With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-␣. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-␣. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed r8 cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in r8 cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP 4± for ADP 3± by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete F o F 1 -ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian r8 cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in r8 cells and an incomplete F o F 1 -ATP synthase was also required. The maximum value of this potential was <110 mV in permeabilized cells and <67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete F o F 1 -ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but <65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of r8 mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete F o F 1 -ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete F o F 1 -ATP synthase in maintaining the mitochondrial membrane potential in r8 cells.Keywords: mammalian r8 cells; mitochondrial membrane potential; adenine nucleotide carrier; F o F 1 -ATP synthase; mitochondrial DNA.Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of respiratory proton pumps and the F o F 1 -ATP synthase, which are all essential for oxidative phosphorylation [1±4]. Cells cultured with ethidium bromide lose their mtDNA and consequently these cells (termed r8 cells) cannot carry out oxidative phosphorylation, require pyruvate and uridine for growth and have glycolysis as their only source of ATP [2±4]. Even so, mitochondria are still essential for r8 cells because of vital metabolic pathways catalysed by mitochondrial proteins encoded by nuclear DNA [4,5]. Import of nuclear-encoded proteins into mitochondria requires a membrane potential, but in r8 cells this must arise by a mechanism other t...

Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (∆-mtDNA) lacking a specific 4977-bp fragment, the common deletion, leads to three related mtDNA diseases : Pearson's syndrome; Kearns-Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of ∆-mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of ∆-mtDNA on cell function. The 4977-bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of ∆-mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (ρ 0 ) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0Ϫ86 % ∆-mtDNA and all had volumes, protein contents, plasma-membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating ∆-mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50Ϫ55% ∆-mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of ∆-mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of ∆-mtDNA in the target tissues of patients with mtDNA diseases.

A trait of vascular fragility, characterized by the formation of abrupt defects within the elastic laminae of the abdominal aorta, has been identified in Brown Norway (BN) rats. These lesions are greatly exacerbated in F(1) rats from a BN x New Zealand genetically hypertensive (GH) intercross, implying that the genetic background provided by the GH rat influences lesion severity. The F(2) progeny of the BN x GH intercross were used to identify susceptibility loci for the lesions as well as exacerbating loci. Two major quantitative trait loci (QTLs) for number of internal elastic lamina lesions were identified on rat chromosomes 5 and 10, with the maximum "log of the odds ratio" (LOD) scores at D5Rat119 (LOD 5.0) and at D10Mit2 (LOD 4.5), respectively, together contributing 33.5% to the genetic variance. Further analysis revealed that the chromosome 10 locus exhibits a dominant mode of inheritance, with BN alleles being associated with increased lesion number (P < 0.0002) compared with GH homozygotes. This locus was in epistasis to a modifier locus on rat chromosome 2 at D2Mit14 (LOD score 2.12). A second major locus was identified on chromosome 5, exhibiting a semidominant mode of inheritance, again with the BN allele being significantly associated with increased lesion number (P < 0.0001). Furthermore, a locus influencing lesion severity was identified on chromosome 3 wherein GH alleles associated with increased severity. This is the first study to identify susceptibility loci for vascular elastic tissue fragility.

Aim To determine the independent and combined effects of three quantitative trait loci (QTL) for blood pressure in the Genetically Hypertensive (GH/Omr) rat by generating and characterizing single and combined congenic strains that have QTL on rat chromosomes (RNO) 2, 6, and 18 from the GH rat introduced into a hypertension resistant Brown Norway (BN) background.Methods Linkage analysis and QTL identification (genome wide QTL scan) were performed with MapMaker/EXP to build the genetic maps and MapMaker/QTL for linking the phenotypes to the genetic map. The congenic strains were derived using marker-assisted selection strategy from a single male F1 offspring of an intercross between the male GH/Omr and female BN/Elh, followed by 10 generations of selective backcrossing to the female BN progenitor strain. Single congenic strains generated were BN.GH-(D2Rat22-D2Mgh11)/Mcwi (BN.GH2); BN.GH-(D6Mit12-D6Rat15)/Mcwi (BN.GH6); and BN.GH-(D18Rat41-D18Mgh4)/Mcwi (BN.GH18). Blood pressure measurements were obtained either via a catheter placed in the femoral artery or by radiotelemetry in the single and combined congenics. Responses to angiotensin II (ANGII), norepinephrine (NE), and baroreceptor sensitivity were measured in the single congenics.Results Transferring one or more QTL from the hypertensive GH into normotensive BN strain was not sufficient to cause hypertension in any of the developed congenic strains. There were no differences between the parental and congenic strains in their response to NE. However, BN.GH18 rats revealed significantly lower baroreceptor sensitivity (β = -1.25 ± 0.17), whereas BN.GH2 (β = 0.66 ± 0.09) and BN.GH18 (β = 0.71 ± 0.07) had significantly decreased responses to ANGII from those observed in the BN (β = 0.88 ± 0.08). ConclusionThe failure to alter blood pressure levels by introducing the hypertensive QTL from the GH into the hypertension resistant BN background suggests that the QTL effects are genome backgrounddependent in the GH rat. BN.GH2 and BN.GH18 rats reveal significant differences in response to ANGII and impaired baroreflex sensitivity, suggesting that we may have captured a locus responsible for the genetic control of baroreceptor sensitivity, which would be considered an intermediate phenotype of blood pressure.