Research ArticleCardiovascular Biology

Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission–associated myocardial senescence

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Science Signaling  13 Nov 2018:
Vol. 11, Issue 556, eaat5185
DOI: 10.1126/scisignal.aat5185
  • Fig. 1 Mitochondrial fission–associated premature myocardial senescence in peri-infarct zone myocardium after MI.

    (A) Myocardial senescence determined by SA-β-gal staining in mouse heart 4 weeks after MI (n = 5 mice per group). IZ, infarct zone; RZ, remote zone. Scale bars, 500 μm (left panels) and 50 μm (middle and right panels). (B) Representative electron micrographs of peri-infarct zone myocardium 1 week after MI depicting two subpopulations of mitochondria: interfibrillar (IF) and perinuclear (PN) mitochondria. N, nucleus (n = 5 mice per group). Scale bars, 1 μm. (C) Quantification of mitochondrial area and circularity (n > 350 or 125 perinuclear or interfibrillar mitochondria from 5 mice per group). (D) Guanosine 5′-triphosphate (GTP)–binding activity and protein abundance of Drp1 in the peri-infarct zone myocardium at 1 week after MI (n = 5 mice per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) GTP-binding activity of Drp1 in NRCMs under normoxia (N) or hypoxia (H) for 16 hours (n = 3 independent experiments). (F) Hypoxia-induced change in mitochondrial morphology. Left: Representative images of mitochondrial morphologies of NRCMs loaded with MitoTracker Green. Right: Mitochondrial morphologies of NRCMs classified into three groups: vesicle, intermediate, and tubule (n = 3 independent experiments). Scale bars, 10 μm (white) and 5 μm (yellow). (G) Effects of Drp1 knockdown on hypoxia/reoxygenation (H/R)–induced cardiomyocyte senescence. NRCMs transfected with negative control small interfering RNA (siRNA) (siNC) or two different siRNAs for Drp1 (siDrp1 #1 and #2) were incubated for 16 hours of hypoxia, followed by 12 hours of reoxygenation (n = 3 independent experiments). Left: Representative images of SA-β-gal staining of NRCMs. The arrowheads show SA-β-gal–positive cells. Right: Quantification of SA-β-gal–positive cells. Scale bars, 50 μm. Data are means ± SEM. *P < 0.05, **P < 0.01 by unpaired t test (D to F) or one-way analysis of variance (ANOVA) (G).

  • Fig. 2 FLNa is a hypoxia-dependent binding partner of Drp1 in peri-infarct zone myocardium after MI.

    (A) Coomassie brilliant blue–stained images of SDS–polyacrylamide gel electrophoresis (PAGE) gels to identify proteins interacting with FLAG-Drp1. HeLa cells expressing FLAG-Drp1 were exposed to normoxia or hypoxia. (B) Hypoxia-dependent interaction of FLAG-Drp1 with endogenous FLNa in HeLa cells. (n = 3 independent experiments). (C) Interaction of Myc-FLNa with FLAG-Drp1 S637D (SD) and S637A (SA) mutants in HeLa cells under hypoxia (n = 3 independent experiments). (D) Colocalization of GFP-Drp1 with mCherry-FLNa in H9c2 cells under normoxia or hypoxia (n = 3 independent experiments). Scale bars, 10 μm. (E) Interaction of Myc-FLNa with FLAG-Drp1 in mitochondrial (Mito) or cytosolic (Cyto) fractions of HeLa cells (n = 3 independent experiments). (F) Representative images and distribution of Drp1 (red) and FLNa (green) in mouse LV myocardium 4 weeks after MI. Lower panels display high magnification images. The boxed regions in peri-infarct zone myocardium of MI-treated heart or sham-treated hearts were magnified and color separated (n = 3 mice per group). Scale bars, 60 μm (white) and 10 μm (yellow).

  • Fig. 3 The interacting regions of Drp1 and FLNa.

    (A) Interaction of truncated FLAG-Drp1 (GTPase) with endogenous FLNa under hypoxia (n = 3 independent experiments). (B) Interaction of FLAG-Drp1 with actin-binding–deficient Myc-FLNa truncated mutants (Ig1-24 or Ig16-24) under hypoxia (n = 3 independent experiments).

  • Fig. 4 FLNa is required for hypoxia-mediated Drp1 activation and mitochondrial fission–associated myocardial senescence.

