Rho kinase inhibitor Y-27632 downregulates IL-1β expression in mice with experimental autoimmune myocarditis

Abstract

Autoimmune myocarditis is the limited or diffuse inflammation of the myocardium due to dysfunctional cellular and humoral immunity mechanisms. We constructed mouse models of experimental autoimmune myocarditis (EAM) using peptide MyHC-α614-629. On the day after secondary immunization, the mice were intraperitoneally injected with Rho kinase (ROCK) inhibitor Y-27632. On day 21, the cardiac tissues were harvested and weighed. The hearts of EAM mice were significantly enlarged and whitened. Furthermore, body weight (BW) slowly increased during the treatment period, the heart weight (HW) and the ratio of HW/eventual BW were increased, and inflammatory infiltration and fibrosis were aggravated in the myocardial tissue. Y-27632 treatment improved the aforementioned phenotypic and pathological features of EAM mice. Mechanistic analysis revealed a significant increase in Notch1, Hes1, Jag2, Dil1, Toll-like receptor (Tlr) 2, and interleukin (IL)-1β expression in the myocardial tissue of EAM mice. Notably, IL-1β expression was correlated with that of Notch1 and Tlr2. Following Y-27632 treatment, the expression of key target genes of the Notch signaling pathway (Notch1, Hes1, Dil1, and Jag2) and Tlr2 were obviously decreased. Y-27632 treatment also decreased the number of monocytes in the spleen of EAM mice. Thus, ROCK inhibitor Y-27632 exerted a protective effect in EAM mice by downregulating IL-1β expression. This study aimed to provide a reference point for the future treatment of myocarditis in clinical settings.

Keywords: Experimental autoimmune myocarditis; IL-1β; Notch/TLR pathway; Y-27632.

Figure 1

Comparison of BW, HW, and HW/e-BW of mice in the EAM, control, Y-27632 2HCl, and saline groups. (A) process of mouse model construction. (B) representative photographs of mouse hearts in the EAM and control groups. (C) HW of mice in the control, EAM, saline, and Y-27632 2HCl groups. n = 10, *P < 0.05, ***P < 0.001. (D) BW of mice in the control, EAM, saline, and Y-27632 2HCl groups. n = 10, ***P < 0.001. (E) HW/e-BW of mice in the control, EAM, saline, and Y-27632 2HCl groups. n = 10, ***P < 0.001.

Figure 2

Changes in myocardial tissue fibrosis in mice in the EAM, control, Y-27632 2HCl, and saline groups. (A) representative plots of mouse hearts in the Y-27632 2HCl and saline groups. (B) representative plots of Masson trichrome staining of mouse myocardial tissues. (C) proportion of myocardial tissue fibrosis in mice in the control, EAM, saline, and Y-27632 2HCl groups. n = 8, ***P < 0.001.

Figure 3

Y-27632 2HCl treatment effectively alleviated the inflammation of myocardial tissue in EAM mice. (A) Representative graph of HE staining of myocardial tissue in mice. (B) Inflammatory infiltration score of myocardial tissue in mice in the control, EAM, saline, and Y-27632 2HCl groups. n = 8, ***P < 0.001. (C) number of mononuclear cells in the spleen of mice in the control, EAM, saline, and Y-27632 2HCl groups. n = 8, ***P < 0.001. EAM experimental autoimmune myocarditis.

Figure 4

Expression levels of pro-inflammatory factors and key genes of the TLR signaling pathway as determined by qRT-PCR. (A–C) qRT-PCR results of the expression of IL-1β, Nos2, and Agr1. n = 8, **P < 0.01, ***P < 0.001. (D–E) qRT-PCR results of the expression of Tlr2 and Tlr4. n = 8, *P < 0.05, ***P < 0.001. ns not significant.

Figure 5

Inhibition of ROCK activity beneficially alleviated the upregulation of IL-1β caused by Notch signaling pathway activation. (A) western blot to detect the expression of Notch1, IL-1β, and Hes1. n = 3. (B,D,F) representative staining plots of mouse myocardial tissue for IL-1β, Hes1, and Tlr2 in the control, EAM, saline, and Y-27632 2HCl groups. (C,E,G) IHC detection of IL-1β-, Hes1-, and Tlr2-positive areas in myocardial tissue in the control, EAM, saline, and Y-27632 2HCl groups. n = 8, **P < 0.01, ***P < 0.001. (H–K) qRT-PCR results of the expression of Notch1, Hes1, Jag2, and Dil1 in the control, EAM, saline, and Y-27632 2HCl groups. n = 8, **P < 0.01, ***P < 0.001.

Figure 6

Proposed model of the Notch-ROCK pathway and its biological significance. The cytoplasmic form of Notch1, independent of its transcriptional activity, triggers caspase-mediated cleavage (activation) of ROCK, which promotes increased expression of inflammatory factors, resulting in the exacerbation of myocardial inflammation.