adPEO mutations in ANT1 impair ADP-ATP translocation in muscle mitochondria

Abstract

Mutations in the heart and muscle isoform of adenine nucleotide translocator 1 (ANT1) are associated with autosomal-dominant progressive external opthalmoplegia (adPEO) clinically characterized by exercise intolerance, ptosis and muscle weakness. The pathogenic mechanisms underlying the mitochondrial myopathy caused by ANT1 mutations remain largely unknown. In yeast, expression of ANT1 carrying mutations corresponding to the human adPEO ones causes a wide range of mitochondrial abnormalities. However, functional studies of ANT1 mutations in mammalian cells are lacking, because they have been hindered by the fact that ANT1 expression leads to apoptotic cell death in commonly utilized replicating cell lines. Here, we successfully express functional ANT1 in differentiated mouse myotubes, which naturally contain high levels of ANT1, without causing cell death. We demonstrate, for the first time in these disease-relevant mammalian cells, that mutant human ANT1 causes dominant mitochondrial defects characterized by decreased ADP-ATP exchange function and abnormal translocator reversal potential. These abnormalities are not due to ANT1 loss of function, because knocking down Ant1 in myotubes causes functional changes different from ANT1 mutants. Under certain physiological conditions, mitochondria consume ATP to maintain membrane potential by reversing the ADP-ATP transport. The modified properties of mutant ANT1 can be responsible for disease pathogenesis in adPEO, because exchange reversal occurring at higher than normal membrane potential can cause excessive energy depletion and nucleotide imbalance in ANT1 mutant muscle cells.

Figure 1.

Human ANT1 expression in C2C12 myotubes. (A) WT ANT1 was expressed in C2C12 myotubes (DM4) for 24 h prior to fixation and immunocytochemistry. Cells were immunostained for the HA epitope tag of recombinant ANT1 (in green) and for the COX1 subunit of the mitochondrial respiratory chain (in red). The merged image shows co-localization of these two mitochondrial IM proteins (in yellow). (B) A high-magnification merged image (colors are as in A) of a multinucleated myotube expressing WT ANT1. (C) Expression of WT ANT1 in DM4 myotubes did not cause apoptosis, as determined by negative staining with the apoptotic markers, Annexin V (in green) and propidium iodide (in red). DM4 myotubes treated with 1 μM staurosporine for 24 h caused apoptosis as shown by annexin V and propidium iodide positive staining. (D) Cell viability assessed by MTT assay in DM4 myotubes expressing exogenous WT, or mutant ANT1 (A114P or V289M), or GFP control, for 48 h, and treated with (+STS, 24 h) or without (−STS) 1 μM staurosporine (n = 4 independent experiments).

Figure 2.

Localization of human ANT1 in the inner mitochondrial membrane. (A) C2C12 myotubes (DM4) expressing ANT1 (WT, A114P and V289M) or GFP were harvested 48 h after transduction to obtain enriched mitochondrial and cytosolic fractions (C). Mitochondria were further subfractionated into mitoplasts (MP), containing IMs and matrix material, and post-mitoplast supernatant (PMS), containing IMS material and outer membranes. Equal amount of proteins from each fraction were resolved by SDS-PAGE, and immunoblotted for exogenous ANT1 with an antibody against HA (top panel). Tim23 was used as a loading control of inner mitochondrial membrane proteins. The membranes were stripped and re-probed with an ANT1 antibody to determine total levels of ANT1. Purified mitochondria from mouse heart (H) were loaded as a positive control. (B) Mitochondrial fractions were treated with alkaline solution to separate membrane-bound (M) and soluble proteins (S). ANT1-HA was exclusively found in the membrane fraction, whereas the soluble matrix protein Hsp60 was completely released in the supernatant.

Figure 3.

Mitochondrial properties of human ANT1 expressing myotubes. (A) ADP-driven state 3 respiration in permeabilized C2C12 myotubes expressing GFP, WT, A114P and V289M ANT1 (DM6). State 3 respiration rates were normalized by COX activity to adjust for differences in the mitochondrial content (n = 4). (B) Mitochondrial ATP synthesis in DM6 ANT1 expressing cells with glutamate and malate as substrates (n = 3). (C) Total cellular ATP content (n = 4). (D) The CATR : state 3 respiration ratio is shown, as a measure of the ANT1 sensitivity to CATR (0.5 μM), in permeabilized DM6 myotubes expressing GFP, WT and mutant ANT1. n = 5 for WT, A114P and GFP, n = 3 for V289M. (E) mtDNA content in DM7 myotubes expressing ANT1 for 4 days. Ratio of the mtDNA gene (COXI) to the nuclear DNA-encoded gene (SDH) relative to naive C2C12 cells was determined by real-time PCR (n = 3).

Figure 4.

ADP–ATP exchange rate/ΔΨm profile of C2C12 myotubes expressing WT and mutant ANT1. Plot of ADP–ATP exchange rate mediated by ANT versus ΔΨm in in situ mitochondria of WT, A114P and V289M C2C12 permeabilized cells depolarized to various voltages by increasing amounts of SF 6847, and normalized to specific citrate synthase activity. Each point in the graph represents the average ADP–ATP exchange rate calculated by linear regression of the magnesium green fluorescence, calibrated and converted to ATP appearing in the medium, as a function of calibrated safranine O values. E_{rev_ANT} points are indicated by arrows. Dashed box highlights the ΔΨm values prior to addition of the uncoupler SF6847, which is smaller for A114P myotubes. Each point is the average of five to seven independent experiments. *: Statistically significant by one-way ANOVA followed by Tukey's post-hoc test versus WT, P = 2.0e⁻³ (ADP), 4.0e⁻³ (10 sf), 0.043 (20 sf) and 0.045 (30 sf).

Figure 5.

Biochemical profiles of Ant1-silenced C2C12 myotubes. (A) Ant1 and Ant2 mRNA levels in Ant1-silenced (shAnt1) and scrambled control (SCR) C2C12 myotubes normalized to β-actin mRNA. *: Statistically significant by unpaired, two-tailed Student's t test, P = 0.007. (B) Western blot for Ant1 expression in shAnt1 and SCR C2C12 myotubes. Translocase of the IM (Tim23) and β-actin levels in the lysate were used as loading controls for mitochondrial and cytosolic proteins, respectively. (C) ADP–ATP exchange rate versus ΔΨm in mitochondria of Ant1-silenced C2C12 cells. Dashed box highlights the ΔΨm values prior to addition of the uncoupler SF6847, which is smaller for ANT1-silenced myotubes. *: Statistically significant by unpaired, two-tailed Student's t test, P = 0.02. Each point is the average of four independent experiments. (D) mtDNA content in Ant1-silenced and scrambled control C2C12 myotubes shows 3-fold higher mtDNA in Ant1-silenced cells (n = 5). *: Statistically significant by unpaired, two-tailed Student's t test, P= 0.034.