Mitochondrial myopathies: clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA

Analysis of mitochondrial DNA (mtDNA) in muscle and blood from 72 patients with mitochondrial myopathy showed that 30 had major deletions of a variable proportion of muscle mtDNA. All of these 30 patients presented with progressive external ophthalmoplegia and limb weakness, and 8 had the additional features of the Kearns-Sayre syndrome. Of the 42 patients without detectable muscle mtDNA deletions, 10 had progressive external ophthalmoplegia and limb weakness, 2 had the Kearns-Sayre syndrome, 11 had limb weakness without extraocular involvement, and 19 had multisystem disorders predominantly affecting the central nervous system. Only 2 patients with mtDNA deletions had clinically affected relatives, compared with 10 of those without deletions. In the 4 patients with polarographic defects exclusively involving complex I (NADH coenzyme Q reductase), the deleted protein-coding genes were confined to those for complex I subunits. Thirteen other patients with apparently identical deletions had variable clinical and biochemical features. Immunoblots of complex I polypeptides from patients with deletions were either indistinguishable from controls or showed only a mild generalized decrease in all identifiable subunits.     

Deletions of mitochondrial DNA in Kearns-Sayre syndrome and ocular myopathies: genetic, biochemical and morphological studies.

Genetic, biochemical and morphological investigations were conducted on skeletal muscle mitochondria from 6 cases of ocular myopathy: 4 cases with Kearns-Sayre syndrome (KSS) and 2 with chronic progressive external ophthalmoplegia. All of these 6 cases showed mitochondrial DNA (mtDNA) deletions in addition to normal sized DNA in the quadriceps muscle. The deletions ranging from 3 to 8 kbp were also mapped between nucleotides 5500 and 16000 by Southern blot. The deleted genes encoded for some subunits of complexes I, IV, V and 5-10 tRNAS. The boundaries of the deletions have been sequenced in three patients. Five patients had mitochondrial respiratory chain deficiency in complex I as shown by the low oxygen consumption in isolated mitochondria using three NAD(+)-linked substrates. Mitochondria with an abnormal ultrastructure were also observed in 2 cases. A good relationship between the cytochrome c oxidase deficiency and the amount of deleted mtDNA was shown in our present investigations.     

Correlative biochemical and morphological studies of myelination in human ontogenesis

Summary Biochemical and morphological observations of nerve roots in six fetuses from the 16th to 34th week of gestation and five infants 1 day to 3 years old are presented. In dorsal roots the process of myelination begins later than in the ventral roots and spinal cord and proceeds much slower. As in the spinal cord during nerve roots myelin maturation profound lipid changes are observed.The study was supported partly by the Polish Academy of Sciences (No. of the agreement 10.4.2.02) and partly by NIH (Bethesda) —Marie Sklodowska-Curie Joint Fund (No. of the agreement J-05094-N)     

Hereditary human myopathies in muscle culture

In this article I illustrated the use of regenerating human muscle cultures for studying the hereditary human myopathies. Although some of the data are still controversial, they do point up the great potential of this “in vitro system”. For hereditary myopathies due to developmentally regulated proteins that are expressed only at a more advanced stage of muscle differentiation, the use of highly differentiated nerve-muscle cocultures might contribute significantly to a better understanding of their developmental pathogenesis. More advanced techniques (permanent human muscle cell lines, heterokaryons, myoblast transfer, gene transfer, myogenic conversion of human non-muscle cells, cybrid clones) may provide a great deal of information at molecular level and may also have practical applications in the diagnosis or even in the treatment of hereditary human myopathies.

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Sommario L'Autore illustra l'utilità e i limiti delle colture di muscolo umano nello studio delle miopatie umane ereditarie. In particolare nelle miopatie dovute a difetti di enzimi-specifici muscolari, l'utilizzo di tecniche di cocoltura muscolo-nervo con avanzato grado di maturazione muscolare permette di delucidare i meccanismi molecolari patogenetici di tali malattie. L'uso di avanzate tecniche di biologia molecolare e cellulare, quali linee permanenti, trapianto di mioblasti, trasferimento genico e di mitocondri, oltre che fornire utili informazioni a livello molecolare potranno trovare applicazione, in futuro, persino nella terapia di molte miopatie ereditarie.

