Iron derived from autophagy-mediated ferritin degradation induces cardiomyocyte death and heart failure in mice

  1. Jumpei Ito
  2. Shigemiki Omiya
  3. Mara-Camelia Rusu
  4. Hiromichi Ueda
  5. Tomokazu Murakawa
  6. Yohei Tanada
  7. Hajime Abe
  8. Kazuki Nakahara
  9. Michio Asahi
  10. Manabu Taneike
  11. Kazuhiko Nishida
  12. Ajay M Shah
  13. Kinya Otsu  Is a corresponding author
  1. The School of Cardiovascular Medicine and Sciences, King’s College London British Heart Foundation Centre of Excellence, United Kingdom
  2. Department of Pharmacology, Faculty of Medicine, Osaka Medical College, Japan
  3. Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, Japan
6 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
Cardiomyocyte-specific Ncoa4 ablation attenuated the development of pressure overload-induced heart failure.

The Ncoa4+/+ and Ncoa4–/– mice were subjected to pressure overload by transverse aortic constriction (TAC) and analyzed 4 weeks after the operation. (A) Representative images of transthoracic M-mode …

Figure 1—source data 1

Source data for Figure 1.

https://cdn.elifesciences.org/articles/62174/elife-62174-fig1-data1-v1.xlsx
Figure 1—source data 2

Physiological and echocardiographic parameters in 8- to 10-week-old Ncoa4+/+ and Ncoa4–/– mice at baseline.

BP, blood pressure; TL, tibia length; LV, left ventricle; LVIDd, end-diastolic left ventricular internal dimension; LVIDs, end-systolic left ventricular internal dimension; IVSd, end-diastolic interventricular septum thickness; LVPWd, end-diastolic left ventricular posterior wall thickness; FS, fractional shortening. The data are the mean ± SEM. n indicates the number of biologically independent samples. The data were evaluated by unpaired, two-tailed Student’s t-test.

https://cdn.elifesciences.org/articles/62174/elife-62174-fig1-data2-v1.docx
Figure 1—source data 3

Source data for data table provided in Figure 1—source data 2.

https://cdn.elifesciences.org/articles/62174/elife-62174-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
Generation of cardiomyocyte-specific nuclear receptor coactivator 4 (NCOA4)-deficient mice.

(A) Targeted modification of the Ncoa4 gene. Schematic structures of the wild-type genomic Ncoa4 sequence, the targeting construct, the targeted allele, the floxed allele after flippase recognition …

Figure 1—figure supplement 2
Cardiac remodeling markers in transverse aortic constriction (TAC)-operated Ncoa4–/– mice.

The Ncoa4+/+ and Ncoa4–/– mice were subjected to TAC. The levels of mRNA were analyzed 4 weeks after the operation (n = 8 biologically independent samples). Gapdh mRNA was used as the loading …

Figure 1—figure supplement 3
Myh6-Cre transgene does not alter the heart response to pressure overload stress.

The Myh6-Cre and Myh6-Cre mice were subjected to TAC and analyzed 4 weeks after the operation. Echocardiographic parameters of the mice (n = 4 biologically independent samples) are shown. LVIDd and …

Figure 2 with 1 supplement
The time course of ferritinophagy in the heart after transverse aortic constriction (TAC).

(A) Western blot analysis of FTH1 in Ncoa4+/+ and Ncoa4–/– hearts 4 weeks after TAC. GAPDH was used as the loading control. The right-hand graphs show the densitometric analysis. The average value …

Figure 2—figure supplement 1
Echocardiographic parameter aftertransverse aortic constriction (TAC).

Time course of changes in the echocardiographic parameters of left ventricular (LV) fractional shortening (FS), the end-diastolic LV internal dimension (LVIDd) and the end-systolic LV internal …

Ablation of Ncoa4 in cardiomyocytes showed defective ferritinophagy.

