Cytochrome C Oxidase Deficient Fibres Research Articles

Extraocular Muscle Reveals Selective Vulnerability of Type IIB Fibers to Respiratory Chain Defects Induced by Mitochondrial DNA Alterations.

PurposeThe purpose of this study was to gain insights on the pathogenesis of chronic progressive external ophthalmoplegia, thus we investigated the vulnerability of five extra ocular muscles (EOMs) fiber types to pathogenic mitochondrial DNA deletions in a mouse model expressing a mutated mitochondrial helicase TWINKLE.MethodsConsecutive pairs of EOM sections were analyzed by cytochrome C oxidase (COX)/succinate dehydrogenase (SDH) assay and fiber type specific immunohistochemistry (type I, IIA, IIB, embryonic, and EOM-specific staining).ResultsThe mean average of COX deficient fibers (COX−) in the recti muscles of mutant mice was 1.04 ± 0.52% at 12 months and increased with age (7.01 ± 1.53% at 24 months). A significant proportion of these COX− fibers were of the fast-twitch, glycolytic type IIB (> 50% and > 35% total COX− fibers at 12 and 24 months, respectively), whereas embryonic myosin heavy chain-expressing fibers were almost completely spared. Furthermore, the proportion of COX− fibers in the type IIB-rich retractor bulbi muscle was > 2-fold higher compared to the M. recti at both 12 (2.6 ± 0.78%) and 24 months (20.85 ± 2.69%). Collectively, these results demonstrate a selective vulnerability of type IIB fibers to mitochondrial DNA (mtDNA) deletions in EOMs and retractor bulbi muscle. We also show that EOMs of mutant mice display histopathological abnormalities, including altered fiber type composition, increased fibrosis, ragged red fibers, and infiltration of mononucleated nonmuscle cells.ConclusionsOur results point to the existence of fiber type IIB-intrinsic factors and/or molecular mechanisms that predispose them to increased generation, clonal expansion, and detrimental effects of mtDNA deletions.

The mutations m.5628T>C and m.8348A>G in single muscle fibers of a patient with chronic progressive external ophthalmoplegia

We identified a double mutation in a patient with chronic progressive external ophthalmoplegia, located in the tRNAAla (m.5628T>C) and tRNALys (m.8348A>G) genes. Both mutations were previously described separately and considered pathogenic, however the same mutations were also reported as polymorphisms or phenotype modulator. We analyzed the proportion of each mutation in isolated muscle fibers by single fiber-polymerase chain reaction to investigate the contribution of each mutation to mitochondrial deficiency. Our findings demonstrated that the mutations were heteroplasmic in skeletal muscle and both mutations were present in all single muscle fibers. The proportions of the m.5628T>C mutation were not significantly different between normal and cytochrome-c-oxidase (COX) deficient fibers. However, a significant higher proportion of the m.8348A>G mutation was observed in COX deficient fibers. Homoplasmic m.8348A>G was only observed in COX negative fibers. In conclusion, we provide a piece of evidence toward the pathogenicity of the m.8348A>G mutation and suggest that m.5628T>C is probably a neutral polymorphism.

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle.

Mitochondrial abnormalities in inclusion-body myositis

Mitochondrial changes are frequently encountered in sporadic inclusion-body myositis (s-IBM). Cytochrome c oxidase (COX)-deficient muscle fibers and large-scale mitochondrial DNA (mtDNA) deletions are more frequent in s-IBM than in age-matched controls. COX deficient muscle fibers are due to clonal expansion of mtDNA deletions and point mutations in segments of muscle fibers. Such segments range from 75 microm to more than 1,000 microm in length. Clonal expansion of the 4977 bp "common deletion" is a frequent cause of COX deficient muscle fiber segments, but many other deletions also occur. The deletion breakpoints cluster in a few regions that are similar to what is found in human mtDNA deletions in general. Analysis in s-IBM patients of three nuclear genes associated with multiple mtDNA deletions, POLG1, ANT1 and C10orf2, failed to demonstrate any mutations. In s-IBM patients with high number of COX-deficient fibers, the impaired mitochondrial function probably contribute to muscle weakness and wasting. Treatment that has positive effects in mitochondrial myopathies may be tried also in s-IBM.

Cytochrome c oxidase deficient muscle fibres: Substantial variation in their proportions within skeletal muscles from patients with mitochondrial myopathy

Mitochondrial DNA (mtDNA) disease is a common cause of myopathy and the presence of histochemically demonstrated cytochrome c oxidase (COX) deficiency is an extremely useful diagnostic feature. However, there is currently no quantitative information regarding the variability of COX deficiency within or between muscles. This study addresses this issue by studying a number of skeletal muscle samples obtained at post-mortem from three patients with mitochondrial disease due to established mitochondrial DNA defects. COX deficient muscle fibres were enumerated in sections of these muscles and analysed according to patient, individual muscle, position within a particular muscle and sample size. Descriptive statistics were generated followed by an analysis of variance (ANOVA) to assess the effect of these parameters on the mean percentage of COX deficient fibres. We observed statistically significant variation in the percentage of COX deficient fibres within individual muscles from each patient for samples sizes of between 100 and 400 fibres. Our results have implications for the way in which biopsies of skeletal muscle are used for the assessment of disease severity, progression and response to treatment.

