Immunohistochemical analysis of periostin in the hearts of Lewis rats with experimental autoimmune myocarditis

Figures

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  Fig. 1.. The representative photos and body weights of normal control and experimental autoimmune myocarditis (EAM)-induced rats. (A) Heart of experimental autoimmune myocarditis (EAM)-induced rats were shown the discolored surface and enlarged, compared to that of normal controls. (B) The body weight significantly decreased in EAM-induced rats from day 10 post-immunization, compared to that of normal controls. Rats were weighed daily. *P<0.05 vs. control. (A) Scale bar, 1 cm.

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  Fig. 2.. Histopathological evaluation of the fibrosis in heart of normal (A) and experimental autoimmune myocarditis (EAM)-affected rats (B and D). The normal rat heart contained no inflammatory cells (A), but such cells were evident in the EAM-affected heart (B and D). The insets (C and E) show inflammatory cells under high magnification. Fibrotic lesions (arrowheads indicating blue in F−H; arrows indicating red in K) were detected in the hearts of EAM-affected rats. (A–E) Hematoxylin-and-eosin staining. (F–H) Masson’s Trichrome staining. (I−K) Sirius Red staining. HE, Hematoxylin-and-eosin staining; D14PI, day 14 post-immunization; D28PI, day 28 post-immunization. (A, B, D, F, G, H, I, J and K) Scale bars, 100 µm. (C and E) Scale bars, 20 µm.

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  Fig. 3.. Western blot analysis of periostin and alpha-smooth muscle actin (αSMA) protein levels in the hearts of control and experimental autoimmune myocarditis (EAM)-induced rats (n=3 per group). (A) Representative immunoblot showing periostin (~100 kDa), alpha smooth actin (αSMA; 43 kDa), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 37 kDa) protein levels. Bar graphs show the quantification of periostin (B) and αSMA (C) protein levels normalized to GAPDH. Periostin and αSMA protein levels were significantly increased in the hearts of experimental autoimmune myocarditis (EAM)-induced rats. *P<0.05, **P<0.01, ***P<0.001 vs. control.

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  Fig. 4.. Immunohistochemical staining of periostin and alpha-smooth muscle actin (αSMA) in the hearts of control and experimental autoimmune myocarditis (EAM)-induced rats. Periostin expression was detected in some inflammatory cells at day 14 post-immunization (C) and in fibroblasts (D and F) of EAM-affected hearts. However, only fibroblasts expressed alpha-smooth muscle actin (αSMA) in the hearts of EAM-affected rats (I and K). Tissues were counterstained with hematoxylin. (A, B, E, G, H, and J) Scale bars, 40 µm. (C, D, F, I, and K) Scale bars, 20 µm.

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  Fig. 5.. Double immunofluorescence staining for periostin and either isolectin-B4 (IB4), ED1 or alpha-smooth muscle actin (αSMA) in experimental autoimmune myocarditis (EAM)-affected hearts (A–I). Periostin-positive inflammatory cells co-expressed isolectin-B4 (IB4) in EAM-affected hearts (arrowheads in A–C). Notably, macrophages doubly stained for periostin and ED1 (arrows in D–F). Some cardiac fibroblasts co-localized with αSMA and periostin (hollow arrows in G–I). (A–I) Scale bars, 20 µm.

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  Fig. 6.. Schematic illustration of periostin-mediated fibrosis in the hearts of experimental autoimmune myocarditis (EAM)-affected rats. When EAM is induced, macrophages infiltrate the heart. Infiltrated inflammatory cells secrete periostin, which activates cardiac fibroblasts and further promotes inflammatory cell infiltration. Activated fibroblasts express high levels of alpha-smooth muscle actin (αSMA) and periostin. Periostin accumulation results in the formation of fibrotic lesions in the heart, which could lead to heart failure.