INTRODUCTION
Chagas’ disease, an ancient infection caused by the protozoan Trypanosoma cruzi, still impacts public health with a prevalence of infection of 10 million people and about 25 million at risk of contracting the disease (WHO, 2012). Human infection can lead to serious or fatal disease, causing significant mortality in children during the acute phase and heart failure in chronically affected patients (Andrade et al. Reference Andrade, Gollob and Dutra2014). Chagas disease is the cause of 8–20% of severe cardiomyopathy cases in Latin America (Bocchi et al. Reference Bocchi, Arias, Verdejo, Diez, Gómez and Castro2013; Malik et al. Reference Malik, Singh and Amsterdam2015), where the infection is traditionally endemic, but due to population migration it is now considered an emerging infection in North America and Europe (Coura and Borges-Pereira, Reference Coura and Borges-Pereira2012).
Transforming growth factor beta (TGF-β), a multifunctional and pleiotropic cytokine, is involved in the establishment and development of chagasic cardiomyopathy (Araújo-Jorge et al. Reference Araújo-Jorge, Waghabi, Soeiro, Keramidas, Bailly and Feige2008, Reference Araújo-Jorge, Waghabi, Bailly and Feige2012). TGF-β signalling pathway is triggered by activation of latent TGF-β which binds to and activates two receptors, TGF-β receptors type II and type I, respectively, that phosphorylate the Smad proteins, resulting in signal transmission into the nucleus and activation of gene transcription (Biernacka et al. Reference Biernacka, Dobaczewski and Frangogiannis2011). Several non-Smad signalling pathways are also activated by TGF-β and may influence the final outcome of TGF-β stimulation (Mu et al. Reference Mu, Gudey and Landström2012). TGF-β participates in the host cell invasion process, since T. cruzi needs functional TGF-β receptors and active TGF-β signalling pathway to enter into mammalian cells (Ming et al. Reference Ming, Ewen and Pereira1995; Hall and Pereira, Reference Hall and Pereira2000; Waghabi et al. Reference Waghabi, Keramidas, Calvet, Meuser, de Nazaré, Soeiro, Mendonça-Lima, Araújo-Jorge, Feige and Bailly2007). Trypanosoma cruzi directly triggers latent TGF-β activation (Waghabi et al. Reference Waghabi, Keramidas, Feige, Araújo-Jorge and Bailly2005a ) and its uptake by intracellular amastigotes, eliciting parasite differentiation. TGF-β activation appears to govern the parasite's intracellular cycle progression (Waghabi et al. Reference Waghabi, Keramidas, Bailly, Degrave, Mendonça-Lima, Soeiro, Meirelles, Paciornik, Araújo-Jorge and Feige2005b ), contributing to the invasion process and pathogenic development of chronic Chagas disease. In addition, the host immune response is regulated by TGF-β during T. cruzi infection, with high level of this cytokine being detected in plasma of α 2-macroglobulin deficient mice (Waghabi et al. Reference Waghabi, Coutinho, Soeiro, Pereira, Feige, Keramidas, Cosson, Minoprio, Van Leuven and Araújo-Jorge2002), in dogs (de Souza et al. Reference De Souza, Vieira, Roatt, Reis, da Silva Fonseca, Nogueira, Reis, Tafuri and Carneiro2014) and in plasma of chronic chagasic patients (Araújo-Jorge et al. Reference Araújo-Jorge, Waghabi, Hasslocher-Moreno, Xavier, Higuchi, Keramidas, Bailly and Feige2002; Rocha Rodrigues et al. Reference Rocha Rodrigues, dos Reis, Romano, Pereira, Teixeira, Tostes and Rodrigues2012), together with phosphorylated Smad 2 detection in cell nuclei and extensive fibrosis in the heart. TGF-β triggers rise in fibronectin (FN), specifically in uninfected cells of T. cruzi-infected cardiomyocyte cultures, while laminin expression remains unaltered (Calvet et al. Reference Calvet, Oliveira, Araújo-Jorge and Pereira2009). Inhibition of the TGF-β signalling pathway by SB-431542 in the same model reduces cardiomyocyte invasion and parasite intracellular cycle progression (Waghabi et al. Reference Waghabi, Keramidas, Calvet, Meuser, de Nazaré, Soeiro, Mendonça-Lima, Araújo-Jorge, Feige and Bailly2007). Recent reports showed that TGF-β signalling inhibitors decreases infection and prevents heart damage and fibrosis in experimental T. cruzi infection in mice (Waghabi et al. Reference Waghabi, de Souza, de Oliveira, Keramidas, Feige, Araújo-Jorge and Bailly2009; de Oliveira et al. Reference De Oliveira, Araújo-Jorge, de Souza, de Oliveira, Degrave, Feige, Bailly and Waghabi2012). This evidence highlights the important role of TGF-β in T. cruzi infection and Chagas disease development and leads to discussion of therapeutic strategies designed to interfere with TGF-β pathway in clinical management of Chagas disease (Araújo-Jorge et al. Reference Araújo-Jorge, Waghabi, Bailly and Feige2012).
