CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis

Figures

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  Fig 1. Impact of CX3CR1-/- on left ventricular chemokine receptor and chemokine expression in Coxsackievirus B3-infected mice. Bar graphs represent the mean ± SEM of gene expression data of LV (A) CCR2, (B) MCP-1, (C) CX3CR1, and (D) CX3CL1, as indicated, after normalization to the housekeeping gene 18S using the 2−ΔCt formula and normalized to the WT group, which was set as 1. Quantitative CX3CL1 protein expression (E) was determined via digital image analysis at a 200x magnification (scale bar = 200 μm). Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, ***p<0.001, ****p<0.0001 with n = 7–12 per group.

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  Fig 2. CX3CR1-/- induces left ventricular cell infiltration in Coxsackievirus B3-infected mice. (A,B) Gene expression level of Ly6C and immunohistological staining of CD68+ cells as marker for monocyte infiltration (C,D) Staining for CD3+ and CD4+ cells in the myocardium after CVB3-infection. The quantitative determination of the immune staining was performed via digital image analysis at a 200x magnification (scale bar = 200 μm). Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with n = 7–12 per group.

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  Fig 3. CX3CR1-/- enhances left ventricular expression of adhesion molecules and pro-inflammatory cytokines in Coxsackievirus B3-infected mice. Induction of ICAM-1 (A) and VCAM-1 (B) as well as of pro-inflammatory cytokines (C-F) in CX3CR1-/- mice compared to WT CVB3 mice. The quantitative determination of ICAM-1 and VCAM-1, indicated by the positive area (%) in relation to the heart area (mm2), was performed via digital image analysis at a 200x magnification (scale bar = 200 μm). Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with n = 7–12 per group.

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  Fig 4. CX3CR1-/- displays increased left ventricular oxidative stress in Coxsackievirus B3-infected mice. Bar graphs represent the mean ± SEM of gene expression data of LV (A) iNOS (B) eNOS, (C) NOX11, and (D) NOX4, as indicated, after normalization to the housekeeping gene 18S using the 2−ΔCt formula and normalized to the WT group, which was set as 1. (A,B) Gene expression analysis of NOS show differences between CX3CR1-/- CVB3 and WT CVB3 mice. Additional, no changes in NOX mRNA expression between CX3CR1-/- CVB3 and WT CVB3 mice could be observed (C,D). Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with n = 7–12 per group.

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  Fig 5. CX3CR1-/- leads to enhancement of myocardial fibrosis in Coxsackievirus B3-infected mice. Bar graphs represent the mean ± SEM of LV (A) TGF-ß1 gene expression, as indicated, after normalization to the housekeeping gene 18S using the 2−ΔCt formula and normalized to the WT group, which was set as 1, and (B) collagen I/III ratio, demonstrated by immunohistological stainings of (C) collagen I and (D) collagen III. Specific epitopes of collagen I and collagen III are coloured red as shown by representative images (scale bar = 200 μm). Quantification of the positive area (%) / heart area (mm2) was performed via digital image analysis. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with n = 7–12 per group.

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  Fig 6. CX3CR1-/- results in changes in titin isoform phosphorylation. (A) Expression of the N2B isoform and the corresponding phosphorylation (B). The composition as well as the phosphorylation state of the cardiac titin isoform N2B was determined using Pro-Q Diamond phospho-protein stain. In contrast to N2B phosphorylation the cardiac titin isoform composition is different in CX3CR1-/- CVB3 mice to WT CVB3 mice, respectively. Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, ***p<0.001 with n = 7–12 per group.

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  Fig 7. CX3CR1-/- increases apoptosis and anti-viral cytokine expression in Coxsackievirus B3-infected mice. (A) The Bax/Bcl-2 ratio as index for cardiomyocyte apoptosis indicates increased cardiomyocyte apoptosis in infected CX3CR1-/- mice compared to the corresponding WT mice. (B) Gene expression of anti-viral IFN-β is increased in CX3CR1-/- CVB3 versus WT CVB3 mice. Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, ***p<0.001, ****p<0.0001 with n = 7–12 per group.

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  Fig 8. CX3CR1-/- impaired left ventricular function in Coxsackievirus B3-infected mice. Evaluation of cardiac function via tip catheter. After inoculation with CVB3 CX3CR1-/- mice show declined heart function as indicated by (A) reduced max. LV pressure (LVPmax), (B) reduced LV contractility (dP/dtmax) and (C) decreased LV relaxation (dP/dtmin). Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with n = 7–12 per group.