Cytochrome c oxidase and cardiolipin alterations in response to skeletal muscle ischaemia and reperfusion.

The effect of 2 and 4 h of tourniquet ischaemia followed by 1 h of reperfusion on the major mitochondrial phospholipids and on the cytochrome c oxidase kinetic parameters has been investigated in rat skeletal muscle. There was no change either in the mitochondrial phospholipid content or in the Vmax and the Km of the enzyme after 2 h of ischaemia with and without subsequent reperfusion. Four hours of ischaemia had no effect on the lecithin and the cephalin content, while the cardiolipin content decreased as well as the Vmax of the enzyme (P less than 0.05). Tissue reperfusion caused a dramatic decrease in both cardiolipin (55% of the control, P less than 0.001) and Vmax (38% of the control, P less than 0.001). The corresponding reduction in lecithin and cephalin contents was 12% and 14% respectively (P less than 0.05). The Km remained unchanged at all conditions. These findings suggest that mitochondrial dysfunction in response to ischaemia and reperfusion could be a consequence of the reperfusion itself following severe ischaemia. The results are discussed in terms of cardiolipin peroxidation and cytochrome oxidase as a functional parameter.

Evolution of cardiac involvement in progressive ophthalmoplegia with deleted mitochondrial DNA.

A 43-year-old woman with progressive external ophthalmoplegia developed a bifascicular block and dilatation of the right ventricle during 4 years of follow-up. Histochemical and electron microscopy studies detected mitochondrial abnormalities in ocular, skeletal muscle and cardiac biopsies. This case registers disease progression from the external ocular to the skeletal and cardiac muscles. Mitochondrial DNA was deleted in relation to the morphological abnormality.

Immunocytochemical identification of beta-protein precursor in cultured vascular cells.

The immunocytochemical and immunochemical identification of beta-protein precursor in cultured vascular cells was undertaken using three types of antibodies to the synthetic predictive peptides (extracellular portion; 275-286, beta-protein portion; 597-620, C-terminal portion; 681-695) of beta-protein precursor. Monoclonal antibody directed toward the beta-protein portion stained the surface membrane of the cultured endothelial cells as well as cytoplasmic organelles. Immunoblotting of the subcellular fractions of the cell homogenate with three antibodies revealed that the plasma membrane associated beta-protein precursor consists of 105-130 kDa protein complexes and that the mitochondria-microsome-associated beta-protein consists of related 30-67 kDa protein complexes. The immunoreactivity of the protein bands was blocked by pretreating the monoclonal antibody with bovine serum albumin-conjugated beta-protein. These results indicate that the form of the beta-protein precursor or beta-protein-related components in cultured vascular cells is heterogeneous in terms of molecular size and subcellular localization.

Directly repeated sequences associated with pathogenic mitochondrial DNA deletions.

We determined the nucleotide sequences of junctional regions associated with large deletions of mitochondrial DNA found in four unrelated individuals with a phenotype of chronic progressive external ophthalmoplegia. In each patient, the deletion breakpoint occurred within a directly repeated sequence of 13-18 base pairs, present in different regions of the normal mitochondrial genome-separated by 4.5-7.7 kilobases. In two patients, the deletions were identical. When all four repeated sequences are compared, a consensus sequence of 11 nucleotides emerges, similar to putative recombination signals, suggesting the involvement of a recombinational event. Partially deleted and normal mitochondrial DNAs were found in all tissues examined, but in very different proportions, indicating that these mutations originated before the primary cell layers diverged.

[Chronic progressive external ophthalmoplegia (CPEO) with deleted mitochondrial DNA].

