MiP2005: Session 7 - Young Investigator Presentation
Mitochondrial Physiology Network 10.9: 90 (2005) - download pdf
Mitochondrial superoxide production is inversely proportional to complex I activity in human complex I deficiency.
Sjoerd Verkaart1,2, W Koopman1,3, LG Nijtmans2, LWPJ van den Heuvel2, JAM Smeitink2, PHGM Willems1
Depts. 1Biochemistry, 2Paediatrics, 3Microscopical Imaging Center of the Nijmegen Center for Molecular Life Sciences and 2Mitochondrial Disorders, Radboud University, Nijmegen, The Netherlands. - firstname.lastname@example.org
Respiratory chain dysfunction lies at the basis of severe clinical syndromes, presenting either at birth or in early childhood, especially affecting organs and tissues with a high-energy demand, including brain, heart and skeletal muscle. Among these disorders, isolated complex I deficiency (OMIM 252010) is the most frequently encountered enzyme defect. Structurally, complex I (NADH:ubiquinone oxidoreductase; E.C. 126.96.36.199) consists of 46 subunits, seven of which are encoded by the mitochondrial DNA and the remainder by the nuclear genome. In addition to defects in the mitochondrial DNA, mutations in nuclear genes (NDUFV1, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8) have been shown to be associated with isolated human complex I deficiency. In order to enhance our understanding of the pathophysiology of disorders of the human oxidative phosphorylation (OXPHOS) system, we study genetically characterized patient skin fibroblasts. A previous study  revealed an increased production of superoxide and enchanced lipid peroxidation in human skin fibroblasts chronically treated with the complex I inhibitor rotenone. Here we determined whether enhanced superoxide generation was associated with complex I deficiency in a collection of 24 pediatric patients. To this end we applied videomicroscopy to measure the superoxide-induced conversion of hydroethidine (HEt) into ethidium (Et) in living cells. Superoxide production was similar in five control fibroblast cell lines of different genetic origin and passage number, indicating that these factors are not major contributors to superoxide production in our assay. Analysis of patient fibroblasts displayed a significantly increased rate of superoxide production relative to control. Strikingly, this rate was inversely proportional to the residual enzymatic complex I but unrelated to complex III activity. This supports the idea that the superoxide detected, originates from complex I. Using Blue Native gel electrophoresis on CI deficient fibroblasts, we show that residual CI activity is linearly correlated to the amount of CI-39 kDa protein in fully assembled CI. Since superoxide production is inversely related to CI activity, which represents fully assembled and active CI protein, these findings support the idea that increased superoxide production is caused by a smaller amount of active (but less stable?) CI and not by normal amounts of ‘leaky’ CI.
1. Koopman WJH, Verkaart S, Visch HJ, van der Westhuizen FH, Murphy MP, van den Heuvel LW, Smeitink JAM, Willems PHGM (2005) Inhibition of complex I of the electron transport chain causes oxygen radical-mediated mitochondrial outgrowth. Am J. Physiol. Cell Physiol. 288: C1440-1450.
2. Koopman WJ, Visch HJ, Verkaart S, van den Heuvel LW, Smeitink JA, Willems PHGM (2005) Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated complex I deficiency. Am. J. Physiol. Cell. Physiol. (in press).