Research ArticleCardiovascular Biology

Depolysulfidation of Drp1 induced by low-dose methylmercury exposure increases cardiac vulnerability to hemodynamic overload

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Science Signaling  25 Jun 2019:
Vol. 12, Issue 587, eaaw1920
DOI: 10.1126/scisignal.aaw1920
  • Fig. 1 Exposure to subneurotoxic dose of MeHg promotes maladaptive cardiac hypertrophy induced by pressure overload.

    (A and B) Changes in body weight (A) and survival rate (B) in mice after TAC. Mice were orally given MeHg (10 ppm, in drinking water) for 1 week before TAC (n = 5 to 11 mice per treatment). (C) The mercury concentrations in the indicated organs from mice given drinking water (vehicle) or MeHg-containing water (MeHg) for 1 week (n = 4 to 8 mice per treatment). (D) Representative images of mouse hearts 1 week after TAC (n = 4 to 6 mice per treatment). Scale bars, 1 μm. (E to G) Heart weight (HW)/body weight (BW) (E), HW/tibia length (TL) (F), and lung weight (LW)/TL (G) in mice 1 week after TAC (n = 4 to 6 mice per treatment). (H) Myocardial cell size in mice 1 week after TAC. Left: Representative images of wheat germ agglutinin staining of LV sections. Scale bars, 200 μm. Right: Relative changes in the average cross-sectional area (CSA) of cardiomyocytes in LV myocardium (n = 3 mice per treatment). Data are shown as means ± SEM. *P < 0.05 and **P < 0.01, one-way analysis of variance (ANOVA) (E to H).

  • Fig. 2 MeHg induces Drp1 activation and mitochondrial fission in cardiomyocytes.

    (A) Representative electron micrographs of LV myocardium in mice 1 week after MeHg exposure (n = 3 mice per treatment). Scale bars, 1 μm. (B) GTP-binding activity of Drp1 in mouse hearts 4 weeks after TAC (n = 5 mice per treatment). (C) Representative images of mitochondrial morphology in NRCMs treated with MeHg (0.03 μM) for 3 days. Right: Mitochondrial morphologies of NRCMs classified into three groups (vesicle, intermediate, and tubule) (n = 3 independent experiments). Scale bars, 10 μm. DMSO, dimethyl sulfoxide. (D) Dose-dependent effect of MeHg on cytotoxicity (circles) and mitochondrial fission (triangles) in NRCMs (n = 3 independent experiments). LDH, lactate dehydrogenase. (E) Effect of Drp1 knockdown on MeHg-induced mitochondrial fission. NRCMs were transfected with negative control siRNA (siNC) or siRNA for Drp1 (n = 3 independent experiments). Right: Percentage of cells with vesicle-type mitochondria. Scale bars, 10 μm. (F) Colocalization of mitochondria and Drp1 in NRCMs treated with MeHg. Green, mitochondria; red, endogenous Drp1; blue, 4′,6-diamidino-2-phenylindole (DAPI) (n = 3 independent experiments). Scale bars, 5 μm. Data are shown as means ± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA (B, C, and E). ATP, adenosine 5′-triphosphate.

  • Fig. 3 MeHg increases Drp1 activity through depolysulfidation of Drp1 proteins.

    (A) Intracellular sulfane sulfur levels in NRCMs. NRCMs pretreated with MeHg (0.5 μM) for 14 hours, NaHS (100 μM) for 14 hours, or Na2S4 (10 μM) for 3 hours were incubated with SSP4 (n = 4 independent experiments). (B) Effect of MeHg on the polysulfidation (polyS) of endogenous Drp1 proteins in the presence or absence of NaHS (n = 3 independent experiments). (C) Effect of MeHg with or without NaHS on the GTP-binding activity of Drp1 (n = 3 independent experiments). (D) Effect of MeHg on Drp1 polysulfidation in mouse hearts 1 week after TAC (n = 4 mice per treatment). Data are shown as means ± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA.

  • Fig. 4 Administration of NaHS rescues MeHg-mediated Drp1 activation and cardiac dysfunction.

    (A and B) Effect of NaHS on Drp1 activation (A) and polysulfidation (B) in MeHg-exposed mouse hearts. Bands corresponding to Drp1 (arrowheads) were quantified (n = 5 mice per treatment). (C) Quantification of GTP-Drp1 (A) and polyS-Drp1 (B). (D) Representative images of echocardiographs from mice 1 week after TAC (n = 5 to 10 mice per treatment). Scale bars, 2 μm. (E to G) Effect of NaHS on ejection fraction (EF) (E), fractional shortening (FS) (F), and LV internal diameters at end-systole (LVIDs) (G) in vehicle-, MeHg-, and NaHS-exposed mice 1 week after TAC (n = 5 to 10 mice per treatment). (H) TUNEL staining to detect apoptosis in myocardium 2 weeks after TAC. Red, TUNEL-positive nuclei; blue, DAPI-stained nuclei. The population of TUNEL-positive apoptotic cells (arrowheads) was quantified (n = 5 mice per treatment). Scale bars, 50 μm. Data are shown as means ± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA (C and E to H).

