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Trypanosoma cruzi infection disrupts vinculin costameres in cardiomyocytes
Eur. J. Cell Biol. 83 (2004); 531 ± 540 http: // www.elsevier.de/ejcb
Trypanosoma cruzi infection disrupts vinculin costameres in cardiomyocytes Tatiana G. Melo, Danielle S. Almeida, Maria de Nazareth S. L. de Meirelles, Mirian Claudia S. Pereira1) Departamento de Ultra-estrutura e Biologia Celular, Laborato¬rio de Ultra-estrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil Received May 28, 2004 Received in revised version August 11, 2004 Accepted August 19, 2004
Cardiomyocytes; Trypanosoma cruzi; Focal adhesion protein; Vinculin
Introduction
Chagas× disease cardiomyopathy is an important manifestation of Trypanosoma cruzi infection, leading to cardiac dysfunction and serious arrhythmias. We have here investigated by indirect immunofluorescence assay the distribution of vinculin, a focal adhesion protein with a major role in the transmission of contraction force, during the T. cruzi-cardiomyocyte infection in vitro and in vivo. No change in vinculin distribution was observed after 24 h of infection, where control and T. cruziinfected cardiomyocytes displayed vinculin localized at costameres and intercalated discs. On the other hand, a clear disruption of vinculin costameric distribution was noted after 72 h of infection. A significant reduction in the levels of vinculin expression was observed at all times of infection. In murine experimental Chagas× disease, alteration in the vinculin distribution was also detected in the infected myocardium, with no costameric staining in infected myocytes and irregular alignment of intercalated discs in cardiac fibers. These data suggest that the disruption of costameric vinculin distribution and the enlargement of interstitial space due to inflammatory infiltration may contribute to the reduction of transmission of cardiac contraction force, leading to alterations in the heart function in Chagas× disease.
Trypanosoma cruzi, the causative agent of Chagas× disease, needs to invade vertebrate host cells to survive and replicate. Therefore, its ability to enter non-professional phagocytic cells is a critical factor to enhance its subsistence. Although many cell types are infected by T. cruzi in vitro (Piras et al., 1983), a defined tropism to muscle cells has been reported (Brener, 1973; Cabrine-Santos et al., 2001; Vera-Cruz et al., 2003), leading to progressive cardiomyopathy and megaesophagus in the chronic phase of Chagas× disease (reviewed by Higuchi et al. (2003), Kirchhoff (1996), Lages-Silva et al. (2001)). Thus, a detailed investigation of the T. cruzi-muscle cell interaction may contribute to the knowledge of biological and molecular events that occur during the infectious process. Changes in the cytoskeleton of host cells have been reported to facilitate several microbe-host cell interactions (reviewed by Gruenheid and Finlay (2003), Smith and Enquist (2002)). Rearrangement of actin is required for many pathogens to invade the host cell (Elliot and Clark, 2000; Mortara et al., 1991; Tan and Andrews, 2002). Once within the host cell, the pathogens may modulate the cytoskeleton dynamics of the host cell to their advantage by remodeling the parasitophorous vacuole (Guerin and De Chastellier, 2000; Meresse et al., 2001) or facilitating their intracellular motility (Frischknecht and Way, 2001; Goldberg, 2001). Focal adhesion proteins have been also reported to participate in many parasite-host cell processes (Shifrin et al., 2002; Teoh et al., 2000; Yilmaz et al., 2003). Vinculin, talin and a-actinin have been identified in association with microfilaments at the parasite-host cell interface (Finlay et al., 1991, 1992; Freeman et al., 2000; Tran Van Nhieu et al., 1997). In addition, vinculin has been reported to play an important role in generating force to Shigella motility within and between adjacent host cells, leading to a systemic disease (Laine et al., 1997). Vinculin, a major protein of adherens-type junctions (Geiger and Ginsberg, 1991), displays several binding sites for many
Abbreviations. Abbreviations. BSA Bovine serum albumin. ± DABCO 1,4Diazabicyclo-(2.2.2)-octane. ± DAPI 4',6-Diamidino-2-phenylindole. ± DMEM Dulbecco×s modified Eagle×s medium. ± dpi Days post infection. ± FBS Fetal bovine serum. ± PIP2 Phosphatidylinositol 4,5-bisphosphate. ± PBS Phosphate-buffered saline. 1)
Corresponding author: Dr. Mirian Claudia S. Pereira, Departamento de Ultra-estrutura e Biologia Celular, Laborato¬rio de Ultra-estrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, RJ, 21045 ± 900, Brazil, e-mail: [email protected], Fax: 5521 2260 4434.
0171-9335/04/083/10-531 $30.00/0
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cytoskeletal proteins. The vinculin primary structure consists of a globular head connected by a proline-rich neck to a long flexible tail (Milam, 1985; Molony and Burridge, 1985; Winkler et al., 1996; Zamir and Geiger, 2001). Once activated by exposure to phosphatidylinositol 4,5-bisphosphate (Gilmore and Burridge 1995, 1996; H¸ttelmaier et al., 1997), which binds to the protein tail (Johnson et al., 1998; Sechi and Wehland, 2000), vinculin exposes its multidomains, allowing the interaction with a-actinin (Wachsstock et al., 1987; Kroemker et al., 1994) and talin (Burridge and Mangeat, 1984; Johnson and Craig, 1994) at the head region, while tail domains bind to Factin, paxillin, VASP, ponsin, vinexin, lipid bilayer and also to the vinculin head in its inactive conformation (Critchley, 2000; Geiger et al., 2001; Jockusch et al., 1995; Johnson and Craig, 1995). Vinculin plays an important role in anchoring microfilaments to the plasma membrane at focal adhesion sites. In muscle cells, this protein is also located at the Z line of myofibrils, termed costameres, in association with the plasma membrane (Pardo et al., 1983a, b; Craig and Pardo, 1983; Koteliansky and Gneushev, 1983), suggesting its participation in the mechanical force transmission during the contractionrelaxation process (Danowski et al., 1992; Imanaka-Yoshida et al., 1994). Chagas× disease cardiomyopathy leads to cardiac damage and dysfunction with ventricular and atrial arrhythmias (Elizari, 1999, 2002). However, the mechanism leading to the evolution of the disease is still unknown. Many studies have reported that mutations in cytoskeletal protein genes are involved in the hypertrophic and dilated cardiomyopathy (Daehmlow et al., 2002; Marian and Roberts 1998, 2001). Although previous data revealed that T. cruzi affects organization of the cytoskeleton and regulation of the actin gene in cardiomyocytes (Pereira et al., 1993, 2000), little is known about actin-binding proteins, such as focal adhesion proteins, during the T. cruzi-host cell interaction. In the present study we report for the first time changes in the expression and distribution of vinculin in cardiomyocytes induced by T. cruzi infection.
