Trypanosoma cruzi activates mouse cardiac fibroblasts in vitro leading to fibroblast-myofibroblast transition and increase in expression of extracellular matrix proteins

Abstract

Background: Cardiac fibrosis is a consequence of chronic chagasic cardiomyopathy (CCC). In other cardiovascular diseases, the protagonist role of fibroblasts in cardiac fibrosis is well established. However, the role of cardiac fibroblasts (CFs) in fibrosis during the CCC is not clear. Here, our aim was to investigate the effect of Trypanosoma cruzi, the etiological agent of Chagas disease on CFs activation.

Methods: Cardiac fibroblasts were purified from primary cultures of mouse embryo cardiac cells. After two passages, cells were infected with T. cruzi (Y strain) and analyzed at different times for determination of infectivity, activation and production of extracellular matrix components (fibronectin, laminin and collagen IV) by immunofluorescence and western blot.

Results: At second passage, cultures were enriched in CFs (95% of fibroblasts and 5% of cardiomyocytes), as revealed by presence of alpha-smooth muscle actin (α-SMA) and discoidin domain receptor 2 (DDR2) and absence of sarcomeric tropomyosin (ST) protein expression. Trypanosoma cruzi infection induced fibroblast-myofibroblast transition, with increased expression of α-SMA after 6 and 24 h post-infection (hpi). Fibronectin was increased at 6, 24 and 48 hpi, laminin was increased at 6 and 24 hpi and collagen IV was increased at 6 hpi.

Conclusions: Our results showed that T. cruzi activates CFs, inducing activation and exacerbates ECM production. Furthermore, our data raise the possibility of the involvement of CFs in heart fibrosis during Chagas disease.

Keywords: Cardiac fibroblasts; Chagas disease; Extracellular matrix; Fibrosis; Trypanosoma cruzi.

Fig. 1

Characterization of culture enriched in cardiac fibroblast. a-f Phase contrast and bright field microscopy showing features of CFs cultures. a, e Primary culture presenting cardiomyocytes clusters (arrow) that showed spontaneous contraction, surrounded by CFs (*) forming a monolayer. b-d Passages 1, 2 and 3, respectively, showing the aspect of CF-enriched cultures. f Fibroblast culture at passage 2, stained with Giemsa, demonstrating typical morphology with elongated cells, cytoplasmic extensions, the oval and large nucleus with apparent nucleoli. g Immunofluorescence showing sarcomeric tropomyosin expression indicating 95% purity of CFs (nuclei were labeled with DAPI). h Immunoblotting for ST revealed that CF cultures were myocyte-free. For positive controls, hearts of mouse embryos were used. Lanes show technical triplicates of three independent experiments (EXP). α-SMA expression, used as load control, can be observed in all samples. i Representative immunoblotting demonstrating DDR2 expression in CFs in different times of culture

Fig. 2

Trypanosoma cruzi intracellular cycle in cardiac fibroblasts. a Schematics of the experimental design: fibroblast cultures were infected at passage 2 and analyzed after 6 to 96 h of infection (MOI 10). b Quantitative data of the infection of CF by T. cruzi. At 6 hpi 9.4% of the host cells are infected by one parasite and from 24 to 72 hpi the infectivity was of 38%. Proliferation of the intracellular amastigotes started at 48 hpi with 5 parasites/infected cell and reached 40 parasites/infected cell at 72 hpi. c-h Bright field microscopy representative images of T. cruzi infected CFs cultures stained with Giemsa showing the parasite intracellular cycle. In c intracellular parasites are visible already after 6 h of infection (arrow). d At 24 h post-infection we can observe the beginning of the amastigotes proliferation process (arrow). e At 48 h an increased number of intracellular amastigotes can be observed. f After 72 h of infection a high number of amastigote forms are seen through all cytoplasm. g At 96 h of infection is possible to see that the parasites differentiated to trypomastigotes (asterisk) and evaded the host cells (arrow). h After evading host cells, the released parasites attach to new CFs to restart a new cycle (arrow)

Fig. 3

Fibroblast-myofibroblast transition induced by T. cruzi infection. a Immunoblotting was performed to evaluate α-SMA expression in CFs cultures. α-SMA expression is increased at 6 h and 24 h of T. cruzi infection, followed by a decrease at 48 h and 72 h of infection. GAPDH was used as loading control. b Quantitative analysis of the percentage of SMA-stained cells in CF cultures at 24 hpi. Uninfected cultures showed an average of 30% positivity, whereas infection led to a 1.5-fold increase in the number of stained cells. c-f Immunofluorescence revealed the labelling pattern of α-SMA filaments in CFs cultures. c, e Uninfected cultures. d, f T. cruzi-infected cultures. d At 24 hpi it is possible to observe an increase in α-SMA signal in comparison to non-infected control (c). f At 72 hpi, α-SMA immunoreactivity was drastically altered when compared to non-infected controls (e). DNA staining with DAPI can be observed in blue. Values are expressed as fold change of infected cultures by their respective controls (NI) ± SEM. *P < 0.05 (stimulus); && P < 0.01 (inhibition); ***P < 0.001 (stimulus). One-way ANOVA with Tukey’s post-hoc test of three independent experiments. Abbreviations: NI, non-infected cultures; Y, T. cruzi-infected cultures

Fig. 4

Trypanosoma cruzi infection alters ECM components expression by CFs. a, d, g Immunoblotting performed to ECM proteins in CFs cultures and their densitometric analyses plotted as graphs. a-c Fibronectin (FN) expression was increased after 6, 24 and 48 h of T. cruzi infection, followed by a decrease at 72 hpi. Immunofluorescence for FN (green) showed that at 24 hpi T. cruzi-infected cultures (c) had higher deposition of this protein (arrows) when compared to uninfected dishes (b). d-f Laminin (LN) is increased after 6 and 24 h of infection as shown by immunoblot. e and f show immunolocalization of LN in CF. In non-infected cultures LN showed a cytoplasmic distribution (in green). At 24 hpi, infected cultures displayed patches of cells with a more intense signal for LN (arrows), that corresponded with parasitized cells (arrowheads for intracellular amastigotes). g-i Collagen IV (COL) is transiently increased by T. cruzi infection. Immunoblot for COL showed a 1.5-fold increase at 6 hpi compared to uninfected cultures (g). COL had a fibrillar pattern in uninfected cultures (arrows in h) while in T. cruzi infection (6 hpi), COL immunoreactivity was increased. GAPDH was used as loading control. DAPI stained the DNA of host cells and of intracellular parasites (arrowheads) and is shown in red (pseudo-color). Values are expressed as fold change of means of the analyzed samples by means of their respective controls (NI) ± SEM. Representative data from three independent experiments. *P < 0.05 (stimulus), **P < 0.01 (stimulus), ***P < 0.001 (stimulus), &&& P < 0.001 (inhibition). One-way ANOVA with Tukey’s post-hoc test. Abbreviations: NI, non-infected cultures; Y, T. cruzi-infected cultures. Scale-bars: 20 μm