Trypanosoma cruzi down-regulates mechanosensitive proteins in cardiomyocytes

Figures

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  Fig. 1:. changes in paxillin induced by Trypanosoma cruzi infection. Double labelling of uninfected and T. cruzi-infected cardiomyocytes with anti-paxillin antibody (white) and To-PRO-3 iodide (green). Cardiac cells cultured for 48 h (A) and 96 h (B) displayed striations as shown by Phalloidin staining (magenta, in D-F) and paxillin staining (white in A, B and C) at focal adhesion sites (arrows in G). Paxillin location was unaltered after 24 h of infection, even in the presence of intracellular amastigotes within the host cell cytoplasm (arrowheads in B and H). Loss of paxillin in focal adhesions was observed in highly infected cells (*), concomitant with myofibrillar breakdown as evidenced by Phalloidin staining (F). Parasites were also labelled by the paxillin antibody (B and C). Paxillin content and expression were also affected at 72 h post-infection (hpi). Representative blots from three independent experiments are shown in J. Densitometric analyses revealed a 34% decrease in paxillin (K) protein content after 72 h of infection. Quantitative real-time polymerase chain reaction (RT-PCR) showed a 26% reduction in the relative expression of the paxillin transcripts (L). *: p < 0.05,…

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  Fig. 2:. focal adhesion kinase (FAK) immunolocalisation in cardiomyocytes during Trypanosoma cruzi infection in vitro. Primary cultures were stained for total FAK protein and co-labelled with Phalloidin-Alex488 and 4’-6-diamidino-2-phenylindole (DAPI) for F-actin and nuclear visualisation, respectively. FAK immunolocalisation was found as punctate dots throughout the cytoplasm of cardiomyocytes (white in A, B and C), showing well developed myofibrils (D, E and F). Infected cells showed no significant changes in FAK immunoreactivity at 24 or 72 h post-infection (hpi) (H and I), whereas actin filaments are absent around intracellular amastigotes at later time of infection (I). Bars: 10 µm.

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  Fig. 3:. Trypanosoma cruzi induces a biphasic effect on focal adhesion kinase (FAK) phosphorylation in cardiac myocytes. Cultures were infected and immunostained for anti-phosphorylated FAK (white in A-C), F-actin (magenta in D-F) and 4’-6-diamidino-2-phenylindole (DAPI) for host cell and parasite chromatin (green). Intense pFAK staining was observed at 24 h post-infection (hpi) (B and H), whereas highly infected cells at 72 hpi (* in C and I) showed a clear reduction of immunoreactivity for pFAK. F-actin staining evidenced the striations of the differentiated myocytes (magenta in D, E and F). Bars: 10 μm.

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  Fig. 4:. Trypanosoma cruzi infection disrupts talin in cardiomyocytes. Cardiac cells, cultivated for 24 h before infection with trypomastigote forms of T. cruzi (Y strain), were fixed at desired times and immunostained with anti-talin specific antibody (white). Nuclear chromatin was stained with To-PRO-3 Iodide (green), allowing the visualisation of host cell nucleus and intracellular parasite nuclei and kinetoplasts (arrowheads in C). Uninfected cells displayed abundant talin immunoreactivity at 48h (A) and 96h (B) in vitro (left panels) and revealed a striated pattern in fully differentiated myocytes (arrows), as well as staining in focal adhesion sites (opened arrows). After 24 h of infection, few intracellular parasites are seen in the host cell cytoplasm and no change is observed in talin spatial distribution (C). Talin location was drastically disturbed in highly infected cells (*), at 72 hpi, in which staining was only noticeable in focal adhesion sites. Uninfected cells in infected cultures maintained the striated talin staining pattern (D). Western blot analysis for talin (E) revealed that infected cultures had a significant decrease (32%) at 72 h post-infection (hpi) (F).…

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  Fig. 5:. Trypanosoma cruzi infection alters integrin and FAK-Tyr397. β-1-integrin (A), focal adhesion kinase (FAK) (C) and FAK-Tyr397 (E) protein expression was analysed by western blot. Densitometric analyses revealed an upregulation of 38 and 50% of integrin (B) and FAK-Tyr397 (F), respectively, at the early stage of infection 24 h post-infection (hpi). A downregulation of 30% in FAK-Tyr397 expression was showed at 72 hpi (F) but no change was seen in β1-integrin (B) and total FAK (D) levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as housekeeping control. **: p < 0.01; ***: p < 0.0001, unpaired Student’s t test.