Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes

Figures

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  Figure 1. Cardiac ventricular wall thinning, rupture and reduced cardiac scar density in Nur77-KO mice. (A) Incidence of myocardial wall thinning and rupture in full-body Nur77-KO, full-body ApoE/Nur77-KO mice, and cardiomyocyte-specific Nur77-KO (CM-KO) mice after 2 weeks of permanent LAD ligation or 7 days of chronic isoproterenol (ISO, 60 mg/kg/day) infusion. (B) A typical example of severe myocardial wall thinning (arrow). (C) A typical example of cardiac rupture (arrow) with a blood clot in the chest cavity (asterisk). (D) Area of the left ventricle (LV) and septum affected by fibrosis upon 7 days of isoproterenol (ISO, 60 mg/kg/day) infusion quantified on Masson trichrome-stained tissue sections. (E) Histologic quantification of empty space between collagen fibrils in cardiac fibrotic patches on Masson trichrome-stained heart sections. (F) Expression levels of matrix metalloproteinase 2 (MMP2) in LV as assessed by qPCR. n = 8–20 mice per group. (G) Typical examples of cardiac fibrotic patches stained with Masson trichrome. Blue is collagen. Data presented as boxplots with whiskers for minimum/maximum values; * p < 0.05, *** p < 0.001.

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  Figure 2. Nur77 knockdown in CFs promotes a MyoFB phenotype. (A) Primary neonatal rat CF in culture were identified by expression of fibroblast marker vimentin. Scale bar represents 100 μm. (B) Induction of Nur77 mRNA expression in CFs after ISO (10 μM) treatment. (C) Decreased Nur77 mRNA expression after siRNA-mediated knockdown in CFs (siNur77) compared to CFs treated with control siRNA (siCon), as measured by qPCR. (D) Number of CF expressing MyoFB marker α-smooth muscle actin (aSMA) as assessed by immunofluorescence. Scale bar represents 20 μm. (E) MyoFB and ECM-related gene expression measured by qPCR. acta2: α-smooth muscle actin, col1a1: collagen type 1, fn1: fibronectin, postn: periostin. (D,E) ISO stimulation (10 μM) was for 24 h. n = 3–4 independent experiments. Data presented as mean + SEM; (B): *** p < 0.001 vs. t = 0; (C–E): # p < 0.05 vs. siCon vehicle; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. siCon same stimulus.

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  Figure 3. Nur77 knockdown in cardiac fibroblasts (CFs) represses MyoFB functional characteristics in CF. (A) CF collagen content as measured by soluble Sirius red assay. (B) CF proliferation as measured by bromodeoxyuridine (BrdU) incorporation. (C) CF wound closure capacity in scratch wound assay; quantification in the right panel. (A) ISO (10 μM) stimulation was for 72 h. (B) ISO (10 μM) stimulation was for 24 h. n = 3–4 independent experiments per group. Data presented as mean + SEM; # p < 0.05 vs. siCon vehicle; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. siCon same stimulus.

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  Figure 4. Paracrine factors from Nur77-silenced cardiomyocytes promote a MyoFB phenotype in CF. (A) Primary neonatal rat ventricular cardiomyocytes in culture were identified by expression of α-actinin. Scale bar represents 100 μm). (B) Nur77 mRNA expression after siRNA-mediated knockdown of Nur77 in neonatal rat ventricular cardiomyocytes, as measured by qPCR. (C) Schematic presentation of the experimental setup. (D) mRNA expression of MyoFB markers in CF after incubation with siCon or siNur77 cardiomyocyte conditioned medium, as assessed by qPCR. (E) Proliferation of CF after incubation with siCon or siNur77 cardiomyocyte conditioned medium, as measured by BrdU incorporation. (D,E) Conditioned medium stimulation (1:2 ratio with fresh medium) was for 24 h. n = 3–4 independent experiments. Data presented as mean + SEM; # p < 0.05 vs. siCon vehicle; * p < 0.05, ** p < 0.01 vs. siCon same stimulus.

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  Figure 5. Nur77-silenced cardiomyocytes promote a MyoFB phenotype in CF via TGF-β. (A) Expression of TGF-β and target genes in CF stimulated with conditioned medium from siCon or siNur77 neonatal rat cardiomyocytes, as assessed by qPCR. (B) Expression of TGF-β target genes and MyoFB genes (C) in CFs stimulated with conditioned medium from siCon or siNur77 neonatal rat cardiomyocytes after 15 min pre-incubation with 10 μM TGF-β receptor I (TGFR1) inhibitor SB431542. Conditioned medium stimulation (1:2 ratio with fresh medium) was for 24 h. tgfb: transforming growth factor—β, ctgf: connective tissue growth factor. Data presented as mean + SEM; * p < 0.05, *** p < 0.001 vs. siCon same stimulus, ns—not significant.

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  Figure 6. Nur77 knockdown in CFs inhibits TGF-β-induced signaling and myofibroblasts (MyoFB) differentiation. (A) Induction of Nur77 mRNA in CFs after TGF-β (10 ng/mL) stimulation. (B) Expression of TGF-β pathway downstream markers in siCon-CFs and siNur77-CFs stimulated with TGF-β, as measured by qPCR. (C) Expression of MyoFB markers in siCon-CFs and siNur77-CFs stimulated with TGF-β, as measured by qPCR. (D) CF collagen production as measured by soluble Sirius red assay. (E) CF proliferation as measured by BrdU incorporation. (B,C,E) TGF-β stimulation (10 ng/mL) was for 24 h. (D) TGF-β stimulation (10 ng/mL) was for 72 h. n = 3–4 independent experiments per group. Data presented as mean + SEM; (A): ** p < 0.01 vs. t = 0; (B–D): # p < 0.05 vs. siCon vehicle; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. siCon TGF-β.

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  Figure 7. Proposed working model. Nur77 in cardiac fibroblasts promotes ISO and TGF-β-induced fibroblast-to-myofibroblast transition, while Nur77 in cardiomyocytes represses the ability of these cells to induce paracrine TGF-β-mediated fibroblast-to-myofibroblast transition, thereby balancing the cardiac fibrotic response. ISO: isoproterenol, TGF-β: transforming growth factor-β.