loop region encode either mRNAs or tRNAs) increases the chance that a mutation event will affect a gene product. MtDNA mutations have been shown, in humans, to result in a number of diseases including myopathies and encephalopathies, a broad class of conditions characterized by muscle weakness and central nervous system dysfunc- tion. Myopathies can be divided into two major groups: (a) those caused by a single mtDNA base substitution, such as Leber’s hereditary optic nerve atrophy and myoclonic epilepsy and ragged red fiber (MERRF) [14,15]; and (b) diseases caused by large mtDNA deletion mutations such as chronic progressive external ophthalmoplegia (CPEO) and Kearns–Sayre Syndrome (KSS) [16–18]. In myopathy patients, the levels of the mutated mtDNA genomes are very high, 73–98% mutated mtDNA in symptomatic MERRF patients [19]. Deletion mutations are generally present as 20–80% of all mtDNA genomes in KSS patients [20]. The first age-associated mtDNA alterations identified where those that were also detected in the mitochondrial myopathies (reviewed by Wallace [1]). Initial studies focussed on the ÔcommonÕ deletion, mtDNA 4977 in humans. Although this common deletion was not detected in normal aged individuals by Southern blot analysis, it was detectable using the more sensitive PCR. This particular deletion mutation was found to accumulate, with age, in a variety of human tissues [21,22]. The highest levels were detected in nerve and muscle tissue [23], the same tissues in which mitochondrial enzyme activities were observed to decline with age. Later studies demonstrated the presence of different deletion mutations that accrued within and between different human tissues [21,24–28]. Subsequent studies identified multiple mtDNA deletion mutations in a variety of species including rhesus monkeys [29], mice [30–33] and rats [34–36]. Using PCR, we analyzed tissue homogenates prepared from specific skeletal muscles of rhesus monkey, mice and rats for the presence of age-associated mtDNA deletion mutations [29,30,35]. Deletion mutations were observed in all three species. In rhesus monkey skeletal muscle, there was a significant increase in the number and frequency of mtDNA deletion mutations with age [29]. The number of deletion mutations increased most dramatically in rhesus monkeys greater than 20 years of age. Some of the deletion products were common among the rhesus monkeys while others were unique. The rodent studies yielded similar information, multiple mtDNA deletion mutations accumu- lated with age. Unlike rhesus monkeys, however, common deletion events were rare in both rats and mice suggesting that they might initiate or accumulate differently in these animals [33,36,37]. DELETION MUTATIONS ACCUMULATE FOCALLY Initial studies utilizing radioactive PCR methods calculated the abundance of the specific deletion mutation, mtDNA 4977 , to be < 0.1% of the total mtDNA present Fig. 1. Analysis of FBN rat rectus femoris muscles from three different age groups. Muscle weight is represented by the black bars using the left axis, fiber number is represented by the white bars using the right axis, muscle cross-sectional area is represented by the width of the black bars using the x-axis. Different letters associated with like bars indicate a significant difference (P <0.05). Ó FEBS 2002 mtDNA deletions and sarcopenia (Eur. J. Biochem. 269) 2011 in the tissue homogenate [22,23]. The low abundance of this deletion led to the speculation that mtDNA deletion mutations are of minor physiological significance. All of the initial quantitative analyses, however, were performed using cellular homogenates, in which thousands of cells were present. Estimating the abundance of mtDNA deletion mutations from homogenates assumed an equal cellular distribution of the deletion-containing genomes. In situ hybridization analyses of aged muscle tissue provided the first evidence of a focal accumulation of mtDNA deletion mutations. The initial in situ hybridization studies of age- associated mtDNA deletion mutations were performed on human skeletal muscle and were focussed on the common deletions [38,39]. Using mtDNA probes located either within or outside the deleted regions, high levels of mtDNA deletion mutations were localized to individual cells. This suggests that deletion mutations accumulate focally and that neither their abundance nor distribution can be accurately assessed in cellular homogenates. Further evidence for the focal and mosaic distribution of the mtDNA deletion mutations came from muscle fiber bundle analyses [40]. In these studies, defined numbers of skeletal muscle fibers were dissected from old rhesus monkeys. Two classes of samples were analyzed for mtDNA deletion products, those containing either: (a) several thousand individual muscle fibers or (b) defined groups of 75 or 10 fibers