Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

Figures

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  Figure 1. Cardiomyocyte-specific overexpression of LRP6 prevents cardiac dysfunction induced by pressure overload. (A), Echocardiographic analysis of fraction shortening (FS) in mice at different time-points (3d, 1w, 2w, and 4w) after TAC. *p < 0.05 vs Sham group; n = 6-10 mice/each group. (B), Western blot analysis of p-LRP6 and LRP6 levels in left ventricular tissues from mice at different time-points (3d, 1w, 2w, and 4w) after TAC. *p < 0.05; **p < 0.01 vs sham group; n = 4 mice/ each group. (C), Summary of the generation of tamoxifen-inducible cardiac-specific LRP6 overexpressing mice. (D), Representative images of Western blot analysis of LRP6 expression in heart and kidney tissues from LRP6CAG/MCM after injection of tamoxifen or DMSO for 3 consecutive days. (E), Representative images of Western blot analysis of LRP6 expression in isolated CMs and non-CMs from tamoxifen-injected LRP6CAG/MCM (LRP6 Over) or -MCM (MCM) mice. CMs: cardiomyocytes. (F), Overall strategy of exploring the effects of cardiac LRP6 overexpression in TAC mice. (G), Echocardiographic analysis of cardiac function in tamoxifen-injected LRP6CAG/MCM (LRP6 Over) or -MCM (MCM) mice at 2 weeks and 4 weeks after TAC or…

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  Figure 2. Cardiomyocyte-specific overexpression of LRP6 attenuates cardiac hypertrophy and fibrosis induced by pressure overload. (A), HE and Masson staining of cross heart sections. Scale bars, 2 mm. (B), Representative images of Masson staining heart tissue were showed and the cardiac interstitial and vascular fibrosis were quantitatively analyzed. Scale bars, 100 um. (C), Western blot analysis of p-LRP6, LRP6, Col1, Col3, α-SMA, MMP2, MMP9, TGF-β1, p-smad2/3 in heart tissues. *p < 0.05; **p < 0.01; ***p<0.001; n = 3-5/each group. (D), Quantitative analysis of the ratio of heart weight/body weight (HW/BW) and heart/tibia (HW/TL). *p < 0.05; **p < 0.01; ***p < 0.001; n = 4-7/each group. Tamoxifen-injected-LRP6CAG/MCM (LRP6 Over) or -MCM (MCM) mice were analyzed at 4 weeks after TAC or sham operation.

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  Figure 3. Cardiomyocyte-specific overexpression of LRP6 inhibits the expression of cardiomyocyte-derived-Wnt5a/11 under pressure overload. (A), Western blot analysis of LRP6 expression in CMs, and Col1, MMP2, MMP9, TGF-β1, α-SMA, and p-smad2/3 expression in CFs. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON), and the culture medium from these CMs was used to stimulate CFs. *p < 0.05; **p < 0.01; ***p < 0.001; n = 4/each group. (B), Western blot analysis of Wnt5a and Wnt11 expression in CMs and the culture medium. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON). **p < 0.01; ***p < 0.001; n = 5-6/each group. (C), Western blot analysis of Wnt5a and Wnt11 expression in tamoxifen-injected-LRP6CAG/MCM (LRP6 Over) or -MCM (MCM) mice after TAC or sham operation. **p < 0.01; n = 5/each group.

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  Figure 4. Wnt5a and Wnt11 are involved in cardiac fibrosis during pressure overload. (A-B), Western blot analysis of Col1, MMP2, MMP9, and TGF-β1 expression in CFs treated with Wnt5a or Wnt11 at 0, 10, or 50 ng/mL. *p < 0.05; **p < 0.01; n = 3/each group. (C), Western blot analysis of Wnt5a and Wnt11 in stretched CMs pre-transfected with si-Scramble (sh-NC) or si-Wnt5a plus si-Wnt11 (si-Wnt5a/11). The conditioned medium from the two groups of CMs was used to treat CFs for 24 h. Col1, MMP9, TGF-β1, α-SMA, and p-smad2/3 were analyzed by Western blotting. **p < 0.01; ***p < 0.001. n = 3/each group.

