TGF-β receptor type II costameric localization incardiomyocytes and host cell TGF-β response isdisrupted by Trypanosoma cruzi infection

CLAUDIA MAGALHÃES CALVET1*, TATIANA ARAÚJO SILVA1,TATIANA GALVÃO DE MELO1, TÂNIA CREMONINI DE ARAÚJO-JORGE2

and MIRIAN CLAUDIA DE SOUZA PEREIRA1

1Laboratório de Ultraestrutura Celular, Fundação Oswaldo Cruz, Av. Brasil 4365, Pav. Carlos Chagas 3° andar,Rio de Janeiro, Brazil2Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365,Manguinhos, Rio de Janeiro, RJ 21040-360, Brazil

(Received 3 August 2015; revised 18 December 2015; accepted 27 January 2016; first published online 21 March 2016)

SUMMARY

Transforming growth factor beta (TGF-β) cytokine is involved in Chagas disease establishment and progression. SinceTrypanosoma cruzi can modulate host cell receptors, we analysed the TGF-β receptor type II (TβRII) expression and dis-tribution during T. cruzi – cardiomyocyte interaction. TβRII immunofluorescent staining revealed a striated organizationin cardiomyocytes, which was co-localized with vinculin costameres and enhanced (38%) after TGF-β treatment.Cytochalasin D induced a decrease of 45·3% in the ratio of cardiomyocytes presenting TβRII striations, demonstratingan association of TβRII with the cytoskeleton. Western blot analysis showed that cytochalasin D significantly inhibitedSmad 2 phosphorylation and fibronectin stimulation after TGF-β treatment in cardiomyocytes. Trypanosoma cruzi infec-tion elicited a decrease of 79·8% in the frequency of cardiomyocytes presenting TβRII striations, but did not interfere sign-ificantly in its expression. In addition, T. cruzi-infected cardiomyocytes present a lower response to exogenous TGF-β,showing no enhancement of TβRII striations and a reduction of phosphorylated Smad 2, with no significant differencein TβRII expression when compared to uninfected cells. Together, these results suggest that the co-localization ofTβRII with costameres is important in activating the TGF-β signalling cascade, and that T. cruzi-derived cytoskeletondisorganization could result in altered or low TGF-β response in infected cardiomyocytes.

Key words: Cardiomyocytes, T. cruzi, TGF-β, TGF-β receptor type II, costameres.

INTRODUCTION

Chagas’ disease, an ancient infection caused by theprotozoan Trypanosoma cruzi, still impacts publichealth with a prevalence of infection of 10 millionpeople and about 25 million at risk of contractingthe disease (WHO, 2012). Human infection can leadto serious or fatal disease, causing significant mortal-ity in children during the acute phase and heart failurein chronically affected patients (Andrade et al. 2014).Chagas disease is the cause of 8–20% of severe cardio-myopathy cases in Latin America (Bocchi et al. 2013;Malik et al. 2015), where the infection is traditionallyendemic, but due to population migration it is nowconsidered an emerging infection in North Americaand Europe (Coura and Borges-Pereira, 2012).Transforming growth factor beta (TGF-β), a

multifunctional and pleiotropic cytokine, is involvedin the establishment and development of chagasic car-diomyopathy (Araújo-Jorge et al. 2008, 2012). TGF-β signalling pathway is triggered by activation of

latent TGF-βwhich binds to and activates two recep-tors, TGF-β receptors type II and type I, respective-ly, that phosphorylate the Smad proteins, resulting insignal transmission into the nucleus and activation ofgene transcription (Biernacka et al. 2011). Severalnon-Smad signalling pathways are also activated byTGF-β and may influence the final outcome ofTGF-β stimulation (Mu et al. 2012). TGF-β partici-pates in the host cell invasion process, since T. cruzineeds functional TGF-β receptors and active TGF-β signalling pathway to enter into mammalian cells(Ming et al. 1995; Hall and Pereira, 2000; Waghabiet al. 2007). Trypanosoma cruzi directly triggerslatent TGF-β activation (Waghabi et al. 2005a) andits uptake by intracellular amastigotes, eliciting para-site differentiation. TGF-β activation appears togovern the parasite’s intracellular cycle progression(Waghabi et al. 2005b), contributing to the invasionprocess and pathogenic development of chronicChagas disease. In addition, the host immune re-sponse is regulated by TGF-β during T. cruziinfection, with high level of this cytokine beingdetected in plasma of α2-macroglobulin deficientmice (Waghabi et al. 2002), in dogs (de Souza et al.2014) and in plasma of chronic chagasic patients

* Corresponding author: Laboratório de UltraestruturaCelular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil4365, Manguinhos 21040-360 Rio de Janeiro, RJ, Brazil.Phone: +5521 25621027. E-mail: cmcalvet@ioc.fiocruz.br

