Nuskova 2015 Abstract MiP2015
|Insufficient energy provision or increased oxidative stress – what matters more in ATP synthase deficiencies?|
Mitochondrial F1Fo-ATP synthase is responsible for most of the ATP production in aerobic organisms. Its deficiencies are associated with severe pathologic phenotypes. To shed light on the functional consequences of ATP synthase deficiencies, we utilised a model of HEK293 cell line and explored the effect of RNAi mediated knockdown of the three subunits (γ, δ and ε) forming the central stalk of the enzyme, which results in an isolated decrease of ATP synthase content.
For functional evaluations of ATP synthase deficiencies, 9 stable knockdown clones with down-regulated subunits γ (ATP5C1 gene), δ (ATP5D gene), or ε (ATP5E gene) have been selected. The residual oligomycin-sensitive ATPase hydrolytic activity in these clones ranges between 2 and 78 % as compared to controls, which is paralleled by a decrease in the content of fully assembled ATP synthase complex.
Examination of cellular respiration and glycolytic flux, using the Seahorse XFe24 analyser, revealed that the clones with less than 30 % of residual ATPase activity switched their metabolism to enhanced glycolysis. There is a decrease in their basal respiration rate relatively to their respiratory capacity (47 vs 61 % in controls) and in parallel, their basal glycolytic rates utilise by up to 20 % more of their glycolytic capacity. These findings clearly demonstrate metabolic adaptations of these cells. On the other hand, the clones with more than 30 % residual ATPase activity showed no change either in the respiration or in their basal glycolytic rate.
As a result of ATP synthase deficiency, the knockdown clones exhibit reduced dissipation of mitochondrial membrane potential (ΔΨm) under ADP stimulation (by up to 20 mV compared to controls). The increase of ΔΨm might then stimulate the production of reactive oxygen species (ROS) that is, indeed, elevated by 20 % in the knockdown clones with the lowest ATPase residual activity. The content of antioxidant enzymes, on the other hand, did not display any correlation to ATPase activity or ROS production.
In conclusion, our data indicate two pathogenic mechanisms of ATPase deficiency – energetic deprivation and increased oxidative stress. Generally, the threshold for defect manifestation and subsequent metabolic remodelling equals to approximately 30 % of ATPase activity.
Labels: MiParea: Genetic knockout;overexpression
Stress:Oxidative stress;RONS Organism: Human Tissue;cell: HEK
Event: B1, Oral MiP2015
Inst Physiology, Czech Acad Sc, Prague, Czech Republic. - [email protected]
This project is supported by the Grant Agency of Charles University (grant 1160214) and the Czech Science Foundation (P303/12/1363, P303/11/0970).