Simvastatin Improves Cardiac Function through Notch 1 Activation in BALB/c Mice with Chronic Chagas Cardiomyopathy

Figures

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  FIG 1. Simvastatin (Simv) increases the expression and activation of the Notch receptor and signaling pathway in chronically T. cruzi-infected mice. BALB/c mice were infected with T. cruzi trypomastigotes and treated with simvastatin on day 60 postinfection for 20 days. (A, C, and E) Representative Notch 1 receptor, NICD, and Hes1 immunohistochemistry in cardiac tissues. (B, D, and F) Semiquantitative analysis of the immunohistochemistry signal obtained from cardiac tissues for Notch 1 receptor, NICD, and HES1, respectively. Data from one experiment with five animals per group are expressed as the number of pixels ± standard errors. One-way ANOVA with Dunnett’s posttest was used to determine significance. **, P ≤ 0.01; ***, P ≤ 0.001, for results compared with those of infected mice; α, P ≤ 0.05, for results comparing with healthy mice.

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  FIG 2. Simvastatin and 15-epi-lipoxin A4 decrease cardiac tissue damage and fibrosis in chronically T. cruzi-infected BALB/c mice. Mice were infected with T. cruzi trypomastigotes and treated with simvastatin on day 60 postinfection for 20 days. (A) Images of hematoxylin-eosin staining of representative cardiac tissues. (B) Semiquantitative analysis of cellularity in the cardiac tissues from five fields per animal. (C) Images of Picrosirius red staining of representative cardiac tissues. (D) Semiquantitative analysis of the red pixels in cardiac tissue, corresponding to the fibrosis indicator, of five fields per animal. (E to G) Concentration of TNF-α, IL-1β, and IL-10, as indicated, in the serum of the infected BALB/c mice. Determinations were performed by ELISAs. Data from one experiment with five animals are expressed as the number of pixels or nuclei ± standard errors. One-way ANOVA with Dunnett’s posttest was used to determine significance. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05, for results compared to those with infected mice; ααα, ≤ 0.001; αα, P ≤ 0.01; α, P < 0.05, for results compared to those with healthy mice.

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  FIG 3. Simvastatin normalizes cardiac function in chronically T. cruzi-infected BALB/c mice. The mice were lightly sedated, and bidimensional echocardiography was performed with a Vivid-i instrument, using an iRL-RS 5- to 13-MHz transducer. (A) Representative bidimensional echocardiograms on day 80 postinfection. (B) Ejection fraction analysis at 0, 40, and 80 days postinfection. (C) Ejection fraction analysis at 80 dpi with the treatments labeled in the graph. (D) Fractional shortening at 0, 40, and 80 dpi. (E) Fractional shortening at 80 dpi with the indicated treatments. (F) Systolic left ventricular diameter at 0, 40, and 80 dpi. (G) Systolic left ventricular diameter at 80 dpi with the indicated treatments. (H) Left ventricular mass at 0, 40, and 80 dpi. (I) Left ventricular mass at 80 dpi with the indicated treatments. Data correspond to the means ± standard errors from one experiment (n = 9). One-way ANOVA with Dunnett’s posttest was used to determine significance. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05, for results compared with those of the infected control; ααα, P ≤ 0.001; αα, P ≤ 0.01, for results compared with those of healthy controls.

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  FIG 4. Simvastatin promotes angiogenesis in chronically T. cruzi-infected BALB/c mice. Mice were infected with T. cruzi trypomastigotes and treated with simvastatin on day 60 postinfection for 20 days. (A) Images of Ki67 and isolectin B4 staining of representative cardiac tissues. (B) Images of NICD and isolectin B4 staining of representative cardiac tissues. Images from one experiment with five animals per condition are shown.

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  FIG 5. Survival analysis and cardiac parasite load in chronically T. cruzi-infected BALB/c mice. Mice were infected with T. cruzi trypomastigotes and treated with simvastatin on day 60 postinfection for 20 days. (A) Kaplan-Meier survival analysis from one experiment (n = 9). (B) Parasite load as determined by qPCR from the hearts of mice on day 80 postinfection. Results are expressed as the number of parasite equivalents per milliliter of heart homogenate. Data are expressed as the means ± standard errors from one experiment (n = 9). One-way ANOVA was performed to identify significant differences. *, P ≤ 0.05.