Cell type-specific roles of PAR1 in Coxsackievirus B3 infection

Figures

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  Figure 1. PAR1 expression in hearts of PAR1ΔCF and PAR1ΔCM mice. PAR1 mRNA expression in (A) uninfected hearts of PAR1ΔCF (PAR1fl/fl;TCF21ETR2Cre) mice and (B) PAR1ΔCM (PAR1fl/fl;Mlc2vCre) mice and their respective control (PAR1fl/fl) littermates was analyzed by RT-PCR. PAR1 mRNA expression was normalized to HPRT mRNA expression. Data (mean ± SEM) of control littermates were set to 100% and analyzed by Student’s t test. *P < 0.05. (A: N = 5–6, B: N = 9–10).

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  Figure 2. Mice with PAR1 deficiency in cardiac fibroblasts or cardiac myocytes have increased levels of CVB3 genomes and inflammatory mediators. Levels of CVB3 genomes (A,D), TNF-α mRNA (B,E) and IL-6 mRNA (C,F) were increased in the hearts of PAR1ΔCF (PAR1fl/fl;TCF21ETR2Cre) mice and PAR1ΔCM (PAR1fl/fl;Mlc2vCre) mice compared with infected control (PAR1fl/fl) littermates 8 days after CVB3 infection. Data are shown as mean ± SEM and analyzed by Student’s t test. *P < 0.05. (N = 7–22).

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  Figure 3. Mice with PAR1 deficiency in cardiac fibroblasts have increased CVB3-induced myocarditis. Representative H&E staining (A) and quantified immune cell infiltration area (B), representative macrophage staining (C) and macrophage staining score of heart sections form PAR1ΔCF (PAR1fl/fl;TCF21ETR2Cre) mice and control (PAR1fl/fl) littermates 8 days after CVB3 infection. Bar = 200 µm. Data are mean ± SEM and analyzed by Student’s t test. *P < 0.05. (B: N = 7–10; D: N = 8–10).

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  Figure 4. Mice with PAR1 deficiency in cardiac myocytes have increased CVB3-induced myocarditis. Representative H&E staining (A) and quantified immune cell infiltration area (B), representative macrophage staining (C) and macrophage staining score of heart sections form from PAR1ΔCM (PAR1fl/fl;Mlc2vCre) mice and control (PAR1fl/fl) littermates 8 days after CVB3 infection. Bar = 200 µm. Data are mean ± SEM and analyzed by Student’s t test. *P < 0.05. (B: N = 18–22; D: N = 8–11).

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  Figure 5. Effect of PAR1 activation on poly I:C induction of CXCL10 expression in rat neonatal cardiac fibroblasts and cardiac myocytes. Rat neonatal cardiac fibroblasts (A) and cardiac myocytes (B) were stimulated with either poly I:C (25 µg/mL), PAR1 agonist peptide (TFLLR, 200 µM), thrombin (IIa, 5 nM) or combinations of agonists for 8 h. Levels of CXCL10 in the supernatant were measure by ELISA in triplicate. Data are mean ± SEM and analyzed by 1-way ANOVA with Bonferroni post-test. *P < 0.05 vs unstimulated control, #P < 0.01 vs poly I:C treated sample. (N = 3).

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  Figure 6. Effect of PAR1 activation on CVB3 replication in mouse embryonic fibroblasts and myocytes. (A) Wild-type (WT) mouse embryonic fibroblasts were infected with CVB3 (multiplicity of infection [MOI] 1) and stimulated with PAR1 agonist peptide (P1AP) (200 µM) to analyze CVB3 replication 6 h after infection. (B) Murine embryonic cardiac myocytes from WT mice were infected with CVB3 (MOI 1) with or without PAR1AP (200 µM) stimulation to analyze the effect of PAR1 activation on virus genome levels 6 h after infection. (C) Levels of active CVB3 virus was analyzed by plaque assay in WT murine embryonic cardiac myocytes infected with CVB3 (MOI 1) and stimulated with/ without P1AP (200 µM) for 24 h. Virus genome levels (A–C) were analyzed by real-time PCR. Virus genomes were normalized to expression of hypoxanthine guanine phosphoribosyltransferase mRNA and set was set to 1 for CVB3 infected control cells of each genotype. Data are mean ± SEM and analyzed by Student’s t test. *P < 0.05.

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  Figure 7. Effect of thrombin stimulation and PAR1 deficiency on autophagy. (A,B) Autophagy flux was determined by measuring levels of LC3-I and LC3-II levels by Western blotting in murine embryonic fibroblasts (MEFs) isolated from wild-type (white bars) and ΔPAR1 (black bars) embryos with or without 10 µM chloroquine (CQ) for 6 h under serum-free conditions. (C) Wild-type MEFs were cultured in serum containing growth media (GM), serum-free media (SFM) with or without thrombin (IIa, 10 nM) for 24 h. Levels of LC3-I/II, p62, ZBTB16 and actin were determined by Western blotting. Densitometry was performed to calculate LC3-II to LC3-I ratios and p62 and ZBTB16 protein expression. Levels of p62 and ZBTB16 were normalized to total β-actin protein levels (D–F). Data are mean ± SEM and analyzed by 1-way and 2-way ANOVA with Bonferroni post-test. *P < 0.05. (B: N = 3; C–E: N = 6).

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  Figure 8. Thrombin reduces autophagy in serum-starved or CVB3 infected H9c2 cells. (A,B) Levels of p62 in H9c2 cells with or without serum deprivation in the presence or absence of thrombin (10 nM) for 24 h. Levels of p62 were normalized to β-actin protein levels. (C,D) Levels of p62 in H9c2 cells infected with CVB3 in the absence and presence of thrombin (10 nM) for 24 h. Levels of p62 were normalized to β-actin protein levels. Data are mean ± SEM and analyzed by 1-way ANOVA. *P < 0.05. (N = 4).