A 19-year-old man with chronic progressive external ophthalmoplegia with deleted mitochondrial DNA was reported. Neurological examination revealed bilateral external ophthalmoplegia, hearing loss of sensorineural type, short stature, mental retardation, muscle atrophy and weakness in the proximal muscles. Lactate and pyruvate levels were elevated in both serum and cerebrospinal fluid (CSF). Protein concentration was slightly increased in CSF. Electromyogram showed myopathic changes on all the muscles examined. Ragged-red fibers were found in biopsied rectus femoris muscle, stained with modified Gomori trichrome. Scattered cytochrome c oxidase deficient fibers were encountered. The computed tomography of the brain showed mild cerebral and cerebellar atrophy without any abnormal calcification or hypo-lucency. Southern blot analysis of the mitochondrial DNA (mtDNA) extracted from the patient's muscle revealed mixed population of mtDNA, consisting of the normal one and partially deleted one. The size of the deletion was about 4.5-kilobase. The region included the sequences coding for at least four subunits of Complex I, one subunit of Complex IV, two subunits of Complex V and five tRNAs. There may be a "hot area" on the mitochondrial genome that is more prone to be deleted than other regions of mtDNA. Southern blot analysis is usefull for the diagnosis of KSS or CPEO.

Tissue-specific mitochondrial proteins.

Mitochondrial proteins from rat brain cortex, muscle, liver, and from neuronal cells in culture were compared on 2-D electrophoregrams. This analysis permitted characterization of certain specificities in the distribution of polypeptides depending on tissue localization. In particular, 16 mit-proteins were found exclusively in the mitochondrion from brain tissue.

Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome.

We investigated the correlations of deletions of mitochondrial DNA in skeletal muscle with clinical manifestations of mitochondrial myopathies, a group of disorders defined either by biochemical abnormalities of mitochondria or by morphologic changes causing a ragged red appearance of the muscle fibers histochemically. We performed genomic Southern blot analysis of muscle mitochondrial DNA from 123 patients with different mitochondrial myopathies or encephalomyopathies. Deletions were found in the mitochondrial DNA of 32 patients, all of whom had progressive external ophthalmoplegia. Some patients had only ocular myopathy, whereas others had Kearns-Sayre syndrome, a multisystem disorder characterized by ophthalmoplegia, pigmentary retinopathy, heart block, and cerebellar ataxia. The deletions ranged in size from 1.3 to 7.6 kilobases and were mapped to different sites in the mitochondrial DNA, but an identical 4.9-kilobase deletion was found in the same location in 11 patients. Biochemical analysis showed decreased activities of NADH dehydrogenase, rotenone-sensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome c oxidase, four enzymes of the mitochondrial respiratory chain containing subunits encoded by mitochondrial DNA. We conclude that deletions of muscle mitochondrial DNA are associated with ophthalmoplegia and may result in impaired mitochondrial function. However, the precise relation between clinical and biochemical phenotypes and deletions remains to be defined.

X-ray microanalysis of calcium in ischemic skeletal muscle cells.

Tissue sections of both transiently and persistently ischemic musculus latissimus dorsi of rabbits show degenerated and necrotic cells in varying degrees. Degenerated mitochondria appearing in the cells contain moderate electron-dense fluffy intramatrical deposits. However, only in the transient ischemia sample can other two kinds of electron-dense granules be seen between myofibrils. According to their ultrastructure, size and distribution, the larger granules are considered the degenerated mitochondria, and the smaller ones the degenerated sarcoplasmic reticulum. X-ray microanalysis proves that these two kinds of granules contain high concentration of calcium, while the fluffy deposits contain little calcium. The findings suggest that the calcium deposits in the degenerated and necrotic cell seen under light microscope occur in mitochondria and sarcoplasmic reticulum of the transiently ischemic muscle cell. The conditions of calcium accumulation in ischemic muscle cells are discussed.

Purification and some properties of glycerol-3-phosphate dehydrogenase from rabbit skeletal muscle mitochondria.

Glycerol-3-phosphate dehydrogenase (E. C. 1. 1. 99. 5) was solubilized from rabbit skeletal muscle mitochondria by Triton X-100 and purified through hydroxyapatite column chromatography, DEAE-Sepharose CL-6B column chromatography and sucrose density gradient ultracentrifugation. The preparation was electrophoretically pure, the total recovery was 10% and the specific activity had been increased 200-fold. The apparent molecular weight of the enzyme polypeptide was 69,000, it existed in the form of enzyme-Triton X-100 complex with a Stokes' radius of 59 A and a sedimentation coefficient of 10.7 S. There were 1.7 mg Triton X-100 and 26 micrograms phospholipid per mg protein of the preparation. The enzyme absorbed at 410 and 460 nm which could be attributed to non-haem iron and FAD respectively. Both of the absorption would be largely diminished by adding the substrate glycerol-3-phosphate.

