Angiotensin II receptor 1 controls profibrotic Wnt/β-catenin signalling in experimental autoimmune myocarditis

Abstract

Aims: Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis.

Methods and results: EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts.

Conclusion: Ang II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway.

Keywords: Angiotensin II; Angiotensin II receptor 1; Cardiac fibrosis; Experimental autoimmune myocarditis; Inflammatory cells; TGF-β signalling; Wnt.

Graphical abstract

Figure 1

EAM was induced in wild-type (WT) and in Agtr1a^-/- mice by αMyHC/CFA immunization at day 0 and 7. Representative histology (H/E) and immunohistochemistry for CD45, CD3, and F4/80 in hearts of indicated recipients at day 21 (inflammatory phase) are presented in (A). Scale bar = 100 μm. Myocarditis severity scores and quantification of heart-infiltrating CD3⁺, CD45⁺, and F4/80⁺ cells in WT (n = 5) and Agtr1a^-/- (n = 5) mice are shown in (B). t-SNE plot presenting cardiac CD45⁺-gated cell subsets identified by flow cytometry (gating strategy shown in FigureS 2) and quantification of the indicated subsets in hearts of WT (n = 6) and Agtr1a^-/- (n = 6) mice at day 21 of EAM are shown in (C). Myocarditis severity scores of vehicle- (control, n = 6) or telmisartan-treated (n = 7) Agtr1a^-/- mice at day 21 are presented in (D). Expression of selected profibrotic genes in cardiac tissue at day 21 is shown in (E). p values calculated with Mann–Whitney U test or unpaired Student’s t-test.

Figure 2

EAM was induced in wild-type (WT) and in Agtr1a^-/- mice by αMyHC/CFA immunization at day 0 and 7. Representative Masson’s Trichrome staining and immunohistochemistry for periostin and vimentin in hearts of indicated recipients at day 40 (fibrotic phase) are shown in (A). Scale bar = 100 μm. Quantifications of positive signals for WT (n = 10) and Agtr1a^-/- (n = 10) mice are presented in (B). Panel (C) shows hydroxyproline contents in cardiac tissue of WT (n = 7) and Agtr1a^-/- (n = 7) mice at day 40. Echocardiography was performed on WT and Agtr1a^-/- mice at day 0 and day 40. Panel (D) shows the differences (day 40–day 0) of measured ejection fraction (EF), fractional shortening (FS), and cardiac output (CO) for WT (n = 7) and Agtr1a^-/- (n = 7) mice. Heart weight/tibia length (HW/TL) ratio of WT (n = 7) and Agtr1a^-/- (n = 7) measured at day 40 are presented in (E). P values for (C–E) calculated with unpaired Student’st-test. Bone marrow (BM) chimeric mice were generated by lethal irradiation of the recipients followed by transplantation of the donor BM. About 6 weeks after BM transplantation, chimeric mice were immunized with αMyHC/CFA, and heart sections were analysed at day 40 of EAM. Panel (F) shows quantifications of Masson’s Trichrome staining and immunopositive signals for periostin and vimentin in the indicated chimeric mice (n = 4–9).p values calculated with one-way ANOVA followed by multiple comparison using the Fisher’s LSD test. *p < 0.05 (post-hoc test).

Figure 3

Inflammatory cells were isolated from hearts of wild-type mice at day 17–21 of EAM, expanded, and stimulated with TGF-β1 (10 ng/mL). Panel (A) shows expression of genes involved in Ang II synthesis in unstimulated cells and treated with TGF-β for 24 h (n = 4). A kinetic of angiotensinogen (Agt) expression is shown in panel (B) (n = 3–4). p value calculated with one-way ANOVA. Panel (C) shows immunoblots of enzymes involved in Ang II production. Quantifications of the respective protein levels (normalized to GAPDH) are presented in panel (D) (n = 7–8). *p < 0.05 (vs. control) calculated with Kruskal–Wallis followed by Dunn’s test. Levels of Ang II measured with mass spectroscopy in cell lysate (n = 4–5) and in supernatants (n = 5) are shown in panel (E). p values calculated with one-way ANOVA followed by multiple comparison using the Fisher’s LSD test. *p < 0.05 (post-hoc test).

