Cthrc1 deficiency aggravates wound healing and promotes cardiac rupture after myocardial infarction via non-canonical WNT5A signaling pathway

Figures

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  Figure 1. Serum levels of CTHRC1 were elevated in MI mice and CTHRC1 expression was increased in cardiac fibroblasts after MI. (A) Serum CTHRC1 levels in MI mice at day 7 after MI were measured by ELISA (n=5-6). (B) Two crucial phases of cardiac repair after MI. (C) Timeline of the experimental design for Figure 1. (D) CTHRC1 expression was analyzed by western blotting in the infarct zone, in the border zone, as well as in the remote zone of WT hearts at different time points post- MI. (E and F) Quantitative analyses of western blotting band intensity in D (n=4-10). (G and H) CTHRC1 mRNA expression was determined in the infarct and the border areas of WT MI-operated hearts and sham controls at different time points post-MI (n=5-10). (I) Representative immunohistochemistry analyses of CTHRC1 in both sham-operated and WT post-MI mice, including the infarct zone, the border zone, as well as the remote zone (bar=50 μm). (J) Immunofluorescence co-staining for CTHRC1 with vimentin in WT hearts at day 7 after MI (bar=50 μm). (K and L) Primary cardiac fibroblasts were isolated from WT hearts at day 7 post-MI, and CTHRC1 protein expression was examined and quantified (n=3). Data are presented as…

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  Figure 2. Induction of CTHRC1 by canonical TGFβ1-Smad2/3 signaling axis in cardiac fibroblasts. (A and B) Immunoblotting of primary cardiac fibroblasts stimulated with serial concentrations of TGFβ1 for 24 h (n=3). (C and D) CTHRC1 up-regulation induced by TGFβ1 (10 ng/ml) in primary cardiac fibroblasts isolated from WT hearts at different time points (n=4). (E) CTHRC1 up-regulation induced by TGFβ1 (10 ng/ml) in primary cardiac fibroblasts isolated from neonatal mouse hearts at different time points. (F-I) Immunoblot analyses of primary cardiac fibroblast treated with TGFβ1 (10 ng/ml) for 24 h, in the presence of LY2109761 (10 μM, F and G) or (E)-SIS3 (10 μM, H and I) (n=3). Data are presented as mean±SEM. *P<0.05. **P<0.01. ****P<0.0001. NS: not significant. Data in B, D, G, and I were analyzed using one-way ANOVA followed by Bonferroni multiple comparison test.

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  Figure 3. CTHRC1 improved wound healing and promoted cardiac fibroblast activation in vitro. (A and B) The migratory capacity of cardiac fibroblasts isolated from WT hearts was assessed by the covering area of the scratch after 24 h (n=3, bar=200 μm). (C and D) α-SMA expression was detected by western blotting in primary cardiac fibroblasts following stimulation with rCTHRC1 (1 μg/ml) or TGFβ1 (10ng/ml) for 24h (n=4). (E and F) α-SMA immunofluorescence staining of primary cardiac fibroblasts treated with rCTHRC1 (1 μg/ml) for 24h was used to evaluate cardiac fibroblast activation (n=3, bar=50 μm). Data are presented as mean±SEM. *P<0.05. ***P<0.001. NS: not significant. Data were analyzed using one-way ANOVA followed by Bonferroni multiple comparison test (D) and unpaired two-tailed Student's t test (B and F).

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  Figure 4. Cthrc1 deficiency aggravated cardiac function after MI. (A) Timeline of the experimental design for Figure 4 and 5. (B and C) Representative echocardiograms obtained from WT and C1KO mice at day 1 (B) and day 14 (C) post-MI or sham operation. (D) Echocardiographic assessment of cardiac function in WT and C1KO mice at day 1 and day 14 post-MI or sham surgery (n=4-8). (E) Representative macroscopic view of heart from MI-operated mice at day 14 after MI. (F) Heart weight to body weight ratio (HW/BW) in WT and C1KO mice subjected to MI for 14 days were analyzed (n=4-8). Data are presented as mean±SEM. *P<0.05. **P<0.01. ***P<0.001. NS: not significant. Data in D and F were compared using two-way ANOVA followed by Bonferroni multiple comparison test.

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  Figure 5. Ablation of Cthrc1 exacerbated MI-induced cardiac rupture and reduced collagen-rich scar formation after MI. (A) Kaplan-Meier survival analysis of mice at different time points after MI. (B) Total cardiac rupture frequency in WT (n=10) and C1KO (n=14) at day 14 after MI. (C) Representative macroscopic view of a hemothorax (left), the presence of blood clots (middle) in the chest cavity and/or in the pericardium, and masson's trichrome staining (right) of cardiac rupture in C1KO mice at day 5 post-MI. (D) Masson's trichrome staining performed on serial heart transverse sections obtained from WT and C1KO mice at day 7 after LAD ligation. (E) Quantitative assessment of infarct size as well as wall thickness at day 7 after MI in WT and C1KO mice (n=6-9). (F) Masson's trichrome (TC) and picrosirius red (PSR) staining performed on heart cross sections at apical as well as papillary level to examine collagen density in the scar of WT and C1KO mice at day 14 after MI. (G) Quantification of fibrotic areas at day 14 after MI in WT and C1KO mice (n=6-7). Data are presented as mean±SEM. *P<0.05. **P<0.01. ****P<0.0001. NS: not significant. Survival rate was calculated by the Kaplan-Meier…