Given the important role of TGF-β in chagasic cardiomyopathy, we investigated the subcellular localization of TGF-β receptor type II (TβRII) in cardiomyocytes and its functional regulation after infection by T. cruzi. We demonstrated that TβRII co-localizes with costameres, striated domains of associated proteins that couple myofibrils to the sarcolemma in muscle cells. These structures were first described with vinculin, and are involved in adhesion, force transmission and signal transduction processes of muscle cells (Craig and Pardo, Reference Craig and Pardo1983; Jaka et al. Reference Jaka, Casas-Fraile, López de Munain and Sáenz2015). Our data also demonstrated that this peculiar localization pattern is important to TGF-β signalling in cardiomyocytes, since its disruption by T. cruzi infection disturbs TGF-β response in these cells.
MATERIALS AND METHODS
Cell culture
Cardiomyocytes were isolated from 18-day-old mouse embryos (Swiss Webster, animal facilities of the Oswaldo Cruz Foundation – FIOCRUZ, Rio de Janeiro, Brazil), by enzymatic dissociation as previously described (Meirelles et al. Reference Meirelles, de Araújo-Jorge, Miranda, de Souza and Barbosa1986). All procedures involving animals were approved by the Committee of Ethics for the Use of Animals from FIOCRUZ (CEUA LW-37/13). Briefly, heart ventricles were fragmented and submitted to sequential dissociation in phosphate-buffered saline (PBS) containing 0·025% trypsin plus 0·01% collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA), pH 7·2. The isolated cells were plated into 24-wells (105 cells/mL) containing glass coverslips or 60 mm Petri dishes (2 × 106 cells) coated with 0·01% of gelatin. The cells were cultivated in Dulbeccós modified Eagle medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% horse serum, 5% fetal bovine serum (FBS; Sigma), 2·5 mm CaCl2, 1 mm L-glutamine, 2% chicken embryo extract and maintained at 37 °C in atmosphere of 5% CO2. After 24 h in culture, most cells were well-spread and synchronously beating.
Parasites and cell culture infection
Bloodstream trypomastigote forms of T. cruzi (Y strain) were obtained from Swiss Webster mice at the peak of parasitemia as previously described (Meirelles et al. Reference Meirelles, Souto-Padrón and De Souza1984). Muscle cell cultures were infected at a multiplicity of infection of 10 parasites per host cell. After 24 h of interaction, free trypanosomes in the medium were removed by washing the cultures with Ringer's solution and fresh medium was added to the culture. The time course of infection was interrupted after 72 h.
TGF-β treatment
After 24 h of culture, cardiomyocytes were washed in Ringer's solution and the TGF-β treatment assays were carried out in serum-free medium. Cardiomyocyte cultures were incubated with 5, 10 or 15 ng/mL of TGF-β, purified from bovine platelets (Promega Corporation, Madison, WI, USA), in DMEM supplemented with 1% bovine serum albumine (BSA; Sigma) and 2·5 mm CaCl2. The cultures were maintained at 37 °C in atmosphere of 5% CO2 and the medium containing TGF-β was daily replaced. After 24 and 48 h of treatment, the cultures were processed for detection of TβRII using indirect immunofluorescence and/or Western blot assays.
Cytoskeleton disruption assays
To inhibit polymerization of actin filaments and microtubules, 96 h-cultured cardiomyocytes were incubated for 4 h at 37 °C with 12·5 µg mL cytochalasin D (Sigma) and/or 15 µg/mL nocodazole (Sigma), respectively, in DMEM supplemented with 1% BSA and 2·5 mm CaCl2. The 4 h long-term incubation was performed to disrupt the myofibrils of cardiomyocytes. After drug treatment, TβRII distribution was analysed by indirect immunofluorescence as described below. For protein detection, cultures treated for 4 h with cythochalasin D and appropriated controls were incubated with 15 ng/mL of TGF-β for 1 or 48 h at 37 °C and submitted to protein extraction and Western blot analysis as further described. The viability of the cytochalasin D treated cells was assessed by determining the number of live cells after trypan blue dye exclusion, in a total of 200 cells, in duplicate in each condition, under Zeiss phase contrast inverted microscopy.
