Defective kinetics of cytochrome c oxidase and alteration of mitochondrial membrane potential in fibroblasts and cytoplasmic hybrid cells with the mutation for myoclonus epilepsy with ragged-red fibres (‘MERRF’) at position 8344 nt

Abstract

We have investigated pathogenic effects of the tRNA(Lys) A8344G mutation associated with the syndrome myoclonus epilepsy with ragged-red fibres (MERRF) by using fibroblasts and fibroblast-derived cytoplasmic hybrid cells harbouring different percentages of mutated mitochondrial DNA (mtDNA). The activity of cytochrome c oxidase (COX) in patient fibroblasts with 89% mutated mtDNA was decreased to 20% of the control levels. COX exhibited altered kinetics, with a decreased V(max) for both the low-affinity and high-affinity phases; however, the K(m) values were not significantly changed. The substrate-dependent synthesis of ATP was decreased to 50% of the control. Analysis of the mitochondrial membrane potential, DeltaPsi, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow cytometry showed a 80% decrease in DeltaPsi at state 4 and an increased sensitivity of DeltaPsi to an uncoupler in fibroblasts from the patient. The investigation of transmitochondrial cytoplasmic hybrid clones derived from the patient's fibroblasts enabled us to characterize the relationship between heteroplasmy of the MERRF mutation, COX activity and DeltaPsi. Within the range of 87-73% mutated mtDNA, COX activity was decreased to 5-35% and DeltaPsi was decreased to 6-78%. These results demonstrate that the MERRF mutation affects COX activity and DeltaPsi in different proportions with regard to mutation heteroplasmy and indicate that the biochemical manifestation of the MERRF mutation exerts a very steep threshold of DeltaPsi inhibition.

Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification

“…Similar results were obtained using rhodamine 123 (data not shown). Thus, knocking down the expression of GTPBP3 causes a mitochondrial dysfunction that is consistent with phenotypic features exhibited by cells harboring MERRF and MELAS mutations (1,16,33,37).…”

“…These mutations cause reduction in oxygen consumption and mitochondrial protein synthesis in the carrier cells (37), which also exhibit reduced ATP production, decreased mitochondrial membrane potential, and increased superoxide levels (1,16,33). Interestingly, tRNAs harboring MERRF and MELAS mutations are deficient in normal taurine-containing modifications at the anticodon wobble position [5- (18,20,44,(51)(52)(53)(54)(55).…”

Retrospective, Multicentric Study of 180 Children with Cytochrome c Oxidase Deficiency

“…The quality of the enzymatic assays protocol was confirmed by the 1st European laboratory ring external quality test (Dr. Gellerich, Martin Luther University, Halle, Germany). Two-dimensional electrophoresis (BN-PAGE/SDS-PAGE) and/or Western blot analysis (17,18) was performed to assess the amount and composition of respiratory chain complexes. In cases of Polish patients, respiratory chain complexes activity was measured in tissue homogenates as above and the procedure was not changed during the study.…”

Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce β-oxidation in white fat

“…Tissue fragments were removed by centrifugation for 10 min at 4°C and 600 g and crude cell membranes were separated from the supernatant by centrifugation for 35 min at 4°C and 100,000 g. Samples of the crude cell membranes were analysed using SDS-PAGE and immunoblots [26]. Polyclonal rabbit antibody [26] was used (1:5,000) to detect cytochrome c oxidase subunits I and VI (MT-CO1 and COX6).…”

“…Tissue fragments were removed by centrifugation for 10 min at 4°C and 600 g and crude cell membranes were separated from the supernatant by centrifugation for 35 min at 4°C and 100,000 g. Samples of the crude cell membranes were analysed using SDS-PAGE and immunoblots [26]. Polyclonal rabbit antibody [26] was used (1:5,000) to detect cytochrome c oxidase subunits I and VI (MT-CO1 and COX6). Monoclonal antibody was used to detect the 70 kDa subunit of complex II (SDHA; 1:2,000; Molecular Probes A-11142, Eugene, OR, USA) and subunit α of F 1 -ATPase (ATP5A1; 1:200,000, lot 20D6 [27]).…”