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  Figure 6. Absence of Cthrc1 impaired wound repair after MI. (A) Timeline of the experimental design for Figure 6-7. (B and C) Representative immunoblots (B) and summarized data (C) of MMP2 as well as MMP9 expression in the infarct areas of WT and C1KO hearts at day 7 after MI or post-sham operation (n=3-6). (D and F) α-SMA (D) and CD31 (F) protein levels were detected in the infarcted heart tissues from WT and C1KO mice after sham operation or day 7 post-MI. (E and G) Quantified data of western blotting band intensity in D and F (n=3-6). (H) Gene expression of collagen I and collagen III were analyzed by qRT-PCR in the infarct zones of WT and C1KO hearts at day 7 after MI (n=3-5). (I and J) Immunofluorescence analyses of collagen I, collagen III, and α-SMA (I) as well as CD31 (J) in the scar tissues obtained from WT and C1KO mice. Data are presented as mean±SEM. *P<0.05. **P<0.01. ***P<0.01. NS: not significant. Data in C, E, G, and H were compared using two-way ANOVA followed by Bonferroni multiple comparison test.

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  Figure 7. CTHRC1 improved cardiac repair after MI via selectively activating non-canonical WNT5A-PCP signaling pathway. (A-D) Western blot analyses for the indicated proteins of non-canonical WNT5A-PCP signaling axis (A and B) and canonical WNT3A-β-catenin signaling pathway (C and D) extracted from the infarcted heart tissues of WT and C1KO mice at day 7 after MI (n=3-6). (E-H) Western blotting of primary cardiac fibroblasts treated with rCTHRC1 (1 μg/ml) for 1h to detected the protein levels of non-canonical WNT5A-PCP signaling axis (E and F) and canonical WNT3A-β-catenin signaling pathway (G and H) (n=5). (I) Immunofluorescence co-staining for CTHRC1 with FZD3, FZD6, ROR2, or WNT5A in primary cardiac fibroblasts followed by treatment with TGFβ1 (10 ng/ml) for 24 h (bar=20 μm). Data are presented as mean±SEM. *P<0.05. **P<0.01. ***P<0.001. NS: not significant. Data were compared using two-way ANOVA followed by Bonferroni multiple comparison test (B and D) and unpaired two-tailed Student's t test (F and H).

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  Figure 8. Cthrc1 loss-induced cardiac rupture after MI was partly reversed by rWNT5A or rCTHRC1 protein. (A) Timeline of the experimental design for Figure 8B-8H. (B) Total cardiac rupture frequency in C1KO mice with PBS (n=10) and with rWNT5A (n=9) at day 7 post-MI. (C) Picrosirius red staining performed on heart cross sections to examine collagen density in the scar of C1KO mice with PBS and with rWNT5A at day 7 after MI (bar=100 μm). (D) Quantitative analysis of fibrotic areas at day 7 after MI in C1KO and rWNT5A-treated mice (n=3). (E and F) α-SMA expression was analyzed by western blotting in the infarct zone of C1KO hearts at day 7 post-MI (n=3-4). (G and H) Western blot analyses for the indicated proteins of non-canonical WNT5A-PCP signaling axis in the infarct area of C1KO hearts at day 7 post-MI (n=3-4). (I) Timeline of the experimental design for Figure 8J-8P. (J) Total cardiac rupture frequency in C1KO mice with PBS (n=10) and with rCTHRC1 (n=10) at day 7 post-MI. (K) Picrosirius red staining performed on heart cross sections to examine collagen deposition in the scar of C1KO mice with PBS and with rCTHRC1 at day 7 post-MI (bar=100 μm). (L) Quantification of fibrotic areas at…

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  Figure 9. Schematic diagram depicting the role of CTHRC1 in cardiac repair after MI. TGFβ1 can be rapidly activated following MI. Active TGFβ1 binds to TGFβ receptors I/II, further promoting the phosphorylation of Smad2/3. Smad4 then binds to activated Smad2/3, which enables this complex to translocate to the nucleus and transcribe Cthrc1 genes. CTHRC1 secreted by cardiac fibroblasts after intracellular synthesis and glycosylation modification acts on other adjacent cardiac fibroblasts in a paracrine manner. CTHRC1 selectively activates the non-canonical WNT5A-PCP signaling pathway by forming a stabilized CTHRC1-WNT5A-FZD3/6-ROR2 complex to enhance the interaction of WNT5A with FZD3/6-ROR2, further boosting the migration and activation of cardiac fibroblasts in the infarcted tissue post-MI and increasing collagen-rich scar formation after MI, thereby improving post-MI cardiac repair and preventing cardiac rupture.