  • Fig. 5 Polysulfidation of Cys624 negatively regulates Drp1 activity.

    (A) Effect of Drp1 overexpression on mitochondrial fission. HeLa cells overexpressed Drp1 WT or the depolysulfidation mimic CS mutants, and mitochondrial morphologies were visualized (n = 3 independent experiments). Right: Percentage of cells with tubule-type mitochondria. Scale bars, 20 μm. GFP, green fluorescent protein. (B) Drp1 activity in response to MeHg in Drp1 (WT)– or Drp1 (C624S)–overexpressing HeLa cells (n = 3 independent experiments). (C) Effect of MeHg on polysulfidation of Drp1 WT or CS mutants. Drp1 8CS(C624); all eight Cys residues except Cys624 were mutated to serine (n = 3 independent experiments). (D) Effect of MeHg on mitochondrial fission in HeLa cells expressing Drp1 WT or the polysulfidation mimic C624W mutant (n = 3 independent experiments). Right: Percentage of cells with tubule-type mitochondria. Scale bars, 20 μm. Data are shown as means ± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA.

  • Fig. 6 FLNa mediates MeHg-induced Drp1 activation.

    (A) Effect of cilnidipine (CIL) on MeHg-induced mitochondrial fission. NRCMs were incubated with CIL (1 μM) for 1 hour before MeHg (0.03 μM) treatment. Right: Percentage of cells with vesicle-type mitochondria (n = 3 independent experiments). Scale bars, 20 μm. (B) Effect of FLNa knockdown on MeHg-induced mitochondrial fission. NRCMs were transfected with negative control siRNA (siNC) or two different siRNAs for FLNa (siFLNa #1 and #2). Right: Percentage of cells with vesicle-type mitochondria (n = 3 independent experiments). Scale bars, 20 μm. (C) Interaction of FLNa and Drp1 WT or C624W. HeLa cells transfected with myc-FLNa and FLAG-Drp1 WT or C624W were treated with MeHg for 3 days (n = 3 independent experiments). (D) Localization of GFP-Drp1 WT or C624W with mCherry-FLNa (mChy-FLNa) in cardiac fibroblasts. Cardiac fibroblasts expressing GFP-Drp1 WT or C624W and mCherry-FLNa were incubated with CIL for 1 hour before MeHg treatment. The arrowheads show punctate where Drp1 colocalizes with FLNa (n = 3 independent experiments). Scale bars, 20 μm. Data are shown as means ± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA (A and B).

  • Fig. 7 Vulnerability of MeHg-exposed rat cardiomyocytes to mechanical stress.

    (A and B) Effect of Mdivi-1 on static stretch (A) or hypotonic-induced (B) cardiac injury in MeHg-exposed NRCMs. NRCMs were preincubated with Mdivi-1 (10 μM) for 30 min and then stretched statically for 12 hours (A) or cultured in hypotonic media for 3 days (B) with or without MeHg (0.05 μM). Cell viability and toxicity were measured by MTT and LDH release assay [n = 4 (A) and 3 (B) independent experiments]. (C and D) Effect of NaHS on static stretch (C) or hypotonic-induced (D) cardiac injury in MeHg-exposed NRCMs. NRCMs were preincubated with NaHS (100 μM) for 24 hours and then stretched statically for 12 hours (C) or cultured in hypotonic media for 3 days (D) with or without MeHg (0.05 μM) (n = 4 independent experiments). (E) Effect of CIL on hypotonic-induced cardiac injury. NRCMs were preincubated with CIL (1 μM) for 1 hour and then cultured in hypotonic media for 3 days with or without MeHg (0.05 μM) (n = 3 independent experiments). (F) Effect of FLNa knockdown on hypotonic-induced cardiac injury. NRCMs were transfected with negative control siRNA (siNC) or two different siRNAs for FLNa (siFLNa #1 and #2) and then cultured in hypotonic media for 3 days with or without MeHg (0.05 μM) (n = 3 independent experiments). (G) Effect of Drp1 polysulfidation on hypotonic-induced cardiac injury. H9c2 cells expressing FLAG-Drp1 WT or C624W were cultured in hypotonic media for 3 days with or without MeHg (n = 4 independent experiments). Data are shown as means ± SEM. **P < 0.01, one-way ANOVA. (H) Working model for cardiac fragility to hemodynamic overload through reactive sulfur-dependent regulation of Drp1 activity. GDP, guanosine diphosphate.