per bundle. We found that the number of amplification products decreased with the reduction in the number of muscle fibers analyzed, but that there was a significant increase in the abundance of specific deletion-containing genomes in the samples containing fewer fibers [40]. These experiments demonstrated that mtDNA deletion mutations are not distributed evenly throughout a muscle group, but rather focally accumulate to high levels in a subset of fibers. ETS ABNORMALITIES ACCRUE WITH AGE Dramatic changes in the activities of specific ETS enzymes were observed in myopathy patients [41–44] and were later demonstrated to occur in humans with age [45,46]. We examined the muscle tissue of young and old rhesus monkeys and rats, histologically, for changes in the activity of two ETS enzymes with age, cytochrome c oxidase (COX) and succinate dehydrogenase (SDH). Several of the COX subunits are encoded by the mitochondrial genome and, thus, the absence of COX activity would be indicative of changes in the mtDNA. Although SDH is entirely encoded by the nuclear genome, decreases in mitochondrial energy output result in a compensatory up-regulation of mito- chondrial synthesis and of nuclear-encoded transcripts. For the rat studies, entire muscles were dissected, cut at the midbelly and embedded. Muscle biopsy samples (vastus lateralis) were embedded for the rhesus monkey studies. Sections were then obtained using a cryostat and, depending on the study, 100–200 serial, 8–10-lm thick sections were produced. Samples were analyzed by staining every seventh slide (i.e. the first, seventh, 14th, etc.) for COX activity and every eighth slide (i.e. the second, eighth, 15th, etc.) for SDH activity. Fibers containing ETS abnormalities would be expected to show a negative reaction for COX and often, but not always, hyper-reactive for SDH (Fig. 2). All stained slides were analyzed by light microscopy and all COX – and/ or SDH ++ fibers were noted. ETS abnormal (COX – and/ or SDH ++ ) were followed along the 1000–2000 lmto determine the length of the ETS abnormal region. Using these two enzymatic stains, we demonstrated the age- associated increase of ETS abnormalities in several different muscles and animal models [36,47]. For example, in the rectus femoris of FBN rats, no COX – /SDH ++ fibers were observed in the 5-month-old animals and only one was found among the nine 18-month-old rats. At 36/38-months of age, however, a total of 184 COX – /SDH ++ regions were observed in 11 rats (Table 1). As only 1 mm of tissue was examined ( 3% of the length of the rectus femoris), extrapolation was used to determine that  7% of the muscle fibers of aged rectus femoris muscles contained ETS abnormal regions. ETS ABNORMAL FIBERS ATROPHY While following these abnormal fibers for 1000–2000 microns, we noted that many of the ETS abnormal fibers displayed an overt decline in cross-sectional area (intrafiber atrophy) within the ETS abnormal region of the fiber. To Fig. 2. Electron transport system abnormalities. Consecutive, serial cross-sections of the rectus femoris from a 36-month-old FBN rat identifying a fiber (indicated by the arrow) that stains negative for cytochrome c oxidase (COX – ) and hyperreactive for succinate dehydrogenase (SDH ++ ). 2012 D. McKenzie et al. (Eur. J. Biochem. 269) Ó FEBS 2002 quantitate the extent of atrophy in these ETS abnormal regions, the cross-sectional area (CSA) of both ETS normal and abnormal regions was measured along the length of the 1000 lm. The smallest (minimum CSA) value of the fiber CSA in the ETS abnormal region was divided by the average value of the fiber CSA in the ETS normal region within the same fiber [36,52]. In the ETS normal fibers, the ratio between minimum CSA and average CSA was determined. The cross-sectional area ratio of the ragged red phenotype is significantly smaller than that observed in either normal fibers or fibers that display a COX – /SDH normal phenotype. These data clearly demonstrate that ETS abnormalities have a localized physiological impact on the cell and can result in fiber atrophy. Subsequent longitudinal analysis of atrophied fibers also showed that some decrease in CSA until they are no longer observable by light microscopy, suggesting that they are broken. The fiber can often be found again several sections later. Our analysis of the cross-sectional area of ETS abnormal fibers also demonstrates that longer ETS abnormal regions are more likely to atrophy than shorter ETS abnormal regions. COX – /SDH normal regions are shorter than COX – /SDH ++ regions and their CSA slightly decrea- ses. The COX – /SDH ++ regions are larger than the COX – /SDH normal regions and the CSA declines to a greater extent. These studies were performed in the rat and suggest a process in the rat quadricep muscle in which the initial phenotype is COX – /SDH normal . The phenotype progresses with time to the COX – /SDH ++ phenotype, associated with fiber atrophy and fiber breakage. A similar process was observed in the rhesus monkey skeletal muscle. ETSABNORMALFIBERSCONTAIN mtDNA DELETION MUTATIONS We have recently defined the mtDNA genotype associated with the abnormal ETS phenotypes in aged muscle fibers. Using laser capture microdissection (LCM), we have amplified the mtDNA from both abnormal and normal regions of fibers. As described by Cao et al.