Differential features of patients with mutations in two COX assembly genes, SURF-1 and SCO2.

We screened 41 patients with undiagnosed encephalomyopathies and cytochrome c oxidase (COX) deficiency for mutations in two COX assembly genes, SURF-1 and SCO2; 6 patients had mutations in SURF-1 and 3 had mutations in SCO2. All of the mutations in SURF-1 were small-scale rearrangements (deletions/insertions); 3 patients were homozygotes and the other 3 were compound heterozygotes. All patients with SCO2 mutations were compound heterozygotes for nonsense or missense mutations. All of the patients with mutations in SURF-1 had Leigh syndrome, whereas the 3 patients with SCO2 mutations had a combination of encephalopathy and hypertrophic cardiomyopathy, and the neuropathology did not show the typical features of Leigh syndrome. In patients with SCO2 mutations, onset was earlier and the clinical course and progression to death more rapid than in patients with SURF-1 mutations. In addition, biochemical and morphological studies showed that the COX deficiency was more severe in patients with SCO2 mutations. Immunohistochemical studies suggested that SURF-1 mutations result in similarly reduced levels of mitochondrial-encoded and nuclear-encoded COX subunits, whereas SCO2 mutations affected mitochondrial-encoded subunits to a greater degree. We conclude that patients with mutations in SURF-1 and SCO2 genes have distinct phenotypes despite the common biochemical defect of COX activity.

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I-III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.

Structural and functional impairment of mitochondria in adriamycin-induced cardiomyopathy in mice: suppression of cytochrome c oxidase II gene expression.

The use of adriamycin (ADR) in cancer chemotherapy has been limited due to its cumulative cardiovascular toxicity. Earlier observations that ADR interacts with mitochondrial cytochrome c oxidase (COX) and suppresses its enzyme activity led us to investigate ADR's action on the cardiovascular functions and heart mitochondrial morphology in Balb-c mice i.p. treated with ADR for several weeks. At various times during treatment, the animals were assessed for cardiovascular functions by electrocardiography and for heart tissue damage by electron microscopy. In parallel, total RNA was extracted from samples of dissected heart and analyzed by Northern blot hybridization to determine the steady-state level of three RNA transcripts encoded by the COXII, COXIII, and COXIV genes. Similarly, samples obtained from the liver of the same animals were analyzed for comparative studies. Our results indicated that 1) treatment of mice with ADR caused cardiovascular arrhythmias characterized by bradycardia, extension of ventricular depolarization time (tQRS), and failure of QRS at high concentrations (10-14 mg/kg body weight cumulative dose); 2) the heart mitochondria underwent swelling, fusion, dissolution, and/or disruption of mitochondrial cristae after several weeks of treatment. Such abnormalities were not observed in the mitochondria of liver tissue; and 3) among the three genes of COX enzyme examined, only COXII gene expression was suppressed by ADR treatment, mainly after 8 weeks in both heart and liver. Knowing that heart mitochondria represent almost 40% of heart muscle by weight, we conclude that the deteriorating effects of ADR on cardiovascular function involve mitochondrial structural and functional impairment.

The fate of human sperm-derived mtDNA in somatic cells.

Inheritance of animal mtDNA is almost exclusively maternal, most likely because sperm-derived mitochondria are actively eliminated from the ovum, either at or soon after fertilization. How such elimination occurs is currently unknown. We asked whether similar behavior could be detected in somatic cells, by following the fate of mitochondria and mtDNAs after entry of human sperm into transformed cells containing mitochondria but lacking endogenous mtDNAs (rho0 cells). We found that a high proportion (10%-20%) of cells contained functioning sperm mitochondria soon after sperm entry. However, under selective conditions permitting only the survival of cells harboring functional mtDNAs, only approximately 1/10(5) cells containing sperm mitochondria survived and proliferated. These data imply that mitochondria in sperm can enter somatic cells relatively easily, but they also suggest that mechanisms exist to eliminate sperm-derived mtDNA from somatic cells, mechanisms perhaps similar to those presumed to operate in the fertilized oocyte.

Effects of hemin on apoptosis, suppression of cytochrome c oxidase gene expression, and bone-marrow toxicity induced by doxorubicin (adriamycin).

We have shown that hemin (iron-protoporphyrin IX) selectively counteracts doxorubicin (Adriamycin, ADR)-induced cytotoxicity on human leukemia K-562 cells by preventing ADR from inhibiting mitochondrial cytochrome c oxidase (COX), a novel target site for anthracyclines. Here, we investigated whether or not (a) treatment with ADR promotes apoptosis and represses the expression of two COX genes (one nuclear and one mitochondrial) in human K-562 cells in the absence and presence of hemin, and (b) injection of hemin preserves bone-marrow cellularity in ADR-myelosuppressed rats. Cultured K-562 cells were incubated with varying concentrations of ADR.HCl (0.2 microM to 5 microM) in the presence and absence of hemin (30 microM) and assessed for DNA degradation, as well as for expression of mitochondrial COXII and nuclear COXIV genes by RNA Northern blot hybridization analysis. In parallel, we investigated whether or not hemin injected i.p. in myelosuppressed rats affected ADR-induced bone-marrow cytotoxicity. These studies have shown the following: (a) ADR caused a dose- and time-dependent DNA fragmentation, characteristic of apoptosis, in K-562 cells; (b) hemin reduced the frequency of cell death caused by ADR: this effect was specific for ADR, because hemin failed to prevent apoptosis induced by methotrexate (MTX) in these cells; (c) ADR suppressed expression of COXIV and COXII genes, and exposure of ADR-treated K-562 cells to hemin did not reverse this suppression; and (d) i.p. injection of hemin in ADR-myelosuppressed rats improved bone-marrow cellularity, promoted colony formation (CFU-GM and CFU-F), and stromal cell outgrowth; moreover, hemin increased WBC counts depressed 12 days after ADR treatment. These studies indicate that hemin is a selective inhibitor of ADR-induced apoptosis of human leukemia cells and preserves bone-marrow cellularity in rats injected with ADR.

Mitochondrial cytochrome c oxidase as a target site for daunomycin in K-562 cells and heart tissue.

Daunomycin and other structurally related anthracyclines can cause myelosuppression and cardiomyopathy. We explored the possible mechanism(s) by which daunomycin (DAU) interacts with target sites in neoplastic hemopoietic cells and heart tissue. We observed that [3H(G)]DAU interacts selectively with mitochondrial hemoproteins isolated from K-562 cells and rat and bovine heart and forms relatively stable protein complexes. Isolation, purification, and chromatographic analysis of the mitochondrial components complexed with [3H(G)]DAU revealed that one of the major components involved is cytochrome c oxidase (COX). Both DAU and ADR caused a dose-dependent inhibition of COX activity in vitro, an event prevented by exogenous hemin. The interaction of DAU with COX appears to occur via more than one site, one of which at least appears to be the prosthetic group of heme. Therefore, mitochondrial COX, a pivotal mitochondrial enzyme for cell respiration, may serve as a potential target site for DAU and other related anthracyclines.