Accelerating the Mdx Heart Histo-Pathology through Physical Exercise

Figures

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  Figure 1. Kinetics of fibrosis establishment and immune infiltration in mdx hearts: (A) representative images of Sirius red collagen staining of heart ventricles cryosections from mdx mice of 1, 3, 6, and 11 months of age. (B) Quantification of ventricular fibrotic area, determined as in (A), expressed as percentage over total area, calculated with ImageJ Color Deconvolution plugin. n = 4 independent samples. The level of ventricular fibrotic area determined in age-matched wild-type mice, as in (A), is also shown. (C) Representative images of cytofluorimetric analysis of cells derived from mdx mice hearts of 1, 3, 6, and 11 months of age, showing the gating for CD45+ cells, as determined after excluding debris, doublets, and dead cells. (D) Quantification of the CD45+ cells infiltrating wild-type or mdx heart at the indicated ages, expressed as number per gram of tissue. n = 5–11 independent samples. Data are shown as mean ± S.E.M; * = p < 0.05, ** = p > 0.01, *** = p < 0.001, **** = p < 0.0001, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons.

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  Figure 2. Description of the exercise protocol: (A) timeline of the exercise protocol: 4-week-old mice were exercised twice a week for 2 months. (B) Description of the exercise session: mice were exercised on the treadmill with no inclination. Each session consists in 1 h of running at moderate speed (max 20 cm/s). After 10 min of acclimation on the stationary treadmill, the exercise starts with 10 min-long «warm-up» session, when the speed is slowly increased from 10 cm/s to 20 cm/s. Two session of exercise at a constant speed of 20 cm/s are spaced out by a 5 min-long pause. Instead of shocker plates, gentle manipulation or physical obstacles are used to force running.

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  Figure 3. Increased fibrotic tissue deposition in exercised mdx heart ventricles: (A) representative images of Sirius red collagen staining of heart ventricles cryosections from wild-type C57/BL10, exercised mdx, and control mdx mice. (B) Quantification of heart ventricular fibrosis, determined by Sirius red collagen staining as in (A). Data are expressed as percentage of ventricular fibrotic area over total ventricular area, as determined with ImageJ Color Deconvolution plugin by G. Landini. n = 4–5 independent samples/group; * = p < 0.05, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons. (C) qRT-PCR on total heart RNA in exercised and control mdx for the pro-fibrotic factors collagen1α1, connective tissue growth factor (CTGF), and TGF-β. Data expressed as fold change (2-ddCT) normalized with GAPDH. Data are shown as media ± S.E.M. n = 4, 6 independent samples; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, unpaired t-test w/Welch’s correction.

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  Figure 4. Increased non-muscle cells’ infiltration area and cardiomyocyte necrosis in exercised mdx heart ventricles: (A) representative H&E staining of ventricular cryosections derived from wild-type, exercised, and control mdx. (B) Quantification of cell infiltration area expressed as percentage over total ventricular area, as determined in H&E stained cryosections. (C) Quantification of the size of cell infiltration patches, expressed in µm2. (D) Quantification of the number of cell infiltration patches, normalized per mm2. Data from B, C, and D are shown as mean ± S.E.M., n = 3–6 independent samples/group; * = p < 0.05, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons. All quantifications were calculated using ImageJ software. (E) Representative images of immunofluorescence of ventricular cryosection from exercised and control mdx using an anti-mouse IgG antibody staining. (F) Quantification of ventricular necrotic area expressed as percentage over total ventricular area, calculated using ImageJ software. Data are shown as mean ± S.E.M n = 3/6 independent samples * = p < 0.05, unpaired t-test w/Welch’s correction.

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  Figure 5. Cytofluorimetric analysis of the immune population isolated from wild-type, exercised mdx, and control mdx hearts: (A) representative images showing the gating for CD45+ infiltrating cells in wild-type exercised and control mdx hearts, after excluding debris, doublets, and dead cells. (B) Quantification of total hematopoietic cells infiltrating wild-type, exercised mdx, and control mdx hearts, identified as CD45 positive and expressed as number of cells normalized per gram of tissue. (C) Quantification of the immune subpopulation infiltrating the heart of wild-type, exercised mdx, and control mdx mice, expressed as number of cells normalized per gram of tissue. The cell populations were identified as indicated: total myeloid cells as CD11b+, macrophages as Ly6c-/F4/80hi, freshly recruited monocytes as Ly6chi/F4/80-, neutrophils as Ly6g+, and T cells as CD3+. Data are shown as mean ± S.E.M., n = 6/8/5 independent samples. ** = p < 0.01, *** = p < 0.01, **** = p < 0.0001, ordinary one-way ANOVA with Bonferroni correction for multiple comparisons.

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  Figure 6. Echocardiographic analysis of left ventricle fractional shortening in exercised and control mdx: (A) representative images of the echocardiographic analysis of exercised and control mdx hearts, as indicated. (B) Left ventricle fractional shortening expressed as percentage and calculated on the short axe. Data are shown as mean ± S.E.M., n = 6/7 independent samples; * = p < 0.05, unpaired t-test w/Welch’s correction.