Figure 4

Inflammatory cells were isolated from hearts of wild-type mice at day 17–21 of EAM, expanded, and stimulated with TGF-β1 (10 ng/mL) for up to 72 h. Panel (A) shows immunoblots of profibrotic α-SMA, TGF-β downstream molecules (Smad, pSmad, TAK1, pTAK1), and G proteins (Gα12 and Gα13). Quantifications of protein levels (normalized to GAPDH) of α-SMA (n = 6–8), pSmad/Smad2 (n = 7–8), pTAK1/TAK1 (n = 7), Gα12 (n = 9–10), and Gα13 (n = 10–11) are shown in panel (B). *p < 0.05 (vs. control) calculated with Kruskal–Wallis followed by Dunn’s test.

Figure 5

Inflammatory cells were isolated from hearts of wild-type (WT) and Agtr1a^-/- mice at day 17–21 of EAM, expanded, and stimulated with TGF-β1 (10 ng/mL). Representative immunofluorescences of α-SMA and fibronectin of wild-type and Agtr1a^-/- cells treated with TGF-β for 14 days are presented in panel (A). Data are representative of three independent experiments. Scale bar = 20 µm. Panel (B) shows transcriptional response to TGF-β (24 h) of WT (white, n = 4) and Agtr1a^-/- (black, n = 5) cells. p values calculated with unpaired Student’s t-test, * p < 0.05. Panel (C) shows immunoblots of indicated proteins in wild-type and Agtr1a^-/- cells unstimulated or stimulated with TGF-β. Quantifications of protein levels (normalized to GAPDH or β-tubulin) of pSmad/Smad2 (n = 4–5), pTAK1/TAK1 (n = 6), and α-SMA (n = 6) are presented in panel (D). p values calculated with unpaired Student’s t-test.

Figure 6

Inflammatory cells were isolated from hearts of wild-type (WT) and Agtr1a^-/- mice at day 17–21 of EAM, expanded, and stimulated with TGF-β1 (10 ng/mL) for 30 min or 24 h. Panel (A) shows anti-RhoA immunoblots of purified GTP-bound Rho protein extracts (RhoA-GTP) and total cell lysates (total RhoA). Panel (B) shows anti-RhoA and anti-AKAP-Lbc immunoblots (IB) of immunoprecipitated (IP) protein extracts with anti-AKAP-Lbc antibodies. Anti-RhoA immunoblots of the whole cell extracts are presented in the bottom panel. Panel (C) shows quantifications of normalized RhoA-GTP levels (normalized to total RhoA, left, n = 4) and amount of RhoA immunoprecipitated with anti-AKAP-Lbc antibodies (normalized to IB AKAP-Lbc, right, n = 3). p values calculated with unpaired Student’s t-test.

Figure 7

Inflammatory cells were isolated from hearts of wild-type (WT) and Agtr1a^-/- mice at day 17–21 of EAM, expanded, and stimulated with TGF-β1 (10 ng/mL). Panel (A) shows GSK3β activity in response to TGF-β (n = 4–6). * p < 0.05 (vs. control) calculated with Kruskal–Wallis followed by Dunn’s test. Cellular localizations of β-catenin in control and TGF-β-treated (1 h) cells are presented in panel (B). DAPI staining visualizes cell nuclei. Arrows indicate nuclear localization of β-catenin. Scale bar = 10 µm. Data are representative of three independent experiments. Panel (C) shows immunohistochemistry for β-catenin (brown) in hearts at day 21 of EAM (inflammatory phase). Scale bar = 100 μm. Pictures are representative for at least three WT and three Agtr1a^-/-mice. Expression of selected Wnt genes in the indicated cardiac tissues at day 21 is shown in (D, n = 4–5). p values calculated with unpaired Student’s t-test.