Impaired cardiomyocytes accelerate cardiac hypertrophy and fibrosis by delivering exosomes containing Shh/N-Shh/Gli1 in angiotensin II infused mice

Figures

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  Fig. 1. Inhibition of exosome release attenuates cardiac hypertrophy and fibrosis in Ang II-treated mice. (A) Quantitative data on the heart weight-to-body weight ratio (HW/BW) in the three groups of mice. (B) Graphical representation of the SBP, the DBP and the MAP of mice in different groups over four weeks. Data are presented as mean ± SEM (n = 6). ∗p ≤ 0.05 vs control; †p ≤ 0.05 vs Ang II. (C) Western blot analyses showing that DMA alleviates cardiac hypertrophy and fibrosis in Ang II-infused mice. Representative Western blot (C) and quantitative data for β-MHC (D), α-Actin (E), Fibronectin, Collagen I, α-SMA (F) are presented. ∗p ≤ 0.05 vs control; †p ≤ 0.05 vs Ang II (n = 6). (G) Representative micrographs showing cardiac hypertrophy and matrix deposition in Ang II-infused mice after DMA treatment. Scale bar, 50 μm. (H) Quantitative data on α-Actin, Collagen I (H), Masson (I) and WGA staining (J) in the heart tissue of the three groups of mice are presented. ∗p ≤ 0.05 vs control; †p ≤ 0.05 vs Ang II (n = 6).

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  Fig. 2. DMA alleviates Ang II-induced cardiac lesions associated with inhibition of exosome secretion and Shh upregulation. (A) Western blot analyses demonstrating that DMA inhibits the exosome secretion and activation of Shh signaling after Ang II infusion. Representative Western blot (A) and quantitative data on CD63 (B), TSG101 (C), Shh (D), N-Shh (E), Gli1 (F) are presented. ∗p ≤ 0.05 vs control; †p ≤ 0.05 vs Ang II (n = 6). (G) Representative micrographs of immunostaining demonstrating the upregulation of Shh in the heart tissue of the Ang II infusion model. Scale bar, 50 μm.

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  Fig. 3. Conditioned media from Ang II-treated cardiomyocytes promotes cardiomyocyte hypertrophy and cardiac fibroblast activation. (A) Experimental design. (B) Western blot analyses showing that protein levels of β-MHC and α-Actin in cardiomyocytes after incubation with conditioned media collected from Ang II-induced cardiomyocytes. (C–D) Quantitative data on β-MHC and α-Actin expression are shown for the indicated groups (n = 3). ∗p ≤ 0.05. (E) Western blot analyses showing Fibronectin and α-SMA expression in cardiac fibroblasts incubated with conditioned media. (F–G) Quantitative data showing the protein levels of Fibronectin and α-SMA in the different groups (n = 3). ∗p ≤ 0.05.

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  Fig. 4. Cardiomyocyte-derived exosomes induce the hypertrophy of cardiomyocytes and activation of cardiac fibroblasts. (A) We further treated cardiomyocytes and cardiac fibroblasts with exosomes isolated from the above-mentioned conditioned media for 24 h. (B) Transmission electron microscopy (TEM) image showing the exosomes isolated from the conditioned media of primary cardiomyocytes. Scale bar, 200 nm. (C–D) Graphical data showing the particle size and concentration of cardiomyocyte-derived exosomes in the different groups. Representative Western blot (E) and quantitative data on the protein levels of β-MHC (F) and α-Actin (G) are shown in different groups as indicated (n = 3). ∗p ≤ 0.05. (H) Micrographs confirming the immunofluorescence staining of β-MHC in cardiomyocytes treated with exosomes isolated from Ang II-induced cardiomyocytes. Arrows indicate positive staining. Scale bar, 50 μm. (I) Western blot analyses show Fibronectin and α-SMA expression in cardiac fibroblasts treated with exosomes separated from conditioned media. (J–K) Quantitative data on protein expression of Fibronectin and α-SMA are shown in different groups as indicated (n = 3). ∗p ≤ 0.05. (L) Representative…

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  Fig. 5. Blocking exosome secretion mitigates cardiac fibroblast activation and proliferation in vitro. (A) A schematic diagram outlines the experimental design. We employed conditioned media obtained from Ang II-induced primary cardiomyocytes with or without DMA to treat cardiac fibroblasts for 24 h. (B) Western blot analysis shows the expression of fibrosis-related proteins and proliferation markers in cardiac fibroblasts after incubation with conditioned media collected from Ang II-induced cardiomyocytes with or without DMA treatment. (C–F) Quantitative data showing Collagen I (C), Fibronectin (D), c-Myc (E) and PCNA (F) expression in the indicated groups. ∗p ≤ 0.05 vs sham control CM; †p ≤ 0.05 vs Ang II CM (n = 3).

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  Fig. 6. Exosomal Shh promotes cardiac fibroblast activation and proliferation in vitro. (A) Western blot analysis confirmed the presence of TSG101 and Shh signaling proteins in exosomes isolated from Ang II-induced cardiomyocytes. (B) Double immunofluorescence staining showing the colocalization of Shh and CD63 in Ang II-induced cardiomyocytes. Arrows indicate positive staining. Scale bar, 25 μm. (C) A schematic diagram outlines the experimental design. We treated cardiac fibroblasts with exosomes isolated from conditioned media for 24 h with or without CPN. (D) Western blot analysis illustrates the expression of fibrotic and proliferative proteins in cardiac fibroblasts incubated with cardiomyocyte-derived exosomes. (E–F) Quantitative data showing the protein expression of Collagen I, Fibronectin, c-Myc, and PCNA in the different groups are shown. ∗p ≤ 0.05 vs sham control Exo; †p ≤ 0.05 vs Ang II Exo (n = 3). Representative images (G) and quantitative data (H) show the Edu staining in the groups mentioned above. Scale bar, 100 μm ∗p ≤ 0.05 vs sham control Exo; †p ≤ 0.05 vs Ang II Exo (n = 3).