[37],the mtDNA deletion mutations were concomitant with the COX – /SDH ++ regions of affected muscle fibers. Single fiber sections, 10-lm thick, of normal and abnormal regions of rat rectus femoris muscle fibers were isolated using LCM. When total DNA from each fiber section was subjected to whole mitochondrial genome PCR, smaller than wild-type amplifications were observed in all of the COX – /SDH ++ regions demonstrating the association of deletion mutations with the ETS abnormal phenotype. These deletion-containing genomes were the only mtDNA genomes detected in the ETS abnormal regions while only wild-type genomes were found in the ETS normal regions. LCM coupled with PCR analysis has clearly demonstrated that age-associated mtDNA deletion mutations are locali- zed to specific cells identified as abnormal by histo- chemical analysis. When the same ETS abnormal region was sampled in two different places from the same fiber (i.e. 70 lm apart), the same deletion product was obtained. The accumulation of the same deletion product in both COX – /SDH ++ regions of skeletal muscle [37] and indi- vidual cardiomyocytes [48] suggests that mtDNA deletion mutations are clonal events. Two hypotheses have been proposed to account for this clonal expansion phenomenon. De Grey [49] proposed that damaged mitochondria degrade more slowly than intact mitochondria. The abnormal mitochondrial would there- fore accumulate in the cell by the Ôsurvival of the slowestÕ. Due to the low proton gradient present in defective mitochondria, the production of free radicals would be decreased and, hence, less damage to the mitochondrial membranes. The second hypothesis, based on the size of the mtDNA deletion mutations observed, presumes that the smaller deletion-containing genomes would have a replica- tive advantage [50]. Support for the replicative advantage of the smaller genomes is provided by re-population kinetic studies using several mtDNA forms after severe mtDNA depletion by ethidium bromide. These studies demonstrated that the replication and maintenance of mtDNA in human cells is highly dependent on molecular features, as partially deleted mtDNA molecules re-populated cells significantly faster than full-length molecules [51]. This work, in total, has led us to propose the following mechanism for the role of mitochondrial DNA deletion mutations in sarcopenia. A portion of the mtDNA genome is deleted by an as yet unknown mechanism, possibly as the result of oxidative damage. As mtDNA genomes carrying a deletion would be smaller, they would have a replicative advantage over wild-type genomes. As the ratio of deletion- containing genomes increased, the mitochondria would become deficient in major subunits of the ETS (i.e. the COX – phenotype) and an energy deficiency would occur (Fig. 3B). Concurrent with the energy deficiency would be increased oxidative damage [36]. Signals from the nuclear genome would then trigger mitochondrial amplification in an effort to overcome the energy deficiencies. This increased synthesis of mitochondria would subsequently result in an increase in the production of the nuclear subunits of the ETS system (i.e. the COX – /SDH ++ phenotype) (Fig. 3C). However, as the deletion mutations would continue to Table 1. Ragged red fiber content of rat rectus femoris muscles. Age No. of animals Fibers examined a (mean±SD) Ragged red fibers (mean±SD) Estimated no. of RRF per muscle (%) b 5 Months 6 10 426 ± 714 0 c 0 18-Months 9 10 075 ± 707 0.11 ± 0.33 c 4 (0.04%) 36/38-Months 11 7606 ± 1257 17.0 ± 14 d 567 (7.5%) a Each fiber was examined through 1000 lm. b Estimates were determined by dividing the mean number of ETS abnormalities found in 1 mm of tissue by 0.03 (3% of the approximate 3.0 cm length of rectus femoris), percentages were determined by dividing the resulting value by the mean number of fibers. c,d Values with different superscripts were significantly different. Ó FEBS 2002 mtDNA deletions and sarcopenia (Eur. J. Biochem. 269) 2013 out-replicate the wild-type mtDNA genomes, both energy deficiencies and oxidative damage would continue to accrue. In response, the fiber atrophies (Fig. 3D) and, eventually, breaks (Fig. 3E). ACKNOWLEDGEMENTS Research in our laboratory is supported by the National Institutes of Health Grant Nos. 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