(A) Western blot analysis of FTH1 in Ncoa4+/+ and Ncoa4–/– hearts 1 week after transverse aortic constriction (TAC). GAPDH was used as the loading control. The right-hand graphs show the …

Figure 4 with 4 supplements
The effect of Ncoa4 ablation on the pathways to iron-dependent cell death.

(A) Tissue levels of total non-heme, ferrous, and ferric iron in Ncoa4+/+ and Ncoa4–/– hearts 4 weeks after transverse aortic constriction (TAC) (n = 6 biologically independent samples). The ratio …

Figure 4—source data 1

Source data for Figure 4.

https://cdn.elifesciences.org/articles/62174/elife-62174-fig4-data1-v1.xlsx
Figure 4—source data 2

Hematological parameters and red cell indices in Ncoa4+/+ and Ncoa4–/– mice.

PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CH, hemoglobin concentration. Data are the mean ± SEM. n indicates the number of biologically independent samples. The data were evaluated by one-way analysis of variance (ANOVA), followed by Tukey–Kramer’s post hoc test. *p=0.0418 and **p=0.0108 versus the corresponding sham-operated group. Exact p-values are provided in Supplementary file 1.

https://cdn.elifesciences.org/articles/62174/elife-62174-fig4-data2-v1.docx
Figure 4—source data 3

Source data for data table provided in Figure 4—source data 2.

https://cdn.elifesciences.org/articles/62174/elife-62174-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Liver iron store and serum inflammatory cytokine in Ncoa4+/+ and Ncoa4–/– mice.

(A) Tissue levels of total non-heme iron in Ncoa4+/+ and Ncoa4–/– livers 4 weeks after TAC (n = 6 biologically independent samples). (B) Serum interleukin 6 (IL-6) levels (n = 8 biologically …

Figure 4—figure supplement 2
Iron regulating proteins in transverse aortic constriction (TAC)-operated Ncoa4–/– hearts.

(A) Homogenates from the heart 4 weeks after TAC were subjected to western blot analysis using antibodies against iron-responsive element-binding protein 2 (IREB2), ferroportin 1 (SLC40A1), and …

Figure 4—figure supplement 3
Lipid reactive oxygen species and anti-oxidant proteins in Ncoa4–/– hearts 4 weeks after transverse aortic constriction (TAC).

(A) 4-Hydroxy-2-noneal (HNE) staining of heart sections. Scale bar, 50 μm. The right panel shows the quantitative analysis of 4-HNE-positive area to whole heart section (n = 5 biologically …

Figure 4—figure supplement 4
The system Xc-/glutathione axis and glutaminolysis pathway in transverse aortic constriction (TAC)-operated Ncoa4–/– hearts.

(A) The mRNA expression level of Slc7a11 in the heart 4 weeks after TAC (n = 8 biologically independent samples). Gapdh mRNA was used as the loading control. (B) The cystine concentration in the …

Figure 5 with 2 supplements
Isoproterenol induces iron-dependent cell death in isolated cardiomyocytes.

(A) Cell death assay. Cell death was estimated using a Live/Dead Viability/Cytotoxicity Assay Kit. Isolated mouse cardiomyocytes from Ncoa4+/+ and Ncoa4–/– hearts were treated with the indicated …

Figure 5—figure supplement 1
Erasin induces cell death in isolated cardiomyocytes.

(A) Cell death assay. Cell death was estimated using a Live/Dead Viability/Cytotoxicity Assay Kit. Isolated mouse cardiomyocytes from Ncoa4+/+ and Ncoa4–/– hearts were treated with the indicated …

Figure 5—figure supplement 2
RSL3 induces cell death in isolated cardiomyocytes.

Cell death assay. Cell death was estimated using a Live/Dead Viability/Cytotoxicity Assay Kit. Isolated mouse cardiomyocytes from Ncoa4+/+ and Ncoa4–/– hearts were treated with the indicated …

Figure 6 with 2 supplements
Inhibition of lipid peroxidation protects hearts from pressure overload.

