Pirfenidone Prevents Heart Fibrosis during Chronic Chagas Disease Cardiomyopathy

Abstract

Cardiac fibrosis is a severe outcome of Chagas disease (CD), caused by the protozoan Trypanosoma cruzi. Clinical evidence revealed a correlation between fibrosis levels with impaired cardiac performance in CD patients. Therefore, we sought to analyze the effect of inhibitors of TGF-β (pirfenidone), p38-MAPK (losmapimod) and c-Jun (SP600125) on the modulation of collagen deposition in cardiac fibroblasts (CF) and in vivo models of T. cruzi chronic infection. Sirius Red/Fast Green dye was used to quantify both collagen expression and total protein amount, assessing cytotoxicity. The compounds were also used to treat C57/Bl6 mice chronically infected with T. cruzi, Brazil strain. We identified an anti-fibrotic effect in vitro for pirfenidone (TGF-β inhibitor, IC50 114.3 μM), losmapimod (p38 inhibitor, IC50 17.6 μM) and SP600125 (c-Jun inhibitor, IC50 3.9 μM). This effect was independent of CF proliferation since these compounds do not affect T. cruzi-induced host cell multiplication as measured by BrdU incorporation. Assays of chronic infection of mice with T. cruzi have shown a reduction in heart collagen by pirfenidone. These results propose a novel approach to fibrosis therapy in CD, with the prospect of repurposing pirfenidone to prevent the onset of ECM accumulation in the hearts of the patients.

Keywords: Chagas disease; heart fibrosis; pirfenidone.

Figure 1

Proteins secreted by T. cruzi in the media (PCM—parasite-conditioned media) and T. cruzi lysate (TCL) stimulate collagen expression in human cardiac fibroblasts. (A) Analysis performed in 96-well format with collagen detection using Sirius Red/Fast Green shows that PCM and TCL treatment increases collagen in human cardiac fibroblasts; (B) the increase in collagen resulting from PCM stimulus is dose-dependent; (C) uninfected fibroblasts treated with PCM (C3–4) compared to untreated (C1–2) show the accumulation of fibronectin (FN) by immunofluorescence. DAPI is shown in blue and FN immunofluorescent staining in red; (D) image processing analysis showed an increase in the area occupied by fibronectin fibrils in the fields. * p ≤ 0.05 vs. control.

Figure 2

Scatter plots of data from Sirius Red/Fast Green assays for collagen detection directly infected with T. cruzi. (A) Raw data showing variation in collagen from uninfected controls to T. cruzi-infected samples. Δ collagen stands for T. cruzi-infected collagen values in µg minus uninfected control sample values (µg). T. cruzi infection robustly stimulates collagen in cardiac fibroblasts and resulted in Z’ = 0.55, S/B 12.3 and% CV 12.36–16.04. Z’- statistical parameter to assess reproducibility of high throughput assays [44]; S/B-signal to background ratio; % CV-percentage of mean by the coefficient of variation (i.e., the ratio of the standard deviation and the mean of the replicates); (B) normalized inhibition data showing that signaling inhibitors induced high inhibition of collagen stimulation by T. cruzi in human cardiac fibroblasts.

Figure 3

T. cruzi modulates collagen expression and inhibits the signaling inhibitors with collagen stimulation. (A) Infection with T. cruzi (Brazil strain), (B) stimulation with serum from uninfected and infected mice, (C) with the interaction of T. cruzi lysates and (D) with parasite-conditioned medium, resulted an increased collagen expression in human cardiac fibroblasts. Treatment with benznidazole, pirfenidone, losmapimod and SP600125 promoted a reduction in collagen expression in human cardiac fibroblast cultures infected by (A) T. cruzi and (B) stimulated with serum from uninfected and T. cruzi-infected mice. (C) The treatment with benznidazole and pirfenidone in human cardiac fibroblasts stimulated with parasite lysate promoted an inhibition of collagen expression in these cultures. (D) Human cardiac fibroblast cultures, treated with benznidazole, pirfenidone, losmapimod and SP600125, revealed that only the treatment with SP600125 inhibited the collagen expression in cells stimulated with parasite-conditioned medium. * p ≤ 0.05 vs. control; # p ≤ 0.05 vs. stimulus.

