Segmental cytochrome c-oxidase deficiency in CPEO: teased muscle fiber analysis.

In an attempt to elucidate the pathogenesis of focal cytochrome c-oxidase (COX) deficiency in skeletal muscle from patients with chronic progressive external ophthalmoplegia (CPEO), we examined the longitudinal distribution of COX activity in single muscle fibers from 6 CPEO patients with muscle mitochondrial DNA (mtDNA) deletions. A new method for teasing single muscle fibers, recently developed in our laboratory, revealed fibers with COX-positive and -negative segments in all 6 patients. The borders between the enzyme-positive and -negative segments in these fibers were sharply delineated, so that the length of each COX-negative segments could be accurately measured. The proportion of the sum of the lengths of the enzyme-negative segments to the total length of the muscle fibers correlated well with the proportion of deleted mtDNA, suggesting that abnormal mitochondria harboring mutant mtDNA may be responsible for the focal loss of COX activity.     

Mitochondrial DNA copy number threshold in mtDNA depletion myopathy

The authors measured the absolute amount of mitochondrial DNA (mtDNA) within single muscle fibers from two patients with thymidine kinase 2 (TK2) deficiency and two healthy controls. TK2 deficient fibers containing more than 0.01 mtDNA/microm3 had residual cytochrome c oxidase (COX) activity. This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome.     

Clinical progression of mitochondrial myopathy is associated with the random accumulation of cytochrome c oxidase negative skeletal muscle fibres

We studied the accumulation of cytochrome c oxidase (COX)-negative skeletal muscle fibres in six patients with a myopathy due to a mitochondrial DNA (mtDNA) defect. Each patient was biopsied on two or more occasions over a period of 3-15 years. Progressive proximal weakness was associated with an increase in the proportion of COX-negative fibres. These fibres were arranged randomly, indicating that each fibre became COX negative independently of the status of neighbouring fibres. The clinical progression of mtDNA myopathy is therefore a consequence of a biochemical defect that develops independently within individual muscle fibres. It is likely that this is due to the clonal expansion of mutant mtDNA.     

Association of myopathy with large-scale mitochondrial dna duplications and deletions: Which is pathogenic?

We identified large-scale heteroplasmic mitochondrial DNA (mtDNA) rearrangements in a 50–year-old woman with an adult-onset progressive myopathy. The predominant mtDNA abnormality was a 21.2–kb duplicated molecule. In addition, a small population of the corresponding partially deleted 4.6–kb molecule was detected. Skeletal muscle histology revealed fibers that were negative for cytochrome c oxidase (COX) activity and had reduced mtDNA-encoded COX subunits. By single-fiber polymerase chain reaction analysis, COX-negative fibers contained a low number of wild-type or duplicated mtDNA molecules (ie, nondeleted). In situ hybridization demonstrated that the abnormal fibers contained increased amounts of mtDNA compared with normal fibers and that most of the genomes were deleted. We concluded that deleted mtDNA molecules were primarily responsible for the phenotype in this patient.     

Analysis of mtDNA deletions in muscle by in situ hybridization

We compared the distribution of deleted mitochondrial DNA (Delta-mtDNA) in skeletal muscle of a patient with autosomal recessive (AR) and another with autosomal dominant (AD) progressive external ophthalmoplegia (PEO) by in situ hybridization (ISH). The patients studied had similar numbers of fibers deficient in cytochrome c oxidase (COX) activity (13.6% and 12.8%) and fibers with mitochondrial proliferation (5.5% and 5.3%). ISH suggested that each COX-deficient fiber contained a single species of Delta-mtDNA. Most deletions ablated the region between the genes encoding adenosine triphosphate (ATP) synthase subunit 8 and cytochrome b. Fibers that appeared to be depleted of mtDNA were also present. We conclude that muscle from patients with autosomally inherited PEO contains not only Delta-mtDNA but also focal depletion of mtDNA and that the distribution of these mtDNA defects appears to be similar. These changes most likely represent the common consequence of whatever genetic factors are responsible for the generation of Delta-mtDNA.     

