Mitochondrial encephalomyopathies and cytochrome c oxidase deficiency: muscle culture study

Summary The populations of cytochrome c oxidase (CCO)-positive and-negative mitochondria were analyzed in the elongated cells containing occasional multiple nuclei (myotubes) in primary muscle cultures derived from patients with various forms of mitochondrial encephalomyopathies with CCO deficiency. Even in control muscle cultures, CCO-positive (79.7%) and -negative (20.3%) mitochondria were distributed randomly, showing intracellular mosaicism. All mitochondria in all muscle cultures from two patients with clinical characteristics of Leigh's disease exhibited faint to negative CCO activity. In these patients no enzyme activity could be detected in any tissue including intrafusal fibers and fibroblasts in muscle biopsies. In patients with the fatal infantile and the encephalomyopathic forms of CCO deficiency, and myoclonic epilepsy with ragged-red fibers, two different types of myotubes containing mostly CCO-positive mitochondria and only negative mitochondria, respectively, representing intercellular mosaicism, were demonstrated. The intercellular mosaicism in biopsied and cultured muscles in the case of CCO deficiency supports the contention that both CCO-positive and -negative mitochondria coexist in the early myogenic cell and later randomly segregated during cell division (mitotic segregation), forming two different cells.Support in part by Grants-in-Aid for Scientific Research (No. 0267032), Scientific Research on Priority Areas (No. 62617523) and Developmental Scientific Research (No. 62870041) from the Ministry of Education, Science and Culture of Japan     

Multiple mitochondrial DNA deletions in hereditary inclusion body myopathy

We have recently described an autosomal dominant hereditary inclusion body myopathy (h-IBM). Clinically it is is characterized by congenital joint contractures and slowly progressive, proximal muscle weakness and ophthalmoplegia. There is deterioration of muscle function between 30 and 50 years of age. While young patients show minor pathological changes in muscle, the middle-aged and old patients show rimmed vacuoles and inclusions of filaments measuring 15–18 nm in diameter. Except for the absence of significant inflammation the histopathology is similar to that found in sporadic inclusion body myositis (s-IBM). In s-IBM mitochondrial alterations including cytochrome c oxidase (COX) -deficient muscle fibers are common. These are due to multiple mitochondrial DNA (mtDNA) deletions. In this study we investigated the occurrence of mitochondrial alterations in autosomal dominant h-IBM. Young affected individuals showed no mitochondrial changes but three patients aged 38, 51 and 59 years, respectively, showed ragged red fibers and COX-deficient muscle fibers. Polymerase chain reaction analysis showed multiple mtDNA deletions. By in situ hybridization clonal expansions of mtDNA with deletions were demonstrated in COX-deficient muscle fibers. Most of the analyzed deletion breakpoints showed nucleotide repeats flanking the deletions. The results show that COX-deficient muscle fibers and somatic mtDNA deletions are present in this family with h-IBM. The same factors may be involved in the development of mtDNA deletions in s-IBM and this family with h-IBM. Received: 13 July 1999 / Revised: 6 October 1999 · Accepted: 12 October 1999     

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     

Fatal infantile mitochondrial myopathy due to cytochrome c oxidase deficiency

A case of cytochrome c oxidase deficiency primarily affecting skeletal muscle is described. The child was admitted at 4 weeks due to failure to thrive and examination at that time revealed weakness and hypotonia. His condition deteriorated until at 11 weeks respiratory arrest necessitated artificial ventilation and death occurred at 14 weeks. Biochemical investigation showed lactic acidaemia and generalised aminoaciduria. Histochemical examination of muscle obtained at biopsy showed strong reactions for some oxidative enzymes, but by contrast cytochrome c oxidase could not be detected. Cytochrome c oxidase activity was less than 5% of control values in an extract of fresh muscle. The reduced-minus oxidised absorption spectra of muscle mitochondrial fractions prepared from postmortem tissue showed an absence of cytochrome aa₃ and a partial deficiency of cytochrome b. Ultra-structural examination showed abnormal mitochondria with loss of cristae and an abnormal granular matrix. The family history suggests autosomal recessive inheritance.     

