Coby Van den Bogert

Altered properties of mitochondrial ATP-synthase in patients with a T → G mutation in the ATPase 6 (subunit a) gene at position 8993 of mtDNA

A family is described with a T-->G mutation at position 8993 of mtDNA. This mutation is located in the ATPase 6 gene of mtDNA which encodes subunit a of the ATP-synthase complex (FlFo-ATPase). Clinically, the patients showed severe infantile lactate acidosis and encephalomyopathy in a form that was different from the classical Leigh syndrome. In 3 affected boys, ranging in age from 3 months to 8 years, the mutation was found in 95-99% of the mtDNA population. The clinical symptoms correlated with the mtDNA heteroplasmy and in the healthy mother 50% of the mtDNA was mutated. The rate of mitochondrial ATP production by cultured skin fibroblasts containing 99% of mutated mtDNA was about 2-fold lower than that in normal fibroblasts. Native electrophoresis of the mitochondrial enzyme complexes revealed instability of the FlFo-ATPase in all the tissues of the patient that were investigated (heart, muscle, kidney, liver). Only a small portion of the ATP-synthase complex was present in the complete, intact form (620 kDa). Incomplete forms of the enzyme were present as subcomplexes with approx. molecular weights of 460, 390 and 150 kDa, respectively, which differed in the content of F1 and Fo subunits. Immunochemical analysis of the subunits of the FlFo-ATPase further revealed a markedly decreased content of the Fo subunit b in mitochondria from muscle and heart, and an increased content of the Fo subunit c in muscle mitochondria, respectively. These results indicate that in this family the T-->G point mutation at position 8993 in the mitochondrial ATPase 6 gene is accompanied by structural instability and altered assembly of the enzyme complex, that are both most likely due to changes in the properties of subunit a of the membrane sector part of the ATP-synthase.

Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases

To study the assembly of mitochondrial F1F0 ATP synthase, cultured human cells were labeled with [35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F1 part appeared to be assembled, and next an intermediate product that contained F1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochrondrial diseases.

Differentiation and proliferation of respiration-deficient human myoblasts

Replication and transcription of mitochondrial DNA were impaired in dividing human myoblasts exposed to ethidium bromide. MtDNA content decreased linearly per cell division and mitochondrial transcript levels declined rapidly, resulting in respiration-deficiency of the myoblasts. Despite the absence of functional mitochondria the cells remained able to proliferate when grown under specific culture conditions. However, the formation of myotubes was severely impaired in respiration-deficient myoblasts. We conclude that differentiation of myoblasts into myotubes is more dependent on mitochondrial function than proliferation of myoblasts.

Expression and fate of the nuclearly encoded subunits of cytochrome-c oxidase in cultured human cells depleted of mitochondrial gene products

Synthesis, import, assembly and turnover of the nuclearly encoded subunits of cytochrome-c oxidase were investigated in cultured human cells depleted of mitochondrial gene products by continuous inhibition of mitochondrial protein synthesis (OP- cells). Immunoprecipitation after pulse labeling demonstrated that the synthesis of the nuclear subunits was not preferentially inhibited, implying that there is no tight regulation in the synthesis of mitochondrial and nuclear subunits of mitochondrial enzyme complexes. Quantitative analysis of the mitochondrial membrane potential in OP- cells indicated that its magnitude was about 30% of that in control cells. This explains the normal import of the nuclearly encoded subunits of cytochrome-c oxidase and other nuclearly encoded mitochondrial proteins into the mitochondria that was found in OP- cells. The turnover rate of nuclear subunits of cytochrome-c oxidase, determined in pulse-chase experiments, showed a specific increase in OP- cells. Moreover, immunoblotting demonstrated that the steady-state levels of nuclear subunits of cytochrome-c oxidase were severely reduced in these cells, in contrast to those of the F1 part of complex V. Native electrophoresis of mitochondrial enzyme complexes showed that assembly of the nuclear subunits of cytochrome-c oxidase did not occur in OP- cells, whereas the (nuclear) subunits of F1 were assembled. The increased turnover of the nuclear subunits of cytochrome-c oxidase in OP- cells is, therefore, most likely due to an increased susceptibility of unassembled subunits to intra-mitochondrial degradation.

Familial mitochondrial DNA depletion in liver: haplotype analysis of candidate genes

Two sons and one daughter of healthy consanguineous parents presented with fatal hepatic failure in association with severe depletion of mitochondrial (mt)DNA in liver; a third son is healthy. Other published cases of mtDNA depletion concern single members of a family, which excludes the use of haplotype analysis. In the family presented here, the inheritance of the genes for mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF-1), mitochondrial single-stranded DNA-binding protein (mtSSBP), and endonuclease G (EndoG) was studied using microsatellite markers linked to these genes. The inheritance of the gene for mtDNA polymerase (pol γ) was studied using a polymorphic CAG repeat present within the coding region of the gene. EndoG and mtSSBP were excluded, but mtTFA remains a candidate. Pol γ or NRF-1 involvement would be compatible only with autosomal dominant inheritance. Coding sequence analysis of NRF-1 and mtTFA revealed no novel mutations in affected individuals.

Demonstration of two isoforms of subunit VIIa of cytochrome c oxidase from human skeletal muscle. Implications for mitochondrial myopathies.