     

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.     

Experimental chloroquine myopathy: morphological and biochemical studies

Morphological and biochemical studies were performed on the soleus muscles of rats receiving a daily intraperitoneal injection of 50 mg chloroquine chloride (CQ) per kilogram of body weight. Light microscopy showed mild to moderate variations in fiber size, numerous dense membranous bodies and vacuoles. The vacuoles were ringed by material that was intensely basophilic in hematoxylin-eosin preparations, resembling rimmed vacuoles in the muscle fibers of distal myopathy. Segmental degradation or necrosis was often observed. The 3H-leucine uptake by myofibrillar and soluble sarcoplasmic fractions in CQ-treated muscles was the same as in the controls. The significant increases in lysosomal (cathepsin B & L, B and D) proteases and thiol protease inhibitor occurred in the earlier stages of CQ-induced myopathy, when hardly any autophagic vacuoles or dense bodies were observable by light microscopy. We conclude that the over-development of autophagic vacuoles and the significant increases in lysosomal protease activity in muscle tissues may be important in the development of the focal degradation and necrosis of CQ-treated muscles.     

Correlative morphological and biochemical studies of the human fetal sciatic nerve

Correlative biochemical and morphological studies are reported concerning the sciatic nerves of 39 human fetuses ranging in age from 9 to 23 weeks. The period of premyelination — 9 to 17 weeks — is marked more by morphological changes than biochemical, with only cholesterol and total phospholipid present. The period of transition between premyelination and myelination — 17 to 20 weeks — is noted by the increasing presence of one axon associated with one Schwann cell, and the beginning of spiraling around the axon, and the appearance of lecithin, cerebrosides, sulfatides and other lipids.     

Mitochondrial abnormalities in the myofibrillar myopathies

Myofibrillar myopathies are a genetically diverse group of skeletal muscle disorders, with distinctive muscle histopathology. Causative mutations have been identified in the genes MYOT, LDB3, DES, CRYAB, FLNC, BAG3, DNAJB6, FHL1, PLEC and TTN, which encode proteins which either reside in the Z‐disc or associate with the Z‐disc. Mitochondrial abnormalities have been described in muscle from patients with a myofibrillar myopathy. We reviewed the literature to determine the extent of mitochondrial dysfunction in each of the myofibrillar myopathy subtypes. Abnormal mitochondrial distribution is a frequent finding in each of the subtypes, but a high frequency of COX‐negative or ragged red fibres, a characteristic finding in some of the conventional mitochondrial myopathies, is a rare finding. Few in vitro studies of mitochondrial function have been performed in affected patients.     

Reducing body myopathy and desmin storage in skeletal muscle: morphological and biochemical findings

We describe clinical, morphological and biochemical findings of a patient with reducing body myopathy (RBM). This 15-year-old patient was affected by severe limb-girdle progressive myopathy with asymmetric distribution. Muscle biopsy showed many fibers with cytoplasmic polymorphic masses, which stained dark purple with modified Gomori's trichrome, associated with proliferation of cytoplasmic bodies. Cytoplasmic polymorphic masses showed marked reducing activity with menadione-nitro blue tetrazolium reaction. Ultrastructurally, there was great amount of highly electron-dense tubular-filamentous structures of 16–17 nm in diameter. Immunohistochemistry showed that many fibers were positive for desmin. Sodium dodecyl sulfate-electrophoresis disclosed an increase in two bands of approximately 53 and 70 kDa, and Western blot demonstrated that the 53-kDa band was desmin. It was not possible to characterize the 70-kDa protein further.Supported by Telethon and institutional funds of the Consiglio Nazionale delle Ricerche     

Recessive carnitine palmityl transferase deficiency: biochemical studies in tissue cultures and platelets