Indirect immunofluorescence
Cardiomyocyte cultures were fixed for 5 min at room temperature with 4% paraformaldehyde (Sigma) in PBS. The cultures were then washed with PBS containing 4% BSA to block unspecific reaction and incubated overnight at 4 °C with primary antibodies. To detect the TGF-β receptor, the cells were incubated with anti-TβRII antibody (developed against the full length molecule, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:300 in PBS. The antigen–antibody complex was revealed by incubation for 1 h at 37 °C with anti-IgG antibodies TRITC or fluorescein isothiocyanate (FITC)-conjugated (Sigma). Cytoskeleton proteins such as actin filaments and vinculin were detected with 4 µg/mL phalloidin-FITC (Sigma) and anti-vinculin antibody (1:300; Sigma), respectively. When cytoskeleton proteins were analysed, cardiomyocytes were permeabilized with PBS containing 0·5% Triton X-100 (Sigma), prior to antibody incubation. DNA was stained with 10 µg mL 4,6-diamidino-2-phenilyndole (DAPI; Sigma). Controls were performed by incubation with homologue serum or by omission of the primary antibody. The cells were mounted in 2·5% 1,4-diazabicyclo-(2,2,2)-octane (DABCO; Sigma) in PBS containing 50% glycerol, pH 7·2 and images were taken using confocal laser scanning microscope (Zeiss LSM Meta 510). Co-localization analysis was performed with Image J software, using the plugin co-localization highlighter (http://www.rsb.info.nih.gov/ij/plugins/colocalization.html). The index of cardiomyocytes presenting striated array of TβRII was determined by counting TβRII striations positive cardiomyocytes in a total of 300 cells in 3 separate experiments under conventional epifluorescence microscopy using a Zeiss Axioplan instrument.
Protein extraction and immunoblotting assay
Total protein was extracted using lysis buffer (50 mm Tris-HCl, NaCl 150 mm, 1% Triton X-100, 1 mm ethylene glycol-bis-(β-amino-ethyl ether) N,N,Ń,Ń-tetra-acetic acid (Sigma), 100 µg/mL phenylmethylsulphonyl fluoride (Sigma), 1 µg/mL pepstatin (Sigma), 1 µg/mL aprotinin, and phosphatase inhibitor cocktail PhoSTOP (Roche Applied Science, Mannheim, Germany), pH 8·0). The cell lysate was mixed with Laemli sample buffer containing 2-mercaptoethanol, and boiled at 100 °C subsequent to protein extraction to avoid protein degradation and dephosphorylation. The amount of protein was determined by the Folin–Lowry method and a total of 20 µg of protein was electrophoretically separated in a 12% polyacrylamide gel, by SDS–PAGE. After transference and blockage, the nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) were incubated 18 h at 4 °C with primary antibodies, diluted in blocking buffer. Cytoskeleton proteins and the TGF-β receptor were detected with anti-vinculin (1:2000; Sigma), anti-α-sarcomeric actin (1:3000; Sigma), anti-α-actinin (1:2000; Santa Cruz Biotechnology), anti-desmin (1:1000; Sigma) and anti-TβRII antibodies. For Western blot analysis, two different antibodies for TβRII were used: one developed against the full length receptor (1:2000, Santa Cruz Biotechnology) and a second to which the immunogenic peptide corresponded to the first 28 N-terminal residues of the mature human TGF-β receptor (1:1000, Merck Millipore, Darmstadt, Germany). Phosphorylated Smad 2 (1:1000; Cell Signaling Technology, Danvers, MA, USA) and FN (1:5000; Sigma) expression were also analyzed. Mouse anti-α-GAPDH (1:50 000; Ambion, Austin, TX, USA) antibodies, added together with primary antibodies, were used as internal pattern. After several washes, the membrane was incubated with anti-mouse (1:30 000; Thermo Scientific, Waltham, MA, USA) or anti-rabbit (1:30 000; Thermo Scientific) antibody conjugated to horseradish peroxidase. The enzyme activity was revealed with SuperSignal West Pico Chemiluminescent kit (Thermo Scientific). The densitometry was performed with Image J software (National Institutes of Health, http://www.rsb.info.nih.gov/ij/) with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band, at ~36 kDa, as internal control. The immunoblotting experiments were performed independently at least three times.
Statistical analysis
Student's t-test was used for comparison of experimental data from quantification of immunofluorescence detection of TβRII, as well as for densitometry of Western blotting bands. Values were considered statistically significant when P ⩽ 0·05.
RESULTS
The subcellular localization of TβRII in cardiomyocytes was determined by confocal microscopy. The TβRII was visualized as punctuated staining widespread over the cell and also in a striated pattern profile (Fig. 1). No cross-reaction with the secondary antibody or cardiomyocyte autofluorescence was observed in the negative controls after omission of anti-TβRII antibody (Supplementary Fig S1, Supplementary material). This peculiar location opened the question whether the TβRII could be associated with cytoskeleton proteins, conferring its localization in subcellular domains and functionality in cardiomyocytes. To address this question, cardiomyocyte cultures were double labelled with anti-TβRII and anti-vinculin antibodies or phalloidin-FITC, which binds to actin filaments. Confocal imaging revealed co-localization of TβRII with costameres of vinculin, showing its characteristic staining at the Z-line of myofibrils associated with the cell membrane (Fig. 1), while no co-localization was observed in I band of myofibrils. The co-localized pixels from TβRII and vinculin staining images were highlighted using the co-localization plugin of Image J software demonstrating TβRII detection in vinculin costameres (Fig. 1).
