Research ArticleHEALTH AND MEDICINE

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing

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Science Advances  15 Apr 2020:
Vol. 6, no. 16, eaba0694
DOI: 10.1126/sciadv.aba0694
  • Fig. 1 Acute HPV is absent in AOX-expressing isolated murine lungs.

    (A) AOX protein expression detected as brownish color in bronchial walls (*) and pulmonary arteries (arrows). (B) PAP response of isolated, buffer-perfused WT and AOX murine lungs ventilated with 1% O2 for time as indicated. Data are shown as means ± SEM of n = 9 experiments. Gray area indicates significant difference with P < 0.05 tested by multiple t tests. (C) PAP response to hypoxic (HOX; 1% O2) challenge with and without AOX inhibitor nPG applied 5 min before sequential HOX. Data are shown as means ± SEM of n = 4 experiments. *P < 0.05, ***P < 0.001 for comparison as indicated, analyzed by two-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test. (D) PAP response to pulmonary artery infusion of the thromboxane mimetic U46619. Data are shown as means ± SEM of n = 6 experiments. (E) Kfc after 90 min of ischemia. Data are shown as means ± SEM of n = 3 experiments. (F) Lung weight gain (retention) during reperfusion after 90 min of ischemia. Data are shown as means ± SEM of n = 3 experiments.

  • Fig. 2 Hypoxia-induced cellular membrane depolarization is decreased in AOX-expressing PASMC.

    (A and B) Representative traces of patch clamp measurements to determine cellular membrane potential (MP) during acute HOX (1% O2) in mouse WT (A) and AOX (B) PASMC. Gray traces depict oxygen concentration in %; blue (WT) and red (AOX) traces indicate MP in millivolts. Addition of AOX inhibitor nPG as indicated. Cellular MP in mouse WT (C) and AOX (D) PASMC during normoxia (NOX) and acute HOX or acute HOX plus nPG. (E) Change of cellular MP compared with NOX in the absence and presence of nPG as indicated. Data of (C) to (E) shown as means ± SEM of n = 6 experiments. Horizontal bars indicate significant difference with P < 0.05 analyzed by repeated-measures one-way ANOVA and Tukey’s multiple comparisons test. (F) Vasoconstriction of isolated pulmonary arteries during superfusion with hypoxic (1% O2) or normoxic KCl-free buffer shown as % of response to 80 mM KCl. Data are shown as means ± SEM of n = 8 experiments. *P < 0.05 WT NOX versus WT HOX; #P < 0.05 WT HOX versus AOX HOX analyzed by two-way ANOVA and Tukey’s multiple comparisons test. (G) Vasoconstriction as in (F) but in the presence of ~20 mM KCl. Data are shown as means ± SEM of n = 8 experiments. *P < 0.05 WT NOX versus WT HOX; #P < 0.05 WT NOX versus AOX HOX analyzed by two-way ANOVA and Tukey’s multiple comparisons test. (H) PAP response of isolated WT and AOX lungs during HOX (10% O2) ventilation before and after infusion of 20 mM KCl. Data are shown as means ± SEM of n = 3 experiments. Horizontal bars indicated significant difference with P < 0.05 analyzed by two-way ANOVA and Sidak’s multiple comparisons test.

  • Fig. 3 AOX inhibits hypoxia-induced superoxide release and mitochondrial membrane hyperpolarization in PASMC and affects the redox state of mitochondrial biomarkers.

