Tnni3k Is Cardioprotective in Viral Myocarditis

Figures

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  Figure 1. Tnni3k KO mice are susceptible to more severe myocarditis. (A). Representative H&E images of C57BL/6J WT, A/J WT, and Tnni3k KO hearts 8 dpi (left) and their corresponding ilastik inflammation “masks” (right). Scale bars: 1 mm. (B). Percent inflammation of C57BL/6J wild-type (C57 WT, n = 10), A/J wild-type (A/J WT, n = 2), and Tnni3k knockout (Tnni3k KO, n = 10) animals 8 dpi. (C). Myocarditis is defined as area of immune cell infiltration divided by total area of ventricular tissue. Percent inflammation at 4, 6, 8, 10, and 12 dpi (C57 WT n = 6, 8, 10, 12, 3; Tnni3k KO n = 6, 9, 10, 9, 3). The same day 8 data shown in B for C57 WT and Tnni3k KO are incorporated into this panel. (D). Relative gene expression (log10) of viral protein gene VP1 at 4, 6, 8, 10, and 12 dpi.

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  Figure 2. Ly6C+ monocytes/macrophages make up the majority of immune infiltration in both genotypes. (A). Density plot showing percentage of viable cells that are CD45+, quantified to the right. (B–D). Representative density plots showing percentage of CD45+ cells that are Ly6G+ (B), Ly6C+ (C), or CD3+ (D), with quantification to the right. n = 6 for each genotype. The x axis in flow cytometry plots is in log10 scale. Gated areas of the plots are indicated by rectangles or ovals.

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  Figure 3. Mac-3 staining supports flow cytometry data, showing macrophages make up a prominent portion of immune infiltrates. (A) Representative H&E images of C57 WT and Tnni3k KO hearts 8 dpi (left, 2x; top right, 20x) and adjacent sections immunostained for Mac-3 (middle, 2x ilastik mask; bottom right, 20x). Scale bars: 1 mm in low-magnification views, 200 µm in high-magnification views. (B) Quantification of Mac-3 staining normalized to tissue area (WT n = 6, KO n = 5). (C) Mac-3 staining normalized to inflammation (based on ilastik masks of adjacent H&E stained sections).

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  Figure 4. Tnni3k KO does not influence chronic myocarditis. (A) Inflammation (defined as in Figure 1) normalized to tissue area at 35 dpi is low and comparable between genotypes. (B) Representative images of C57 WT and Tnni3k KO hearts 35 dpi stained with picrosirius red for fibrosis (left) and of the ilastik masks generated for quantification (right). Scale bars: 1 mm. (C) Quantification of picrosirius red staining at 35 dpi (C57 WT n = 7, Tnni3k KO n = 8).

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  Figure 5. Tnni3k K489R kinase-dead mice and full null mice are equally susceptible to severe myocarditis. Percent inflammation at 10 dpi (C57 WT n = 12, Tnni3k KO n = 9, Tnni3k K489R n = 10). Data for WT and KO are the same as in Figure 1C.