Summary In a new case of carnitine palmityl transferase (CPT) deficiency the defect was documented in muscle and muscle cultures with an isotope exchange reaction, using different concentrations of palmityl-dl-carnitine and a forward reaction with and without albumin. The defect was expressed in cultured skin fibroblasts only by the reverse and hydroxamate reactions. The parents and the patient's daughter had intermediate levels of the enzyme in platelets and fibroblasts, supporting the concept that CPT deficiency has an autosomal recessive pattern of inheritance. The growth pattern and development of muscle cultures in this CPT-deficient patient indicate that CPT activity may be sufficient to allow normal muscle differentiation in culture without lipid storage.     

Human spinal cord neurons in dissociated monolayer cultures: morphological, biochemical, and electrophysiological properties

The preparation of dissociated monolayer cultures from embryonic human spinal cord is described. Optimal survival was achieved with embryonic tissue between the eighth and ninth week. The neurons survive for as long as 7 weeks in culture and they grow in a standard tissue culture medium which contains 13% decomplemented human serum. The neurons have been identified by indirect immunofluorescence techniques using antibodies to tetanus toxin and neurofilament protein. Our biochemical studies demonstrate the presence of cholinergic and GABAergic neurons. Cholinergic neurons develop in culture and are more numerous in the cultures prepared from the anterior part of the spinal cord as compared to those from the posterior part. Therefore, it is possible that a large part of the cholinergic neurons derive from the motoneuron pool. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin (TTX). Voltage clamp experiments combined with the use of pharmacological blocking agents revealed the presence of several voltage- and time-dependent currents: a sodium current sensitive to TTX, a potassium current made up of two components, sensitive to tetraethylammonium and 4-aminopyridine, and a calcium current sensitive to cobalt. From a biochemical and electrophysiological point of view the properties of human spinal cord neurons in culture closely resemble the properties of spinal cord neurons from other species.     

Toxic myopathies: muscle biopsy features

Several drugs and toxic substances can cause muscular abnormalities and are frequent causes of acquired myopathies. We present a series of 32 patients, predominance of young adult patients, diagnosed with toxic myopathy. The most common substances inducing myopathy were corticosteroids (56.2%) followed by the propoxyphene, neuroleptics, zidovudine and drug-induced hypokalemia. The investigation showed normal serum creatine kinase levels in 65.4%, myopathic pattern of the needle electromyography in 40% and the more frequent histological diagnosis of the muscle biopsy was type 2 fiber atrophy (59.3%). Clinical features, etiology, course of the disease, serum levels of muscular enzymes, electromyographic features and, especially, muscle biopsy features are discussed.     

Serum-free aggregate cultures of rat CNS glial cells: biochemical, immunocytochemical and morphological characterization

Aggregate cultures of mixed glial cells, as well as of enriched astrocytes and oligodendrocytes were prepared, and maintained in serum-free medium for up to 25 days. Biochemical measurements of both neuron-specific and glia-specific enzyme activities showed that these three types of aggregate cultures were virtually devoid of neurons. Astrocyte-enriched cultures were greater than 95% pure, with oligodendrocytes as the only apparent contaminant, whereas oligodendrocyte-enriched cultures still contained a considerable proportion of astrocytes. In all these neuron-free aggregate cultures both astrocytes and oligodendrocytes attained a high degree of maturation. These findings were confirmed by morphological examinations, and by immunofluorescence studies. Furthermore, ultrastructural as well as immunocytochemical investigations using antibodies to myelin basic protein revealed that all three types of glial cell aggregate cultures contained myelin membranes, indicating that the presence of axons is not a prerequisite for the formation of myelin.     