Leigh syndrome with cytochrome-c oxidase deficiency and a single T insertion nt 5537 in the mitochondrial tRNATrp gene

We report a nine-year-old boy with the features of Leigh syndrome (LS) and a severe cytochrome-c oxidase (COX) deficiency with a single thymidine insertion at nucleotide position 5537 (T 5537i) in the tRNA Trp gene of mitochondrial DNA. During infancy the boy was irritable and hypotonus was noticed. Early motor development was delayed, although mental development seemed normal until eight months of age. Early neurological signs were nystagmus, hypertonus and optic atrophy. Severe seizures and mental retardation developed subsequently. Major findings on neuroradiological investigation were from the brainstem, thalami and white matter compatible with LS. Spectrophotometric analysis of skeletal muscle mitochondria showed a profound COX deficiency and a marked complex I deficiency. Enzyme-histochemical analysis showed reduced COX activity in the majority of the muscle fibres. There were no ragged red fibres. The T 5537i mutation was found in a high proportion (> 95 %) in blood, liver and muscle tissue of the patient and in blood of the patient's mother (81 %). This mutation has previously been described in one family in which one child had a very high proportion of the T 5537i mutation and clinical features of LS. We conclude that, although mtDNA mutations are considered to be rare in LS with COX deficiency, the T 5537i mutation should be screened for in cases of LS with COX deficiency when SURF1 gene mutations have been excluded, especially when complex I activity is also decreased.     

Mitochondrial DNA deletions and depletion within paraspinal muscles

G. R. Campbell, A. Reeve, I. Ziabreva, T. M. Polvikoski, R. W. Taylor, R. Reynolds, D. M. Turnbull and D. J. Mahad (2013) Neuropathology and Applied Neurobiology 39, 377–389 Mitochondrial DNA deletions and depletion within paraspinal muscles Aims: Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age‐related histopathological changes were previously reported. Methods: Cervical and lumbar paraspinal muscles were obtained peri‐operatively from 13 patients and from six post mortem control cases (age range 18–82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory‐deficient muscle fibres (lacking complex IV with intact complex II activity). Real‐time polymerase chain reaction, long‐range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry. Results: The density of respiratory‐deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory‐deficient (range 0–10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory‐deficient fibres with and without mtDNA deletions or mtDNA depletion. Conclusions: Paraspinal muscles appeared to be particularly susceptible to age‐related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age‐related postural abnormalities.     

Patterns of abnormal histochemical fibre type differentitation in human muscle biopsies.

The histochemical profile of individual human skeletal muscle fibres was analysed by correlating mitochondrial oxidative enzyme activity and that of myofibrillar ATPase at pH 9.5 and after pre-incubation at pH 4.3 and pH 4.6. In normal control muscle, only a small percentage of fibres did not conform to one or other of the normal variants of Type I and Type II fibres. In biopsies from early cases of Werdnig-Hoffmann disease, the denervated fibre populations contained many abnormal Type I and Type II fibres, including "IIc" fibres, but the basic distinction between Type I and Type II was preserved. However, in infantile spinal muscular atrophy patients aged two years and over, this distinction was progressively lost, leading to the total dedifferentiation of the atrophied fibres. In the Kugelberg-Welander form of spinal muscular atrophy, many of the constituent fibres of re-innervated groups displayed normal or near-normal histochemical profiles, but chronically denervated fibres became totally dedifferentiated. In Duchenne dystrophy, the spectrum of histochemical types appeared to be more continuous due to the emergence of fibres with properties intermediate between those of the normal variants, but the basic distinction between Type I and Type II fibres was preserved in the majority of cases. The percentage of severely abnormal fibres was higher in Type II than Type I and probably contributed to the observed decrease in the overall proportion of Type II fibres. Although very small atrophied fibres were observed in biopsies from cases of Becker and Duchenne dystrophy, these did not show the total dedifferentiation seen in the chronically denervated fibres in cases of spinal muscular atrophy.     

