Mitochondrial Physiology Network 10.9: 114 (2005) - download pdf

Two components in pathogenic mechanism of mitochondrial ATPase deficiency: Energy deprivation and ROS production.

Josef Houštěk¹, T Mráček¹, P Pecina¹, A Vojtíšková¹, O Šebesta¹, M Vrbacký¹, H Mayr², M  Kalous¹, P Ješina¹

¹Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic; ²Department of Paediatrics, General Hospital Salzburg, Müllner Hauptstraße 48, A-5020 Salzburg, Austria – houstek@biomed.cas.cz

    Isolated defects of mitochondrial ATP-synthase (ATPase) due to diminished biosynthesis of the enzyme represent new class of severe mitochondrial diseases, characterized by selective, 70-90 % decrease in cellular content of ATPase [1, 2]. The primary cause is a mutation in nuclear gene (s) affecting the initial stage of enzyme biogenesis - assembly of the F₁ catalytic part of the enzyme [1, 3]. Our studies of cellular respiration (OROBOROS Oxygraph-2k) and ATP synthesis in fibroblasts from several unrelated patients with ATPase deficiency showed that the content of the enzyme is insufficient, or without any reserve capacity to maintain basal mitochondrial energy provision. The cells exhibited altered discharge of mitochondrial membrane potential Δψ_m (cytofluorometry with TMRM), which was increased at state 4 and especially at state 3-ADP. ATPase-deficient cells further showed several-fold increase in mitochondrial ROS production analyzed by CM-H₂DCFDA fluorescence (using fluorometry or confocal microscopy) that was fully abolished by uncoupler FCCP. Activated ROS production was associated with a variable increase of mitochondrial superoxide dismutase (MnSOD), and small changes in cellular content of glutathione, but inhibition of glutathione synthase with buthionine sulphoximide (BSO) significantly activated ROS production. Aurovertin and Oligomycin titration studies and replacement of glucose by galactose in culture medium demonstrated marked decrease in viability of ATPase-deficient cells when they depend on mitochondrial oxidative metabolism rather than on glycolysis.

    Our studies demonstrate that altered discharge and high levels of Δψ_m activate ROS production in ATPase-deficient cells. The resulting oxidative stress can be counteracted by activity of MnSOD and glutathione levels, however, activated mitochondrial ROS production is lethal in patient cells.

1.  Houstek J, Klement P, Floryk D, Antonicka H, Hermanska J, Kalous M, Hansikova H, Hout'kova H, Chowdhury SK, Rosipal T, Kmoch S, Stratilova L, Zeman J (1999) A novel deficiency of mitochondrial ATPase of nuclear origin. Hum. Mol. Genet. 8: 1967-1974.

2.  Mayr JA, Paul J, Pecina P, Kurnik P, Förster H, Fötschl U, Sperl W, Houstek J (2004) Reduced respiratory control with ADP and changed pattern of respiratory chain enzymes due to selective deficiency of the mitochondrial ATP synthase. Pediatr. Res. 55: 1-7.

3.  Houstek J, Mracek T, Vojtiskova A, Zeman J (2004) Mitochondrial diseases and ATPase defects of nuclear origin. Biochim Biophys Acta. 1658: 115-121.