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Mitochondrial DNA–Deletion Mutations Accumulate Intracellularly to Detrimental Levels in Aged Human Skeletal Muscle Fibers

Skeletal muscle-mass loss with age has severe health consequences, yet the molecular basis of the loss remains obscure. Although mitochondrial DNA (mtDNA)-deletion mutations have been shown to accumulate with age, for these aberrant genomes to be physiologically relevant, they must accumulate to high levels intracellularly and be present in a significant number of cells. We examined mtDNA-deletion mutations in vastus lateralis (VL) muscle of human subjects aged 49-93 years, using both histologic and polymerase-chain-reaction (PCR) analyses, to determine the physiological and genomic integrity of mitochondria in aging human muscle. The number of VL muscle fibers exhibiting mitochondrial electron-transport-system (ETS) abnormalities increased from an estimated 6% at age 49 years to 31% at age 92 years. We analyzed the mitochondrial genotype of 48 single ETS-abnormal, cytochrome c oxidase-negative/succinate dehydrogenase-hyperreactive (COX-/SDH++) fibers from normal aging human subjects and identified mtDNA-deletion mutations in all abnormal fibers. Deletion mutations were clonal within a fiber and concomitant to the COX-/SDH++ region. Quantitative PCR analysis of wild-type and deletion-containing mtDNA genomes within ETS-abnormal regions of single fibers demonstrated that these deletion mutations accumulate to detrimental levels (>90% of the total mtDNA).

Early-onset calorie restriction conserves fiber number in aging rat skeletal muscle

The purpose of this work was to determine the effect of early‐onset calorie restriction on sarcopenia in the aging rat. Ad libitum (AL) fed animals were examined at 5, 18, 21, and 36 months of age. Calorie‐restricted (CR) rats, 40% restricted since 4 months of age, were examined at 21 and 36 months of age. By 36 months, vastus lateralis, rectus femoris and soleus muscles, from AL‐fed rats, had significant muscle mass and fiber loss, and reduced muscle cross‐sectional area. Mean fiber diameter decreased with age in the vastus lateralis and rectus femoris but not the soleus of AL‐fed rats. The number of Type I fibers significantly increased in the vastus lateralis with age. Calorie restriction did not prevent muscle mass loss with age; however, it significantly reduced muscle mass loss between 21 and 36 months of age compared with age‐ matched AL cohorts. Calorie restriction prevented fiber loss with age, and this conservation of fiber number reduced muscle mass loss with age.

Mitochondrial DNA mutations as a fundamental mechanism in physiological declines associated with aging.

The hypothesis that mitochondrial DNA damage accumulates and contributes to aging was proposed decades ago. Only recently have technological advancements, which facilitate microanalysis of single cells or portions of cells, revealed that mtDNA deletion mutations and, perhaps, single nucleotide mutations accumulate to physiologically relevant levels in the tissues of various species with age. Although a link between single nucleotide mutations and physiological consequences in aging tissue has not been established, the accumulation of deletion mutations in skeletal muscle fibres has been associated with sarcopenia. Different, and apparently random, deletion mutations are specific to individual fibres. However, the mtDNA deletion mutation within a phenotypically abnormal region of a fibre is the same, suggesting a selection, amplification and clonal expansion of the initial deletion mutation. mtDNA deletion mutations within a muscle fibre are associated with specific electron transport system abnormalities, muscle fibre atrophy and fibre breakage. These data point to a causal relationship between mitochondrial DNA mutations and the age‐related loss of muscle mass.

Mitochondrial DNA Deletion Mutations and Sarcopenia

This manuscript summarizes our studies on mitochondrial DNA and enzymatic abnormalities that accumulate, with age, in skeletal muscle. Specific quadricep muscles, rectus femoris in the rat and vastus lateralis in the rhesus monkey, were used in these studies. These muscles exhibit considerable sarcopenia, the loss of muscle mass with age. The focal accumulation of mtDNA deletion mutations and enzymatic abnormalities in aged skeletal muscle necessitates a histologic approach in which every muscle fiber is examined for electron transport system (ETS) enzyme activity along its length. These studies demonstrate that ETS abnormalities accumulate to high levels within small regions of aged muscle fibers. Concomitant with the ETS abnormalities, we observe intrafiber atrophy and, in many cases, fiber breakage. Laser capture microdissection facilitates analysis of individual fibers from histologic sections and demonstrates a tight association between mtDNA deletion mutations and the ETS abnormalities. On the basis of these results, we propose a molecular basis for skeletal muscle fiber loss with age, a process beginning with the mtDNA deletion event and culminating with muscle fiber breakage and loss.

Calorie restriction limits the generation but not the progression of mitochondrial abnormalities in aging skeletal muscle.