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  Figure 5. Knockdown of Wnt5a and Wnt11 inhibits cardiac fibrosis in mice under pressure overload. (A), Overall strategy to explore cardiac remodeling after knockdown of Wnt5a and Wnt11 in mice under pressure overload. (B), Fraction shortening (FS) in mice at 4 weeks after TAC. Mice were injected with sh-Wnt5a/Wnt11-AAV9 (sh-Wnt5a/Wnt11) or sh-scramble-AAV9 (shNC) by tail vein two weeks before TAC or sham operation. *p < 0.05; n = 6-10/each group. (C), Masson staining of heart tissue from mice in (B) and fibrosis area was quantitatively analyzed. Bar = 100 µm. *p < 0.05; ***p < 0.001. n = 4-9/each group. (D), Western blot analysis of Wnt5a, Wnt11, Col1, Col3, MMP9, TGF-β1, α-SMA, and p-smad2/3 expression in heart tissue from TAC or sham mice pre-injected with sh-Wnt5a/Wnt11-AAV9 (sh-Wnt5a/Wnt11) or sh-scramble-AAV9 (sh-NC) by tail vein. **p < 0.01; ***p < 0.001. n = 4-6 mice/each group. (E), Western blot analysis of Col1, MMP2, MMP9, TGF-β1 and α-SMA expression in CFs stimulated with the culture medium from control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6), Wnt5a, Wnt11, Wnt5a+Wnt11 at 10 ng/mL, or PBS were supplemented with the culture medium from Ad-LRP6-CMs under MS and treated with…

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  Figure 6. Cardiomyocyte-specific overexpression of LRP6 promotes the degradation of Wnt5a and Wnt11 by interaction with LRP6 and CTSD during pressure overload. (A), Western blot analysis of LRP6 and CTSD expression in tamoxifen-injected LRP6CAG/MCM (LRP6 Over) or -MCM (MCM) mice at 4 weeks after TAC or sham operation. *p < 0.05; **p < 0.01; n = 4 mice/each group. (B), Western blot analysis of LRP6 and CTSD expression in control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6) with or without MS. ***p < 0.001; n = 4-6/each group. (C), Interaction of LRP6 and CTSD was analyzed by IP analysis in control (Ad-CON) or LRP6-overexpressing CMs (Ad-LRP6) with or without MS. M: marker. (D), Western blot analysis of Wnt5a, Wnt11, and CTSD expression in stretched control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6) after treatment with si-CTSD or si-scramble. *p < 0.05; ***p < 0.001; n = 3/each group. (E), Western blot analysis of Wnt5a and Wnt11 expression in stretched control (Ad-CON) or LRP6-overexpressing CMs (Ad-LRP6) after treatment with PBS or leupeptin (50 µg/ml). *p < 0.05; **p < 0.01; ***p < 0.001; n = 3 /each group.

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  Figure 7. Protease inhibitor attenuates the interaction of LRP6 and CTSD in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. (A), Schematic of the experimental procedure in (B). A protease inhibitor, leupeptin (40 mg/kg), was intraperitoneally injected into LRP6 Over or -MCM mice from 2 to 4 weeks (every other day) after TAC; PBS treatment was used as a control. (B), IP analysis of the interaction among LRP6, CTSD, Wnt5a, and Wnt11. The experiment was repeated for at least 3 times.

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  Figure 8. Protease inhibitor attenuates cardiac protection in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. (A), Western blot analysis of p-LRP6, LRP6, Wnt5a, Wnt11, Col1, Col3, MMP9 and TGF-β1 in the heart tissue. *p < 0.05; **p < 0.01; ***p < 0.001; n = 3/each group. (B), Masson staining of heart tissue and quantitative analysis of fibrosis area. The upper lane: bar = 2 mm; The lower lane: bar = 100 µm, *p < 0.05; **p < 0.01; n = 7-10 /each group. (C), Fraction shortening (FS) in mice. *p < 0.05; **p < 0.01; n = 8/ each group.

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  Figure 9. Mutants of LRP6 or CTSD inhibit the interaction of LRP6 and CTSD. (A), Sequencing results of LRP6 and CTSD mutations. (B), Schematic diagram of LRP6 and CTSD showing approximate locations of the mutants. (C), IP analysis of the interaction among LRP6 WT, LRP6 (P1104S), or LRP6 (P1427Q) and CTSD WT or CTSD (G136R). LRP6 WT and CTSD WT or their mutant plasmids were transfected into 293T cells for 48 h. The experiment was repeated 4 times. (D), Western blot analysis of LRP6, CTSD, Wnt5a, Wnt11, and MESD in stretched COS7 cells transfected with LRP6 WT and CTSD WT or their mutant plasmids as in (C). ****P < 0.0001, n = 3/each group.

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  Figure 10. Proposed model of cardiac protection mediated by cardiac-specific LRP6 overexpression in response to pressure overload. LRP6 overexpression suppresses β-catenin activation, which may contribute to the inhibition of cardiac hypertrophy. LRP6 overexpression inhibits cardiomyocyte-derived Wnt5a/Wnt11 to inhibit cardiac fibrosis. Cardiomyocyte-expressed LRP6 interacts with CTSD, which may promote the degradation of Wnt5a and Wnt11.