  • Fig. 8 MeHg-mediated cardiac vulnerability to mechanical stress in human cardiomyocytes.

    (A) Effect of CIL on MeHg-induced mitochondrial hyperfission. Human iPS cardiomyocytes were preincubated with CIL (0.5 μM) for 1 hour and then treated with MeHg (0.05 μM) for 3 days. Left: Representative images of mitochondrial morphology (green) in human cardiomyocytes with DAPI (blue). Right: Percentage of cells with vesicle-type mitochondria (n = 3 independent experiments). Scale bars, 50 μm. (B) Effect of CIL and NaHS on hypotonic-induced cytotoxicity of human cardiomyocytes. Cells were preincubated with CIL (0.5 μM) for 1 hour or NaHS (100 μM) for 24 hours and then cultured in hypotonic media for 2 days with or without MeHg (n = 3 independent experiments). (C) Effect of CIL and NaHS on hypotonic stress–induced apoptosis of human cardiomyocytes. Left: Representative images of TUNEL-positive apoptotic cells (green) merged with DAPI (blue). The proportion of TUNEL-positive nuclei (arrowheads) was quantified (n = 3 independent experiments). Scale bars, 50 μm. (D) Effect of Drp1 polysulfidation on hypotonic stress–induced cytotoxicity. Cells were transfected with FLAG-Drp1 WT or C624W and then cultured in hypotonic media for 2 days with or without MeHg. FLAG-Drp1 WT or C624W-positive human cardiomyocytes were visualized with DsRed fluorescence (n = 3 independent experiments). (E) Effect of Drp1 polysulfidation on hypotonic stress–induced apoptosis. The proportion of TUNEL-positive nuclei in DsRed-expressing cells (arrowheads) was calculated (n = 3 independent experiments). Scale bars, 50 μm. Data are shown as means ± SEM. **P < 0.01, one-way ANOVA.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/587/eaaw1920/DC1

    Fig. S1. Effect of MeHg (100 ppm) on body weight and protein amounts.

    Fig. S2. Subneurotoxic dose of MeHg promotes induction of maladaptive gene expression and remodeling after pressure overload.

    Fig. S3. Changes in the abundance of mitochondrial fusion- and fission-related proteins.

    Fig. S4. Effect of MeHg exposure on cardiomyocyte apoptosis.

    Fig. S5. MeHg exposure minimally affects Drp1 phosphorylation in the heart.

    Fig. S6. Depolysulfidation of recombinant Drp1 by MeHg.

    Fig. S7. Hypotonic stress–induced apoptosis of MeHg-exposed NRCMs.

    Fig. S8. Uncropped Western blots for Figs. 2 to 4.

    Fig. S9. Uncropped Western blots for Figs. 5 and 6.

    Fig. S10. Uncropped Western blots for fig. S1.

    Fig. S11. Uncropped Western blots for figs. S2 and S3.

    Fig. S12. Uncropped Western blots for figs. S4 to S7.

    Table S1. Echocardiographic parameters of mice with 100-ppm MeHg exposure at 1 or 2 weeks.

    Table S2. Echocardiographic parameters of TAC-operated mice with MeHg exposure.

    Table S3. Millar catheter analysis of TAC-operated mice given MeHg and NaHS.

  • This PDF file includes:

    • Fig. S1. Effect of MeHg (100 ppm) on body weight and protein amounts.
    • Fig. S2. Subneurotoxic dose of MeHg promotes induction of maladaptive gene expression and remodeling after pressure overload.
    • Fig. S3. Changes in the abundance of mitochondrial fusion- and fission-related proteins.
    • Fig. S4. Effect of MeHg exposure on cardiomyocyte apoptosis.
    • Fig. S5. MeHg exposure minimally affects Drp1 phosphorylation in the heart.
    • Fig. S6. Depolysulfidation of recombinant Drp1 by MeHg.
    • Fig. S7. Hypotonic stress–induced apoptosis of MeHg-exposed NRCMs.
    • Fig. S8. Uncropped Western blots for Figs. 2 to 4.
    • Fig. S9. Uncropped Western blots for Figs. 5 and 6.
    • Fig. S10. Uncropped Western blots for fig. S1.
    • Fig. S11. Uncropped Western blots for figs. S2 and S3.
    • Fig. S12. Uncropped Western blots for figs. S4 to S7.
    • Table S1. Echocardiographic parameters of mice with 100-ppm MeHg exposure at 1 or 2 weeks.
    • Table S2. Echocardiographic parameters of TAC-operated mice with MeHg exposure.
    • Table S3. Millar catheter analysis of TAC-operated mice given MeHg and NaHS.

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