Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.     

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).     

Mitochondrial DNA deletion mutations: a causal role in sarcopenia.

Mitochondrial DNA (mtDNA) deletion mutations accumulate 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 investigators to suggest that these mutations do not have any physiological 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 dysfunctional 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 process contained within a very small region of a muscle fiber can result in breakage and loss of muscle fiber from the tissue.     

Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation‐containing electron transport chain‐deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.     

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.     

Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys

Rhesus monkey vastus lateralis muscle was examined histologically for age-associated electron transport system (ETS) abnormalities: fibers lacking cytochrome c oxidase activity (COX(-)) and/or exhibiting succinate dehydrogenase hyperreactivity (SDH(++)). Two hundred serial cross-sections (spanning 1600 microm) were obtained and analyzed for ETS abnormalities at regular intervals. The abundance and length of ETS abnormal regions increased with age. Extrapolating the data to the entire length of the fiber, up to 60% of the fibers were estimated to display ETS abnormalities in the oldest animal studied (34 years) compared to 4% in a young adult animal (11 years). ETS abnormal phenotypes varied with age and fiber type. Middle-aged animals primarily exhibited the COX(-) phenotype, while COX(-)/SDH(++) abnormalities were more common in old animals. Transition region phenotype was affected by fiber type with type 2 fibers first displaying COX(-) and then COX(-)/SDH(++) while type 1 fibers progressed from normal to SDH(++) and then to COX(-)/SDH(++). In situ hybridizations studies revealed an association of ETS abnormalities with deletions of the mitochondrial genome. By measuring cross-sectional area along the length of ETS abnormal fibers, we demonstrated that some of these fibers exhibit atrophy. Our data suggest mitochondrial (mtDNA) deletions and associated ETS abnormalities are contributors to age-associated fiber atrophy.     

Role of Direct Repeat and Stem-Loop Motifs in mtDNA Deletions: Cause or Coincidence?

Deletion mutations within mitochondrial DNA (mtDNA) have been implicated in degenerative and aging related conditions, such as sarcopenia and neuro-degeneration. While the precise molecular mechanism of deletion formation in mtDNA is still not completely understood, genome motifs such as direct repeat (DR) and stem-loop (SL) have been observed in the neighborhood of deletion breakpoints and thus have been postulated to take part in mutagenesis. In this study, we have analyzed the mitochondrial genomes from four different mammals: human, rhesus monkey, mouse and rat, and compared them to randomly generated sequences to further elucidate the role of direct repeat and stem-loop motifs in aging associated mtDNA deletions. Our analysis revealed that in the four species, DR and SL structures are abundant and that their distributions in mtDNA are not statistically different from randomized sequences. However, the average distance between the reported age associated mtDNA breakpoints and their respective nearest DR motifs is significantly shorter than what is expected of random chance in human (p<10(-4)) and rhesus monkey (p = 0.0034), but not in mouse (p = 0.0719) and rat (p = 0.0437), indicating the existence of species specific difference in the relationship between DR motifs and deletion breakpoints. In addition, the frequencies of large DRs (>10 bp) tend to decrease with increasing lifespan among the four mammals studied here, further suggesting an evolutionary selection against stable mtDNA misalignments associated with long DRs in long-living animals. In contrast to the results on DR, the probability of finding SL motifs near a deletion breakpoint does not differ from random in any of the four mtDNA sequences considered. Taken together, the findings in this study give support for the importance of stable mtDNA misalignments, aided by long DRs, as a major mechanism of deletion formation in long-living, but not in short-living mammals.     

