Myeloid MyD88 Mediates Macrophage Infiltration and Activation in Ang II ‐Induced Cardiac Hypertrophy

Figures

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  FIGURE 1. Ang II treatment increases the expression of MyD88 in mouse heart tissues. C57BL/6 mice were continuously challenged with Ang II (1 μg/kg/min) or normal saline using micro‐osmotic pumps for 4 weeks. (A) Protein levels of MyD88 in heart tissues from normal (Ctrl) and Ang II‐treated (Ang II) mice were measured using Western blot analysis. GAPDH was used as the loading control. Representative Western blot images (left panel) and densitometric quantifications (right panel) are shown. (B–D) The cellular distribution of MyD88 in heart tissues from normal and Ang II‐infused mice was determined using the immunofluorescence assay. Representative immunofluorescence images of MyD88 (red) in α‐actinin staining‐positive cardiomyocytes (green), vimentin staining‐positive fibroblasts (green), and CD68 staining‐positive macrophages (green) are shown. Sections were counterstained with DAPI (blue). Scale bar = 20 μm. Statistical data were presented as mean ± SEM, n = 5; **p < 0.01 compared to the control group (Ctrl).

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  FIGURE 2. Cardiac‐specific knockout of MyD88 exhibits no significant therapeutic effect on Ang II‐induced cardiac hypertrophy. MyD88f/f and MyD88f/f Myh6‐Cre mice were continuously challenged with Ang II (1 μg/kg/min) or normal saline using micro‐osmotic pumps for 4 weeks. (A) Representative images of heart tissues. (B) Haematoxylin & Eosin staining showing myocardial fibre arrangements in heart tissues [scale bar = 30 μm]. (C) WGA‐FITC staining showing the cross‐sectional area of cardiomyocytes in heart tissues [scale bar = 20 μm]. (D) Masson's staining showing the interstitial fibrotic area in heart tissues [scale bar = 30 μm]. (E) Sirius Red staining showing the heart tissue fibres [scale bar = 30 μm]. (F) Anti‐F4/80 staining showing macrophage infiltration in heart tissues [scale bar = 30 μm]. (G–I) RT‐qPCR analysis of Il1b (G), Il6 (H) and Tnfa (I) in heart tissues. Data were presented as mean ± SEM, n = 6; **p < 0.01 compared to the MyD88f/f group; ns = not significant, compared to the MyD88f/f + Ang II group.

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  FIGURE 3. Macrophage‐specific knockout of MyD88 attenuates Ang II‐induced cardiac hypertrophy. MyD88f/f and MyD88f/f Lyz2‐Cre mice were continuously challenged with Ang II (1 μg/kg/min) or normal saline using micro‐osmotic pumps for 4 weeks. (A) Representative images of heart tissues. (B) Haematoxylin & Eosin staining showing myocardial fibre arrangements in heart tissues [scale bar = 30 μm]. (C) WGA‐FITC staining showing the cross‐sectional area of cardiomyocytes in heart tissues [scale bar = 20 μm]. (D) Masson's staining showing the interstitial fibrotic area in heart tissues [scale bar = 30 μm]. (E) Sirius Red staining showing the heart tissue fibres [scale bar = 30 μm]. (F) Anti‐F4/80 staining showing macrophage infiltration in heart tissues [scale bar = 30 μm]. (G–I) RT‐qPCR analysis of Il1b (G), Il6 (H), and Tnfa (I) in heart tissues. Data were presented as mean ± SEM, n = 5; *p < 0.05; **p < 0.01, compared to the MyD88f/f group; # p < 0.05 compared to the MyD88f/f + Ang II group.

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  FIGURE 4. MyD88 inhibitor ameliorates Ang II‐induced cardiac hypertrophy in vivo. C57BL/6 mice were continuously challenged with Ang II (1 μg/kg/min) using micro‐osmotic pumps for 4 weeks. Three weeks after Ang II infusion, mice were administered with MyD88 inhibitor, LM8 (5 or 10 mg/kg), or vehicle control (CMC‐Na) via oral gavage daily for 2 weeks. (A) Representative images of heart tissues. (B) Haematoxylin & Eosin staining showing myocardial fibre arrangements in heart tissues [scale bar = 30 μm]. (C) WGA‐FITC staining showing the cross‐sectional area of cardiomyocytes in heart tissues [scale bar = 20 μm]. (D) Masson's staining showing the interstitial fibrotic area in heart tissues [scale bar = 30 μm]. (E) Sirius Red staining showing the heart tissue fibres [scale bar = 30 μm]. (F) Anti‐F4/80 staining showing macrophage infiltration in heart tissues [scale bar = 30 μm]. (G–I) RT‐qPCR analysis of Il1b (G), Il6 (H) and Tnfa (I) in heart tissues. Data were presented as mean ± SEM, n = 6; ***p < 0.001, compared to the Ctrl group; # p < 0.05; ## p < 0.01; ### p < 0.001, compared to the Ang II group.

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  FIGURE 5. MyD88 mediates Ang II‐induced macrophage infiltration via regulating chemokine secretion. (A) RT‐qPCR analysis of inflammatory chemokines and cytokines in heart tissues from MyD88 cardiac‐specific knockout (upper panel) and MyD88 macrophage‐specific knockout mice (lower panel) with or without Ang II treatment. (B) RT‐qPCR validation of Cxcl1 and Ccl2 mRNA levels in heart tissues of Ang II‐challenged mice with or without LM8 treatment. (C) RT‐qPCR analysis of Cxcl1 and Ccl2 mRNA levels in MyD88‐deficient macrophages challenged with 1 μM Ang II for 6 h. (D) Macrophage adhesion experiments. Macrophages derived from MyD88f/f Lyz2‐Cre mice were incubated with CFSE for 30 min, followed by stimulation with 1 μM Ang II for 6 h. The stimulated MyD88‐deficient macrophages were added to the cultured H9c2 cells for 4 h. The non‐attached macrophages were washed three times with PBS. The attached macrophages were measured by detecting CFSE immunofluorescence [scale bar = 100 μm]. (E) Representative Western blot images of the NF‐κB pathway in cardiac tissues from MyD88f/f Lyz2‐Cre mice infused with Ang II. The right panel shows the densitometric quantifications. (F) Representative Western blot…

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  FIGURE 6. Conditioned medium from MyD88‐deficient macrophages fails to induce cardiomyocyte hypertrophy. (a) Schematic illustration of the experimental design of the conditioned medium study. Primary macrophages derived from MyD88f/f and MyD88f/f Lyz2‐Cre mice were treated with LPS (50 μg/mL) or PBS for 24 h. The cell culture medium was collected as the conditioned medium. H9c2 cells were treated with different conditioned medium for 24 h. (b) The protein levels of β‐Myhc, Col‐1, and TGF‐β in primary cardiomyocytes were evaluated using Western blot analysis. Representative images (upper panel) and the densitometric quantifications (lower panel) are shown. (c) Representative images of Rhodamine phalloidin staining in H9c2 cells [scale bar = 30 μm] (left panel). The statistical quantification of cellular hypertrophy was shown (right panel). (d) Schematic demonstrating mechanisms involved in Myeloid MyD88 mediates macrophage infiltration and activation in Ang II‐induced cardiac hypertrophy. Statistical data were presented as mean ± SEM, n = 3 for in vitro data; *p < 0.05 compared to group 1; # p < 0.05 compared to group 2.