Research ArticleCELL BIOLOGY

Inhibition of mitochondrial permeability transition by deletion of the ANT family and CypD

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Science Advances  28 Aug 2019:
Vol. 5, no. 8, eaaw4597
DOI: 10.1126/sciadv.aaw4597
  • Fig. 1 Tissue-specific expression and compensation of the ANT family.

    (A) Western blots of isolated mitochondria from various mouse tissues for ANT1, ANT2, ANT4, and a loading control for electron transport chain complex V (ComV). ANT2 protein is up-regulated in all five tissues analyzed to compensate for the loss of the Ant1 gene. ANT2 is up-regulated in testis in response to loss of the Ant4 gene. These results are representative of three independent experiments. (B) Western blots for ANT2 and ANT4 from isolated liver mitochondria from the indicated gene-targeted mice showing that ANT4 is induced for the first time in the absence of the Ant1 and Ant2 genes. A loading control for ComV is shown. These results are representative of three independent experiments. (C) Quantification of ANT4 expression normalized to ComV expression, as in (B) (n = 3; *P ≤ 0.05). (D) Western blots of ANT1, ANT2, ANT4, and a loading control for ComV, performed using extracts of isolated liver mitochondria from Ant1−/− Ant4−/− Ant2fl/fl (ANT2-only) and Ant1−/− Ant4−/− Ant2fl/fl-Alb-Cre (Ant-triple-null) mice. Data are representative of three independent experiments. (E) Quantification of ANT2 expression normalized to ComV expression, as in (D) (n = 4; *P ≤ 0.05). (F) Representative fluorescence-activated cell sorting analysis of TMRE-stained mouse liver mitochondria isolated from Ant1−/− Ant4−/− Ant2fl/fl versus Ant1−/− Ant4−/− Ant2fl/fl-Alb-Cre mice. The TMRE fluorescence threshold is shown in the bottom of the panel versus when no fluorescence signal is detected above background. FCCP is a potent uncoupler of mitochondrial respiration, which is provided to depolarize mitochondria. Results are representative of three independent experiments.

  • Fig. 2 Loss of the ANT family or CypD desensitizes the MPTP, while Ant family deletion with CsA completely inhibits MPTP opening.

    (A) Ca2+ retention capacity (CRC) assay with Calcium Green-5N fluorescence indicator in the buffer was performed using purified liver mitochondria isolated from wild-type (WT), Ant1−/− Ant2fl/fl-Alb-Cre, or Ant1−/− Ant4−/− Ant2fl/fl-Alb-Cre mice treated with or without 5 μM cyclosporine A (CsA). Two milligrams of mitochondria were used in each assay, and 40 μM pulses of CaCl2 were continually given (represented by each peak in fluorescence in the traces) until mitochondria either underwent MPTP or displayed saturated Ca2+ uptake. The arrowhead shows 40 μM alamethicin addition, a membrane-permeabilizing agent. (B) Simultaneous with the CRC assay shown in (A), light absorbance was recorded during the course of Ca2+ additions to measure mitochondrial swelling represented by a decrease in absorbance. The results shown are representative traces from at least three independent assays. AU, arbitrary units. (C) CRC assay using conditions similar to those of (A) performed on liver mitochondria isolated from WT or Ppif−/− mice treated with or without 50 μM ADP. (D) Absorbance-based mitochondria swelling was simultaneously measured in the CRC assay samples shown in (C). The results shown are representative traces from at least three independent assays.

  • Fig. 3 Genetic deletion of the Ant family and Ppif completely inhibits MPTP opening.