Wild-type C57BL/6J mice were subjected to transverse aortic constriction (TAC) and analyzed 4 weeks after the operation. Ferrostatin-1 (Fer-1) or saline was intraperitoneally administered daily …

Figure 6—figure supplement 1
Cardiac remodeling markers and 4-HNE staining in ferrostatin-1-treated transverse aortic constriction (TAC)-operated wild-type hearts.

The wild-type C57BL/6J mice were subjected to TAC. Ferrostatin-1 (Fer-1) was intraperitoneally administered daily starting one day before TAC. (A) The levels of mRNA were analyzed 4 weeks after the …

Figure 6—figure supplement 2
Ferrostatin-1 does not provide additional protection from cardiac remodeling in Ncoa4–/– mice.

The Ncoa4–/– mice were subjected to TAC. Ferrostatin-1 (Fer-1) or saline was intraperitoneally administered daily starting one day before TAC. (A) Echocardiographic parameters of the mice (n = 5 …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (male Mus musculus)C57BL/6JEnvigo
UK
C57BL/6OlaHsd
Strain, strain background (male Mus musculus)Ncoa4flox/flox;Myh6-Cre+This paperSee Materials and methods
Strain, strain background (male Mus musculus)Ncoa4flox/flox;Myh6-CreThis paperSee Materials and methods
Strain, strain background (male Mus musculus)Myh6-Cre+Nishida et al., 2004See Materials and methods
AntibodyMouse monoclonal antibody to NCOA4Sigma-AldrichSAB1404569, Lot: HC071-1F11, RRID:AB_10759525(1:1000)
AntibodyRabbit polyclonal antibody to FTH1Cell Signaling Technology3998, Lot: 2, RRID:AB_1903974for western blots, (1:1000); for immunofluorescence, (1:100)
AntibodyMouse monoclonal antibody to LC3BCell Signaling Technology83506, Lot: 1, RRID:AB_2800018(1:100)
AntibodyRat monoclonal antibody to LAMP2aAbcamab13524, Lot: GR3317907-1, RRID:AB_2134736(1:100)
AntibodyRat monoclonal antibody to GPX4MilliporeMABS1274, Lot: Q2633070, RRID:AB_2885039(1:1000)
AntibodyRabbit polyclonal antibody to 4-HNEMillipore393207, Lot: 3167038, RRID:AB_566310(1:500)
AntibodyRabbit polyclonal antibody to IREB2Thermo Fisher ScientificPA1-16543, Lot: TK2666362A, RRID:AB_2126583(1:250)
AntibodyRabbit polyclonal antibody to SLC40A1Alpha Diagnostic InternationalMTP11-A, Lot: 1169899A3-L, RRID:AB_1619475(1:1000)
AntibodyMouse monoclonal antibody to TFRCThermo Fisher Scientific13–6800, Lot: TI275369, RRID:AB_2533029(1:1000)
AntibodyRabbit polyclonal antibody to SOD2Abcamab13534, Lot: GR33618-66, RRID:AB_2191667(1:1000)
AntibodyRabbit polyclonal antibody to HO-1Enzo Life SciencesADI-SPA-895, Lot: 03301708, RRID:AB_10618757(1:1000)
AntibodyMouse monoclonal antibody to GAPDHSigma-AldrichG8795, Lot: 056M4856V, RRID:AB_1078991(1:10000)
AntibodyMouse immunoglobulinSanta Cruz biochemistrysc-2025, RRID:AB_737182
AntibodyRabbit IgGAbcamab37415, RRID:AB_2631996
AntibodyFluorescein isothiocyanate-conjugated lectinSigma-AldrichL4895
AntibodyAlexa Fluor 488 donkey-anti-mouseThermo Fisher ScientificA21202, RRID:AB_141607(1:500)
AntibodyAlexa Fluor 568 donkey-anti-rabbitThermo Fisher ScientificA10042, RRID:AB_2534017(1:500)
AntibodyAlexa Fluor 488 goat-anti-ratThermo Fisher ScientificA11006, RRID:AB_2534074(1:500)
AntibodyIRDye 680LT Donkey anti-Mouse IgG Secondary AntibodyLI-COR Biosciences926–68020, RRID:AB_10706161(1:10,000)