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Parasitology (2016), 143, 704–715. © Cambridge University Press 2016doi:10.1017/S0031182016000299

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(Araújo-Jorge et al. 2002; Rocha Rodrigues et al.2012), together with phosphorylated Smad 2 detectionin cell nuclei and extensive fibrosis in the heart. TGF-β triggers rise in fibronectin (FN), specifically inuninfected cells of T. cruzi-infected cardiomyocytecultures, while laminin expression remains unaltered(Calvet et al. 2009). Inhibition of theTGF-β signallingpathway by SB-431542 in the samemodel reduces car-diomyocyte invasion and parasite intracellular cycleprogression (Waghabi et al. 2007). Recent reportsshowed that TGF-β signalling inhibitors decreases in-fection and prevents heart damage and fibrosis in ex-perimental T. cruzi infection in mice (Waghabi et al.2009; de Oliveira et al. 2012). This evidence highlightsthe important role of TGF-β in T. cruzi infection andChagas disease development and leads to discussion oftherapeutic strategies designed to interfere with TGF-β pathway in clinical management of Chagas disease(Araújo-Jorge et al. 2012).Given the important role of TGF-β in chagasic car-

diomyopathy, we investigated the subcellular localiza-tion of TGF-β receptor type II (TβRII) incardiomyocytes and its functional regulation after in-fection by T. cruzi. We demonstrated that TβRIIco-localizes with costameres, striated domains of asso-ciated proteins that couple myofibrils to the sarco-lemma in muscle cells. These structures were firstdescribed with vinculin, and are involved in adhesion,force transmission and signal transduction processesof muscle cells (Craig and Pardo, 1983; Jaka et al.2015). Our data also demonstrated that this peculiarlocalization pattern is important to TGF-β signallingin cardiomyocytes, since its disruption by T. cruzi in-fection disturbs TGF-β response in these cells.

MATERIALS AND METHODS

Cell culture

Cardiomyocytes were isolated from 18-day-oldmouse embryos (Swiss Webster, animal facilities ofthe Oswaldo Cruz Foundation – FIOCRUZ, Riode Janeiro, Brazil), by enzymatic dissociation as pre-viously described (Meirelles et al. 1986). All proce-dures involving animals were approved by theCommittee of Ethics for the Use of Animals fromFIOCRUZ (CEUA LW-37/13). Briefly, heart ven-tricles were fragmented and submitted to sequentialdissociation in phosphate-buffered saline (PBS) con-taining 0·025% trypsin plus 0·01% collagenase(Worthington Biochemical Corporation, Lakewood,NJ, USA), pH 7·2. The isolated cells were platedinto 24-wells (105 cells/mL) containing glass cover-slips or 60 mm Petri dishes (2 × 106 cells) coatedwith 0·01% of gelatin. The cells were cultivated inDulbecco´s modified Eagle medium (DMEM;Sigma, St. Louis, MO, USA) supplemented with10% 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 inatmosphere of 5% CO2. After 24 h in culture, mostcells were well-spread and synchronously beating.

Parasites and cell culture infection

Bloodstream trypomastigote forms of T. cruzi(Y strain) were obtained from Swiss Webster miceat the peak of parasitemia as previously described(Meirelles et al. 1984). Muscle cell cultures wereinfected at a multiplicity of infection of 10 parasitesper host cell. After 24 h of interaction, free trypano-somes in the medium were removed by washing thecultures with Ringer’s solution and fresh mediumwas added to the culture. The time course of infec-tion was interrupted after 72 h.

TGF-β treatment

After 24 h of culture, cardiomyocytes were washedin Ringer’s solution and the TGF-β treatmentassays were carried out in serum-free medium.Cardiomyocyte cultures were incubated with 5, 10or 15 ng/mL of TGF-β, purified from bovine plate-lets (Promega Corporation, Madison, WI, USA), inDMEM supplemented with 1% bovine serum albu-mine (BSA; Sigma) and 2·5 mM CaCl2. The cultureswere maintained at 37 °C in atmosphere of 5% CO2and the medium containing TGF-β was dailyreplaced. After 24 and 48 h of treatment, the cultureswere processed for detection of TβRII using indirectimmunofluorescence and/or Western blot assays.

Cytoskeleton disruption assays

To inhibit polymerization of actin filaments andmicrotubules, 96 h-cultured cardiomyocytes wereincubated for 4 h at 37 °Cwith 12·5 µg mL cytochala-sin 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 incuba-tion was performed to disrupt the myofibrils of cardi-omyocytes. After drug treatment, TβRII distributionwas analysed by indirect immunofluorescence asdescribed below. For protein detection, culturestreated for 4 h with cythochalasinD and appropriatedcontrols were incubated with 15 ng/mL of TGF-β for1 or 48 h at 37 °C and submitted to protein extractionand Western blot analysis as further described. Theviability of the cytochalasin D treated cells wasassessed by determining the number of live cellsafter trypan blue dye exclusion, in a total of 200cells, in duplicate in each condition, under Zeissphase contrast inverted microscopy.

