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Electron leak from NDUFA13 within mitochondrial complex I attenuates ischemia-reperfusion injury via dimerized STAT3

  1. Jian’an Wanga,2
  1. aCardiovascular Key Laboratory of Zhejiang Province, Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, Zhejiang Province, China;
  2. bClinical Research Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China;
  3. cInstitute of Translational Medicine, Zhejiang University, Hangzhou 310029, China
  1. Edited by J. G. Seidman, Harvard Medical School, Boston, MA, and approved September 1, 2017 (received for review March 23, 2017)

  1. Fig. S1.

    (A) TMRM was used to measure mitochondrial membrane potential for H9C2 cells that were transfected with different concentrations of siRNA targeting rat NDUFA13 as described in Fig. 1A. These siRNA-NDUFA13–treated H9C2 cells then exposed to either a normoxia culture condition or a H/R indult. (B) The cells were collected for the quantification of p-ASK1 at Thr845, or p-JNK levels by Western blotting where β-actin was used as a loading control (n = 3 mice per group).

  2. Fig. 2.

    Three groups of mice, including cHet (Cre+flox/-), cHomo (Cre+flox/flox), and CON (Cre-flox/-), were studied. (A) The time course of changes in NDUFA13 expression in the heart were evaluated at the indicated time points by Western blot (representative bands shown with its expression level relative to that in Cre-flox/- mice. β-Actin as a loading control, n = 3 mice per group). (B) ATP levels (micromoles per liter) quantified at the indicated time points (*P < 0.05; #P < 0.05 vs. Cre-flox/- group, respectively); (C) Kaplan–Meier survival curves were generated for the three groups of mice over a prospective observation period of 240 d (n = 10 mice per group. *P < 0.05 vs. Cre-flox6/- group). “Injection” indicates the date of the completion of tamoxifen administration. (D) Echocardiography performed for both cHet and CON mice on day 28 after tamoxifen administration; ejection fraction (EF) and LVIDd quantified in the bar graphs (n = 9 mice per group). (E) TEM performed on samples from Cre+flox/- and Cre-flox/- mice on day 28 after tamoxifen administration. (Scale bars: Left, 5 μm; Center, 2 μm; Right, 0.5 μm.) (F) Different components of the mitochondrial respiratory complexes were quantified by Western blot in both group mice with representative bands shown (n = 3 per group). NDUFA13 expression levels were quantified in other tissue with β-actin as a loading control (n = 3 mice per group). (G) OCR was measured by O2k-Fluorometry (*P < 0.05 vs. Cre-flox/- mice; #P < 0.05, comparison within Cre-flox/- group mice).

  3. Fig. S2.

    (A) Both cHet (Cre+flox/-) and CON (Cre-flox/-) mice underwent echocardiographic examination at day 28 after tamoxifen therapy. Parasternal long axis view was shown and M-mode image acquired for assessing cardiac size and function (n = 9 mice per group). (B) Bar graphs for the densitometric quantification for all of the protein bands detected by Western blotting as described in Fig. 2B (n ≥ 3 mice per group). (C) Representative traces of oxygen consumption rate measured by O2k-Fluorometry (*P < 0.05 vs. Cre-flox/- mice). cHet (Cre+flox/-) denotes Myh6Cre+NDUFA13flox/- mice; CON (Cre-flox/-) denotes Myh6Cre?NDUFA13flox/- mice.

  4. Fig. 3.

    (A) Both cHet (Cre+flox/-) and CON (Cre-flox/-) mice underwent either the I/R injury or the sham operation. IS was analyzed for both groups of mice with a representative 2,3,5-triphenyltetrazolium chloride (TTC) staining image and quantified in bar graph (n = 5 mice per group. **P < 0.01 vs. Cre-flox/- mice). (B) TUNEL staining performed (white arrow indicates TUNEL-positive nucleus) and the quantification is shown in the bar graph (**P < 0.01 vs. Cre-flox/- group). (C) Cleaved caspase-3 was also detected by Western blot with representative bands (α-tubulin was used as a loading control; **P < 0.01 vs. Cre-flox/- I/R group, n = 3 mice per group). (D) Cytochrome c release was quantified by Western blot; VDAC, a mitochondrial marker was used as a quality control for the cytosol isolation, and α-tubulin used as a cytosol loading control (n = 3 mice per group. *P < 0.05 vs. Cre-flox/- I/R mice).

