Prostaglandin-E2 receptor-4 stimulant rescues cardiac malfunction during myocarditis and protects the heart from adverse ventricular remodeling after myocarditis

Figures

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  Figure 1. Myocardial inflammation in the experimental autoimmune myocarditis (EAM). (A) Representative Hematoxylin–Eosin staining and (B) statistical analyses of inflamed area (n = 6/each) and macroscopic score (n = 10/each) in ventricular cross-sections and hearts, respectively on day 21 obtained from EAM treated daily with vehicle or selective prostaglandin E2 receptor 4 (EP4) antagonist (CJ-42794) from day 14 to day 21. Scale bars: 500 μm. Inflamed area was calculated by affected area/ventricular area × 100%. (C) Western blot (cropped gel) and (D) its densitometric analysis (n = 6/each) of the EAM inducible Th17-specific master transcription factor, retinoic acid receptor-related orphan nuclear receptor (ROR γt) on day 21 hearts from EAM mice treated with vehicle or CJ-42794. Full-length blots of ROR γt and GAPDH are presented in Supplementary Fig. 4A. *P < 0.05 (vs. vehicle) calculated with nonparametric 2-tailed Mann–Whitney U test.

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  Figure 2. Myocardial collagen deposition in the experimental autoimmune myocarditis (EAM) mice. (A) Representative picrosirius red staining and (B) statistical analyses of collagen deposition area in ventricular cross-sections on day 56 obtained from non-EAM mice treated daily with vehicle (n = 5) or from EAM mice treated daily with vehicle (n = 8), selective prostaglandin E2 receptor 4 (EP4) agonist (ONO-0260164) (n = 8), or both ONO-0260164 and selective EP4 antagonist (CJ-42794) (n = 7) starting on day 14. Scale bars: 500 μm. Collagen deposition area was calculated by affected area/ventricular area × 100%. (C) Reverse transcription-quantitative PCR for gene expression of collagen type I and type III in bulk heart tissues on day 56 obtained from non-EAM mice (n = 5) or from EAM mice treated daily with vehicle (n = 6), ONO-0260164 (n = 6), or both CJ-42794 and ONO-0260164 (n = 9). *P < 0.05 (vs. vehicle alone-treated EAM) or **P < 0.05 (vs. non-EAM) or #P < 0.05 (vs. both CJ-42794 and ONO-0260164-treated EAM) calculated with 2-way ANOVA followed by the Bonferroni-Dunn post hoc testing method.

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  Figure 3. Expression and activation of matrix metalloproteinases (MMPs) in the heart of experimental autoimmune myocarditis (EAM) mice. (A) Reverse transcription-quantitative PCR, (B) representative gelatin zymography (cropped gel), and (C) densitometric analysis of MMPs including MMP-2 and MMP-9 in bulk heart tissues on day 56 obtained from non-EAM mice treated daily with vehicle (n = 5) or from EAM mice treated daily with vehicle (n = 6) or selective prostaglandin E2 receptor 4 (EP4) agonist (ONO-0260164) (n = 7) starting on day 14. The proteolytic bands of 62, 66, and 92 kDa corresponding to the active form of MMP-2, Pro-MMP-2, and Pro-MMP-9, respectively were scanned using a Photo scanner. Full-length gel of MMP-2 and MMP-9 is presented in Supplementary Fig. 4B. *P < 0.05 (vs. vehicle alone-treated EAM) or **P < 0.05 (vs. non-EAM) calculated with 2-way ANOVA followed by the Bonferroni-Dunn post hoc testing method.

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  Figure 4. Endogenous regulators of matrix metalloproteinase (MMP) in the heart of experimental autoimmune myocarditis (EAM) mice. (A,B) Reverse transcription-quantitative PCR for membrane type (MT)-1 MMP and tissue inhibitors of metalloproteinases (TIMPs) and (C) representative western blot (cropped blot) and (D) densitometric analysis of the TIMP-3 in bulk heart tissues on day 56 obtained from non-EAM mice treated daily with vehicle (n = 5) or from EAM mice treated daily with vehicle (n = 6) or selective prostaglandin E2 receptor 4 (EP4) agonist (ONO-0260164) (n = 7) starting on day 14. Full-length blots of TIMP-3 and GAPDH are presented in Supplementary Fig. 4C. *P < 0.05 (vs. vehicle alone-treated EAM) or **P < 0.05 (vs. non-EAM) calculated with 2-way ANOVA followed by the Bonferroni-Dunn post hoc testing method.