    (A) Representative ESR spectra from mouse WT and AOX PASMC using the probe CMH and pSOD for control. (B) Superoxide production in mouse WT and AOX PASMC during exposure to normoxia (NOX) and hypoxia (HOX, 1% O2) for 5 min. A.U., arbitrary units. Data are shown as means ± SEM of n = 4 experiments. The horizontal bar indicates significant difference with P < 0.05 analyzed by two-way ANOVA and Sidak’s multiple comparisons test. (C) Superoxide production in WT mouse PASMC transfected with plasmids encoding native Ciona AOX or a catalytically inactive mutant AOX exposed to NOX and HOX. Data are shown as means ± SEM of n = 12 experiments. The horizontal bar indicates significant difference with P < 0.05 analyzed by two-way ANOVA and Sidak’s multiple comparisons test. No significant differences were detected between the genotypes. Data in (B) and (C) are depicted as the portion of CMH signal inhibited by pSOD. (D) Representative mouse WT and AOX PASMC stained with JC-1 under normoxia and hypoxia (1% O2) as indicated. (E) Mitochondrial MP (Mito MP) determined as JC-1 red/green ratio under acute HOX as indicated. Data are shown as means ± SEM of n = 10 (WT) and n = 12 (AOX) experiments. Gray area depicts significant difference with P < 0.05 analyzed by two-way ANOVA and uncorrected Fisher’s least significant difference (LSD). (F) Oxygen-dependent respiratory rate of 100,000 to 300,000 intact rPASMC per experiment transfected with an empty vector or AOX encoding plasmid. Data are shown as means ± SEM in % of rate at normoxia (max) with P < 0.0001 indicating significant difference between WT and AOX analyzed by paired t test. (G) Normalized fluorescence-corrected Savitzky-Golay reconstructed Raman spectra taken from WT (n = 4) and AOX (n = 6) PASMC before (black line = NOX) and after (gray line) exposure to acute hypoxia (HOX, 5% O2). Background spectrum from microfluidic system and buffer solution is shown as dashed line (n = 25). Differences between NOX and HOX are highlighted with a difference spectrum for each corresponding sample type (WT, blue; AOX, red). Peak locations for mitochondrial biomarkers NAD+ (3: 1033 cm−1), NADH (2: 1000 cm−1), ubiquinol (QH2; 5: 1167 cm−1), and cytochrome b (Cyt b; 7: 1337 cm−1) as well as peaks associated with reduced (1: 750 cm−1; 4: 1127 cm−1; 6: 1313 cm−1; 9: 1505 cm−1) and oxidized forms of Cyt c (8: 1369 cm−1; 10: 1638 cm−1) are shown as vertical dashed lines. (H) Raman intensity for individual biomarkers are shown as means ± SEM of n ≥ 3 experiments. ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.001 analyzed by two-way ANOVA and uncorrected Fisher’s LSD.

  • Fig. 4 Adaptation processes upon chronic hypoxia in WT and AOX transgenic mice.

    (A) Right ventricular systolic pressure (RVSP) after normoxia (NOX) or hypoxia (HOX, 10% O2) for 28 days. Data are shown as means ± SEM of n ≥ 9 experiments. Horizontal bars indicate significant difference with P < 0.05 analyzed by two-way ANOVA and Tukey’s multiple comparisons test. (B) Ratios of right ventricle (RV) and left ventricle (LV) plus septum (S) (Fulton index). Data are shown as means ± SEM of n = 10 experiments. Horizontal bars indicate significant difference with P < 0.05 analyzed by two-way ANOVA and Tukey’s multiple comparisons test. (C) Cardiac output (CO) measured by echocardiography. Data are shown as means ± SEM of n = 10 experiments. Horizontal bars indicate significant difference with P < 0.05 analyzed by two-way ANOVA and Tukey’s multiple comparisons test. (D) Vascular remodeling quantified as degree of muscularization of small (20- to 70-μm diameter) pulmonary arterial vessels. Vessel muscularization categorized as non, partial, or full after immunostaining against α-smooth muscle actin as marker for PASMC and von Willebrand factor to discard endothelium. Data are shown as means ± SEM of n ≥ 4 experiments. ****P < 0.0001 NOX versus HOX analyzed by two-way ANOVA and Tukey’s multiple comparisons test. Note, no difference observed between WT versus AOX. (E) Western blots showing HIF-1α and β-actin expression in WT and AOX transgenic PASMC under normoxia and hypoxia. (F) Quantification of protein expression shown in (E). Data are shown as means ± SEM of n = 3 experiments. Horizontal bars indicate significant difference with P < 0.05 analyzed by two-way ANOVA and Sidak’s multiple comparisons test. (G) Proliferation assay of WT and AOX PASMC cultured under normoxia (NOX) or hypoxia (HOX, 1% O2) for 48 hours. Data are shown as means ± SEM of n = 9 experiments. Horizontal bars indicate significant difference with P < 0.05 analyzed by two-way ANOVA and Tukey’s multiple comparisons test. BrdU, bromodeoxyuridine.

