myxothiazol and Mitochondrial-Myopathies

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Topics: Amino Acid Substitution; Antimycin A; Blotting, Western; Cytochrome b Group; DNA Mutational Analysis; DNA, Mitochondrial; Electron Transport Complex III; Gene Frequency; Genetic Variation; Haplotypes; Humans; Methacrylates; Mitochondrial Myopathies; Mutation; Point Mutation; Thiazoles; Ubiquinone

We have studied the control by complex III of both succinate-cytochrome c reductase and of oxidative flux measured polarographically in rat muscle and liver mitochondria using the specific inhibitor, myxothiazol, to induce partial inhibitions of complex III activity. Complex III exerted a low degree of control on electron flux through succinate-cytochrome c reductase, and a 30-50% decrease in complex III activity remained undetected by this assay. However, when overall oxidative flux was measured polarographically there was a considerable difference in the effect of lowered complex III activity on this pathway between rat muscle and liver mitochondria. Small changes in complex III activity (approximately 5% inhibition) in muscle mitochondria caused marked changes in succinate-stimulated respiration, whereas in liver mitochondria complex III had to be inhibited by about 45% before changes in maximum oxidation rates could be detected. These differences between muscle and liver mitochondria occurred despite rat liver mitochondria having at least a 4-fold lower complex III activity. This suggests that when considering the biochemical consequences of defects of the respiratory chain an important factor is the degree to which an individual complex can be lowered before major changes in overall flux occur. In addition, since many patients with respiratory chain disease present with predominantly muscle symptoms, this latter finding suggests that an understanding of the control of mitochondrial oxidations by different tissues may be important when considering the tissue-specific nature of defects of the respiratory chain.

Topics: Animals; Antifungal Agents; Cytochrome b Group; Electron Transport Complex III; Humans; Kinetics; Methacrylates; Mitochondria, Liver; Mitochondria, Muscle; Mitochondrial Myopathies; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Oxygen Consumption; Rats; Species Specificity; Thiazoles