Indirect immunofluorescence

Cardiomyocyte cultures were fixed for 5 min at roomtemperature with 4% paraformaldehyde (Sigma) in

705T. cruzi disrupts TβRII costameres and TGF-β signalling in cardiomyocytes

PBS. The cultures were then washed with PBS con-taining 4% BSA to block unspecific reaction and incu-bated overnight at 4 °C with primary antibodies. Todetect the TGF-β receptor, the cells were incubatedwith anti-TβRII antibody (developed against thefull length molecule, Santa Cruz Biotechnology,Inc., Santa Cruz, CA, USA) diluted 1:300 in PBS.The antigen–antibody complex was revealed by incu-bation for 1 h at 37 °C with anti-IgG antibodiesTRITC or fluorescein isothiocyanate (FITC)-conju-gated (Sigma). Cytoskeleton proteins such as actinfilaments and vinculin were detected with 4 µg/mLphalloidin-FITC (Sigma) and anti-vinculin antibody(1:300; Sigma), respectively. When cytoskeleton pro-teins were analysed, cardiomyocytes were permeabi-lized with PBS containing 0·5% Triton X-100(Sigma), prior to antibody incubation. DNA wasstained with 10 µg mL 4,6-diamidino-2-phenilyndole(DAPI; Sigma). Controls were performed by incuba-tion with homologue serum or by omission of theprimary antibody. The cells were mounted in 2·5%1,4-diazabicyclo-(2,2,2)-octane (DABCO; Sigma) inPBS containing 50% glycerol, pH 7·2 and imageswere taken using confocal laser scanning microscope(Zeiss LSM Meta 510). Co-localization analysis wasperformed with Image J software, using the pluginco-localization highlighter (http://www.rsb.info.nih.gov/ij/plugins/colocalization.html). The index of car-diomyocytes presenting striated array of TβRII wasdetermined by counting TβRII striations positivecardiomyocytes in a total of 300 cells in 3 separateexperiments under conventional epifluorescence mi-croscopy using a Zeiss Axioplan instrument.

Protein extraction and immunoblotting assay

Total protein was extracted using lysis buffer (50 mMTris-HCl, NaCl 150 mM, 1% Triton X-100, 1 mMethylene glycol-bis-(β-amino-ethyl ether) N,N,N´,N´-tetra-acetic acid (Sigma), 100 µg/mL phenyl-methylsulphonyl fluoride (Sigma), 1 µg/mL pepstatin(Sigma), 1 µg/mL aprotinin, and phosphatase inhibi-tor cocktail PhoSTOP (Roche Applied Science,Mannheim, Germany), pH 8·0). The cell lysate wasmixed with Laemli sample buffer containing 2-mercaptoethanol, and boiled at 100 °C subsequentto protein extraction to avoid protein degradationand dephosphorylation. The amount of protein wasdetermined by the Folin–Lowry method and a totalof 20 µg of protein was electrophoretically separatedin a 12% polyacrylamide gel, by SDS–PAGE. Aftertransference and blockage, the nitrocellulose mem-branes (Bio-Rad, Hercules, CA, USA) were incu-bated 18 h at 4 °C with primary antibodies, dilutedin blocking buffer. Cytoskeleton proteins and theTGF-β receptor were detected with anti-vinculin(1:2000; Sigma), anti-α-sarcomeric actin (1:3000;Sigma), anti-α-actinin (1:2000; Santa CruzBiotechnology), anti-desmin (1:1000; Sigma) and

anti-TβRII antibodies. For Western blot analysis,two different antibodies for TβRII were used: onedeveloped against the full length receptor (1:2000,Santa Cruz Biotechnology) and a second to whichthe immunogenic peptide corresponded to the first28 N-terminal residues of the mature human TGF-β receptor (1:1000, Merck Millipore, Darmstadt,Germany). Phosphorylated Smad 2 (1:1000; CellSignaling Technology, Danvers, MA, USA) andFN (1:5000; Sigma) expression were also analyzed.Mouse anti-α-GAPDH (1:50 000; Ambion, Austin,TX, USA) antibodies, added together with primaryantibodies, were used as internal pattern. Afterseveral washes, the membrane was incubated withanti-mouse (1:30 000; Thermo Scientific, Waltham,MA, USA) or anti-rabbit (1:30 000; ThermoScientific) antibody conjugated to horseradish perox-idase. The enzyme activity was revealed withSuperSignal West Pico Chemiluminescent kit(Thermo Scientific). The densitometry was per-formed with Image J software (National Institutes ofHealth, http://www.rsb.info.nih.gov/ij/) with the gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH)band, at ∼36 kDa, as internal control. The immuno-blotting experiments were performed independentlyat least three times.