Analysis of TβRII and sarcomeric proteins localization in cardiomyocytes. Double labelling of cardiomyocyte cultures with anti-TβRII (A) and anti-vinculin antibody (B) revealed a co-localization of TβRII and vinculin in costameres (C). The co-localization plugin from Image J software was utilized to highlight co-localized pixels from TβRII and vinculin staining images (D), demonstrating TβRII detection in vinculin costameres. Detection of TβRII (E) and actin filaments by phalloidin staining (F), showing myofibrils, demonstrated TβRII localized in Z-line of myofibrils (G). Insets in (C) and (G) show TβRII localization in Z-line of myofibrils from areas delimited in the squares in higher magnification. Bar = 10 µm
Given the similarity of TGF-β receptor reaction to the typical periodicity of sarcomeric proteins within myofibrils, we investigated the specificity of TβRII antibody by Western blotting assay. Our results demonstrated a protein band with molecular mass of 70 kDa and no cross-reaction with sarcomeric cytoskeletal proteins, such as α-sarcomeric actin, α-actinin or desmin, neither with vinculin (Supplementary Fig. S2, Supplementary material). In order to investigate whether the TβRII localization is directly associated with the cytoskeleton arrangement, we treated the cardiomyocyte cultures with drugs known to depolymerize actin filaments and microtubules. Incubation of cardiomyocytes with 12·5 µg/mL of cytochalasin D, which depolymerizes actin filament and triggers the disorganization of myofibrils after long-term treatment, affected the striated distribution of TβRII, showing mainly a punctuated widespread labelling (Fig. 2). In contrast, microtubule disruption, induced by nocodazole treatment, resulted in no major effect on this receptor distribution (Fig. 2). The percentage of cardiomyocytes displaying striated array of TβRII was also determined after treatment of the cultures with cytoskeleton depolymerization agents. Cytochalasin D treatment leads to a significant reduction of 45·3% (P ⩽ 0·033) in the frequency of TβRII striations, whereas nocodazole treatment did not affect this rate (Fig. 3).
Analysis of TβRII distribution after treatment of cardiomyocyte cultures with cytoskeleton depolymerization agents. Untreated cardiomyocytes displaying regular TβRII striations (A and C; red) co-localized with the Z-line of myofibrils stained with phalloidin-FITC (B and C; green). Inset in (B) shows intact microtubules in cardiomyocytes revealed by anti-tubulin antibodies in untreated cultures. DAPI (blue), DNA dye, was used to stain nuclei (A–I). Cytochalasin D treatment, which depolymerize actin filaments (E and F; green), disrupts the costameric distribution of TβRII. (D and F; red). Treatment with cytochalasin does not affect microtubules as shown in inset in (E). Nocodazole treatment does not affect TβRII striated distribution (G and I; red) besides the depolymerization of microtubules (H and I; green). Nocodazole treated cultures still show an intact actin cytoskeleton (H, inset). Bar = 20 µm
(A) The quantification of the percentage of cardiomyocytes showing TβRII striations revealed a significant 45% decrease in TβRII striations after cytochalasin treatment, while no alteration was observed after nocodazole treatment. (B) Variation index (V.I., i.e. normalization of densitometry values considering control = 1) of PS2 detection in cardiomyocytes demonstrates that cytochalasin treatment prior to TGF-β addition inhibits 86% of the PS2 stimulation obtained in untreated controls. (C) Cytochalasin induced cytoskeleton disorganization also affects long-term responses of cardiomyocytes to TGF-β, since previous addition of this drug to culture inhibits FN enhancement induced by TGF-β after 48 h incubation detected by Western blot. *P ⩽ 0·05
To assess the functional role of striated organization of TβRII, cardiomyocyte cultures were pre-treated with 12·5 µg/mL cytochalasin D, stimulated with TGF-β and then phosphorylated Smad 2 (PS2) content was determined in total protein extracts by Western blot analysis. Cytochalasin D treatment alone had no effect on PS2 activation in control cells. As expected, addition of TGF-β (1 h) elicited a significant rise in PS2 expression. In contrast, PS2 levels of cythocalasin D pre-treated cardiomyocytes after TGF-β addition are similar to non-stimulated control cells (P ⩽ 0·018; Fig 3B). These data suggest that the subcellular localization of TβRII in costameres, which is disrupted by cytochalasin D treatment, contribute to the activation of TGF-β signalling pathway. FN expression was also inhibited by myofibrillar disruption induced by cytochalasin D, even after 48 h of TGF-β stimulation (P ⩽ 0·012; Fig 3), confirming the functional role of striated array of TβRII on TGF-β signalling. The viability of cytochalasin D treated cells was determined through quantification of trypan blue stained cells, showing no significant loss of viability under the treatment conditions (Supplementary Fig S3, Supplementary material).
