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.

Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells

“…As in Kawamata et al 56, ANT1 expression in shRNA-treated cells was undetectable by Western blotting (see panel Fig. 6A), and qPCR for mRNA was reduced by over 90% (not shown).…”

“…In order to evaluate the role of ANT1 on voltage-sensing properties of the mPT in a different cell context, we knocked-down its gene by stably expressing lentiviral shRNA targeted against mouse Ant1 as performed in56, and swelling of in situ mitochondria of these versus scramble RNA (scr) transfected cells were compared during Ca 2+ overload induced by addition of calcimycin, as a function of various metabolic conditions. As in Kawamata et al 56, ANT1 expression in shRNA-treated cells was undetectable by Western blotting (see panel Fig.…”

Adenine Nucleotide Translocase, Mitochondrial Stress, and Degenerative Cell Death

“…These authors evaluated the effect of Ant1 A114P and Ant1 V289M on mitochondrial function in the mouse C2C12 myotube cells [86]. Exogenous Ant1 mutant proteins were confirmed to be localized on the mitochondrial inner membrane.…”

Patent Highlights October–November 2016

“…The inventors also describe the functional role of 4qA β-satellite repeats (BSR) in facioscapulohumeral dystrophy (FSHD) [23,24]. SLC25A4 is implicated in muscle function protecting the cells from apoptosis and mitochondrial membrane instability [25,26], and is related to the hypersensitivity of FSHD muscles to oxidative stress. An individual BSR acts as an active unit participating in the transcriptional regulation of genes at locus 4q35.…”