Statistical analysis

Student’s t-test was used for comparison of experi-mental data from quantification of immunofluores-cence detection of TβRII, as well as for densitometryof Western blotting bands. Values were consideredstatistically significant when P⩽ 0·05.

RESULTS

The subcellular localization of TβRII in cardiomyo-cytes was determined by confocal microscopy. TheTβRII was visualized as punctuated staining wide-spread over the cell and also in a striated patternprofile (Fig. 1). No cross-reaction with the second-ary antibody or cardiomyocyte autofluorescencewas observed in the negative controls after omissionof anti-TβRII antibody (Supplementary Fig S1,Supplementary material). This peculiar locationopened the question whether the TβRII could beassociated with cytoskeleton proteins, conferring itslocalization in subcellular domains and functionalityin cardiomyocytes. To address this question, car-diomyocyte cultures were double labelled withanti-TβRII and anti-vinculin antibodies or phalloid-in-FITC, which binds to actin filaments. Confocalimaging revealed co-localization of TβRII with cost-ameres of vinculin, showing its characteristic stain-ing at the Z-line of myofibrils associated with thecell membrane (Fig. 1), while no co-localizationwas observed in I band of myofibrils. The co-loca-lized pixels from TβRII and vinculin staining

706Claudia Magalhães Calvet and others

http://www.rsb.info.nih.gov/ij/plugins/colocalization.htmlhttp://www.rsb.info.nih.gov/ij/plugins/colocalization.htmlhttp://www.rsb.info.nih.gov/ij/plugins/colocalization.htmlhttp://www.rsb.info.nih.gov/ij/http://www.rsb.info.nih.gov/ij/

images were highlighted using the co-localizationplugin of Image J software demonstrating TβRIIdetection in vinculin costameres (Fig. 1).Given the similarity of TGF-β receptor reaction to

the typical periodicity of sarcomeric proteins withinmyofibrils, we investigated the specificity of TβRIIantibody by Western blotting assay. Our resultsdemonstrated a protein band with molecular massof 70 kDa and no cross-reaction with sarcomeric cyto-skeletal proteins, such as α-sarcomeric actin, α-actininor desmin, neither with vinculin (SupplementaryFig. S2, Supplementary material). In order to inves-tigate whether the TβRII localization is directly asso-ciated with the cytoskeleton arrangement, we treatedthe cardiomyocyte cultures with drugs known to de-polymerize actin filaments and microtubules.Incubation of cardiomyocytes with 12·5 µg/mL ofcytochalasin D, which depolymerizes actin filamentand triggers the disorganization of myofibrils afterlong-term treatment, affected the striated distributionof TβRII, showing mainly a punctuated widespreadlabelling (Fig. 2). In contrast, microtubule disrup-tion, induced by nocodazole treatment, resulted inno major effect on this receptor distribution(Fig. 2). The percentage of cardiomyocytes display-ing striated array of TβRII was also determinedafter treatment of the cultures with cytoskeleton de-polymerization agents. Cytochalasin D treatmentleads to a significant reduction of 45·3% (P⩽ 0·033)

in the frequency of TβRII striations, whereas nocoda-zole treatment did not affect this rate (Fig. 3).To assess the functional role of striated organization

of TβRII, cardiomyocyte cultures were pre-treatedwith 12·5 µg/mL cytochalasin D, stimulated withTGF-β and then phosphorylated Smad 2 (PS2)content was determined in total protein extracts byWestern blot analysis. Cytochalasin D treatmentalone had no effect on PS2 activation in control cells.As expected, addition of TGF-β (1 h) elicited asignificant rise in PS2 expression. In contrast, PS2levels of cythocalasin D pre-treated cardiomyocytesafter TGF-β addition are similar to non-stimulatedcontrol cells (P⩽ 0·018; Fig 3B). These data suggestthat the subcellular localizationofTβRII in costameres,which is disrupted by cytochalasin D treatment, con-tribute to the activation of TGF-β signalling pathway.FN expression was also inhibited by myofibrillardisruption induced by cytochalasin D, even after 48 hof TGF-β stimulation (P⩽ 0·012; Fig 3), confirmingthe functional role of striated array of TβRII onTGF-β signalling. The viability of cytochalasin Dtreated cells was determined through quantificationof trypan blue stained cells, showing no significantloss 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 fre-quency of the striation pattern of TβRII in