Chronic progressive external ophthalmoplegia: A correlative study of mitochondrial DNA deletions and their phenotypic expression in muscle biopsies

Deleted mitochondrial DNA (mtDNA) has been shown to coexist with normal mtDNA (heteroplasmy) in muscles from chronic progressive external ophthalmoplegia, including Kearns-Sayre syndrome. In this study, we correlated heteroplasmic mtDNA abnormality with clinical, biochemical and histological findings with the following results: (1) large deletions ranging from 1.8 to 8.8 kb in 22 muscle specimens from 28 patients who had ophthalmoplegia clinically and focal cytochromec oxidase (CCO) deficiency by histochemistry, (2) no difference in clinical and biochemical findings between patients with and without mtDNA deletions, (3) no relationship between the size, site or populations of deleted mtDNA and respiratory chain enzyme activities in muscles, (4) positive correlation between the number of CCO-deficient fibers and the populations of deleted mtDNA, and (5) higher incidence of CCO-negative fibers in patients with deleted mtDNA than in those with no deletion of mtDNA. These results suggest that deleted mtDNA is, at least in part, responsible for focal CCO deficiency as a phenotypic expression and that the investigation on pathogenetic mechanism of focal CCO deficiency may provide a clue to understanding the underlying pathophysiology in this disorder.     

Cytochrome c oxidase deficiency in the muscle of patients with zidovudine myopathy is segmental and affects both mitochondrial DNA- and nuclear DNA-encoded subunits

Zidovudine (AZT) can induce a mitochondrial disorder associated with mitochondrial (mt) DNA depletion affecting skeletal muscle, heart, and liver. Zidovudine myopathy is characterized by ragged-red fibers and partial cytochrome c oxidase (COX) deficiency. We evaluated at a single fiber level the expression of COX II (mtDNA-encoded) and COX IV (nuclear DNA-encoded) subunits in 12 HIV-infected patients with zidovudine myopathy. We also evaluated COX activity on longitudinal muscle sections in one patient. In all patients, evaluation of the expression of COX II and COX IV subunits showed focal deficiency. All fibers negative for COX II or COX IV were negative by COX histochemistry; 32–92% (median 61%) of COX-negative fibers were negative for COX II antigens, and 7–58% (median 28%) were negative for COX IV antigens. One hundred and thirty-nine of 317 COX-negative fibers 139 (43.8%) were selectively negative for COX II; 28 of 317 (8.8%) COX-negative fibers were selectively negative for COX IV. A study of longitudinal distribution of COX activity demonstrated that COX deficiency was segmental with blurred borders, as previously observed in patients with myoclonus epilepsy with ragged-red fibers. We conclude that proteins encoded by mtDNA are predominantly, but not exclusively, involved in zidovudine myopathy. Our results confirm the value of single muscle fiber evaluation in the assessment of mitochondrial abnormalities related to zidovudine. Received: 8 July 1999 / Revised: 6 October 1999 / Accepted: 12 October 1999     

Mutations in mtDNA-encoded cytochrome c oxidase subunit genes causing isolated myopathy or severe encephalomyopathy

We report on clinical, histological and genetic findings in two patients carrying novel heteroplasmic mutations in the mitochondrial cytochrome c oxidase subunit genes COII and COIII. The first patient, a 35 year-old man had a multisystemic disease, with clinical symptoms of bilateral cataract, sensori-neural hearing loss, myopathy, ataxia, cardiac arrhythmia, depression and short stature and carried a 7970 G>T (E129X) nonsense mutation in COII. A sudden episode of metabolic encephalopathy caused by extremely high blood lactate lead to coma. The second patient developed exercise intolerance and rhabdomyolysis at age 22 years. A heteroplasmic missense mutation 9789 T>C (S195P) was found in skeletal muscle, but not in blood and myoblasts pointing to a sporadic mutation. Our report of two patients with isolated COX deficiency and new mutations in COX subunit genes may help to draw more attention to this type of mtDNA defects and provide new aspects for counselling affected families.     

