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

Periostin plays a critical role in tissue regeneration and homeostasis. The aim of this study was to evaluate the changes in periostin levels in the hearts of rats with experimental autoimmune myocarditis (EAM). Western blot analysis revealed that the expression levels of periostin and alpha-smooth muscle actin were significantly increased at day 14 post-immunization. Immunohistochemical analysis indicated that periostin was expressed in macrophages and fibroblasts in the hearts of EAM-induced rats. In conclusion, these results suggest that increased periostin expression in macrophages and fibroblasts promotes cardiac fibrosis in EAM-induced rats, potentially by enhancing immune cell infiltration. Therefore, periostin should be further investigated as a candidate therapeutic target for myocarditis.

Immunohistochemistry was performed using the avidin-biotin complex method (Vectastain Elite ABC Kit; Vector Laboratories) as described in our previous study [13]. Rabbit anti-periostin and mouse anti-αSMA were used as primary antibodies. Sections were then incubated with the appropriate biotinylated secondary antibody. Peroxidase activity was determined using diaminobenzidine (DAB) substrate (Vector Laboratories).
The enlarged heart of EAM-induced rat was shown the discolored surface, compared to that of normal control (Fig. 1A). The body weight of EAM-affected rats was significantly lower, commencing on D10PI (P<0.05), than that of normal control rats (Fig. 1B). Although inflammatory cells were absent in the hearts of control rats ( Fig. 2A), the hearts of EAM-affected rats were infiltrated with inflammatory cells at D14PI ( Fig. 2B and 2C). At D28PI, fibrotic lesions were evident in the hearts of EAMinduced rats, while some inflammatory cells were still present ( Fig. 2D and 2E). The intensity of Masson's trichrome stain was higher in the hearts of EAM-induced rats at D14PI (  significantly higher compared with those in the hearts of control rats (1.00 ± 0.07, P<0.05; Fig. 3A and 3B). The expression levels of periostin were further increased during EAM progression (D21PI, 6.17 ± 1.12, P<0.01 vs. control; D28PI, 9.48 ± 1.86, P<0.001 vs. control). Furthermore, the expression pattern of αSMA was similar to that of periostin during EAM progression ( Fig. 3A and 3C). Immunohistochemical stainings revealed that periostin was expressed at low levels in fibroblasts of control rats (Fig. 4A). On the other hand, periostin was strongly expressed in inflammatory cells and the fibrotic lesions (including the fibrotic stroma and fibroblasts) of EAM-induced rats on D14PI (Fig. 4B-D). Compared to the hearts of control rats, periostin was expressed at higher levels in the fibrotic lesions of the hearts of test animals on D28PI (Fig. 4E and 4F). To assess the relationship between the levels of periostin and the representative fibrosis marker αSMA, we performed immunohistochemical staining of rat hearts. Although αSMA-positive cells were scarce in control hearts (Fig. 4G), the number of αSMA-positive fibroblasts in the hearts increased from D14PI to D28PI (Fig. 4H-K).
To gain further insight into the phenotype of cells infiltrating the hearts of EAM-induced rats, we conducted double immunofluorescence staining in the hearts taken at D14PI. Periostin-IB4 double-positive cells (arrowheads in Fig. 5A-C) were observed in inflammatory lesions, and ED1-positive macrophages were co-localized with periostin-positive cells (arrows in Fig. 5D-F). Furthermore, the periostin-expressing cells were co-localized with αSMA-positive fibroblasts (hollow arrows in Fig. 5G-I). These results suggest that fibroblasts and inflammatory cells are the main cell types expressing periostin in the fibrotic lesions of rats with EAM.
Although periostin has been implicated in various autoimmune diseases including peripheral polyneuropathy and myocardial infarction of mice [2,6], this is the first study to show that periostin is upregulated in the hearts of EAM-induced rats, which model human myocarditis. Moreover, we found that periostin and αSMA, both of which are secreted by fibroblasts, were co-localized in the hearts of EAM-induced animals. These results suggest that upregulation of the extracellular matrix protein periostin is associated with early infiltration of inflammatory cells and subsequent fibrosis in autoimmune heart failure (Fig. 6).
We found that EAM-associated periostin upregulation was predominantly confined to inflammatory cells, consistent with our previous work showing that periostin levels significantly increased in spinal cord inflammatory cells and fibroblasts of mice with experimentally induced autoimmune encephalomyelitis [6]. EAM initiation is associated with heart infiltration of macrophages 1545-1550, 2020 and autoreactive T cells [4]. After the acute inflammatory stage, excessive activation of cardiac fibroblasts triggers fibrosis and subsequent cardiac dysfunction [16]. Periostin neutralization or knockdown reduced glioma stem cell survival and suppressed cellular invasion and growth [18]. Thus, periostin upregulation in the heart may indicate inflammatory cell infiltration and increased risk for myocarditis. However, the precise mechanism underlying periostin-mediated fibrosis in the hearts of EAM-affected rats 1545-1550, 2020    requires further investigation.
In the present study, we found that αSMA levels were increased in EAM fibrotic lesions. αSMA is widely used as a marker for fibroblast activation, and its expression is induced in myocardial infarction by Prrx2-Wnt5a signaling and transforming growth factor (TGF)-β [3]. Furthermore, TGF-β and Smad signaling play important roles in fibrosis by regulating fibroblast activation and matrix deposition [17]. Notably, in an inflammatory dilated cardiomyopathy mouse model, TGF-β/Smad signaling induced myofibroblast development during heart tissue remodeling [5]. TGF-β has also been shown to promote periostin expression in primary osteoblasts [9]. Hence, we believe that αSMA upregulation in cardiac fibroblasts is essential for cardiac remodeling and fibrosis.
In conclusion, we demonstrated that periostin and αSMA are upregulated in the hearts of EAM-induced rats during disease initiation and progression. Our findings also suggest that periostin expression in infiltrating inflammatory cells and αSMA-positive fibroblasts plays an important role in cardiac fibrosis. Thus, periostin is a promising therapeutic target for myocarditis.
ACKNOWLEDGMENT. This work was supported by the 2020 education, research and student guidance grant funded by Jeju National University.