Fig. 1. Analysis of TβRII and sarcomeric proteins localization in cardiomyocytes. Double labelling of cardiomyocytecultures 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 andvinculin staining images (D), demonstrating TβRII detection in vinculin costameres. Detection of TβRII (E) and actinfilaments by phalloidin staining (F), showing myofibrils, demonstrated TβRII localized in Z-line of myofibrils (G). Insetsin (C) and (G) show TβRII localization in Z-line of myofibrils from areas delimited in the squares in higher magnification.Bar = 10 µm

707T. cruzi disrupts TβRII costameres and TGF-β signalling in cardiomyocytes

cardiomyocyte cultures (Fig. 4). The quantificationof cells containing striated distribution of TβRIIrevealed a significant increase of 38% (P⩽ 0·009)after 48 h-stimulation of cardiomyocytes withTGF-β (Fig. 5). The analysis of T. cruzi-infectedcardiomyocytes (72 h) presenting high numbers ofintracellular amastigotes revealed anti-TβRII stain-ing in parasites (Fig. 4). The co-localization ofTβRII with costameres was rarely visualized inT. cruzi-infected cardiomyocyte culture, which

presented mostly TβRII punctuated staining oncell surface, being difficult to observe the host cellspecific distribution due to elevated number ofTβRII stained amastigotes (Fig. 4). At this stage ofinfection, the actin cytoskeleton is disrupted inhighly infected cells (Supplementary Fig. S4,Supplementary material). The quantification ofTβRII striations in T. cruzi-infected culturesrevealed a dramatic 79·8% reduction (P⩽ 0·009) inthe percentage of cells presenting costameric

Fig. 2. Analysis of TβRII distribution after treatment of cardiomyocyte cultures with cytoskeleton depolymerizationagents. Untreated cardiomyocytes displaying regular TβRII striations (A and C; red) co-localized with the Z-line ofmyofibrils stained with phalloidin-FITC (B and C; green). Inset in (B) shows intact microtubules in cardiomyocytesrevealed 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 ofTβRII. (D and F; red). Treatment with cytochalasin does not affect microtubules as shown in inset in (E). Nocodazoletreatment does not affect TβRII striated distribution (G and I; red) besides the depolymerization of microtubules (H andI; green). Nocodazole treated cultures still show an intact actin cytoskeleton (H, inset). Bar = 20 µm

708Claudia Magalhães Calvet and others

TβRII distribution (Fig. 5). The punctuatedistribution of TβRII remained unaltered inT. cruzi-infected cardiomyocytes even after TGF-βtreatment (Fig. 4). This cytokine was not able tostimulate the reorganization of TβRII in costameresin T. cruzi-infected cultures (Fig 4), which still dis-played a statistically significant reduction in the per-centage of cells presenting striated array of TβRII(Fig. 5). The disorganization of TβRII costameresin cardiomyocyte cultures induced by T. cruzi infec-tion (72 h) also seems to impair host cell response toTGF-β, since addition of 15 ng/mL of TGF-β (1 h)inhibited PS2 stimulation observed in uninfectedcultures (Fig. 5). In T. cruzi-infected cultures,TGF-β treatment did not elicit any statistical differ-ence in PS2 levels when compared to untreated car-diomyocytes (Fig. 5).To analyse TβRII expression in control and

T. cruzi-infected cardiomyocyte protein extracts,we utilized a different anti-TβRII antibody raisedagainst the N-terminal region of the receptorwhich did not recognize purified parasite extracts(data not shown), guaranteeing that no cross-reac-tion would occur with intracellular amastigotes ortrypomastigotes. Although attempts to visualizeTβRII distribution by immunofluorescence withthis second antibody were not successful, theWestern blot analysis of TβRII expression in cardi-omyocyte cultures showed one specific band of 70kDa (Fig 6). Our results show that the levels ofTβRII in uninfected cultures after TGF-β or cyto-chalasin D treatment remained unaltered (Fig. 6).In the same fashion, TβRII expression after 72 hT. cruzi infection did not show statistically signifi-cant alteration (Fig. 6).

DISCUSSION

TGF-β has been implicated in tissue remodelling bycontrolling the synthesis and degradation of theextracellular matrix (Leask, 2007; Kong et al.2014) and is involved in Chagas disease fibrosis de-velopment (Araújo-Jorge et al. 2002, 2008, 2012).Nevertheless, few reports demonstrate the distribu-tion of TGF-β receptors in cardiomyocytes, and noinformation is available about its interaction withT. cruzi.Our results revealed a striated pattern of receptor

distribution in cardiomyocytes, which was signifi-cantly increased in 38% after TGF-β stimulation.This distribution suggests an association of the re-ceptor with cytoskeleton proteins in cardiomyocytes.TβRII organization in cardiomyocytes has been pre-viously reported as widespread dots on cell surfacewithout striated pattern (He et al. 2011). This con-trasting result may be related to their lower level ofculture differentiation revealed by the absence ofwell-developed myofibrils. In our data, the striatedpattern of TβRII was clearly demonstrated to