  • Fig. 5 Integration of present findings into current concepts of oxygen sensing and HPV in the murine lung.

    Acute HPV depends on a central oxygen sensor within the mitochondrial respiratory chain. Hypoxia induces mitochondrial membrane hyperpolarization, an increase in mitochondrial superoxide release, and subsequent inhibition of cellular potassium channels (KV), which leads to cellular membrane depolarization and activation of voltage-gated calcium channels. This sequence of events results in intracellular calcium increase and HPV. AOX prevents HPV in mouse PASMC by preventing mitochondrial ROS production and release. This places mitochondria and electron flux through the mitochondrial respiratory chain at the top level in the hierarchy of oxygen sensing and signaling controlling HPV. ADP, adenosine diphosphate; FAD, flavin adenine dinucleotide.

  • Table 1 List of reagents and resources.

    ReagentsSourceIdentifier
    Antibodies
    AOX antiserum (custom raised in rabbit)21st Century Biochemicals, Marlborough, MA, USA(42)
    HIF-1α (C-Term) polyclonal antibodyCayman Chemical10006421
    Anti-beta actin antibody [mAbcam 8226]—loading
    control (HRP)
    Abcamab20272
    Anti-rabbit IgG (H + L), HRP conjugatePromegaW4011
    Anti-mouse IgG (H + L), HRP conjugatePromegaW4021
    Lentiviral particles
    Lentiviral Ciona intestinalis AOXThis paperN/A
    Lentiviral catalytic inactive (mutant) AOXThis paper(20)
    Chemicals
    n-Propyl gallate (nPG, AOX inhibitor)Sigma-AldrichP3130
    U46619 (prostaglandin H2/thromboxane A2
    receptor agonist)
    Enzo Life Sciences, Exeter, UKPG-023
    Sodium chloride (NaCl)Sigma-Aldrich31434
    Potassium chloride (KCl)Carl Roth, Karlsruhe, Germany5346.1
    Calcium chloride (CaCl2)Carl RothCN93.1
    Magnesium chloride (MgCl2)Carl RothKK36.1
    Sodium dihydrogen phosphate dihydrate
    (NaH2PO4·2H2O)
    Merck Millipore, Darmstadt, Germany1063421000
    Sodium hydrogen carbonate (NaHCO3)Carl Roth8551
    d(+)-GlucoseCarl RothX997.1
    l-Aspartic acid potassium salt (l-aspartate)Sigma-AldrichA6558
    Adenosine 5′-triphosphate magnesium salt, ATPSigma-AldrichA9187
    Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-
    tetraacetic acid (EGTA)
    Sigma-Aldrich03777
    Hepes (PUFFERAN®)Carl Roth9105.3
    Potassium hydroxide (KOH)Carl Roth6751
    1-Hydroxy-3-methoxycarbonyl
    −2,2,5,5-tetramethylpyrrolidine (CMH spin
    probe)
    Noxygen, Elzach, GermanyNOX-02.2
    Superoxide dismutase–polyethylene glycol (pSOD,
    PEG-SOD)
    Sigma-AldrichS9549
    5,5′,6,6’-Tetrachloro-1,1′,3,3′-tetraethyl-
    imidacarbocyanine iodide (JC-1)
    ThermoFisher ScientificT3168
    Penicillin-streptomycinGibco, ThermoFisher Scientific1507–063
    Normocin—antimicrobial reagentInvivoGen, San Diego, USAant-nr-1
    Assays
    ZytoChem Plus (HRP) Polymer Bulk KitZytomed Systems GmbH, Berlin, GermanyPOLHRP-100
