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. 2020 Jun 3;39(14):e103812. doi: 10.15252/embj.2019103812

Figure 5. Hindering the ATP synthase activity feeds back to OXPHOS dysfunction by inhibiting CII and generating a SDHA‐dependent burst in ROS.

Figure 5

  • A
    Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD (wt, n = 4; LowOXPHOS, n = 4; wt + HFD, n = 4; LowOXPHOS + HFD, n = 4).
  • B
    Polarographic profiles of isolated mitochondria from wt (lower trace) and LowOXPHOS (upper trace) animals using succinate (left graph) or palmitoyl‐carnitine (right graph) as a substrate. Quantification of maximal respiration in the right histogram. Bars are the mean ± SEM of n = 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A.
  • C–E
    2D‐PAGE of Skm extracts (C, D) and acetylated proteins (E) from wt (n = 4) and LowOXPHOS (n = 3) mice. The isoelectric point (pI) of the CII subunits SDHB (C) and SDHA (D) was calculated by protein migration in pH 3–10 NL strips.
  • F
    Immunocapture (IP) of SDHA blotted with anti‐acetyl‐K antibody (upper panel) and of acetylated proteins blotted with anti‐SDHA antibody (middle panel) in Skm isolated mitochondria from wt (n = 3) and LowOXPHOS (n = 3) mice.
  • G, H
    PyMOL cartoon representations of acetylated lysines in SDHA related to CII activity. Six acetylated‐K are shown.
  • I
    MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each ETC inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± SEM of n = 3 experiments, 12 replicas/condition.
  • J
    MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each CII inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± SEM of n = 3 experiments, 12 replicas/condition.
  • K
    Representative blue‐native immunoblots (BN) and clear‐native in‐gel activity (CN) of Skm mitochondrial membrane proteins from wt (n = 3) and LowOXPHOS (n = 3) 2‐month‐old mice. The migration of the respiratory complexes/supercomplexes CIV is indicated. Subassembly of CI (with no activity, suggesting degradation) was observed in LowOXPHOS mice. VDAC is shown as a loading control.
Data information: *,# P < 0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t‐test. See also Fig EV5.Source data are available online for this figure.