Long-term treatment of cardiomyocytes with TGF-β (48 h) led to an enhancement in the frequency of the striation pattern of TβRII in cardiomyocyte cultures (Fig. 4). The quantification of cells containing striated distribution of TβRII revealed a significant increase of 38% (P ⩽ 0·009) after 48 h-stimulation of cardiomyocytes with TGF-β (Fig. 5). The analysis of T. cruzi-infected cardiomyocytes (72 h) presenting high numbers of intracellular amastigotes revealed anti-TβRII staining in parasites (Fig. 4). The co-localization of TβRII with costameres was rarely visualized in T. cruzi-infected cardiomyocyte culture, which presented mostly TβRII punctuated staining on cell surface, being difficult to observe the host cell specific distribution due to elevated number of TβRII stained amastigotes (Fig. 4). At this stage of infection, the actin cytoskeleton is disrupted in highly infected cells (Supplementary Fig. S4, Supplementary material). The quantification of TβRII striations in T. cruzi-infected cultures revealed a dramatic 79·8% reduction (P ⩽ 0·009) in the percentage of cells presenting costameric TβRII distribution (Fig. 5). The punctuate distribution of TβRII remained unaltered in T. cruzi-infected cardiomyocytes even after TGF-β treatment (Fig. 4). This cytokine was not able to stimulate the reorganization of TβRII in costameres in T. cruzi-infected cultures (Fig 4), which still displayed a statistically significant reduction in the percentage of cells presenting striated array of TβRII (Fig. 5). The disorganization of TβRII costameres in cardiomyocyte cultures induced by T. cruzi infection (72 h) also seems to impair host cell response to TGF-β, since addition of 15 ng/mL of TGF-β (1 h) inhibited PS2 stimulation observed in uninfected cultures (Fig. 5). In T. cruzi-infected cultures, TGF-β treatment did not elicit any statistical difference in PS2 levels when compared to untreated cardiomyocytes (Fig. 5).
Detection of TβRII in cardiomyocytes by indirect immunofluorescence. (A) Cardiomyocyte cultures display punctuate staining and striated array (*) of TβRII organization. (B) TGF-β treatment (48 h) resulted in an increase in the frequency of cells containing TβRII striations (*). (C and D) Trypanosoma cruzi-infected cardiomyocytes present preferentially punctuate distribution of TβRII, with a marked reduction in the number of cells displaying striated pattern of TβRII expression. Confocal microscopy also revealed an intense staining for TβRII at intracellular amastigotes. (E and F) TGF-β addition (48 h) does not influence TβRII expression or distribution in T. cruzi-infected cardiomyocytes, which still displays punctuated, non-striated array of TβRII. (D and F) Merge from TβRII staining and DIC images of T. cruzi-infected cardiomyocytes, showing the localization of infected cells and high number of intracellular amastigotes. Bar = 20 µm
(A) The quantification of the percentage of cardiomyocytes displaying TβRII striations showed a raise of 38% (P ⩽ 0·032) of TβRII striations after TGF-β treatment. Trypanosoma cruzi infection elicited a dramatic 79·8% reduction (P ⩽ 0·009) in the percentage of cells showing costameric TβRII distribution. Trypanosoma cruzi-infected, TGF-β treated cultures still show a statistically significant reduction that reached a maximum of 96% in the percentage of cells displaying striated array of TβRII. (B) Western blot analysis revealed that T. cruzi infection (72 h) prior to addition of 15 ng/mL of TGF-β (1 h) inhibited the PS2 stimulation observed in uninfected cultures, since PS2 levels in T. cruzi-infected cultures after TGF-β treatment were not statistically different from untreated and infected cardiomyocytes.* #P ⩽ 0·05.
To analyse TβRII expression in control and T. cruzi-infected cardiomyocyte protein extracts, we utilized a different anti-TβRII antibody raised against the N-terminal region of the receptor which did not recognize purified parasite extracts (data not shown), guaranteeing that no cross-reaction would occur with intracellular amastigotes or trypomastigotes. Although attempts to visualize TβRII distribution by immunofluorescence with this second antibody were not successful, the Western blot analysis of TβRII expression in cardiomyocyte cultures showed one specific band of 70 kDa (Fig 6). Our results show that the levels of TβRII in uninfected cultures after TGF-β or cytochalasin D treatment remained unaltered (Fig. 6). In the same fashion, TβRII expression after 72 h T. cruzi infection did not show statistically significant alteration (Fig. 6).
Western blot analysis of TβRII effect on cardiomyocyte cultures. (A) TGF-β or cytochalasin treatment did not result in relevant alterations in TβRII expression. (B) TβRII protein levels in T. cruzi-infected cardiomyocyte culture, treated or not with TGF-β, also did not show statistically significant differences. GAPDH was used as internal control. Data was normalized considering densitometry values from untreated and uninfected controls as 1.
DISCUSSION
TGF-β has been implicated in tissue remodelling by controlling the synthesis and degradation of the extracellular matrix (Leask, Reference Leask2007; Kong et al. Reference Kong, Christia and Frangogiannis2014) and is involved in Chagas disease fibrosis development (Araújo-Jorge et al. Reference Araújo-Jorge, Waghabi, Hasslocher-Moreno, Xavier, Higuchi, Keramidas, Bailly and Feige2002, Reference Araújo-Jorge, Waghabi, Soeiro, Keramidas, Bailly and Feige2008, Reference Araújo-Jorge, Waghabi, Bailly and Feige2012). Nevertheless, few reports demonstrate the distribution of TGF-β receptors in cardiomyocytes, and no information is available about its interaction with T. cruzi.