    VECTOR NovaRED Peroxidase (HRP) Substrate KitVector Laboratories, Burlingame, USASK-4800
    Enhanced chemiluminescence ECL Prime Western
    Blotting System
    GE HealthcareRPN2232
    Cell Proliferation ELISA, BrdU (colorimetric) assaySigma-Aldrich11647229001
    Primary cells
    Mouse PASMC from precapillary pulmonary arterial
    vessels
    (30)N/A
    Rat PASMC from precapillary pulmonary arterial
    vessels
    (45)N/A
    Rodent models
    Mouse: Rosa26-Aox (aka AoxRosa26)(10)N/A
    Mouse: C57BL/6JCharles River, Sulzfeld, GermanyStrain Code 632
    Rat: Sprague-Dawley (Crl:SD)Charles RiverStrain Code 400
    Plasmid and vectors
    pWPXL (constitutive lentiviral plasmid)Addgene, Boston, MA, USAPlasmid #12257
    pMD2.G (envelope vector)AddgenePlasmid #12259
    psPAX2 (packing vector)AddgenePlasmid #12260
    Software and algorithms
    Patchmaster softwareHEKA, Lambrecht, Germanyv2x90
    IGOR Pro softwareWavemetrics, Lake Oswego, OR, USAversion 6.37
    DatLab softwareOroboros Instruments, Innsbruck, AustriaDatLab 5
    Prism softwareGraphPad Software; San Diego, CA, USAversion 8.2.0
    Other
    Multi Wire Myograph SystemDanish Myo Technology A/S, Aarhus, Denmark620 M
    N2-balanced gas (21% O2, 5% CO2)Praxair, Danbury, CT, USA592091 (custom made)
    N2-balanced gas (1% O2, 5% CO2)Praxair650329 (custom made)
    Optical Oxygen Meter (FireStingO2)Pyro Science, Aachen, GermanyFSO2–2
    Culture dishesGreiner Bio-One, Frickenhausen, Germany627860
    Multi-line in-line solution heaterWarner Instruments, Hamden, CT, USASHM-8
    Borosilicate glass capillary tubesSutter Instrument, Novato, CA, USABF150–86-10HP
    DMZ universal electrode pullerZeitz, Martinsried, GermanyN/A
    EPC 10 USB patch clamp amplifierHEKA895000
    EMXmicro ESR spectrometerBruker Biospin GmbH, Rheinstetten, GermanyN/A
    OROBOROS-2k oxygraphOroboros InstrumentsN/A
    Media 199—Mammalian Cell CultureGibco, ThermoFisher Scientific31150–022
    Vevo2100 high-resolution imaging system
    equipped with a 40-MHz transducer
    VisualSonics, Toronto, CanadaN/A
    Raman spectrometer Shamrock 303iAndor, Technology, Belfast, UKSR-303i-A
    LaserAltechna, Vilnius, LithuaniaDPSS 532
    Raman filter cube with dichroic mirrorSemrock, Rochester, NY, USAzt532/640rpc
    532-nm EdgeBasic best-value long-pass edge filterSemrockBLP01-532R-25
    ×40 magnification objectiveOlympus, Tokyo, JapanLUCPLFLN

Supplementary Materials

  • Supplementary Materials

    Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensings

    Natascha Sommer, Nasim Alebrahimdehkordi, Oleg Pak, Fenja Knoepp, Ievgen Strielkov, Susan Scheibe, Eric Dufour, Ana Andjelković, Akylbek Sydykov, Alireza Saraji, Aleksandar Petrovic, Karin Quanz, Matthias Hecker, Manish Kumar, Joel Wahl, Simone Kraut, Werner Seeger, Ralph T. Schermuly, Hossein A. Ghofrani, Kerstin Ramser, Thomas Braun, Howard T. Jacobs, Norbert Weissmann, Marten Szibor

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