    (A) CRC assay performed in purified liver mitochondria isolated from Ant1−/− Ant4−/− Ant2fl/fl (ANT2-only) or Ant1−/− Ant4−/− Ppif−/− Ant2fl/fl-Alb-Cre (quadruple-null) mice. Two milligrams of mitochondria per assay were used, and 40 μM pulses of CaCl2 were continually given until mitochondrial Ca2+ uptake no longer occurred or the mitochondria underwent MPT as evidenced by irreversible increase in Calcium Green-5N fluorescence, which was saturated in the presence of 40 μM alamethicin (arrowhead). Similar results were observed across three independent assays. (B) Simultaneous with the CRC assay shown in (A), light absorbance was recorded during the course of Ca2+ additions to measure mitochondrial swelling represented by a decrease in absorbance. The results shown are representative traces from at least three independent assays. (C) Electron microscopy images of isolated liver mitochondria treated with or without 800 μM CaCl2 from Ant1−/− Ant4−/− Ant2fl/fl (ANT2-only controls), Ant1−/− Ant4−/− Ant2fl/fl-Alb-Cre (Ant-triple-null), and Ppif−/−, and Ant1−/− Ant4−/− Ppif−/− Ant2fl/fl-Alb-Cre (quadruple-null) mice. The red arrowheads show quadruple-null mitochondria that were refractory to swelling after treatment with 800 μM CaCl2. Representative images that are consistent across all fields analyzed from two independent isolations are shown.

  • Fig. 4 The ANT family is the pore-forming component of the MPTP in MEFs.

    (A) Western blots of ANT1, ANT2, ANT4, and complex II (ComII) loading control from extracts of isolated mitochondria from MEFs that are WT or Ant1−/− Ant4−/− Ant2fl/fl that have been treated with or without adenovirus-expressing Cre recombinase (AdCre will delete Ant2). (B) Oxygen consumption rate (OCR) of stable MEF lines that were Ant1−/− Ant4−/− Ant2fl/fl (ANT2-only) versus Ant1−/− Ant4−/− Ant2fl/fl(AdCre) (Ant-triple-null) (n = 5). Respiration was challenged with sequential treatment of 2 μM oligomycin (Oligo), 2 μM FCCP, and 0.5 μM rotenone (Rote) (n = 5). (C) Confocal microscopic images of TMRE staining of the same MEF lines described in (B). Similar profiles of TMRE fluorescence were observed in three independent experiments. (D) Cell death analysis of the Ant-triple-null MEFs versus the Ant1−/− Ant4−/− Ant2fl/fl (ANT2-only) controls, subjected to glucose-containing (normal) media or glucose-free, galactose-containing media for 24 hours. Cell death was determined by loss of plasma membrane integrity (n = 3; *P ≤ 0.05). (E) Representative patch clamp current traces of mitoplasts isolated from the MEFs described in (A) in the presence of 250 μM CaCl2. The high conductance openings in the WT and ANT2-only lines were sensitive to the addition of 1 mM ADP/Mg2+ (right). (F) Scatterplot of the permeability in nanosiemens (nS) from all recorded patches. The number of patches analyzed is represented by the data plots (circles). The horizontal lines indicate average permeability. Solid circles represent channels that were either sensitive (gray) or insensitive (black) to the addition of 1 mM ADP/MgCl2 [WT versus Ant1−/− Ant4−/− Ant2fl/fl(AdCre), P = 0.069]. Open circles represent ADP response untested. (G) Bar histograms of average (±SD) change in permeability after bath perfusion with 1 mM ADP/MgCl2 [WT, n = 13; Ant1−/− Ant4−/− Ant2fl/fl, n = 12; Ant1−/− Ant4−/− Ant2fl/fl(AdCre), n = 8; *P ≤ 0.05].

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaaw4597/DC1

    Fig. S1. Loss of Ant1/2/4 genes in the liver does not compromise mitochondrial energetics or cause tissue pathology.

    Fig. S2. Adenine nucleotide levels and transport in liver and MEF mitochondria lacking ANT proteins.

    Fig. S3. Mitochondria from quadruple null mice fail to undergo Ca2+-induced MPTP, although no defects in energetics or mitochondrial ultrastructure were observed.

    Fig. S4. Model of the MPTP.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Loss of Ant1/2/4 genes in the liver does not compromise mitochondrial energetics or cause tissue pathology.
    • Fig. S2. Adenine nucleotide levels and transport in liver and MEF mitochondria lacking ANT proteins.
    • Fig. S3. Mitochondria from quadruple null mice fail to undergo Ca2+-induced MPTP, although no defects in energetics or mitochondrial ultrastructure were observed.
    • Fig. S4. Model of the MPTP.

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