Our results revealed a striated pattern of receptor distribution in cardiomyocytes, which was significantly increased in 38% after TGF-β stimulation. This distribution suggests an association of the receptor with cytoskeleton proteins in cardiomyocytes. TβRII organization in cardiomyocytes has been previously reported as widespread dots on cell surface without striated pattern (He et al. Reference He, Fu, Zhang, Yuan, Li, Lv, Zhang and Fang2011). This contrasting result may be related to their lower level of culture differentiation revealed by the absence of well-developed myofibrils. In our data, the striated pattern of TβRII was clearly demonstrated to co-localize with costameres, suggesting that the localization of the TβRII in this mechanical transduction site in cardiomyocytes, previously reported as a regulatory signalling domain (VanWinkle et al. Reference VanWinkle, Snuggs, De Hostos, Buja, Woods and Couchman2002), may potentiate TGF-β signalling. The association of TβRII with costameres was also demonstrated by myofibrillar disruption after cytochalasin D treatment, which resulted in significant loss of TβRII striation, suggesting that the integrity of contractile apparatus is essential to regulate the TβRII subcellular localization.
TβRII localization in costameres is also supported by the fact that several proteins known to interact or modulate TGF-β signalling such as syndecans (Chen et al. Reference Chen, Klass and Woods2004), integrins (Hayashida, Reference Hayashida2010), β-catenin (Tian and Phillips, Reference Tian and Phillips2002), filamin (Sasaki et al. Reference Sasaki, Masuda, Ohta, Ikeda and Watanabe2001; Razinia et al. Reference Razinia, Mäkelä, Ylänne and Calderwood2012) and spectrin (Lim et al. Reference Lim, Baek, Jang, Choi, Lee, Lee, Lim, Kim, Kim, Kim, Mishra and Kim2014) present striated distribution in cardiomyocytes (Craig and Pardo, Reference Craig and Pardo1983; Koteliansky et al. Reference Koteliansky, Glukhova, Gneushev, Samuel and Rappaport1986; Wu et al. Reference Wu, Sung, Chung and DePhilip2002; Stevenson et al. Reference Stevenson, Cullen, Rothery, Coppen and Severs2005). The TGF-β receptor also has two subunits that need to dimerize for activation of the signalling cascade, plus accessories receptors, which would have their association facilitated or enhanced by a concentrated location of the receptors in signalling domains such as costameres.
An increase in the level of TβRII striation in cardiomyocytes was evidenced after TGF-β stimulation. TGF-β is a well-known modulator of cellular differentiation (Massagué and Xi, Reference Massagué and Xi2012), and the increase of TβRII striations may reflect a higher level of differentiated cells. In addition, TGF-β induces reorganization of the actin cytoskeleton in several models (Hubchak, Reference Hubchak2003; Vardouli et al. Reference Vardouli, Moustakas and Stournaras2005; Assinder and Cole, Reference Assinder and Cole2011) by activation and mobilization of Cdc42, RhoA and LIM-kinase (Edlund et al. Reference Edlund, Landström, Heldin and Aspenstro2002; Hubchak, Reference Hubchak2003), and also triggers the synthesis of muscle specific proteins (Eghbali et al. Reference Eghbali, Tomek, Woods and Bhambi1991; Li et al. Reference Li, Georgakopoulos, Lu, Hester, Kass, Hasday and Wang2005; Singla et al. Reference Singla, Kumar and Sun2005). Therefore, the increase in the percentage of cells presenting TβRII striations may also be related to a direct remodelling of the cytoskeleton triggered by this cytokine.