  5. Fig. 4.

    (A) H2O2 measured in freshly isolated mitochondria from Cre+ERtamNDUFA13flox/- or Cre-ERtamNDUFA13flox/- mice using Amplex Red as an indicator of fluorescence. Different substrates and blockers were used to differentiate the origin or mechanism of ROS generation induced by NDUFA13 down-regulation (*P < 0.05). (B) NMCMs isolated from NDUFA13flox/- mice were treated with either Ad-NC or Ad-Cre and then used for quantification of superoxide generation at both the basal state and after an exposure to the H/R injury. The fluorescence intensity by mitoSOX red was measured by a microplate reader (**P < 0.01, vs. Ad-NC–treated NMCMs exposed to H/R). (C) The same NMCMs used in B were infected with adenovirus containing either cyto-HyPer or mito-HyPer to measure H2O2 levels either at the basal state or after the H/R insult (**P < 0.01, vs. Ad-NC–treated NMCMs at the same condition).

  6. Fig. S3.

    (A) Mitochondria were freshly isolated from cHet (Cre+flox/-) and CON (Cre-flox/-) mice as shown in Fig. 3. H2O2 levels and OCR were simultaneously monitored for later analysis when different substrates and blockers for the mitochondrial respiratory complexes. Representative tracings were shown for both group mice (n = 3 mice per group). (B) Efficiency of infection for Ad-Cre or Ad-NC was tested in NMCMs that were obtained from NDUFA13flox/- mice by Western blotting to detect the expression levels of NDUFA13. (C) Representative imaging for NMCMs that were infected with adenovirus containing either cyto-HyPer or mito-HyPer, which can target the H2O2 in the cytosol and mitochondrion, respectively. (D) Fluorescence by mitoSOX red for measuring superoxide was also read at setting of excitation = 408 nm/emission = 560 nm (**P < 0.05 vs. Ad-NC–treated NMCMs after a H/R exposure).

  7. Fig. S4.

    (A) Schematic illustration of various truncated NDUFA13 mutants with a deletion of different segment of NDUFA13. Adenoviruses containing various HA-tagged truncated NDUFA13 mutants, including Ad-1 (a segment for amino acid 40–50 deleted), Ad-2 (a segment for amino acid 70–80 deleted), and Ad-3 (a segment for amino acid 110–120 deleted), wild-type full-length NDUFA13 (Ad-NDUFA13) as normal control, and an empty vector as a negative control (Ad-Vector) were designed and constructed. To test the role for each segment of NDUFA13 in maintaining MMP, NMCMs were isolated from NDUFA13flox/flox mice and then infected with either Ad-Cre to deplete the endogenous NDUFA13 or Ad-NC as controls. (B) The efficiency of Ad-Cre was confirmed by Western blot. (C) TMRM staining was then performed for these NMCMs with representative images, demonstrating that the depletion of endogenous NDUFA13 in NMCMs resulted in a loss of mitochondrial membrane potential, which was then confirmed by the flow cytometry (D). (E) After NDUFA13-depleted NMCMs were infected with adenovirus containing different mutants as described above, the efficiency for the infection was also confirmed by measuring the levels of NDUFA13. (F) Immunofluorescence imaging showed subcellular colocalization of the mitochondrial component TOMM20 (green) with the truncated NDUFA13 mutant protein (detected by HA in red; DAPI-stained nuclei in blue); the colocalization was only absent when Ad-1 was put back into NDUFA13-depleted NMCMs, indicating the importance of the α-helix structure of NDUFA13 for this molecule attached to the mitochondria. (G) The membrane potential was also tested in knockdown and restored NMCMs by flow cytometry using TMRM, further confirming that only Ad-1 failed to recapitulate a normal mitochondrial membrane potential. (H) The NMCMs obtained from NDUFA13flox/flox mice were treated with Ad-Cre (Ad-NC as a control) to deplete endogenous NDUFA13, exogenous wild-type NDUFA13, and truncated NDUFA13 mutants including Ad-1, Ad-2, and Ad-3 were then put back via adenovirus transfection the same way as shown above, with Ad-Vector and DMEM as controls. These cells were finally measured for cytoplasmic H2O2 levels using the Cyto-Hyper method as described above. *P < 0.05 vs. Ad-NC; #P < 0.05, vs. Ad-Cre treated with DMEM.