Variability of the expression of muscle mitochondrial damage in ocular mitochondrial myopathy

In this study we comparatively analysed deltoid histochemistry, biochemistry and mitochondrial DNA (mtDNA) in two groups of ten sporadic ocular mitochondrial myopathies (OMM), respectively with and without ragged red fibres (RRF). (1) All but one RRF − patients presented the mild form of OMM with blepharoptosis but without ophthalmoplegia; (2) the occurrence of cytochrome c oxidase deficient (COX −) fibres was significantly higher in the RRF + group, but four RRF − cases also showed COX − fibres; (3) no difference was observed in biochemical findings between the groups; (4) two RRF − patients without COX − fibres showed mtDNA heteroplasmy; (5) in two RRF − patients without deltoid mtDNA deletion, biopsy of an eyelid muscle showed significant mitochondrial alterations. These results suggest that the expression of a mitochondrial defect can vary and that the absence of RRF in a skeletal muscle biopsy does not necessarily rule out the diagnosis of OMM, if other data support that.     

Cytochromec oxidase deficiency in infancy

Summary Five children with early onset of muscle weakness, lactic acidosis and deficient cytochromec oxidase staining in the muscle biopsy were studied. By oximetric assay of the respiratory chain of isolated mitochondria, cytochromec oxidase deficiency was confirmed in four of the cases, while one case showed only a slight decrease of cytochromec oxidase activity but considerably reduced activity when assayed spectrophotometrically. The muscle biopsies exhibited mitochondrial structural abnormalities and lipid storage in the four cases with oximetrically confirmed cytochromec oxidase deficiency, while the biopsy of the case with markedly reduced activity of cytochromec oxidase only in the enzyme-histochemical and spectrophotometrical assays had normal morphology. The light microscopical staining of cytochromec oxidase in the four cases with oximetrically confirmed deficiency showed deficient staining of the enzyme in all extrafusal fibres in three cases but one of the cases had normal enzyme-histochemical activity of cytochromec oxidase in about 25% of the fibres. In two cases muscle spindles were included in the biopsy. The intrafusal fibres showed normal enzyme-histochemical activity of cytochromec oxidase. Ultrastructural examination of the enzyme distribution in two of the cases revealed great heterogeneity of the mitochondria. The structurally abnormal mitochondria were usually deficient of enzyme activity. The mitochondria of endothelial cells appeared to have normal activity. Immunohistochemical staining with polyclonal antibodies to cytochromec oxidase revealed presence of immunoreactive material corresponding to the localisation of mitochondria in all cases. The results show that enzyme-histochemical staining of cytochromec oxidase is a useful technique to reveal deficiency of the enzyme and to study the distribution of the deficiency within the tissue both at the light microscopical and ultrastructural levels. However, the results of one of the cases show that deficiency revealed by the enzyme-histochemical technique is not completely reliable. Oximetric studies on isolated mitochondria are necessary to confirm the suspected deficiency and to reveal combined defects of the respiratory chain.Supported by grants from the Swedish Medical Research Council (proj. 03X 585 and 07122), Barnhusfonden Göteborg L 174/87 and The First of May Flower Annual Campaign for Childrens Health     

Residual muscle cytochrome c oxidase activity accounts for submaximal exercise lactate threshold in chronic progressive external ophthalmoplegia