TGF-β signalling is known to be linked to the cytoskeleton (Moustakas and Heldin, Reference Moustakas and Heldin2008), and our data suggest that this linkage might be related to the localization of TβRII in costameres. The classic TGF-β signalling pathway (via Smads) can interact with other pathways such as mitogen-activated protein (MAP) kinases, FAK and Src (Galliher and Schiemann, Reference Galliher and Schiemann2007; Wang et al. Reference Wang, Xiang, Zent, Quaranta, Pozzi and Arteaga2009; Mu et al. Reference Mu, Gudey and Landström2012), which are triggered by integrins (Wu et al. Reference Wu, Sung, Chung and DePhilip2002; Samarel, Reference Samarel2005). Therefore, considering that syndecans and integrins are also found in costameres, the idea that this region may function as a TGF-β signalling domain in cardiomyocytes is not only plausible, but is also suggested by other authors (Samarel, Reference Samarel2005; Moustakas and Heldin, Reference Moustakas and Heldin2008). In uninfected cardiomyocytes, the Smad 2 phosphorylation and TGF-β induced FN stimulation was reduced after cytochalasin treatment, which also disorganized the striated distribution of TβRII. Cytochalasin treatment likewise impairs Smad 2 phosphorylation in mesangial cells (Hubchak, Reference Hubchak2003), and FAK/Src signalling and receptor clustering in human breast cancer cell lines (Wang et al. Reference Wang, Xiang, Zent, Quaranta, Pozzi and Arteaga2009), demonstrating the dependence of TGF-β signalling on an intact actin cytoskeleton in different cells. Spectrin, a cytoskeleton protein that has striated localization in costameres of cardiomyocytes (Stevenson et al. Reference Stevenson, Cullen, Rothery, Coppen and Severs2005), also is linked to TGF-β signalling. Disruption of spectrin by gene knockout results in phenotypes similar to Smad deficiency (Tang et al. Reference Tang, Katuri, Dillner, Mishra, Deng and Mishra2003), suppressing Smad signalling and cardiomyocyte differentiation, leading to cell cycle deregulation and apoptosis in heart muscle cells (Lim et al. Reference Lim, Baek, Jang, Choi, Lee, Lee, Lim, Kim, Kim, Kim, Mishra and Kim2014).
Costameric distribution of TβRII was significantly reduced in T. cruzi-infected cardiomyocytes. The disorganization of TβRII striations is likely to be a result of the myofibrillar breakdown (Pereira et al. Reference Pereira, Costa, Chagas Filho and de Meirelles1993; Taniwaki et al. Reference Taniwaki, Machado, Massensini and Mortara2006), with a clear disorganization of vinculin costameric distribution and disarray of sarcomeric α-actinin caused by T. cruzi infection (Melo et al. Reference Melo, Almeida, Meirelles and Pereira2004, Reference Melo, Almeida, Meirelles and Pereira2006), since the striated organization of the receptor depends on the cytoskeleton integrity. One striking feature was the immunolabelling of intracellular amastigotes revealed by anti-TβRII antibody, corroborating previous studies showing TGF-β binding to intracellular amastigotes, both in the heart tissue and cardiomyocyte cultures, while trypomastigotes showed no reaction (Waghabi et al. Reference Waghabi, Keramidas, Bailly, Degrave, Mendonça-Lima, Soeiro, Meirelles, Paciornik, Araújo-Jorge and Feige2005b ). An intriguing fact is the failure to identify the TGF-β gene in T. cruzi genome associated with the detection of TGF-β in the surface membrane, flagellar pocket, intracellular vesicles or cytoplasm of either the intracellular and axenic amastigotes, suggesting that the parasite could capture TGF-β from the host and use it for its intracellular differentiation (Waghabi et al. Reference Waghabi, Keramidas, Feige, Araújo-Jorge and Bailly2005a , Reference Waghabi, Keramidas, Bailly, Degrave, Mendonça-Lima, Soeiro, Meirelles, Paciornik, Araújo-Jorge and Feige b ). This idea was reinforced by reports showing that the treatment of cardiomyocytes with SB-431542, a selective inhibitor of ALK5 (a TGF-β type I receptor), hindered the proliferation of intracellular amastigotes and the release of trypomastigotes (Waghabi et al. Reference Waghabi, Keramidas, Calvet, Meuser, de Nazaré, Soeiro, Mendonça-Lima, Araújo-Jorge, Feige and Bailly2007). The ability of T. cruzi to biologically respond to TGF-β was also shown by a phosphoproteomic approach that detected both phosphorylated proteins and up- or downregulated proteins after TGF-β interaction (Ferrão et al. Reference Ferrão, de Oliveira, Degrave, Araujo-Jorge, Mendonça-Lima and Waghabi2012), suggesting that the parasite has a TGF-β responsive molecule on its surface.