AntibodyIRDye 680LT Donkey anti-Rabbit IgG Secondary AntibodyLI-COR Biosciences926–68023, RRID:AB_10706167(1:10,000)
AntibodyIRDye 680LT Goat anti-Rat IgG Secondary AntibodyLI-COR Biosciences926–68029, RRID:AB_10715073(1:10,000)
Commercial assay or kitPierce Protein G Magnetic BeadsThermo Scientific88848
Commercial assay or kitRNeasy Fibrous Tissue Mini KitQIAGEN74704
Commercial assay or kitSuperScript IV First-Strand Synthesis SystemThermo Fisher Scientific18091050
Commercial assay or kitPowerUp SYBR Green Master MixThermo Fisher ScientificA25742
Commercial assay or kitMasson's Trichrome Stain KitPolysciences, Inc25088–1
Commercial assay or kitVECTASTAIN Elite ABC-HRP Kit, Peroxidase (Rabbit IgG)Vector Laboratories IncPK-6101
Commercial assay or kitDAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3'-diaminobenzidine)Vector Laboratories IncSK-4100
Commercial assay or kitnormal donkey serumAbcamab7475, RRID:AB_2885042
Commercial assay or kitnormal goat serumAbcamab7481, RRID:AB_2716553
Commercial assay or kitProLong Gold Antifade Reagent with DAPILife TechnologiesP36935
Commercial assay or kitMouse Ferritin ELISA Kit (FTL)Abcamab157713
Commercial assay or kitPointe Scientific Iron/TIBC ReagentsPointe Scientific23-666-320
Commercial assay or kitELISA Kit for Troponin T Type 2, Cardiac (TNNT2)Cloud-CloneSED232Mu
Commercial assay or kitMouse IL-6 Quantikine ELISA KitR and D SystemsM6000B
Commercial assay or kitIron Assay KitAbcamab83366
Commercial assay or kitLipid Peroxidation (MDA) Assay KitAbcamab118970
Commercial assay or kitGSSG/GSH Quantification KitDojindoG257
Commercial assay or kitGlutamine Assay KitAbcamab197011
Commercial assay or kitGlutamate Assay KitAbcamab83389
Commercial assay or kitElectrophoretic Mobility-Shift Assay (EMSA) KitInvitrogenE33075
Commercial assay or kitZero Blunt TOPO PCR Cloning KitInvitrogen451245
Commercial assay or kitHiScribe T7 Quick High Yield RNA Synthesis KitNew England BiolabsE2050S
Chemical compound, drugFerrostatin-1Sigma AldrichSML0583
Chemical compound, drugIsoprenaline hydrochlorideSigma AldrichI5627
Chemical compound, drugErastinSigma AldrichE7781
Chemical compound, drug1S,3R-RSL 3Sigma AldrichSML2234
Chemical compound, drugcalcein-AMInvitrogenC1430
Chemical compound, drugethidium homodimer-1InvitrogenE1169
Chemical compound, drug2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA)InvitrogenD399
Chemical compound, drugC11-BODIPYInvitrogenD3861
Chemical compound, drugpyridoxal isonicotinoyl hydrazine (PIH)Abcamab145871
Software, algorithmImageJNational Institutes of HealthVersion 1.51 r, RRID:SCR_003070
Software, algorithmGraphPad Prism 8GraphPad SoftwareRRID:SCR_002798
Software, algorithmEZChrom EliteAgilent TechnologiesVersion 3.3.2.

Additional files

Supplementary file 1

Quantification and statistical analysis.

The number of independent biological repeats (n) is shown in the figure legends. P values are shown below.

https://cdn.elifesciences.org/articles/62174/elife-62174-supp1-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/62174/elife-62174-transrepform-v1.docx

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