Cytochrome c oxidase deficiencies in the muscle of patients with inflammatory myopathies

We studied mitochondrial function in inflammatory myopathies, using cytochrome c oxidase (COX) reaction on muscle biopsy samples from 30 patients (15 with dermatomyositis, 12 with polymyositis, and 3 with inclusion body myositis) and 30 age-matched controls. We also performed immunocytochemistry for COX II and COX IV subunits in 7 of these patients who had COX deficiency. COX-deficient fibers were a constant finding in patients or controls older than 65 years and the percentage of COX-deficient fibers correlated with age in both patients and controls. Focal COX deficiency was found in 24 patients (13 of 15 with dermatomyositis, 8 of 12 with polymyositis, and 3 of 3 with inclusion body myositis) and 18 controls. The percentages of COX-deficient fibers were higher in patients with inflammatory myopathies (range: 0–4.7%; mean: 1.2%) than in age-matched controls (range: 0–1.9%; mean: 0.4%) (P < 0.01). In the subgroup of patients under age 65, COX-deficient fibers were more frequent in dermatomyositis than in polymyositis (mean: 0.8% vs 0.2%, P = 0.02). In patients with dermatomyositis, capillary loss correlated positively with COX deficiency (P < 0.02). Immunocytochemistry for COX II and IV showed that 82% of COX-negative fibers were COX II-negative and 26% were COX IV-negative, suggesting that proteins encoded by mitochondrial DNA are predominantly, but not exclusively, involved in COX deficiency. We conclude that mitochondrial dysfunction and COX deficiency can occur in inflammatory myopathies. Such a mitochondrial dysfunction is not solely related to the aging process. We suggest that muscle ischemia contributes to mitochondrial dysfunction in dermatomyositis. Received: 16 October 1995 / Revised, accepted: 10 November 1995     

Mitochondrial myopathies: divergences of genetic deletions,biochemical defects and the clinical syndromes

Summary Genomic Southern analysis of muscle mitochondrial (mt) DNA from 16 patients with mitochondrial myopathies was performed; 14 of 16 patients had chronic progressive external ophthalmoplegia (CPEO), while 2 patients had mitochondrial myopathies without CPEO. Eleven patients with CPEO, including 5 who exhibited the complete triad of symptoms characteristic of the Kearns-Sayre syndrome (i.e. CPEO, retinal degeneration and heart block) had hetero-plasmic mtDNA with deletions ranging from 2.0 to 8.0 kb in length. There was no clear-cut correlation between the size and location of the deletions, on the one hand, and the histo-chemical and biochemical data or the severity of the disease, on the other.     

Cytochrome c oxidase deficient fibres in the limb muscle and diaphragm of man without muscular disease: An age-related alteration

Cytochromec oxidase (complex IV of the respiratory chain) was studied histochemically in human limb muscle (n = 109) and diaphragm (n = 115) obtained at autopsy revealing randomly distributed muscle fibres without enzyme activity. The defects were present both in normal type I and type II fibres and in ragged red like fibres with increased content of mitochondria. In both organs an age associated manifestation of the defect was observed. First defects occurred sporadically in the 3rd and 4th decade, but were present from the 6th to 9th decade in 66–83% of the limb muscles and 75–100% of the diaphragms. Also the number of defects/cm² (defect density) increased with age from approx. 5, and 7 in limb muscle and diaphragm below the 6th decade to 54 and 60 defects in the 8th–9th decade (P = 0,000). Between both muscles no statistically significant difference in defect density(P > 0.15) existed. Irrespective of the defect density the defect typically affected isolated fibres showing normal histochemical reactivity for succinate dehydrogenase (complex II). The results indicate that cytochromec oxidase deficient muscle fibres in normal skeletal muscle represent an age related phenomenon which probably results from cellular ageing and might be involved in the reduction of muscle mass and strength during senescence.     

Tubular aggregates and partial cytochrome c oxidase deficiency in skeletal muscle of patients with AIDS treated with zidovudine

Summary We report on two patients, who had myalgias while receiving long-term zidovudine treatment for an HIV infection, in whom muscle biopsy findings included a partial cytochrome c oxidase (CCO) deficiency, a feature of zidovudine myopathy, and tubular aggregates, a finding hitherto unreported in HIV-infected patients. The CCO deficit was observed in 28% and 24% of muscle fibers, respectively. Tubular aggregates were the prominent histopathological feature in patient 1, and were detected by systematic electron microscopy in patient 2. Inflammation and myonecrosis were not detected. In patient 1, the typical mitochondrial and myofibrillar changes of zidovudine myopathy were present and 12% of fibers showed tubular aggregates. The aggregates were not stained at CCO reaction, and 96% of myofibers enclosing tubular aggregates showed a decreased CCO activity. This suggested more than a chance association between mitochondrial dysfunction and the formation of tubular aggregates. We conclude that tubular aggregates are detected in some patients treated by zidovudine, and that the finding could be related to the long-term administration of the drug.     