Normal levels of wild-type mitochondrial DNA maintain cytochrome c oxidase activity for two pathogenic mitochondrial DNA mutations but not for m.3243A-->G

Mitochondrial DNA (mtDNA) mutations are a common cause of human disease and accumulate as part of normal ageing and in common neurodegenerative disorders. Cells express a biochemical defect only when the proportion of mutated mtDNA exceeds a critical threshold, but it is not clear whether the actual cause of this defect is a loss of wild-type mtDNA, an excess of mutated mtDNA, or a combination of the two. Here, we show that segments of human skeletal muscle fibers harboring two pathogenic mtDNA mutations retain normal cytochrome c oxidase (COX) activity by maintaining a minimum amount of wild-type mtDNA. For these mutations, direct measurements of mutated and wild-type mtDNA molecules within the same skeletal muscle fiber are consistent with the "maintenance of wild type" hypothesis, which predicts that there is nonselective proliferation of mutated and wild-type mtDNA in response to the molecular defect. However, for the m.3243A-->G mutation, a superabundance of wild-type mtDNA was found in many muscle-fiber sections with negligible COX activity, indicating that the pathogenic mechanism for this particular mutation involves interference with the function of the wild-type mtDNA or wild-type gene products.     

Apoptosis and necrosis mediate skeletal muscle fiber loss in age‐induced mitochondrial enzymatic abnormalities

Sarcopenia, the age‐induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age‐dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age‐dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC‐abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.     

Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA

Large-scale deletions of mitochondrial DNA (mtDNA) have been described in patients with progressive external ophthalmoplegia (PEO) and ragged red fibers. We have determined the exact deletion breakpoint in 28 cases with PEO, including 12 patients already shown to harbor an identical deletion; the other patients had 16 different deletions. The deletions fell into two classes. In Class I (9 deletions; 71% of the patients), the deletion was flanked by perfect direct repeats, located (in normal mtDNA) at the edges of the deletion. In Class II (8 deletions; 29% of patients), the deletions were not flanked by any obviously unique repeat element, or they were flanked by repeat elements which were located imprecisely relative to the breakpoints. Computer analysis showed a correlation between the location of the deletion breakpoints and sequences in human mtDNA similar to the target sequence for Drosophila topoisomerase II. It is not known how these deletions originate, but both slipped mispairing and legitimate recombination could be mechanisms playing a major role in the generation of the large mtDNA deletions found in PEO.     

肝癌组织中线粒体DNA D-Loop区碱基变异与ROS水平

为了探讨ROS水平与突变的关系,对原发性肝癌线粒体DNA区的突变情况进行研究,同时对原发性肝癌患者组织细胞内ROS进行测定。选择20例原发性肝癌组织及其邻近的癌旁组织,用PCR方法将线粒体DNA D-Loop扩增后测序。组织内ROS的水平采用流式细胞技术测定。结果表明在20对原发性肝癌组织中存在8对mtDNA突变,突变率为40%,共发现突变位点53个,包括2个插入,11个缺失,40个点突变,其中T-C,C-T的转换占75%,4个属于微卫星结构。癌组织突变一般伴有癌旁组织突变,癌组织突变位点高于癌旁组织。发现一例标本的癌组织和癌旁组织均有大片段丢失。原发性肝癌组织内ROS水平明显高于癌旁对照( P<0.01),同时我们发现在区发生突变的患者的组织中ROS水平明显高于未发生突变的肝癌组织标本（P<0.01）,发生突变的癌旁组织内ROS水平明显高于未发生突变的癌旁组织（P<0.01）。结论 (1)线粒体DNA D-Loop区是一个高度多态性和突变性的区域,在原发性肝癌中突变率较高。(2)肝癌患者组织细胞内ROS异常,提示肝癌的线粒体DNA发生的点突变及肝癌的发生可能与ROS升高有关。     

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

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.     

Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA

Autosomal dominant and/or recessive progressive external ophthalmoplegia (ad/arPEO) is associated with mtDNA mutagenesis. It can be caused by mutations in three nuclear genes, encoding the adenine nucleotide translocator 1, the mitochondrial helicase Twinkle or DNA polymerase γ (POLG). How mutations in these genes result in progressive accumulation of multiple mtDNA deletions in post- mitotic tissues is still unclear. A recent hypothesis suggested that mtDNA replication infidelity could promote slipped mispairing, thereby stimulating deletion formation. This hypothesis predicts that mtDNA of ad/arPEO patients will contain frequent mutations throughout; in fact, our analysis of muscle from ad/arPEO patients revealed an age-dependent, enhanced accumulation of point mutations in addition to deletions, but specifically in the mtDNA control region. Both deleted and non-deleted mtDNA molecules showed increased point mutation levels, as did mtDNAs of patients with a single mtDNA deletion, suggesting that point mutations do not cause multiple deletions. Deletion breakpoint analysis showed frequent breakpoints around homopolymeric runs, which could be a signature of replication stalling. Therefore, we propose replication stalling as the principal cause of deletion formation.     

Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks

To investigate mtDNA recombination induced by multiple double strand breaks (DSBs) we used a mitochondria-targeted form of the ScaI restriction endonuclease to introduce DSBs in heteroplasmic mice and cells in which we were able to utilize haplotype differences to trace the origin of recombined molecules. ScaI cleaves multiple sites in each haplotype of the heteroplasmic mice (five in NZB and three in BALB mtDNA) and prolonged expression causes severe mtDNA depletion. After a short pulse of restriction enzyme expression followed by a long period of recovery, mitochondrial genomes with large deletions were detected by PCR. Curiously, we found that some ScaI sites were more commonly involved in recombined molecules than others. In intra-molecular recombination events, deletion breakpoints were close to or upstream of ScaI cleavage sites, confirming the recombinogenic character of DSBs in mtDNA. A region adjacent to the D-loop was preferentially involved in recombination of all molecules. Sequencing through NZB and BALB haplotype markers in recombined molecules enabled us to show that in addition to intra-molecular mtDNA recombination, rare inter-molecular mtDNA recombination events can also occur. This study underscores the role of DSBs in the generation of mtDNA rearrangements and supports the existence of recombination hotspots.     

Segregation patterns of a novel mutation in the mitochondrial tRNA glutamic acid gene associated with myopathy and diabetes mellitus.

We have identified a novel mtDNA mutation in a 29-year-old man with myopathy and diabetes mellitus. This T-->C transition at mtDNA position 14709 alters an evolutionarily conserved nucleotide in the region specifying for the anticodon loop of the mitochondrial tRNA(Glu). The nt-14709 mutation was heteroplasmic but present at very high levels in the patient's muscle, white blood cells (WBCs), and hair follicles; lower proportions of mutated mtDNA were observed in WBCs and hair follicles of all examined maternal relatives. In the patient's muscle, abnormal fibers showed mitochondrial proliferation, severe focal defects in cytochrome c oxidase activity, and absence of cross-reacting material for mitochondrially synthesized polypeptides. These fibers had higher levels of mutated mtDNA than did surrounding "normal" fibers. Although the percentage of mutated mtDNA in WBCs from family members were distributed around the percentage observed in the mothers, the pattern was different in hair follicles, where the mutated population tended to increase in subsequent generations. PCR/RFLP analysis of single hairs showed that the intercellular variations in the percentage of mutated mtDNA differed among family members, with younger generations having a more homogeneous distribution of mutated mtDNA in different hair follicles. These results suggest that the intercellular distribution of the mutated and wild-type mtDNA populations may drift toward homogeneity in subsequent generations.     

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 in adult mouse cardiac side population cells

We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.     

The mtDNA mutation spectrum of the progeroid Polg mutator mouse includes abundant control region multimers

Polg mtDNA mutator mice are important models for investigating the role of acquired mtDNA mutations in aging. Despite extensive study, there remains little consensus on either the etiology of the progeroid phenotype or the mtDNA mutation spectrum induced by disrupted polymerase-γ function. To investigate the latter, we have developed a novel, pragmatic approach we term "Mito-seq," applying next-generation sequencing to enriched, native mtDNA. Regardless of detection parameters we observed an increase of at least two orders of magnitude in the number of mtDNA single nucleotide variants in Polg mutator mice compared to controls. We found no evidence for the accumulation of canonical mtDNA deletions but multimers of the mtDNA control region were identified in brain and heart. These control region multimers (CRMs) contained heterogeneous breakpoints and formed species that excluded the majority of mtDNA genes. CRMs demonstrate that polymerase-γ 3'-5' exonuclease activity is required for preserving mtDNA integrity.