The data from histological, biochemical, and mitochondrial DNA (mtDNA) studies of muscle biopsies from 10 patients affected with chronic progressive external ophthalmoplegia (CPEO) were related to dynamic and metabolic parameters of incremental submaximal exercise. Maximum power output was reduced in all patients as compared to controls. Analysis of the venous lactate curve during exercise revealed a lactate threshold at exercise levels ranging from 40 to 50% of the predicted maximal power output. An earlier significant increase in lactate could be detected by calculating the mean δ lactate. Lactate values were inversely correlated with the cytochrome c oxidase (COX) activity of isolated muscle mitochondria. No relationship was found between lactate values and the number of ragged red fibers, or cytochrome c oxidase-negative fibers or the proportion of deleted mtDNA measured in muscle biopsy specimens. The discussion underscores the value of lactate kinetics in assessing skeletal muscle function, as well as the use of muscle COX levels to predict the effectiveness of wild-type complementation of deleted skeletal muscle mtDNA in in vivo contractile performance of CPEO subjects. © 1996 John Wiley & Sons, Inc.     

Ageing muscle: clonal expansions of mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function

Although mitochondrial DNA deletions have been shown to accumulate in cytochrome c oxidase deficient muscle fibres of ageing muscle, this has not been demonstrated for point mutations. In this study, we investigated the occurrence of mitochondrial DNA alterations (point mutations and deletions) in cytochrome c oxidase deficient muscle fibres from 14 individuals, without muscle disease, aged 69-82 years. Immunohistochemical investigation showed that the majority of the cytochrome c oxidase deficient muscle fibres expressed reduced levels of subunit II of cytochrome c oxidase, which is encoded by mitochondrial DNA, whereas there was normal or increased expression of subunit IV of cytochrome c oxidase, which is encoded by nuclear DNA. This pattern is typical for mitochondrial DNA mutations causing impaired mitochondrial translation. Single muscle fibres (109 cytochrome c oxidase deficient and 109 normal fibres) were dissected and their DNA extracted. Mitochondrial DNA point mutations were searched for in five tRNA genes by denaturing gradient gel electrophoresis while deletions were looked for by polymerase chain reaction amplification. High levels of clonally expanded point mutations were identified in eight cytochrome c oxidase deficient fibres but in none of the normal ones. They included the previously described pathogenic tRNALeu(UUR)A3243G and tRNALysA8344G mutations and three original mutations: tRNAMetT4460C, tRNAMetG4421A, and a 3-bp deletion in the tRNALeu(UUR) gene. Four different large-scale mitochondrial DNA deletions were identified in seven cytochrome c oxidase deficient fibres and in one of the normal ones. There was no evidence of depletion of mitochondrial DNA by in situ hybridisation experiments. Our data show that mitochondrial DNA point mutations, as well as large-scale deletions, are associated with cytochrome c oxidase deficient muscle fibre segments in ageing. Their focal accumulation causes significant impairment of mitochondrial function in individual cells in spite of low overall levels of mitochondrial DNA mutations in muscle.     

Postnatal differentiation and growth of skeletal muscle fibres in normal and undernourished rats. A histochemical and morphometric study.

The postnatal differentiation and growth patterns of the different histochemical muscle fibre types were studied at frequent age intervals in the extensor digitorum longus (EDL) muscle of normal rats and animals subjected to pre- and postnatal protein-calorie undernutrition. Three stepwise changes were seen in the fibre type composition of the EDL muscle during maturation. In newborn normal rats 2 histochemically and morphometrically distinct fibre populations occurred: small foetal type (type F) fibres or myotubes (about 90%), with the staining characteristics of the so-called type 2C fibres, and larger type 1 fibres (10%). At 5 days of age the percentage of type F fibres had dropped to 50 simultaneously with the emergence of a new population (40%) of type 2B fibres, intermediate in cross-sectional area between the remaining type F fibres and type 1 fibres. Between 15 and 20 days the small type F fibres practically disappeared with the emergence of a corresponding percentage (40%) of type 2A fibres, smaller than both type 1 and type 2B fibres; Between 20 and 60 days the percentage of type 1 rose from 10 to almost 30, type 2B fibres correspondingly diminishing in number but growing at a faster rate than either type 1 or type 2A fibres. In the undernourished rats the histochemical differentiation was retarded at birth, all fibres or myotubes being of the foetal type; However, large type 1 and type 2B fibres were seen at the age of 5 days and the histochemical maturation proceeded almost normally at later stages. On the other hand, at all ages there was a proportional reduction in the cross-sectional area of all fibre types, amounting to about 50% at 180 dyas, and not fully restituted by nutritional rehabilitation. These observations suggest that type F (type 2C) fibres are the undifferentiated precursors of all mature muscle fibres, their stepwise histochemical transformation raising the question of maturational differences in the different types of motoneurones.     