Fig. 3. (A) The quantification of the percentage ofcardiomyocytes showing TβRII striations revealed asignificant 45% decrease in TβRII striations aftercytochalasin treatment, while no alteration was observedafter nocodazole treatment. (B) Variation index (V.I., i.e.normalization of densitometry values considering control= 1) of PS2 detection in cardiomyocytes demonstrates thatcytochalasin treatment prior to TGF-β addition inhibits86% of the PS2 stimulation obtained in untreated controls.(C) Cytochalasin induced cytoskeleton disorganizationalso affects long-term responses of cardiomyocytes toTGF-β, since previous addition of this drug to cultureinhibits FN enhancement induced by TGF-β after 48 hincubation detected by Western blot. *P⩽ 0·05

709T. cruzi disrupts TβRII costameres and TGF-β signalling in cardiomyocytes

co-localize with costameres, suggesting that the lo-calization of the TβRII in this mechanical transduc-tion site in cardiomyocytes, previously reported as aregulatory signalling domain (VanWinkle et al.2002), may potentiate TGF-β signalling. The associ-ation of TβRII with costameres was also demon-strated by myofibrillar disruption after cytochalasinD treatment, which resulted in significant loss ofTβRII striation, suggesting that the integrity of con-tractile apparatus is essential to regulate the TβRIIsubcellular localization.

TβRII localization in costameres is also supportedby the fact that several proteins known to interact ormodulate TGF-β signalling such as syndecans(Chen et al. 2004), integrins (Hayashida, 2010),β-catenin (Tian and Phillips, 2002), filamin (Sasakiet al. 2001; Razinia et al. 2012) and spectrin (Limet al. 2014) present striated distribution in cardio-myocytes (Craig and Pardo, 1983; Kotelianskyet al. 1986; Wu et al. 2002; Stevenson et al. 2005).The TGF-β receptor also has two subunits thatneed to dimerize for activation of the signalling

Fig. 4. Detection of TβRII in cardiomyocytes by indirect immunofluorescence. (A) Cardiomyocyte cultures displaypunctuate staining and striated array (*) of TβRII organization. (B) TGF-β treatment (48 h) resulted in an increase in thefrequency of cells containing TβRII striations (*). (C and D) Trypanosoma cruzi-infected cardiomyocytes presentpreferentially punctuate distribution of TβRII, with a marked reduction in the number of cells displaying striated pattern ofTβRII 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, whichstill 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

710Claudia Magalhães Calvet and others

cascade, plus accessories receptors, which wouldhave their association facilitated or enhanced by aconcentrated location of the receptors in signallingdomains such as costameres.An increase in the level of TβRII striation in

cardiomyocytes was evidenced after TGF-β stimula-tion. TGF-β is a well-known modulator of cellulardifferentiation (Massagué and Xi, 2012), and the in-crease of TβRII striations may reflect a higher levelof differentiated cells. In addition, TGF-β inducesreorganization of the actin cytoskeleton in severalmodels (Hubchak, 2003; Vardouli et al. 2005;Assinder and Cole, 2011) by activation and mobiliza-tion of Cdc42, RhoA and LIM-kinase (Edlund et al.

2002; Hubchak, 2003), and also triggers the synthesisof muscle specific proteins (Eghbali et al. 1991; Liet al. 2005; Singla et al. 2005). Therefore, the increasein the percentage of cells presenting TβRII striationsmay also be related to a direct remodelling of thecytoskeleton triggered by this cytokine.TGF-β signalling is known to be linked to the

cytoskeleton (Moustakas and Heldin, 2008), andour data suggest that this linkage might be relatedto the localization of TβRII in costameres. Theclassic TGF-β signalling pathway (via Smads) caninteract with other pathways such as mitogen-activated protein (MAP) kinases, FAK and Src(Galliher and Schiemann, 2007; Wang et al. 2009;Mu et al. 2012), which are triggered by integrins(Wu et al. 2002; Samarel, 2005). Therefore, consid-ering that syndecans and integrins are also found in

Fig. 5. (A) The quantification of the percentage ofcardiomyocytes displaying TβRII striations showed a raiseof 38% (P⩽ 0·032) of TβRII striations after TGF-βtreatment. Trypanosoma cruzi infection elicited a dramatic79·8% reduction (P⩽ 0·009) in the percentage of cellsshowing costameric TβRII distribution. Trypanosomacruzi-infected, TGF-β treated cultures still show astatistically significant reduction that reached a maximumof 96% in the percentage of cells displaying striated array ofTβRII. (B) Western blot analysis revealed that T. cruziinfection (72 h) prior to addition of 15 ng/mL of TGF-β(1 h) inhibited the PS2 stimulation observed in uninfectedcultures, since PS2 levels inT. cruzi-infected cultures afterTGF-β treatment were not statistically different fromuntreated and infected cardiomyocytes.* #P⩽ 0·05.