Our data showed that T. cruzi infection disrupted TGF-β signalling, with infected cardiomyocytes displaying low phosphorylation of Smad 2 after short-term TGF-β addition, together with disorganization of TβRII costameric distribution. Previous data of our group demonstrated that T. cruzi-infected cardiomyocytes present lower FN expression (Calvet et al. Reference Calvet, Meuser, Almeida, Meirelles and Pereira2004), while the addition of TGF-β results in increased ECM expression only in uninfected cells of the infected culture, suggesting that T. cruzi infection prevented FN stimulation by this cytokine (Calvet et al. Reference Calvet, Oliveira, Araújo-Jorge and Pereira2009). These data might be related to the lower capacity of host cell to respond to TGF-β stimulation, which is associated with TβRII delocalization. Although T. cruzi modulates the expression of several surface and cytokine receptors in immune cells (Majumder and Kierszenbaum, Reference Majumder and Kierszenbaum1996; Albareda et al. Reference Albareda, Perez-Mazliah, Natale, Castro-Eiro, Alvarez, Viotti, Bertocchi, Lococo, Tarleton and Laucella2015) and cardiomyocytes (Soeiro et al. Reference Soeiro, Paiva, Barbosa, Meirelles and Araújo-Jorge1999), TβRII protein levels did not show significant alterations in our model, and the lower response in the infected cell is likely to be due to cytoskeleton breakdown induced by the parasite. In addition, T. cruzi also seems to interfere directly in the signalling pathways of TGF-β, since infected cells not exposed to TGF-β exhibit phosphorylation of Smad 2/3 (Unnikrishnan and Burleigh, Reference Unnikrishnan and Burleigh2004; Waghabi et al. Reference Waghabi, Keramidas, Calvet, Meuser, de Nazaré, Soeiro, Mendonça-Lima, Araújo-Jorge, Feige and Bailly2007), and parasite factors antagonize TGF-β dependent induction of CTGF in fibroblasts (Unnikrishnan and Burleigh, Reference Unnikrishnan and Burleigh2004). Other signalling pathways also are modulated by T. cruzi infection such as Erk 1/2 in different cell types (Mukherjee et al. Reference Mukherjee, Huang, Petkova, Albanese, Pestell, Braunstein, Christ, Wittner, Lisanti, Berman, Weiss and Tanowitz2004; Magdesian et al. Reference Magdesian, Tonelli, Fessel, Silveira, Schumacher, Linden, Colli and Alves2007) and in cardiac cells (Adesse et al. Reference Adesse, Lisanti, Spray, Machado, Meirelles, Tanowitz and Garzoni2010). Molecules of the parasite itself may also interfere directly in host cell signalling pathways. A mucin from T. cruzi inhibits the activation of p38, extracellular signal-regulated kinase (ERK) and Jun amino-terminal kinase (JNK) in macrophages, although not preventing the activation of p38 induced by tumor necrosis factor (TNF)-α (Alcaide and Fresno, Reference Alcaide and Fresno2004). In contrast, cruzipain, a major cysteine protease from T. cruzi, induces p38 in macrophages (Stempin et al. Reference Stempin, Garrido, Dulgerian and Cerbán2008), and it was recently shown that cruzipain is also an important activator of latent TGF-β and thereby triggers TGF-β-mediated events crucial for the development of Chagas disease (Ferrão et al. Reference Ferrão, d'Avila-Levy, Araujo-Jorge, Degrave, Gonçalves, Garzoni, Lima, Feige, Bailly, Mendonça-Lima and Waghabi2015). Moreover, addition of purified cruzipain enhanced the invasive activity of trypomastigotes, an effect that can be inhibited by a neutralizing anti-TGF-β antibody, leading to the conclusion that the activities of cruzipain and TGF-β are functionally linked in the process of cell invasion, and the engagement of TβRII in this process is mandatory.
Altogether, our data shows that TβRII presents a striated distribution in cardiomyocytes that co-localizes with costameres, in an organization that is dependent of cytoskeleton integrity. This costameric localization of TβRII is important in triggering the canonical TGF-β signalling pathway, since cytoskeleton disorganization results in lower Smad 2 phosphorylation (Fig. 7). Trypanosoma cruzi-infected cardiomyocytes present a reduction of TβRII striations, together with a lower response to added TGF-β, suggesting that cytoskeleton disorganization triggered by the parasite could result in altered or low TGF-β response localized in highly infected cardiomyocytes (Fig. 7).
Proposal of TGF-β receptor II functional association with cardiomyocyte cytoskeleton. TGF-β receptor II is localized in cardiomyocyte's costameres, which are also rich in vinculin. The localization of the receptor in these signalling domains of cardiomyocyte membranes, that are closely associated with the cytoskeleton, potentiate Smad 2 phosphorylation (A). Once T. cruzi infection is established, the cytoskeleton is disorganized, disrupting TβRII striations and decreasing Smad 2 phosphorylation after interaction with TGF-β (B). In the tissue context, we suggest that the highly infected cardiomyocytes would be specifically less responsive to exogenous TGF-β stimulation.
SUPPLEMENTARY MATERIAL
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ACKNOWLEDGEMENTS
The authors thank Danielle Almeida, Liliane Mesquita and Alanderson Nogueira for technical support; Pedro Paulo de A. Manso and Carlos Bizarro for confocal image acquisition; Dr Jean Jacques Feige and Sabine Bailly (Commissariat à l'Energie Atomique, Grenoble, France) for helpful discussion of the results; Francisco O. de Oliveira Jr for assistance with the figures; Mr Potter Wickware for proofing the manuscript.
FINANCIAL SUPPORT
This work was supported by grants from Fundação Oswaldo Cruz (FIOCRUZ) to C.M.C, T.A.S.. T.G.M., T.C.A.J., M.C.S.P.; Programa Estratégico de Apoio à Pesquisa em Saúde (PAPES) to C.M.C, T.A.S., T.G.M., M.C.S.P.; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) to C.M.C, T.A.S., T.G.M., T.C.A.J., M.C.S.P. and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to C.M.C, T.A.S., T.G.M., T.C.A.J. and M.C.S.P.
CONFLICT OF INTEREST
No conflict of interest to declare.