Cytochrome c oxidase deficiency in zidovudine myopathy affects perifascicular muscle fibres and arterial smooth muscle cells

In order to assess the pathogenesis of myopathological alterations induced by zidovudine, we studied muscle samples from 21 patients infected by human immunodeficiency virus with zidovudine myopathy. Cytochrome c oxidase histoenzymatic reaction was evaluated in slreletal muscle fibres and arterial smooth muscle cells. Other investigations included immunocytochemistry for membrane attack complex and endomysial capillary counts. All patients had partial cytochrome c oxidase deficiency. A perifascicular distribution of cytochrome c oxidasedeficient fibres was found in 14 of 21 patients. Cytochrome c oxidase-deficient fibres were significantly more frequent in perifascicular areas than in the complete muscle sections (28% vs 12%, P<0.001). Cytochrome c oxidase-deficient arteries were found in 11 patients, of whom 10 also had a perifascicular deficiency. Mono-nuclear microvascular inflammation was obsenred in four patients and membrane attack complex deposition in capillary walls in two patients. The capillary counts were not significantly different in the patients and in the controls. These results suggest that, in addition to a direct action of zidovudine on mitochondrial DNA, chronic muscle ischaemia related to zidovudine-induced vascular dysfunction might be implicated at the inception of muscle damage in zidovudine myopathy.     

Mosaicism of mitochondria in mitochondrial myopathy: an electronmicroscopic analysis of cytochrome c oxidase

Summary Electron microscopic histochemistry was applied to the study of cytochrome c oxidase activity in each mitochondrion of biopsied muscles from four patients with mitochondrial myopathy [one case of fatal infantile mitochondrial myopathy, one case of myoclonus epilepsy associated with ragged-red fibers (MERRF), and two cases of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS)]. In the patient with fatal infantile mitochondrial myopathy, intercellular heterogeneity of mitochondria was recognized. In the three patients with either MERRF or MELAS, cytochrome c oxidase activity was segmentally changed from positive to negative within single muscle fibers. In the two patients with MELAS, small groups of positive-stained mitochondria were located among negative-stained mitochondria in the negative segment of a few muscle fibers. These findings revealed that there were heterogeneous populations of normal and abnormal mitochondria intracellularly or intercellularly within the muscles of these patients.Supported in part by Grant-in-Aid for Scientific Research 63570422 from the Ministry of Education, Science and Culture, and Grant 62A-5-08 from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Partial deficiency of complexes I and IV of the mitochondrial respiratory chain in skeletal muscle of two patients with mitochondrial myopathy

Summary Respiratory chain enzymes were studied in isolated mitochondria of two patients with mitochrondrial myopathy. Both patients had been suffering from chronic progressive external ophthalmoplegia and abnormal muscular fatigability since late childhood. One of the patients exhibited the complete triad of symptoms characteristic of Kearns-Sayre syndrome. Venous lactate levels at rest and during minimal exercise were increased in both patients. Histochemical examination of muscle revealed ragged red fibres and intermingled fibres negative for cytochrome c oxidase. Biochemical studies showed decreased activities of complex I and complex IV of the respiratory chain in both patients. Reduced minus oxidized spectra of mitochondrial cytochromes revealed a decreased content of cytochrome aa₃ in only one patient, but a normal content in the other. A combined deficiency of complexes I and IV in muscle might either be due to a deficiency of a single subunit common to both complexes or to a coincidental deficiency of both complexes expressed either in the same or in different fibres.     

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.     