Fig. 6. Western blot analysis of TβRII effect oncardiomyocyte cultures. (A) TGF-β or cytochalasintreatment did not result in relevant alterations in TβRIIexpression. (B) TβRII protein levels in T. cruzi-infectedcardiomyocyte culture, treated or not with TGF-β, alsodid not show statistically significant differences. GAPDHwas used as internal control. Data was normalizedconsidering densitometry values from untreated anduninfected controls as 1.

711T. cruzi disrupts TβRII costameres and TGF-β signalling in cardiomyocytes

costameres, the idea that this region may function asa TGF-β signalling domain in cardiomyocytes is notonly plausible, but is also suggested by other authors(Samarel, 2005; Moustakas and Heldin, 2008). Inuninfected cardiomyocytes, the Smad 2 phosphoryl-ation and TGF-β induced FN stimulation wasreduced after cytochalasin treatment, which also dis-organized the striated distribution of TβRII.

Cytochalasin treatment likewise impairs Smad 2phosphorylation in mesangial cells (Hubchak,2003), and FAK/Src signalling and receptor cluster-ing in human breast cancer cell lines (Wang et al.2009), demonstrating the dependence of TGF-β sig-nalling on an intact actin cytoskeleton in differentcells. Spectrin, a cytoskeleton protein that hasstriated localization in costameres of cardiomyocytes

Fig. 7. Proposal of TGF-β receptor II functional association with cardiomyocyte cytoskeleton. TGF-β receptor II islocalized in cardiomyocyte’s costameres, which are also rich in vinculin. The localization of the receptor in these signallingdomains of cardiomyocyte membranes, that are closely associated with the cytoskeleton, potentiate Smad 2phosphorylation (A). Once T. cruzi infection is established, the cytoskeleton is disorganized, disrupting TβRII striationsand decreasing Smad 2 phosphorylation after interaction with TGF-β (B). In the tissue context, we suggest that the highlyinfected cardiomyocytes would be specifically less responsive to exogenous TGF-β stimulation.

712Claudia Magalhães Calvet and others

(Stevenson et al. 2005), also is linked to TGF-β sig-nalling. Disruption of spectrin by gene knockoutresults in phenotypes similar to Smad deficiency(Tang et al. 2003), suppressing Smad signallingand cardiomyocyte differentiation, leading to cellcycle deregulation and apoptosis in heart musclecells (Lim et al. 2014).Costameric distribution of TβRII was signifi-

cantly reduced in T. cruzi-infected cardiomyocytes.The disorganization of TβRII striations is likely tobe a result of the myofibrillar breakdown (Pereiraet al. 1993; Taniwaki et al. 2006), with a clear disor-ganization of vinculin costameric distribution anddisarray of sarcomeric α-actinin caused by T. cruziinfection (Melo et al. 2004, 2006), since the striatedorganization of the receptor depends on the cyto-skeleton integrity. One striking feature was the im-munolabelling of intracellular amastigotes revealedby anti-TβRII antibody, corroborating previousstudies showing TGF-β binding to intracellularamastigotes, both in the heart tissue and cardiomyo-cyte cultures, while trypomastigotes showed no reac-tion (Waghabi et al. 2005b). An intriguing fact isthe failure to identify the TGF-β gene in T. cruzigenome associated with the detection of TGF-β inthe surface membrane, flagellar pocket, intracellularvesicles or cytoplasm of either the intracellular andaxenic amastigotes, suggesting that the parasitecould capture TGF-β from the host and use it forits intracellular differentiation (Waghabi et al.2005a, b). This idea was reinforced by reportsshowing that the treatment of cardiomyocytes withSB-431542, a selective inhibitor of ALK5 (a TGF-β type I receptor), hindered the proliferation ofintracellular amastigotes and the release of trypo-mastigotes (Waghabi et al. 2007). The ability ofT. cruzi to biologically respond to TGF-β was alsoshown by a phosphoproteomic approach thatdetected both phosphorylated proteins and up- ordownregulated proteins after TGF-β interaction(Ferrão et al. 2012), suggesting that the parasitehas a TGF-β responsive molecule on its surface.Our data showed that T. cruzi infection disrupted