Mitochondrial myoneuropathy with respiratory failure and myoclonic epilepsy. A case report with biochemical studies.

A 55-year-old man is presented who developed severe multifocal myoclonus and tonic clonic seizures in his early thirties, and progressive limb weakness in his mid forties, when a ragged red fibre myopathy was diagnosed. He went on to develop a distal motor neuropathy and respiratory failure. Respiratory function tests indicated respiratory failure secondary to respiratory muscle weakness and a central hypoventilation syndrome. CT scan revealed brain stem atrophy and brain stem evoked responses were abnormal. A sural nerve biopsy showed severe axonal degeneration. Cytochrome difference spectra and polarographic studies on isolated intact muscle mitochondria were normal. This study reports the association of respiratory failure and sleep apnoea with Fukuhara's syndrome and presents biochemical data suggesting that the mitochondrial respiratory chain may be intact in some patients with this syndrome.

Analyses of muscle proteins in a patient with a mitochondrial myopathy.

Using the small amounts of muscle available from biopsy (approximately 100 mg), from both normal controls and a patient with a previously identified defect of the mitochondrial electron transfer protein complex III, we analyzed both structural and mitochondrial proteins. The myosin light chains were found to be unchanged with respect to charge or size between patient and control. Two prominent proteins detected after two dimensional gel electrophoresis were present in the patient's total homogenised muscle protein but were not detected in the controls. One protein was positively identified as cytochrome c oxidase subunit II and the other tentatively as a component of the ATP synthetase. We suggest that the increased amounts of these proteins represents a response of the patients muscle cells to the ATP deficiency caused by the primary lesion in complex III.

Electrophoretic and ultrastructural analysis of the rabbit's striated external urethral sphincter.

The male rabbit's external urethral sphincter was investigated with O'Farrell's 2-dimensional electrophoretic analysis of myosin light chains and electron microscopy. Its pattern of myosin light chains was different from that of the soleus (predominantly slow twitch muscle) but was very similar to that of the psoas (predominantly fast twitch muscle). Ultrastructurally it was shown to be red muscle resembling the soleus. Therefore the fiber type of the rabbit's external urethral sphincter was determined to be the red (fast) type.

Adaptations of skeletal muscle to endurance exercise and their metabolic consequences.

Regularly performed endurance exercise induces major adaptations in skeletal muscle. These include increases in the mitochondrial content and respiratory capacity of the muscle fibers. As a consequence of the increase in mitochondria, exercise of the same intensity results in a disturbance in homeostasis that is smaller in trained than in untrained muscles. The major metabolic consequences of the adaptations of muscle to endurance exercise are a slower utilization of muscle glycogen and blood glucose, a greater reliance on fat oxidation, and less lactate production during exercise of a given intensity. These adaptations play an important role in the large increase in the ability to perform prolonged strenuous exercise that occurs in response to endurance exercise training.

Localization of calcium in skeletal and cardiac muscle.

The requirement of calcium (Ca2+) in the excitation-contraction coupling of both skeletal and cardiac muscle is well established. However, the exact location of the intracellular storage sites of Ca2+ is not firmly established. We report here on the ultrastructural ultrastructural distribution of Ca2+ in white and red skeletal muscle and in cardiac muscle of the rat using combined phosphate-pyroantimonate (PPA) and oxalate-pyroantimonate (OPA) procedures. The methods are based on (a) stabilization and/or trapping of Ca2+ during the primary fixation step in glutaraldehyde by potassium phosphate or oxalate; (b) subsequent wash-out of all non-trapped cations such as Na+ and Mg2+ in potassium phosphate or oxalate; (c) conversion of the complexed or trapped Ca2+ into an electron-dense calcium pyroantimonate salt in 100 micron-thick tissue sections; and (d) wash-out of the excess potassium pyroantimonate at alkaline pH. With the OPA procedure, mitochondria of all muscle types showed little precipitate. The junctional sarcoplasmic reticulum was strongly reactive in relaxed white skeletal muscle, negative in contracted white fibres and negative in red skeletal and cardiac muscle, independent of the state of relaxation-contraction. Other organelles were essentially free of deposits. With the PPA method, the precipitate was almost exclusively confined to the sarcolemma and its T-tubular invaginations in cardiac and slow skeletal muscle, and was absent in fast skeletal muscle. Apart from occasional deposits in mitochondria, all other organelles were free of muscle. Apart from occasional deposits in mitochondria, all other organelles were free of precipitate. The sarcolemma-associated deposits were clearly confined to the inner leaflet of the lipid bilayer. The amount of precipitate varied within the contraction cycle, relaxed cells possessing the highest density. Exposure of the tissue to La3+ resulted in the complete absence of sarcolemma-bound precipitate suggesting that the Ca2+ is exchangeable. Furthermore, these cytological data suggest a basic difference in Ca2+ storage between white skeletal muscle on the one hand, and red skeletal and cardiac muscle on the other.

