Inhibition of PKCθ Improves Dystrophic Heart Phenotype and Function in a Novel Model of DMD Cardiomyopathy

Figures

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  Figure 1. C20 twice-a-week administration increases running performance in the mdx mice. (A) A schematic description of the C20 treatment and exercise protocol. (B) Percentage of mice exhausted per exercise session, expressed as mean +/− S.E.M.; ** p < 0.01, unpaired t-test w/Welch’s correction. N = 10 mice/exercise session.

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  Figure 2. Reduced immune infiltration in C20-treated exercised mdx hearts compared with controls. (A) Representative images showing the gating for CD45+ heart immune infiltrating cells in control mdx, vehicle-treated, and C20-treated exercised mdx. (B–G) Quantification of total CD45+ cells (B) CD3+ T cells (C), CD11b+ myeloid cells (D), F4/80hi macrophages (E), Ly6chi-F4/80− inflammatory monocytes (F), and Ly6g+ neutrophils (G) in control mdx, vehicle-treated, and C20-treated exercised mdx hearts, expressed as number of cells normalised per gram of tissue. Data are shown as media +/− S.E.M. n = 5–9 independent samples per group ** p < 0.01, *** p < 0.001, **** p < 0.0001, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons.

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  Figure 3. Decreased cell infiltration area in C20-treated exercised mdx. (A) Representative H&E staining showing myocardium morphology and the infiltrating cell patches in control mdx and vehicle or C20-treated exercised mdx. (B) Quantification of cell infiltration area expressed as percentage over the total ventricular area, as determined in H&E stained cryosections. (C) Quantification of the size of cells infiltration patches expressed in µm2. (D) Quantification of the number of cells infiltration patches, normalised per mm2. Data from (B,D) are expressed as mean +/− S.E.M.; n = 5, 9, five independent samples per group. All quantifications were calculated using ImageJ software, * p < 0.05, ** p < 0.01, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons.

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  Figure 4. Decreased cardiomyocytes necrosis in C20-treated exercised mdx ventricles. (A) Representative images showing ventricular necrosis in control mdx and vehicle or C20-treated exercised mdx, as detected by anti-mouse IgG staining. (B) Quantification of the ventricular necrotic area expressed as percentage over the total ventricular area, calculated using ImageJ software. Data are shown as mean +/− S.E.M, n = 3, 7, 5 independent samples per group. * p < 0.05, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons.

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  Figure 5. Decreased fibrotic tissue deposition in C20-treated exercised mdx ventricles. (A) Representative images of ventricular fibrosis in the hearts of control mdx vehicle-treated or C20-treated exercised mdx mice, evidenced by Sirius red collagen staining. (B) Quantification often ventricular fibrotic area over the total ventricular area expressed as percentage and calculated by ImageJ Colour Deconvolution plugin. n = 5, 10, 5 independent samples per group. * p < 0.05, ** p < 0.01, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons. (C,D) qRT-PCR on total heart RNA for Collagen1α (n = 6 independent samples per group) and IL-6 mRNA (n = 5 independent samples per group). * p < 0.05, unpaired t-test w/Welch’s correction. Data from (B,D) are shown as mean +/− S.E.M.

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  Figure 6. Prevented left ventricular loss of function in C20 treated ex mdx. (A) Representative images of the echocardiographic analysis of control mdx and vehicle or C20-treated ex mdx. (B) Left ventricle fractional shortening expressed in percentage and calculated on the short axe. n = 10, 8, 10 independent samples per group. * p < 0.05, ** p < 0.01, one-way ordinary ANOVA with Bonferroni correction for multiple comparisons.

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  Figure 7. Proposed mechanism of C20 effect. The loss of functional dystrophin causes cardiomyocytes sarcolemma leaks in response to cardiac workload (mimicked by the exercise protocol), promoting immune cells infiltration. Immune cell infiltration amplifies cardiac damage, leading to exacerbated fibrotic tissue deposition. C20 treatment, inhibiting T cells activity, blunts the inflammation in response to CMCs cell death, leading to a decrease in myeloid infiltration and protecting the tissue by the amplification of the damage.