TGF-β signalling, with infected cardiomyocytes dis-playing low phosphorylation of Smad 2 after short-term TGF-β addition, together with disorganizationof TβRII costameric distribution. Previous data ofour group demonstrated thatT. cruzi-infected cardio-myocytes present lower FN expression (Calvet et al.2004), while the addition of TGF-β results inincreased ECM expression only in uninfected cellsof the infected culture, suggesting that T. cruzi infec-tion prevented FN stimulation by this cytokine(Calvet et al. 2009). These data might be related tothe lower capacity of host cell to respond to TGF-βstimulation, which is associated with TβRII delocal-ization. Although T. cruzi modulates the expressionof several surface and cytokine receptors in immunecells (Majumder and Kierszenbaum, 1996; Albareda

et al. 2015) and cardiomyocytes (Soeiro et al. 1999),TβRII protein levels did not show significant altera-tions in our model, and the lower response in theinfected cell is likely to be due to cytoskeleton break-down induced by the parasite. In addition, T. cruzialso seems to interfere directly in the signalling path-ways of TGF-β, since infected cells not exposed toTGF-β exhibit phosphorylation of Smad 2/3(Unnikrishnan and Burleigh, 2004; Waghabi et al.2007), and parasite factors antagonize TGF-β depend-ent induction of CTGF in fibroblasts (Unnikrishnanand Burleigh, 2004). Other signalling pathways alsoare modulated by T. cruzi infection such as Erk 1/2in different cell types (Mukherjee et al. 2004;Magdesian et al. 2007) and in cardiac cells (Adesseet al. 2010). Molecules of the parasite itself may alsointerfere directly in host cell signalling pathways. Amucin from T. cruzi inhibits the activation of p38,extracellular signal-regulated kinase (ERK) and Junamino-terminal kinase (JNK) in macrophages, al-though not preventing the activation of p38 inducedby tumor necrosis factor (TNF)-α (Alcaide andFresno, 2004). In contrast, cruzipain, a major cysteineprotease from T. cruzi, induces p38 in macrophages(Stempin et al. 2008), and it was recently shown thatcruzipain is also an important activator of latentTGF-β and thereby triggers TGF-β-mediatedevents crucial for the development of Chagas disease(Ferrão et al. 2015). Moreover, addition of purifiedcruzipain enhanced the invasive activity of trypomas-tigotes, an effect that can be inhibited by a neutralizinganti-TGF-β antibody, leading to the conclusion thatthe activities of cruzipain and TGF-β are functionallylinked in the process of cell invasion, and the engage-ment 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 isdependent of cytoskeleton integrity. This costamericlocalization of TβRII is important in triggering thecanonical TGF-β signalling pathway, since cytoskel-eton disorganization results in lower Smad 2 phos-phorylation (Fig. 7). Trypanosoma cruzi-infectedcardiomyocytes present a reduction of TβRII stria-tions, together with a lower response to addedTGF-β, suggesting that cytoskeleton disorganiza-tion triggered by the parasite could result in alteredor low TGF-β response localized in highly infectedcardiomyocytes (Fig. 7).

SUPPLEMENTARY MATERIAL

To view supplementary material for this article,please visit http://www.dx.doi.org/10.1017/S0031182016000299

ACKNOWLEDGEMENTS

The authors thank Danielle Almeida, Liliane Mesquitaand Alanderson Nogueira for technical support; Pedro

713T. cruzi disrupts TβRII costameres and TGF-β signalling in cardiomyocytes

http://www.dx.doi.org/10.1017/S0031182016000299http://www.dx.doi.org/10.1017/S0031182016000299http://www.dx.doi.org/10.1017/S0031182016000299

Paulo de A. Manso and Carlos Bizarro for confocal imageacquisition; Dr Jean Jacques Feige and Sabine Bailly(Commissariat à l’Energie Atomique, Grenoble, France)for helpful discussion of the results; Francisco O. deOliveira Jr for assistance with the figures; Mr PotterWickware for proofing the manuscript.

FINANCIAL SUPPORT

This work was supported by grants from FundaçãoOswaldo Cruz (FIOCRUZ) to C.M.C, T.A.S.. T.G.M.,T.C.A.J., M.C.S.P.; Programa Estratégico de Apoio àPesquisa em Saúde (PAPES) to C.M.C, T.A.S., T.G.M., M.C.S.P.; Fundação de Amparo à Pesquisa doEstado 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 deDesenvolvimento 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.

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715T. cruzi disrupts TβRII costameres and TGF-β signalling in cardiomyocytes

TGF-β receptor type II costameric localization in cardiomyocytes and host cell TGF-β response is disrupted by Trypanosoma cruzi infectionINTRODUCTIONMATERIALS AND METHODSCell cultureParasites and cell culture infectionTGF-β treatmentCytoskeleton disruption assaysIndirect immunofluorescenceProtein extraction and immunoblotting assayStatistical analysis

RESULTSDISCUSSIONSupplementary materialACKNOWLEDGEMENTSFINANCIAL SUPPORTCONFLICT OF INTERESTReferences