Mitochondrial dysfunction in adult-onset myopathies with structural abnormalities

Three patients with chronic progressive external ophthalmoplegia of adult-onset, generalized muscle atrophy and myalgia are described. Two patients fulfilled the histological criteria for centronuclear myopathy, the third those for fiber-type disproportion. Additionally, typical ragged red fibers were found in all muscle specimens, and several muscle fibers were cytochrome c oxidase negative. NADH and succinate dehydrogenase stains showed increased subsarcolemmal accumulation of mitochondria. To determine whether these findings are coincidental or whether they indicated an additional mitochondrial disorder, all patients were investigated using biochemical analysis of the respiratory chain, molecular genetics, magnetic resonance spectroscopy of quadriceps muscle and ergometry. These tests suggested an additional mitochondrial dysfunction. Mitochondrial dysfunction seems to be more common in this group of myopathies than previously estimated, and may be of importance in the pathogenesis of these disorders.Part of this work was supported by the Deutsche forschungsgemeinschaft (Re 265/8-2). Authors are grateful to Professor Schröder, University of Aachen, for neuropathological examination of case 3     

Mitochondrial and ocular myopathies (62 cases)

The authors report the clinical signs and histological findings in 62 patients with ocular myopathies divided into two groups: (a) ocular myopathies with mitochondrial abnormalities, themselves divided into pure (19 cases) or progressive (15 cases) ophthalmoplegia, and multisystemic myopathies (14 cases); (2) oculopharyngeal myopathies (15 cases) in elderly subjects, with constant intranuclear tubulofilamentous inclusions. Among myopathies with mitochondrial abnormalities, ocular forms with a favourable prognosis coexisted with earlier and more severe multisystemic lesions.     

Growth patterns of primary cultures dissociated from 3-day-old chick embryos: morphological and biochemical comparisons

Cultures were prepared by dissociating 3-day-old whole chick embryos and plating the dispersed cells on poly-L-lysine-coated dishes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum. By 48 hr in culture, aggregates and neuritic sprouting were observed. Long neuritic bundles connecting cell aggregates were evident by 4 days in culture. Consistent patterns throughout the lifespan of the cultures were contacts between neurites, and flat isolated cells, presumptively glial, emerged. Throughout the lifespan of the cultures, the cholinergic cell population was characterized histochemically by the method of Karnovsky and Roots and biochemically by assaying choline acetyltransferase. By 4 days in culture, all aggregates showed light cholinesterase-positive staining; however, with days in culture, several aggregates had no staining, and some positive-stained aggregates were interconnected with other aggregates showing only spotted positive staining. Choline acetyltransferase activity showed a developmental profile in agreement with the histological findings. The early presence of choline acetyltransferase activity is taken as indication of the early commitment of cholinergic neurons.     

Adult-onset lysosomal storage disease in a Schipperke dog: clinical,morphological and biochemical studies

Summary An adult-onset lysosomal storage disorder was diagnosed in a 5-year-old Schipperke dog with progressive cerebellar and central vestibular signs. It was characterized by cerebellar atrophy with extensive loss of Purkinje and granular cells, and hydrocephalus. Enlarged and vacuolated neurons were observed in spinal cord and brain; pancreatic centrolobular and islet cells were also vacuolated. Ultrastructurally, enlarged secondary lysosomes laden with lamellated membrane structures were present in neurons and empty enlarged vacuoles were found in pancreatic centroacinar, ductal, and islet cells. On frozen sections neurons stained with Ricinus communis agglutinin-I and wheat germ agglutinin. On paraffin sections neurons stained with luxol fast blue, periodic acid-Schiff, Concanavalia ensiformis agglutinin, and were autofluorescent. These findings indicate an accumulation of glycolipids containing terminal -galactosyl and -sialyl residues, and N-linked oligosaccharides. Tissue activity of lysosomal -galactosidase was 50% of normal and the activity of -hexosaminidase was elevated. Brain lipid-bound sialic acid was twice normal, with a small increase of G_M1-ganglioside, but there was a significant elevation of G_M2 (G_D2) and G_M3 (G_D3). In addition, significant elevations of sialylated and non-sialylated oligosaccharides were noted. These clinical, biochemical and pathological findings are similar to those observed in human patients with adult-onset galactosialidosis.