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

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…

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:…

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…