    (A) Morphological changes in actin cytoskeleton of H9c2 cells in response to the actin-depolymerizing drug LatB (20 nM, 18 hours). Actin cytoskeleton was labeled with Alexa 594–phalloidin (n = 3 independent experiments). (B) Effects of LatB on hypoxia-induced mitochondrial fission. H9c2 cells were treated with 20 nM LatB for 1 hour before being exposed to hypoxia (n = 3 independent experiments). Scale bars, 50 μm. DMSO, dimethyl sulfoxide. (C) Effect of FLNa knockdown on hypoxia-induced mitochondrial fission. H9c2 cells were treated with negative control siRNA (siNC) or two different siRNAs for FLNa (siFLNa #1 and #2) (n = 3 independent experiments). Scale bars, 50 μm. (D) Effect of FLNa knockdown on hypoxia/reoxygenation-induced accumulation of p53 in NRCMs. The population of senescent cells near p53 punctae in α-actinin–positive NRCMs was quantified (n = 3 independent experiments). Scale bars, 20 μm. (E) Effects of FLNa siRNA on the localization of GFP-Drp1 and mitochondria in cardiac fibroblasts under normoxia or hypoxia. Mitochondria were stained with MitoTracker Red (n = 3 independent experiments). Scale bars, 10 μm. Data are shown as the means ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA.

  • Fig. 5 FLNa drives Drp1 activation and mitochondrial fission in coordination with actin filaments.

    (A) Protein abundances of actin and FLNa in peri-infarct zone myocardium at 4 weeks after MI (n = 3 mice per treatment). (B) Effect of FLNa or β-actin overexpression on Drp1 activation in HEK293 cells (n = 3 biological replicates). (C) The time course of GTP hydrolysis by Drp1 (600 nM) in the absence or presence of FLNa (200 nM) or EDTA. Right: Calculated GTP hydrolysis rate of Drp1 (n = 3 independent experiments). (D) Interaction of Myc-FLNa with FLAG-Drp1 WT or K38A (KA) under hypoxia (n = 3 independent experiments). (E) Effect of actin-unbound FLNa mutant on Drp1 activation in HEK293 cells (n = 3 biological replicates). (F) Effect of FLNa overexpression on mitochondrial fission. H9c2 cells expressing mCherry-FLNa wild-type (WT) or A1545T were incubated under normoxia and stained with MitoTracker Green (n = 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. *P < 0.05, **P < 0.01 by unpaired t test (A) or one-way ANOVA (B, C, E, and F).

  • Fig. 6 Identification of cilnidipine as an inhibitor of hypoxia-induced mitochondrial fission by disrupting Drp1-FLNa complex.

    (A) Effects of antihypertensive drugs on mitochondrial fission. Cardiac fibroblasts were treated with each drug (1 μM) for 1 hour before being exposed to hypoxia. The percentages of cells with vesicle-type mitochondrial fragmentation were quantified (n = 3 to 6 independent experiments). Cilnidipine, CIL. (B) Effects of cilnidipine on hypoxia-mediated mitochondrial fragmentation. Left: Representative images of mitochondrial morphologies of NRCMs loaded with MitoTracker Green. Right: Mitochondrial morphologies of NRCMs treated with cilnidipine (1 μM) for 1 hour before 16 hours of normoxia (N) or hypoxia (H) (n = 3 independent experiments). Scale bars, 10 μm. (C) Effects of cilnidipine on hypoxia/reoxygenation-induced cardiomyocyte senescence. NRCMs were treated with 1 μM cilnidipine or amlodipine (AML) for 1 hour before 16 hours of hypoxia, followed by 12 hours of reoxygenation (n = 5 independent experiments). Scale bars, 20 μm. (D and E) Interaction of FLAG-Drp1 with endogenous FLNa under hypoxia and effects of cilnidipine (D) or Mdivi-1 (E) treatment. HeLa cells expressing FLAG-Drp1 were treated with hypoxia with or without cilnidipine (3 μM) or Mdivi-1 (10 μM) [n = 5 (D) and n = 3 (E) independent experiments]. Data are shown as the means ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA.

  • Fig. 7 Improvement of cardiac function after MI through suppression of Drp1-mediated myocardial senescence by cilnidipine.

