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Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing
A M James1, P W Sheard, Y H Wei
1Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Abstract:
Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudy et al. (1995) PNAS, 92, 729-733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60-88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+-ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+-ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2-20-fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.
Insights
Mitochondrial DNA mutations in MELAS and MERRF syndromes impair ATP synthesis. Cells with these mutations are uniquely vulnerable to increased energy demand, potentially leading to neuronal excitotoxicity.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Mutations in mitochondrial DNA (mtDNA) cause MELAS and MERRF syndromes.
- These mutations impact respiratory chain function without altering cellular ATP levels.
Purpose of the Study:
- Investigate mitochondrial ATP synthesis in MELAS and MERRF patient fibroblasts.
- Determine if mitochondrial defects manifest under increased cellular energy demand.
Main Methods:
- Assessed mitochondrial ATP synthesis rates in patient-derived fibroblasts.
- Simulated high ATP demand using gramicidin to stimulate ATP hydrolysis.
- Measured ATP/ADP ratios and plasma membrane potential.
Main Results:
- MELAS and MERRF mitochondria showed 60-88% reduced ATP synthesis rates.
- Quiescent fibroblasts masked the defect, but gramicidin revealed sensitivity.
- Patient cells exhibited a 2-20 fold lower gramicidin threshold for ATP depletion and membrane depolarization.
Conclusions:
- Cells with mtDNA mutations are acutely sensitive to increased ATP demand.
- This sensitivity explains how mitochondrial dysfunction contributes to disease.
- Neurons with mitochondrial defects are susceptible to excitotoxic cell death due to membrane depolarization.