Mitochondrial myopathy and rhabdomyolysis associated with a novel nonsense mutation in the gene encoding cytochrome c oxidase subunit I

Mitochondrial DNA (mtDNA) mutations associated with rhabdomyolysis are rare but have been described in sporadic cases with mutations in the cytochrome b and cytochrome c oxidase (COX) genes and in 3 cases with tRNALeu mutation. We report a novel heteroplasmic G6708A nonsense mutation in the mtDNA COI gene encoding COX subunit I in a 30-year-old woman with muscle weakness, pain, fatigue, and one episode of rhabdomyolysis. Histochemical examination of muscle biopsy specimens revealed reduced COX activity in the majority of the muscle fibers (approximately 90%) and frequent ragged red fibers. Biochemical analysis showed a marked and isolated COX deficiency. Analysis of DNA extracted from single fibers revealed higher levels of the mutation in COX-deficient fibers (> 95%) compared with COX-positive fibers (1%-80%). The mutation was not detected in a skin biopsy, cultured myoblasts, or blood leukocytes. Nor was it identified in blood leukocytes from the asymptomatic mother, indicating a de novo mutation that arose after germ layer differentiation. Western blot analysis and immunohistochemical staining revealed that reduced levels of COX subunit I were accompanied by reduced levels of other mtDNA encoded subunits, as well as nuclear DNA encoded subunit IV, supporting the concept that COX subunit I is essential for the assembly of complex IV in the respiratory chain.     

Parabiotic reinnervation in normal and dystrophic mice. Part 2. Morphological studies.

The technique of parabiotic reinnervation has been used to test directly the neurogenic theory of the aetiology of muscular dystrophy in mice. Dystrophic muscles contain significantly fewer muscle fibres than their normal controls; they also have a much broader spectrum of fibre size because of a much higher proportion of very small fibres and are poorly differentiated into histochemical fibre types. These criteria were used to assess whether there was any amelioration of the dystrophic process in response to the introduction of a normal nerve supply, or whether dystrophic changes were induced in normal muscle reinnervated with a dystrophic nerve. Self-reinnervated normal and dystrophic TA and EDL muscles contained the same numbers of fibres as unoperated controls. The process of parabiosis alone resulted in no changes in normal or dystrophic muscles. In the process of parabiotic reinnervation, the efficiency of the reinnervation process was not affected by the parabiotic state. The parabiotic reinnervation of dystrophic muscle by normal nerve resulted in no significant increase in fibre numbers and the spectrum of fibre sizes was essentially the same as in unoperated dystrophic muscle. The parabiotic reinnervation of normal muscle by dystrophic nerve resulted in a reduction of fibre numbers in only some of the muscles examined. However, the spectrum of fibre diameters remained essentially normal, and the differentiation of the fibres into histochemical fibre types was characteristic of reinnervated normal muscle. There was a marked absence of necrosis or of other histological signs of dystrophy in these muscles. Since there was no positive evidence to show that conversion of normal to dystrophic, or dystrophic to normal muscle occurred under the influence of parabiotic nerve transposition, two alternative conclusions were admissible. Firstly, the influence of dystrophic nerve upon muscle may be operative in fetal or neonatal life and may be irreversible by means of the subsequent introduction of a normal nerve supply. Secondly, the dystrophic state in muscle may be determined by genetic factors independent of nerve supply.     