The effect of early‐onset calorie restriction and aging on the accumulation of electron transport system (ETS) abnormalities was studied in rat skeletal muscle. Rectus femoris and vastus lateralis muscle fibers were analyzed for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) enzyme activities. Fibers displaying COX negative and SDH hyper reactive (COX–/SDH++) phenotype were followed through 1000–2000 micrometers to determine the frequency and length of these abnormalities as well as the physiological impact on fiber structure. Calorie restricted rats had fewer ETS abnormal muscle fibers. The mean length of ETS abnormal regions in ad libitum rat muscle fibers was similar to calorie restricted rat muscles. ETS abnormal fibers from both diet groups exhibited intra‐fiber atrophy. A negative correlation between ETS abnormality length and fiber cross‐sectional area (CSA) ratio was observed in both ad libitum and calorie‐ restricted rats. Although calorie restriction reduced the number of ETS abnormalities, it did not affect the length or associated fiber atrophy of ETS abnormal regions once the abnormality was established. Thus, calorie restriction affects the onset but not the progression of electron transport system abnormalities, thereby, limiting a process that ultimately results in fiber breakage and fiber loss.

Rebuttal to Jacobs: The mitochondrial theory of aging: alive and well

We agree with much of what Dr Jacobs argues but would like to comment on some of his statements. He makes many valid comments, comments that were very relevant 4–5 years ago. Studies based on tissue homogenates are, as he mentions, very difficult to interpret and it is exceedingly difficult to correlate mitochondrial changes with physiological outcomes based on those experiments. We would, however, argue that advances in methodologies along with increased attention to the fate of specific cells has greatly enhanced our ability to link changes in the mitochondrial genome with the aging process. The relatively high abundance of cells affected in the mitochondrial myopathies makes it much easier to envision how cellular dysfunction results in tissue dysfunction in these human diseases. The lower frequency of cells affected by mtDNA deletion mutations in aging tissues has resulted in many researchers questioning whether these mtDNA deletion mutations have any physiological relevance. In the past few years, however, there has been a considerable shift in experimental design. Instead of focusing on tissue homogenates, studies have emphasized the analysis of individual cells. These studies indicate that mtDNA deletion mutations and single nucleotide mutations clonally accumulate to high levels within single cells. Our studies indicate that mitochondrial DNA deletion mutations have a physiological impact on skeletal muscle, leading to fibre atrophy and, ultimately, loss of the cells. Rather than rely on in vitro approaches to the analysis of the role of mtDNA mutations in the aging process, we have taken a two-pronged approach in our in vivo studies. The nature of the mutation event and accumulation makes it impossible to identify and then follow a specific mutation event with time (due to the focal and segmental accumulation of the mutations). We use two different animal models, rhesus monkeys and rats, and follow both through aging. For the rhesus monkeys, muscle biopsies are collected at 3-year intervals. Our initial analysis of these samples suggests that the number of ETS abnormal fibres and the number of mtDNA deletion mutations increase with increasing age. For the rat studies, cohorts of animals have been followed longitudinally starting at 18 months of age and analysed at 3-month intervals. In these studies, a variety of different muscle groups (selected based on the degree to which they undergo sarcopenia) have been analysed with respect to mtDNA deletion mutation abundance as well as the number of ETS abnormal fibres. For all samples, a number of tissue sections are characterized such that 1000 μ m of tissue, at 10μ m intervals, are examined. Not only have we demonstrated that the extent of sarcopenia (i.e. degree of muscle fibre loss) is correlated with the number of ETS abnormal fibres and fibre atrophy, we have also clearly demonstrated the link between the presence of ETS abnormalities and the mtDNA deletion mutation (Cao et al ., 2001; Wanagat et al ., 2001; Bua et al ., 2002). The long-term effect of the mtDNA mutations is often subtle, with, in the case of skeletal muscle, a relatively small number of fibres being affected at any one time. Aging is a gradual process. We and others would not argue that the ‘loss of a single neurone might destroy the integrity of a complex neural circuit’ but rather that, with age, the cumulative effect of the loss of neurones would result in physiological declines. A rapid loss of large numbers of neurones, skeletal muscle fibers or other cells would not be aging, but disease. The declines observed with aging are, by definition, much subtler than those observed in disease. The mechanism by which the mtDNA deletion mutations occur is not yet known. It is a very difficult question to address in aging tissues as the event is probably random and occurs at a very low frequency in vivo . Owing to the relatively low frequency of the deletion events in aging tissue, it would be impossible to identify, using currently available methods, the initial deletion event. We have, however, circumstantial evidence to suggest that the initial mutation event is due to oxidative damage. It is generally accepted that caloric restriction decreases oxidative damage and, hence, increasing lifespan. When muscle from rats that have been calorically restricted are compared to age-matched controls, the number of ETS abnormal fibres and the number of mtDNA deletion mutations are reduced. Caloric restriction does not, however, affect the cascade of events that follow a deletion event, i.e. ETS abnormalities, fibre atrophy, etc., all accrue in a pattern similar if not identical to that of ad libitum fed animals. Our studies in rat muscle have also demonstrated a correlation between fibre atrophy and increased levels of oxidative damage, as indicated by the presence of 8-hydroxydeoxyguanosine (Wanagat et al ., 2001). At this time, however, we do not know Correspondence Judd M. Aiken, Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA. Tel.: (608) 262 7362; fax: (608) 262 7420; e-mail: aiken@ahabs.wisc.edu