Analysis of sarcoplasmic reticulum proteins in patients susceptible to malignant hyperthermia.

It has been suspected that the cause of malignant hyperthermia (MH) is an abnormality in the sarcoplasmic reticulum of skeletal muscle. We isolated the sarcoplasmic reticulum from malignant hyperthermia-susceptible (MHS) patients and controls and analysed the protein composition with sodium dodecyl sulfate polyacrylamide gel electrophoresis. There were no remarkable changes in the sarcoplasmic reticulum protein composition profile of the scanned gel of the patients. Quantitative measurement of the relative proportion of each band in the gel, however, revealed a slight decrease in calsequestrin and a slight increase in protein of molecular weight 23,000. (Ca2+ -Mg2+)ATPase had no altered subfragments in MHS patients. Crude mitochondrial proteins and myoplasmic proteins showed minor alterations in composition in some patients. The data supported the thesis that malignant hyperthermia is due to defects in several different cell membranes including the sarcoplasmic reticulum and the mitochondria.

Biochemical studies in the liver and muscle of patients with Zellweger syndrome.

Biochemical studies have been performed in muscle, liver, leukocytes, and fibroblasts from patients suffering from the Zellweger syndrome. Oxidation rates of [1-14C]pyruvate, [U-14C]malate, and [1-14C]2-oxoglutarate were strongly reduced in skeletal muscle homogenate. Oxygen consumption in isolated skeletal muscle mitochondria could only be stimulated by ADP in the presence of ascorbate and N,N,N1,N1-tetramethyl-p-phenylenediamine. Cytochrome contents in heart muscle and liver mitochondria were normal. A very low activity of succinate-ubiquinone oxidoreductase was found in liver homogenate of two patients. From the effect of 2-thenoyltrifluoroacetone on the succinate-phenazine methosulphate oxidoreductase activity, a nearly competitive inhibition with respect to phenazine methosulphate was demonstrated in contrast with a non-competitive inhibition in controls. Normal oxidation rate of [1-14C]pyruvate and [2-14C]pyruvate was found in leucocytes and fibroblasts. Lactate and pyruvate levels were normal in serum and cerebrospinal fluid and beta-hydroxybutyrate and acetoacetate levels were normal in blood. The ratios lactate/pyruvate and beta-hydroxybutyrate/acetoacetate were normal as well. These findings point to a defect in the electron transport chain at the succinate-ubiquinone oxidoreductase level. This defect might be related to the absence of peroxisomes in the cells of Zellweger patients.

Skeletal muscle NAD+(P) and NADP+-dependent malic enzyme in Friedreich's ataxia.

Malic enzymes were studied in skeletal muscle from seven patients with Friedreich's ataxia (FA) and nine controls. Muscle contained three different malic enzymes. There were two strictly NADP+-dependent enzymes, one in the cytosol and one in mitochondria. These two enzymes are not allosteric. In FA muscle, activity of the mitochondrial NADP+-linked enzyme was significantly low and the cytosol NADP+-linked enzyme was significantly increased. A third malic enzyme, NAD+(P)-dependent, was found in the mitochondrial fraction. That enzyme had allosteric properties, and its activity was about the same in FA and control muscle.