Two different isoforms of subunit VIIa have been found in cytochrome c oxidase isolated from human skeletal muscle. The first 22 residues of the N‐terminal amino acid sequences showed 5 differences. Our results provide the first conclusive evidence for the existence of cytochrome c oxida isoenzymes in man. Since the two cytochrome c oxidase isoforms were both present in skeletal muscle tissue, though not necessarily in the same cell type, this suggests that human cytochrome c oxidase isoforms are not strictly tissue‐specific. These findings may have important implications for the elucidation of genetic diseases in man in which a deficiency of cytochrome c oxidase is restricted to certain tissues.

β-Oxidation of fatty acids in cultured human skin fibroblasts devoid of the capacity for oxidative phosphorylation

Prolonged treatment of cultured cells with ethidium bromide results in loss of the capacity for oxidative phosphorylation. Because of the tight coupling between mitochondrial beta-oxidation of fatty acids and the activity of the respiratory chain, such cells may be used to study the contribution of mitochondria and peroxisomes to fatty acid beta-oxidation. To investigate this, human skin fibroblasts were cultured in the presence of ethidium bromide for at least 10 cell generations, resulting in a virtually complete absence of oxidative phosphorylation as demonstrated directly in digitonin-permeabilized fibroblasts. The cells showed a lowered ATP/ADP ratio, most likely as the consequence of the inability to generate ATP via oxidative phosphorylation. The loss of the capacity for oxidative phosphorylation was also reflected in an increased cytosolic NADH/NAD+ ratio: the cells showed a highly elevated lactate/pyruvate ratio in the suspending medium when incubated with glucose. The beta-oxidation of octanoic and palmitic acid was dramatically decreased, suggesting that the beta-oxidation of these fatty acids takes place predominantly (> 90%) in mitochondria, at least in the cells studied. In contrast, the rates of pristanic and cerotic acid beta-oxidation were only slightly decreased, suggesting that this is mainly a peroxisomal process. The reduction of beta-oxidation of cerotic and pristanic acid, 27% and 15%, respectively, is most likely due to a lowered ATP level and an increased NADH/NAD(+)-redoxstate in these cells. We conclude that fibroblasts subjected to prolonged treatment with ethidium bromide can be used as a model system to study the substrate specificity and functional characteristics of the peroxisomal beta-oxidation system.

Meta-iodobenzylguanidine inhibits complex I and III of the respiratory chain in the human cell line Molt-4.

In this paper we report the effects of meta-iodobenzylguanidine (MIBG), a structural analogue of norepinephrine, on cell proliferation and several parameters related to mitochondrial respiration in Molt-4 cells. In micromolar concentrations, MIBG completely inhibited the proliferation of Molt-4 cells. In intact Molt-4 cells, a progressive increase in the lactate to pyruvate ratio was observed after incubation of these cells with glucose and increasing concentrations of MIBG. In Molt-4 cells permeabilized with digitonin, MIBG inhibited mitochondrial ATP synthesis when malate was used as a substrate. Succinate-driven synthesis of ATP was also inhibited by MIBG, although higher concentrations were required. These results indicate that apart from inhibition of complex I, MIBG inhibits at least one other complex of the respiratory chain. Measurement of the activities of the individual enzyme complexes in the presence of MIBG revealed that complex III is the other enzyme complex susceptible to inhibition by MIBG. Although maximal inhibition of ATP synthesis was observed at a concentration of 10 microM, maximal inhibition of cell proliferation was observed at a concentration of 50 microM of MIBG. This suggests that MIBG also influences other cellular processes apart from mitochondrial oxidative phosphorylation, resulting in additional inhibition of cell proliferation.

Subunit IV of human cytochrome c oxidase, polymorphism and a putative isoform

As part of our study of isoenzyme forms of human cytochrome c oxidase, we purified subunit IV from human heart and skeletal muscle with reversed-phase HPLC and determined the N-terminal amino acid sequences and the electrophoretic mobility. The N-terminus of human heart subunit IV proved to be ragged with 30% of the protein lacking the first three residues. Also a Tyr/Phe polymorphism was observed at residue 16. No differences in N-terminal sequence and electrophoretic mobility were observed between subunit IV of cytochrome c oxidase from human heart and skeletal muscle. Therefore, our results suggest that identical subunits IV are present in cytochrome c oxidase from human heart and skeletal muscle. A putative isoform of subunit IV with a blocked N-terminus was purified from human heart cytochrome c oxidase, which proved to have a different retention time on a reversed-phase column and also a slightly higher electrophoretic mobility on an SDS-polyacrylamide gel compared to the native subunit IV. We could not demonstrate the existence of isoforms of subunit IV in human skeletal muscle.

Cultured human muscle cells and respiratory chain deficiencies

Cultured muscle cells are useful in the study of respiratory chain disorders. Muscle tissue is affected in most cases and muscle biopsies are often taken for diagnostic purposes. Small samples of the biopsies can provide large numbers of muscle cells. In contrast with most other cell types, the muscle cells can differentiate in culture. Upon differentiation, multinuclear myotubes are formed by fusion of myoblasts and muscle-specific proteins are synthesized in the myotubes. Muscle cells can be used for diagnostic purposes, e.g. by measurement of the lactate and pyruvate synthesis, although with the limitation that not each form of respiratory chain deficiency may be expressed in culture. Muscle cells have been studied to obtain insight into mitochondrial DNA disorders, e.g. by investigation of cells after exposure to inhibitors of expression of mitochondrial DNA. Furthermore, cultured muscle cells have been studied to explore the potential of new therapeutic possibilities such as myoblast transfer and gene therapy.