Figure 4

Dose-response effect of pirfenidone (A), losmapimod (B) and SP600125 (C) in collagen stimulation induced by T. cruzi in human cardiac fibroblasts. The Sirius Red/Fast Green assay for collagen detection allowed EC50 value calculation for collagen inhibition (Sirius Red readout, shown in red), while Fast Green allowed cytotoxicity assessment and calculation of CC50 for the host cell (Fast Green, shown in blue).

Figure 5

Trypanocidal activity in human cardiac fibroblasts infected with T. cruzi, treated with different compounds. Sigmoid dose-response curves for inhibition of T. cruzi infection revealed that only benznidazole (A,E) promoted a reduction in T. cruzi infection in human cardiac fibroblasts. Pirfenidone (B,F), losmapimod (C,G) and SP600125 (D,H) did not show the same efficacy in trypanocidal activity observed in human cardiac fibroblasts infected with T. cruzi (Brazil strain).

Figure 6

Proliferation of human cardiac fibroblasts evaluated by BRDU incorporation measured through ELISA. Proliferation of human cardiac fibroblasts infected with T. cruzi (A), stimulated with serum from T. cruzi-infected mice (B), T. cruzi lysate (C) or with parasite-conditioned medium (D) was analyzed by BRDU incorporation measured through ELISA. All conditions tested promoted an increase in human cardiac fibroblast proliferation. The treatment with benznidazole (100 μM), pirfenidone (1000 μM), losmapimod (30 μM) and SP600125 (10 μM) did not result in a reduction in human cardiac fibroblasts proliferation. * p ≤ 0.05 vs. control; unpaired Student’s t-test.

Figure 7

Parasitic load in cardiac tissue. Quantitative PCR revealed a parasitic load in the heart higher than the average plus 3 standard deviations of uninfected mice, showing infection, but at low levels. Treatment with signaling inhibitors such as pirfenidone, losmapimod and SP600125 did not modulate infection in cardiac tissue of C57BL/6 mice.

Figure 8

(A–H)Collagen analysis in cardiac tissue in C57BL/6 mice infected with T. cruzi. Heart tissue from C57BL/6 mice infected with T. cruzi (Brazil strain) was stained with Sirius red/Fast Green to label collagen (purple) and total proteins (green). Treatment with pirfenidone at 60 dpi reduced the total collagen expression in the ECM. The other inhibitors were not effective in reducing the total collagen after the treatment with 60 and 100 dpi. (I) Histological image analysis was performed using Image J software and the percentage of area occupied by total collagen staining is represented normalized as Variation Index (V.I.) when values are divided by the average of the values from uninfected animals (which are then considered = 1). The data showed that only pirfenidone at 60 dpi reduced total collagen in cardiac tissue. The other inhibitors did not show the same profile of the reduction in total collagen in the extracellular matrix in cardiac tissue infected with T. cruzi.

Figure 9

Collagen I expression in hearts of mice infected with T. cruzi. (A) Quantitative data by Western blot corroborated with qualitative data obtained by image processing analysis in which only pirfenidone prevented the increase in collagen in the ECM in treated cardiac tissue. (B) Measurement of the collagen I expression in the heart by quantitative PCR showed that only pirfenidone promoted an inhibition of collagen I gene expression after the treatment in the cardiac tissue infected with T. cruzi. * p ≤ 0.05 vs. uninfected (western blot); ** p ≤ 0.05 vs. infected/untreated; # p ≤ 0.05 vs. uninfected (qPCR); ## p ≤ 0.05 vs. Solutol.