    (A) Effect of cilnidipine on ejection fraction (EF) in mice 4 weeks after MI. An osmotic pump filled with cilnidipine (20 mg/kg per day) or amlodipine (2.5 mg/kg per day) was implanted intraperitoneally 7 days after MI (n = 5 to 8 mice per treatment). (B) Effects of cilnidipine on the heart weight (HW)/body weight (BW) ratio in mice 4 weeks after MI. Cilnidipine (30 or 100 mg/kg per day) was infused at 1 day after MI (n = 4 to 10 mice per treatment). (C) Hematoxylin and eosin–stained images of hearts at 4 weeks after MI. Scale bars, 1.5 mm. Right: Cell surface area (CSA) of cardiomyocytes (n = 4 to 5 mice per treatment). (D) Effects of cilnidipine (30 mg/kg per day) on increased heart weight/body weight ratio in Cav2.2+/+ and Cav2.2−/− mice 4 weeks after MI (n = 3 to 6 mice per treatment). (E) Effects of cilnidipine (30 mg/kg per day) on mitochondrial morphology in peri-infarct zone myocardium 1 week after MI. Left: Representative images of interfibrillar mitochondria. Right: Quantification of mitochondrial circularity. n > 200 mitochondria from three mice per group. Scale bars, 1 μm. (F) Effects of cilnidipine (30 mg/kg per day) on Drp1 activity in peri-infarct zone myocardium 1 week after MI (n = 3 mice per treatment). (G) Effect of cilnidipine (30 mg/kg per day) on the proportion of SA-β-gal–positive area in peri-infarct zone myocardium 4 weeks after MI (n = 3 to 4 mice per treatment). Scale bars, 100 μm. (H) Picrosirius red–stained images of peri-infarct zone myocardium 4 week after MI. Scale bars, 100 μm. Right: Collagen volume fractions (CVFs) (n = 4 mice per treatment). (I) mRNA amounts of hypertrophy-related (ANP and α-SKA) and fibrosis-related (ACE, Periostin, Collagen 1a1, Collagen 3a1, TGF1, TGF2, TGF3, and CTGF) genes in peri-infarct zone myocardium at 1 week after MI with or without cilnidipine (30 mg/kg per day) (n = 3 to 4 mice per treatment). (J) Effects of cilnidipine on protein abundances in peri-infarct zone myocardium at 1 week after MI (n = 3 mice per treatment). *P < 0.05, **P < 0.01 by one-way ANOVA (A to H and J); ††P < 0.01 compared to vehicle sham, #P < 0.05, ##P < 0.01 compared to vehicle MI by one-way ANOVA (I).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/556/eaat5185/DC1

    Fig. S1. Mitochondrial fission and premature myocardial senescence in peri-infarct zone myocardium after MI.

    Fig. S2. Changes in protein abundances and activities in peri-infarct zone myocardium after MI.

    Fig. S3. Mitochondrial morphology in NRCMs.

    Fig. S4. Reoxygenation-induced mitochondrial ROS production plays an indispensable role in myocardial senescence.

    Fig. S5. Effects of gene silencing and pharmacological inhibition of Drp1 on mitochondrial morphology and ROS production.

    Fig. S6. Identification of FLNa by mass spectrometric analysis.

    Fig. S7. No interaction between Drp1 and the Ig24 dimerization domain of FLNa.

    Fig. S8. Effect of FLNa knockdown on hypoxia-induced mitochondrial fission and function.

    Fig. S9. Mitochondrial morphology in cardiac fibroblasts.

    Fig. S10. Mortality in mice after MI.

    Table S1. Echocardiographic parameters of mice with cilnidipine administration.

    Table S2. Cardiac parameters of mice with cilnidipine administration.

    Table S3. Echocardiographic parameters of Cav2.2(+/+) or Cav2.2(−/−) mice with cilnidipine administration.

    Table S4. List of Taqman probe and primer sets for real-time PCR.

  • This PDF file includes:

    • Fig. S1. Mitochondrial fission and premature myocardial senescence in peri-infarct zone myocardium after MI.
    • Fig. S2. Changes in protein abundances and activities in peri-infarct zone myocardium after MI.
    • Fig. S3. Mitochondrial morphology in NRCMs.
    • Fig. S4. Reoxygenation-induced mitochondrial ROS production plays an indispensable role in myocardial senescence.
    • Fig. S5. Effects of gene silencing and pharmacological inhibition of Drp1 on mitochondrial morphology and ROS production.
    • Fig. S6. Identification of FLNa by mass spectrometric analysis.
    • Fig. S7. No interaction between Drp1 and the Ig24 dimerization domain of FLNa.
    • Fig. S8. Effect of FLNa knockdown on hypoxia-induced mitochondrial fission and function.
    • Fig. S9. Mitochondrial morphology in cardiac fibroblasts.
    • Fig. S10. Mortality in mice after MI.
    • Table S1. Echocardiographic parameters of mice with cilnidipine administration.
    • Table S2. Cardiac parameters of mice with cilnidipine administration.
    • Table S3. Echocardiographic parameters of Cav2.2(+/+) or Cav2.2(−/−) mice with cilnidipine administration.
    • Table S4. List of Taqman probe and primer sets for real-time PCR.

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