A. M. Stadhouders

Differential investigation of the capacity of succinate oxidation in human skeletal muscle.

Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6-dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder.

A mitochondrial encephalomyopathy: the first case with an established defect at the level of coenzyme Q

A patient is presented who had therapy-resistant epileptic seizures from the 7th day of life. Examination at the age of 17 months revealed a mentally retarded boy with epileptic seizures, generalised myoclonic contractions, and abnormal ocular movements. A cerebral CT scan showed central and cortical atrophy. Lactate levels in serum, cerebrospinal fluid and urine were elevated, the pyruvate level was raised in serum. A quadriceps muscle biopsy revealed aspecific morphologic signs of a myopathy. Biochemical analysis showed decreased substrate oxidation rates in the mitochondria associated with low rates of ATP production. Total and free carnitine levels were decreased. Investigation of the respiratory chain revealed a defect in the proximal part of respiratory chain revealed a defect in the proximal part of respiratory chain involving the region of coenzyme Q. Based on clinical and chemical data it is likely that the patient is suffering from a multi-system disorder.

Congenital cataract and mitochondrial myopathy of skeletal and heart muscle associated with lactic acidosis after exercise

Congenital cataract involving the nucleus, cortex, and capsule of the lens, and cardiomyopathy were found in seven of 22 children from three unrelated families. Histologic examination showed a mitochondrial myopathy of skeletal and heart muscle with storage of lipid and glycogen. When the patients performed submaximal exercise for 60 minutes they developed metabolic acidosis with lactic acidemia.

Estimation of NADH oxidation in human skeletal muscle mitochondria

Assay procedures are described for the detection of defects in the process of NADH oxidation by the respiratory chain in human skeletal muscle biopsy specimens. The procedures allow determination of rotenone-sensitive NADH: O2 oxidoreductase and NADH: ubiquinone-1 oxidoreductase activity not only in isolated mitochondria but also in post-nuclear supernatants. The use of ferricyanide as electron acceptor for estimation of NADH dehydrogenase activity is inadequate when only applied on a disrupted mitochondrial preparation.

Pyruvate oxidation in rat and human skeletal muscle mitochondria.

Abstract Pyruvate oxidation in rat and human skeletal muscle mitochondria was studied by measuring the rate of 14 CO 2 production from [1- 14 C]pyruvate in the presence of 1 m m pyruvate, an excess of ADP, and varying amounts of citric acid cycle intermediates or carnitine. The rate of pyruvate oxidation is controlled by the availability of acetyl-CoA acceptor since addition of citric acid cycle intermediates or carnitine results in a stimulation of pyruvate oxidation. Pyruvate oxidation proceeds at its maximal rate in the presence of malate since no further stimulation is observed by addition of carnitine. It is concluded that pyruvate dehydrogenase is the rate-limiting step during pyruvate oxidation in the presence of malate. In human skeletal muscle mitochondria pyruvate oxidation proceeds maximally both in the presence of malate or carnitine. Parallel incubations of these mitochondria with [1- 14 C]pyruvate plus malate and [1- 14 C] pyruvate plus carnitine may allow one to establish disturbances in pyruvate oxidation and to discriminate between defects in the pyruvate dehydrogenase complex and in the activity of the citric acid cycle.

Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure

A method is presented which allows the investigation of almost the complete mitochondrial content of small human skeletal muscle biopsy specimens. Thorough mechanical disruption with a chopper apparatus results in the release of about 50% of the mitochondrial content. Subsequent treatment of the 600 x g sediment with trypsin releases another 30% of the total mitochondrial population. The biochemical characteristics of the two mitochondrial fractions obtained in these two successive steps have been compared. No obvious differences could be established. The procedure is well suited for biochemical investigation of muscle biopsy specimens from patients suspected of suffering from a mitochondrial myopathy.

Deficiency of cytochromes b and aa3 in muscle from a floppy infant with cytochrome oxidase deficiency

A girl was presented suffering from generalised weakness and cardiorespiratory insufficiency. She succumbed at the age of 5 months. Lactate levels were elevated in serum, cerebrospinal fluid and urine. Histopathological examination revealed a mitochondrial myopathy. In muscle tissue the cytochrome oxydase activity was strongly reduced. The content of cytochromes b and aa3 was very low. At autopsy a cardiomyopathy was found.

Morphological observations in skeletal muscle from patients with a mitochondrial myopathy.

Mitochondrial metabolic dysfunction is considered to be the cause of certain congenital myopathies and a number of multisystem disorders in humans. The morphological hallmark of these diseases is the ‘ragged red’ fibre, which shows abnormally intensive oxidative enzyme reactions. Electron microscopy reveals that the numerically increased mitochondria in these fibres are often markedly enlarged and possess aberrant configurations of cristae. The mitochondrial matrix often contains lipid-like inclusions or shows vacuolation. The most characteristic mitochondrial abnormality is the occurrence of highly ordered inclusions in the intracristal or intermembrane space. These inclusions appear to be true crystals, composed of proteinaceous material. It is argued that the activity of accumulation of proteins in the mitochondria is related to the nuclear and nucleolar hypertrophy noticeable in the ragged red fibres. Since protein crystals in mitochondria in particular occur when an increased capillary density around the ragged red fibres is present, it is suggested that oxygen free radicals and lipid peroxidation processes are involved in the ragged red fibre pathology.

Differentiation of human skeletal muscle cells in culture: maturation as indicated by titin and desmin striation.

SummaryThis report describes a phenotyping study of differentiating human skeletal muscle cells in tissue culture. Satellite cells (adult myoblasts), isolated from biopsy material, showed a proliferative behaviour in high-nutrition medium, but fused to form myotubes when grown in low-nutrition medium. The expression and structural organization of the intermediate filament proteins desmin and vimentin as well as the sarcomeric constituents α-actin, α-actinin, nebulin, myosin and especially titin during myofibrillogenesis in vitro, were studied by means of indirect immunofluorescence assays. The proliferating myoblasts contained both desmin and vimentin, α-actinin and the filamentous form of actin. Shortly after the change of medium, expression of titin, sarcomeric myosin and skeletal muscle α-actin was found in mononuclear cells in a diffuse, filamentous (titin, myosin, α-actin) or punctate (titin, myosin) pattern. Four to 10 days after the medium change, mature myotubes showed desmin, titin, α-actinin, nebulin, sarcomeric myosin and actin cross-striations, while vimentin was no longer detected. We conclude that human skeletal muscle cell cultures are an appropriate model system to study the molecular basis of myofibrillogenesis. Especially the presence of desmin in a striated fashion points to a high degree of maturation of the muscle cell cultures.

A mitochondrial myopathy with a defective respiratory chain and carnitine deficiency

A boy presented suffering from generalised weakness, exercise intolerance and lactic acidosis. The weakness became evident at 2 years. A cerebral CT-scan showed cerebellar atrophy and central and peripheral atrophy of both hemispheres. With trichrome staining about 20% of the muscle fibres showed large areas containing redstaining granular material. Electron microscopic examination showed that this material consisted of areas of mitochondrial proliferation, most of the mitochondria having abnormal ultrastructural characteristics. Pyruvate dehydrogenase complex and citric acid cycle activities were determined by measuring 14CO2 production from various labelled substrates. Diminished oxidation rates were found with the patients muscle homogenate for all substrates tested, indicating a defect in the respiratory chain. The cytochromes were present in normal quantities. Succinate cytochrome c reductase activity was very decreased. Carnitine concentration was decreased in serum and in muscle as well.