In situ hybridization of muscle mitochondrial mRNA in mitochondrial myopathies

Summary To determine whether a mitochondrial mRNA deficiency exists in mitochondrial myopathies, muscle biopsies from a patient with chronic progressive external ophthalmoplegia (CPEO) and a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) were studied using in situ hybridization. Histochemistry and immunohistochemistry were performed along with hybridization. Hybridization reactions were widely distributed over the sarcoplasm of all muscle fibers in the patient with MELAS. In the patient with CPEO, 80% of the fibers showed a marked decrease in density of autoradiographic grains. This marked decrease corresponded to the histochemical and immunohistochemical findings of a very weak staining of cytochromec oxidase (CCO). The isotope-labeled cDNA probe used in in situ hybridization in this study complements a part of subunit I of CCO and a part of subunit II of complex I in the mitochondrial gene. Our results suggest a defect in the mRNA in this CPEO patient.Supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education and by a grant (62-2-05) from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Skeletal muscle involvement in human immunodeficiency virus infection

Summary In addition to muscle changes due to peripheral nervous system involvement, primary myopathic changes associated with the human immunodeficiency virus (HIV) have also been described. We studied seven cases: two had developed an acquired immunodeficiency syndrome (AIDS) four had seroconverted to HIV but were otherwise asymptomatic, one was HIV seronegative when the biopsy was performed and one was biopsied twice. Besides the HIV no other infectious agent was detected. Muscle biopsies showed: (a) muscle fiber necrosis and regeneration; (b) inflammatory changes with moderate perivascular infiltration; and (c) unusual myofibrillary disorganization. Immunocytochemical techniques using anti-HIV monoclonal antibodies showed the presence of the virus in one biopsy. HIV-RNA was detected by in situ hybridization in the same biopsy. With both techniques the HIV was detected in isolated mononuclear cells in the muscle endomysium and not within the muscle fibers. Muscle involvement associated with HIV infection may be related, at least in some cases, to the presence of the virus in interstitial cells.Supported by a grant from Agence Nationale de Recherches sur le SIDA (A.N.R.S.)Presented in part at the joint MRC/INSERM meeting on HIV infection and the nervous system, Paris, France, January 19–20, 1989     

Two point mutations in mitochondrial DNA of cytochrome c oxidase coexist with normal mtDNA in a patient with Alzheimer’s disease

The mitochondrial cytochrome c oxidase (CO) gene sequence was determined on a patient with Alzheimer’s disease (AD). Compared to the standard Cambridge sequence to identify base changes,two missense mutations were found in the patient with AD. The mutations were a G to T transition at np 8206 and an A to T transition at np 8224. The np 8206 mutation changed a Met to an Ile and np 8224 mutation changed a Leu to a Phe. The normal bases and the mutations of mitochondrial DNA (mtDNA) coexist in the patient. Further studies will be required to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenisis of Alzheimer’s disease.     

The role of cytochrome c oxidase deficient hippocampal neurones in Alzheimer's disease

Defects of mitochondrial function have been proposed as a potential mechanism in the development and pathogenesis of Alzheimer's disease (AD) and neuronal apoptosis. Mitochondrial enzyme-deficient pyramidal neurones are found in greater quantities in the hippocampus of AD patients than in age-matched controls. The presence of these neurones indicates that high levels of mutant mtDNA (mitochondrial DNA), sufficient to cause a biochemical deficiency within individual neurones, occur more frequently in AD than in normal ageing. This study analyses the relationship of cytochrome c oxidase (COX)-deficient neurones with the neuropathological markers of AD, neurofibrillary tangles (NFTs) and amyloid plaques, as well as markers of neuronal apoptosis known to occur in AD brains. Frozen sections of hippocampi from three AD patients were used to directly colocalize in situ the presence of histochemically COX-deficient neurones with immunohistology for the classical neuropathological markers of AD, tau and beta-amyloid. In addition, we also directly colocalized these mitochondrial-enzyme deficient neurones using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and cleaved caspase-3. The distribution of amyloid plaques is anatomically distinct from the COX-deficient hippocampal pyramidal neurones and the neurones that contained NFTs or apoptotic labelling were always COX-positive. COX-deficient, succinate dehydrogenase-positive hippocampal neurones indicative of high mtDNA mutation load do not appear to be prone to apoptosis or to directly participate in the over production of tau or beta-amyloid. Biochemically significant mitochondrial defects do occur in AD and are likely to contribute to the overall central nervous system dysfunction in impairing neuronal function and possibly causing neurodegeneration via mechanisms other than apoptosis.