An age-associated correlation between cellular bioenergy decline and mtDNA rearrangements in human skeletal muscle

Post-mitotic tissues such as skeletal muscle develop a tissue bioenergy mosaic during the process of normal aging that eventually culminates into a bioenergetically diverse tissue containing cells ranging in their oxidative phosphorylation capacity from normal to grossly defective. The mosaic is postulated to develop continuously from birth with the relative proportions of cytochrome c oxidase (COX) proficient (positive) and COX deficient (negative) muscle fibers differing dramatically as a function of age. Generally, young individuals only display the rare fiber deficient in COX activity while aged individuals show a significantly higher proportion of negative fibers. There appears to be a random element governing which cells will be affected. Consequently, adjacent cells within a given tissue may exhibit vastly differing COX activities. Multiple mitochondrial DNA (mtDNA) deletions also appear to accumulate in skeletal muscle, similarly displaying a dramatic disparity as a function of age. Our previous findings have indicated that the accumulation of multiple mtDNA deletions, along with a concurrent decrease in wild-type mtDNA, strongly correlates with the age-associated decrease in COX activity observed in skeletal muscle. Although no definitive associations were established at the cellular level, an important prediction arose from this study. Cells that accumulate large numbers of mitochondrial mutations and have reduced levels of full-length mtDNA would be expected to be severely affected and show reduced COX activity as a consequence. Cells that accumulate fewer mutations or retain adequate amounts of wild-type mtDNA would be predicted to be less affected or even retain normal oxidative metabolism. In order to establish a link associating COX activity to the status of mtDNA within individual fibers, we developed single cell extra-long PCR (XL-PCR). The procedure was used to assess the relative concentration of full-length mtDNA with respect to any mtDNA deletions detected in individual human skeletal muscle fibers of `pre-established' COX activity. Single cell XL-PCR analysis of COX positive fibers dissected from a 5-year old and 90-year old individual showed that 80% or more of the fibers contained full length mtDNA and few, if any, mtDNA rearrangements. COX deficient or COX intermediate fibers taken from the same individuals, by contrast, depicted a heterogeneous population of rearranged mtDNA species with no detectable full-length mtDNA. The data presented here indicates that COX deficient muscle fibers extracted from individuals, regardless of age, were accompanied by extensive mtDNA rearrangements and reduced levels of full-length mtDNA. This provides compelling evidence linking mtDNA mutations to COX activity decline in skeletal muscle and has important implications when considering the molecular basis of the aging process.

Deltoid Human Muscle MTDNA Is Extensively Rearranged in Old Age Subjects

Extra long PCR analysis of mitochondrial DNA (mtDNA) isolated from skeletal muscle of humans of different ages revealed three phenomena: (i) the amount of normal length mtDNA (16.5 kb) was progressively reduced with age, such that the cells of old age individuals (about 90 years) contained little, or undetectable amounts of normal length mtDNA; (ii) the total amount of mtDNA did not appear to be greatly decreased rather the extent of mtDNA deletions greatly increased; (iii) in old age subjects, considerable amounts of over-sized mtDNA (more than 16.5 kb) was observed. Enzyme histochemical analysis of cytochrome-c oxidase (COX) activity in the muscle tissue of all subjects evidenced a cellular bioenergy mosaic with cells ranging from high to zero detectable enzyme activity in the muscle samples. The frequency of COX deficient muscle fibres was highly dependent on the age of the subject. We have found that the extent of the mtDNA mutational changes strongly correlate with the observed progressive decrease in COX activity. Therefore, it was suggested that the total extent of mtDNA mutation is very large in old age subjects and is sufficient to account for the decline in cellular COX activity with age and for a progressive decrease of overall mitochondrial bioenergetic capacity.

Mitochondrial DNA deletions and cytochrome c oxidase deficiency in muscle fibres

We have studied cytochrome c oxidase (COX) deficient muscle fibre segments in 6 patients with mitochondrial myopathy and deletions of mitochondrial DNA (mtDNA). The distribution of transcripts of normal and mutated mtDNA in skeletal muscle sections was studied by in situ hybridization. The results were compared with the enzyme histochemical activity of COX and the immunohistochemical distribution of mtDNA encoded and nuclear DNA encoded subunits of COX. In all cases a proportion of the muscle fibres (< 1–30% of the fibres in cross-sections) and had low COX activity and high activity of succinate dehydrogenase (COX deficient muscle fibres). Transcripts of normal and deleted mtDNA showed the same distribution within the tissue as the corresponding mtDNA, indicating that the deleted mtDNA is transcribed. The COX deficient muscle fibres showed accumulation of transcripts of deleted mtDNA, which had a similar distribution as the accumulated mitochondria within these fibres. With few exceptions, there was a low level of transcripts of normal mtDNA in these COX deficient fibres. Immunohistochemical analysis revealed low levels of immunoreactive material using antiserum to the mtDNA encoded subunits II/III as well as the nuclear DNA encoded subunit IV of COX in all COX deficient muscle fibres. The fraction of deleted mtDNA in muscle ranged from 43 to 87%. There was no correlation between the proportion of COX deficient muscle fibres and the fraction of deleted mtDNA. In 2 cases the deletion did not involve any COX gene. One of these cases had 87% deleted mtDNA but less than 1% COX deficient muscle fibres. This case had several muscle fibres with accumulation of transcripts of deleted mtDNA concomitant with normal COX activity. The other of these 2 cases had 10% COX deficient muscle fibres. Our results show that the proportion of COX deficient muscle fibres is not correlated to the fraction of deleted mtDNA in muscle tissue and that a high proportion of COX deficient fibres may be present even if COX subunit genes are not affected by the deletion. Reduced enzyme histochemical activity of COX in muscle fibre segments is correlated to low levels of normal mtDNA in these fibre segments.