  8. Fig. 5.

    NB-PAGE assay detected STAT3 oligomerization in heterozygous mice (Myh6Cre+NDUFA13flox/-STAT3WT) that could be abolished by either NAC (ROS scavenger) or simultaneous STAT3 knockdown (Myh6Cre+NDUFA13flox/-STAT3flox/-). NDUFA13, STAT3, GPX, PRX2, and Bcl2 expression levels were also quantified in each group of mice by Western blot, with β-actin as a loading control. **P < 0.01 vs. Myh6Cre+NDUFA13WTSTAT3WT mice.

  9. Fig. S5.

    NB-PAGE assay detected Stat3 oligomerization in heart tissue obtained from cardiac-specific STAT3 heterozygous (Myh6Cre+STAT3-lox/-) and control (Myh6Cre+STAT3WT) mice that were treated with tamoxifen. STAT3 expression level was significantly down-regulated in STAT3 heterozygous knockout mice, but no significant differences in polymerized or dimerized STAT3 were detected, the same was true for PRX and Bcl2 expression levels measured by Western blot.

  10. Fig. 6.

    (A) The NDUFA13 heterozygous (Myh6Cre+NDUFA13flox/-STAT3WT) and NDUFA13 and STAT3 double heterozygous (Myh6Cre+NDUFA13flox/-STAT3flox/-) mice were studied the same way as described in Fig. 3 and IS quantified (**P < 0.01 between the two groups). (B) TUNEL staining performed in the peri-infarct area and the percentage of TUNEL-positive cells (marked by white arrows) over total nuclei shown in the bar graph (n = 5 mice per group; **P < 0.01 vs. NDUFA13 heterozygous I/R group). (C) STAT3 and cleaved caspase-3 expression levels measured by Western blot using the tissue of peri-infarct area from both group mice with α-tubulin as a loading control (n = 3 mice per group).

  11. Fig. S6.

    Cardiac-specific STAT3 heterozygous (Myh6Cre+STAT3-lox/-, Cre+STAT3flox/-) and control (Myh6Cre+STAT3WT, Cre+STAT3WT) mice, that had been treated with tamoxifen, experienced an I/R insult or underwent a sham-operated procedure as described above. (A) The IS was then measured the same way as described above for the two group mice. (B) TUNEL staining was performed in the peri-infarct area, and the percentage of TUNEL-positive cells (marked by white arrows) over total nuclei is shown in the bar graph (n = 5 mice per group). (C) Cleaved caspase-3 and STAT3 expression levels were measured by Western blot in both groups, and β-actin was used as a loading control (n = 3 mice per group).

  12. Fig. S7.

    Schematic mechanisms of NDUFA13, demonstrating the major components of the electron transfer chain in complex I and the location where NDUFA13 is anchored. In wild-type mice (the left CI of ETC), the usual response to I/R injury involves a significant amount of superoxide production, leading to cell damage. In mice with a moderate loss of NDUFA13 (moderate knockdown, shown on the right CI), a decent amount of electron leak occurs. Due to the specific location of NDUFA13, where a lower electrochemical potential exists at segments of complex I, a moderate amount of hydroxyl peroxide is generated in the basal state. This, in turn, activates PRX2 and results in STAT3 dimerization. Of note, there was no change in GPX expression. Based on the database (PDB ID code: 5LDX), the first 33 aa of NDUFA13 extend along the dorsal side of the CoQ binding chamber after penetrating the inner membrane and are parallel to the last three FeS clusters (N2, N6b, and N6a), being ~31 ? apart. The enlarged tail of NDUFA13 remains on the intermembrane side of ND1 and ND2.

  13. Fig. S8.

    (A) A vector was generated where the exon 3 of NDUFA13 was flanked by one pair of loxPs, with one site loxP following a Frt-Pgk-Neo-polyA-Frt cassette. The linearized targeting vector was then introduced into SCR012 ES cells by electroporation, and G418-resistant clones were screened for homologous recombination. Targeted ES clones were microinjected into ICR eight-cell stage embryos and transferred into pseudo pregnant ICR females. The resulting chimeras were bred with C57BL/6 mice, and heterozygous offsprings were mated with FLP mice to get neo-free NDUFA13flox/- mice. (B) PCR analysis of the genomic DNA derived from mouse tail for Myh6-CreERtam, NDUFA13, and STAT3.

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