oligomycins and Mitochondrial-Myopathies

Summary Human mitochondrial DNA (mtDNA) encodes 13 of the polypeptides associated with the process of oxidative phosphorylation (OXPHOS), the cells most important ATP generating pathway. Until recently, the effects of mtDNA rearrangements on male fertility have been largely ignored. However, it is becoming increasingly evident that both point mutations and large-scale deletions may have an impact on sperm motility and morphology. We discuss the implications of these rearrangements in the context of the clinical setting. We further discuss the possible consequences resulting from the transmission of sperm mtDNA deletions to the offspring. The role of nucleo-cytoplasmic interaction is investigated in the context of nuclear transcription and replication factors that regulate mtDNA transcription and replication.

Topics: Animals; DNA-Binding Proteins; DNA, Mitochondrial; Gene Deletion; Humans; Infertility, Male; Male; Mitochondrial Myopathies; Mitochondrial Proteins; Nuclear Proteins; Oligomycins; Oxidative Phosphorylation; Potassium Cyanide; Rotenone; Sperm Motility; Testis; Transcription Factors

Mitochondrial (mt) DNA-associated NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) syndrome is due to mutation in the MT-ATP6 gene. We report the case of a 18-year-old man who presented with deafness, a myoclonic epilepsy, muscle weakness since the age of 10 and further developed a retinitis pigmentosa and ataxia. The whole mtDNA analysis by next-generation sequencing revealed the presence of the 2 bp microdeletion m.9127-9128 del AT in the ATP6 gene at 82% heteroplasmy in muscle and to a lower load in blood (10-20%) and fibroblasts (50%). Using the patient's fibroblasts, we demonstrated a 60% reduction of the oligomycin-sensitive ATPase hydrolytic activity, a 40% decrease in the ATP synthesis and determination of the mitochondrial membrane potential using the fluorescent probe tetramethylrhodamine, ethyl ester indicated a significant reduction in oligomycin sensitivity. In conclusion, we demonstrated that this novel AT deletion in the ATP6 gene is pathogenic and responsible for the NARP syndrome.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Base Sequence; Carrier Proteins; Cells, Cultured; DNA Mutational Analysis; DNA, Mitochondrial; High-Throughput Nucleotide Sequencing; Humans; Male; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondrial Myopathies; Mitochondrial Proton-Translocating ATPases; Oligomycins; Retinitis Pigmentosa; Sequence Deletion; Syndrome; Young Adult

Inhibitor titrations were applied to characterize functional changes in mitochondrial energy metabolism in the skeletal muscle of patients with mitochondrial diseases. For this we titrated the maximal mitochondrial respiration rate of saponin-skinned muscle fibers isolated from the skeletal muscle biopsy with the specific inhibitors of mitochondrial oxidative phosphorylation complexes I, IV and V-rotenone, azide and oligomycin. For three patients with deletions of mitochondrial DNA and one patient with a complex I deficiency the titrations revealed at rather normal respiration activities of saponin-skinned fibers significant differences to healthy controls: (i) The inhibitor titration curves of the affected enzyme were much steeper and (ii) for almost complete inhibition of respiration a smaller amount of the inhibitor is necessary. The detailed analysis of the titration curves within the framework of metabolic control theory indicated elevated flux control coefficients of the respective complex of respiratory chain. On the other hand, for one patient with a mitochondrial DNA depletion syndrome, decreased respiration activities of skinned fibers but no redistribution of flux control was observed. We conclude, therefore, that application of inhibitor titrations and the quantitative description of the titration curve can be a valuable approach to elucidate functional defects of mitochondrial oxidative phosphorylation.

Topics: Adult; Azides; Biopsy; Electron Transport Complex I; Enzyme Inhibitors; Female; Humans; In Vitro Techniques; Male; Mitochondrial Myopathies; NADH, NADPH Oxidoreductases; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Proton-Translocating ATPases; Rotenone

A nuclear genome delivery system was developed to deduce the modes of inheritance of the clinical phenotypes observed in patients with mitochondrial diseases by transfer of pure nuclei from normal cells to fibroblasts from the patients. The problem of possible contamination of the nuclei with a small amount of mtDNA was overcome by using mtDNA-less (rho0) human cells as nuclear donors. In this study, intercellular transfer of pure nuclei was carried out by simple fusion of rho0 HeLa cells with 533 fibroblasts from a patient with a fatal mitochondrial disease, which were deficient in cytochrome c oxidase and succinate dehydrogenase activities. The results showed that the cytochrome c oxidase and succinate dehydrogenase activities were restored by the introduction of pure HeLa nuclei, suggesting that the observed phenotypes of mitochondrial dysfunction were not due to mtDNA mutations but to nuclear, recessive mutations. Thus, our nuclear transfer system is effective for determining whether a mitochondrial or nuclear genome of a patient is responsible for a disease and whether deficiency of mitochondrial enzymes, including enzymes exclusively encoded by nuclear genomes, is transmitted in a nuclear recessive or nuclear dominant way, providing the parents of the patients with valuable information for genetic counseling on the risk of mitochondrial diseases in their next babies.

Topics: Adenosine Triphosphatases; Cell Nucleus; Cytochrome-c Oxidase Deficiency; DNA, Mitochondrial; Enzyme Inhibitors; Fibroblasts; HeLa Cells; Humans; Infant; Mitochondrial Myopathies; Oligomycins; Succinate Dehydrogenase