Fibre hybrids in type group: An investigation of human muscle biopsies

Change of fibre type caused by reinnervation implies change in a series of metabolic processes. As long as these changes are in progress the histochemical pattern in muscle fibres may demonstrate deviations from the normal characteristics. The present histochemical study was undertaken to evaluate in human neurogenic muscle disease the completeness of conversion of presumably reinnervated muscle fibres.At least a number of muscle fibres in type groups is reinnervated. Type grouping of non-atrophic fibres was found in 27 of 42 muscle biopsies from patients with denervating diseases. The myosin ATPase activity in these groups was often strikingly even. In a varying degree and in a varying number of muscle fibres myosin ATPase-uniform groups showed intermediate capacity of aerobic and/or glycolytic metabolism; this finding was considered compatible with conversion due to reinnervation. Two main kinds of fibre hybrids were observed. One kind showed low myosin ATPase activity and an apparently low capacity for aerobic metabolism. The other kind also showed low myosin ATPase activity but the capacity of glycolytic metabolism was high, aerobic metabolism in these fibres being intermediate or high.It has been suggested that low capacity of aerobic metabolism in fibre hybrids of the first kind is related to hypertrophy of the muscle fibres. The appearance of fibre hybrids of the second kind would be conceivable as a stage in a process of conversion, if at least changes in the capacities of the 2 metabolic pathways can develop at a markedly different pace. However, groups or fascicles of fibre hybrids of this kind are present is some cases. These configurations point to a steady state rather than to a dynamic process of conversion; a lack of plasticity in the muscle fibre apparently prevents completion of conversion.     

Histochemical and histopathological changes in skeletal muscle in late-onset hereditary distal myopathy (Welander).

Histochemical and histopathological staining methods were applied to muscle biopsy material from 13 patients with distal myopathy of late onset. Six cases showed slight to moderate histopathological changes and the normal distinction between Type I and Type II muscle fibres, based on their staining characteristics for myofibrillar ATPase, was well preserved. A selective Type I atrophy and an irregular distribution of oxidative enzyme and fat staining in Type I fibres were evident. In the other 7 cases, with moderate to advanced histopathological changes, there was a marked blurring of the normal difference observed in ATPase activity between Type I and TYpe II fibres. Thus, both types of fibre exhibited a high intensity of staining for myofibrillar ATPase at pH 9.4 without inhibition by acid preincubation (pH 4.3). These changes in phosphatase activity were found not only in atrophic fibres but also in normal-sized fibres without other signs of degeneration. Nuclear proliferation in chains and "ring fibres" were found. The early histopathological and histochemical changes in distal myopathy are strikingly similar to those of myotonic dystrophy.     

Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level

In situ hybridization combined with immunohistochemical techniques has been applied to study patients affected by mitochondrial myopathies with large mitochondrial (mt)DNA deletions. All patients' muscle biopsies showed ragged red fibers (RRFs) and cytochrome oxidase (COX) deficiency. Two digoxygenin-labeled, polymerase chain reaction (PCR)-amplifed DNAs were used as probes. One probe was designed to hybridize only with wild-type mtDNAs, while the other recognized both wild-type and deleted mtDNAs. Concomitant immunocytochemical analysis using antibodies against subunits II, III, (encoded by mtDNA) and IV (encoded by nuclear DNA) of COX was carried out. In our patients deleted mtDNAs are overexpressed in COX-negative RRFs, while wild-type mtDNAs are decreased in the same fibers. Immunohistochemistry studies show that COX IV is overexpressed in RRFs and that COX II and COX III subunits are still present. Deleted mtDNAs are spatially segregated in muscle fibers, where they interfere with the local population of normal mitochondrial genomes, causing a regional deficiency of the mitochondrial respiratory activity.This work was supported by the Associazione Amici del Centro Dino Ferrari