Materials and methods Animals Swiss Webster male mice weighing 18 ± 20 g were obtained from the animal facilities of the Oswaldo Cruz Institute ± FIOCRUZ, Rio de Janeiro, Brazil. A total of 60 mice, divided in 3 different experimental groups, were intraperitoneally inoculated with 102 bloodstream trypomastigote forms of Trypanosoma cruzi, Y strain. Parasitemia was individually checked by counting the number of parasites/5 ml of blood at different days post-infection (dpi) (Brener, 1962; Luz et al., 1995). Cardiac tissue was obtained from 3 uninfected and 3 infected animals sacrificed at different stages of the acute infection: at negative parasitemia, at the peak of parasitemia, and after parasitemia decline. The hearts were embedded in Tissue-Tek (Sakura Finetek, CA, USA) and frozen in liquid nitrogen.
Cell culture Primary cultures of cardiomyocytes were obtained from 18-day-old mouse embryos as previously described (Meirelles et al., 1986). Briefly, cardiac fragments were dissociated in phosphate-buffered saline (PBS, pH 7.2) containing 0.025% trypsin (Sigma; St. Louis, MO, USA) plus 0.01% collagenase (Worthington, New Jersey, USA) and plated (1 105 cells/ml) into 24-well culture dishes containing glass coverslips previously coated with 0.01% gelatin (Sigma). For biochemical analysis,
the isolated cells were plated at a density of 2.5 106 cells in 60-mm culture dishes. The cells were cultivated in Dulbeccos× modified Eagle medium (DMEM; Sigma) supplemented with 10% horse serum (Sigma), 5% fetal bovine serum (FBS; Sigma), 2.5 mM CaCl2, 1 mM L-glutamine (Sigma), 2% chicken embryo extract, and maintained at 37 8C in a 5% CO2 atmosphere.
Parasites and infection of cultured cells Bloodstream trypomastigote forms of Trypanosoma cruzi, Y strain, were obtained from Swiss mice at the peak of parasitemia as described (Meirelles et al., 1984). Cardiac cell cultures were infected at a multiplicity of 10 parasites per host cell. After 24 h of interaction, free trypomastigotes were removed by washing the cultures with Ringer×s solution (154 mM NaCl, 56 mM KCl, 17 mM Na2HPO4, pH 7.0). The time course of infection was interrupted after 24 and 72 h.
Indirect immunofluorescence Uninfected and T. cruzi-infected cardiomyocytes were fixed for 20 min at 4 8C with 4% paraformaldehyde (Sigma) in PBS. Thereafter, the cells were washed and permeabilized with PBS containing 0.5% Triton X-100 (Sigma). The cultures were then washed with PBS containing 4% bovine serum albumin (BSA; Sigma) to block unspecific reactions and incubated overnight at 4 8C with anti-vinculin antibody (Sigma) diluted 1 : 300 in PBS. The antigen-antibody complex was revealed by incubation for 1 h at 37 8C with TRITC-conjugated anti-mouse IgG (Sigma). Controls were performed by incubation with mouse serum or by omission of the primary antibody. Actin filaments and DNA were detected with 4 mg/ml phalloidin-FITC (Sigma) and 10 mg/ml 4,6diamidino-2-phenylindole (DAPI; Sigma), respectively. The cells were mounted in 2.5% 1,4-diazabicyclo-(2,2,2)-octane (DABCO; Sigma) in PBS containing 50% glycerol, pH 7.2. The percentage of cells (n 500 cells, from 3 different experiments) displaying a costameric distribution of vinculin was evaluated in controls and T. cruzi-infected cells after 24 and 72 hours of infection. The fluorescence images were obtained using either an Axioplan 2 microscope equipped with epifluorescence or a confocal laser scanning microscope (BX51 Olympus). The cardiac tissue cryosections were fixed for 10 min at 20 8C in acetone and processed as described above.
Protein extraction and immunoblotting assay Total proteins were extracted using a lysis solution (50 mM Tris-HCl containing 1% Triton X-100 plus 10 mM E-64 (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma) and 1 mM pepstatin (Sigma), pH 8.0). The amount of protein was determined by the Bradford method (Bradford, 1976) and a total of 20 mg of protein was eletrophoretically separated in a 12% polyacrylamide gel, by SDS-PAGE. The proteins were then transferred at 30 V (60 mA) overnight at 4 8C to a Polyvinylidene difluoride (PVDF) membrane (Bio-Rad, California, USA) in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol, pH 8.3). After protein transfer, the membrane was blocked with 0.5% I-Block (Sigma), 0.1% Tween 20 (Sigma) in PBS and incubated overnight at room temperature with anti-vinculin antibody diluted 1 : 2000 in blocking solution. After several washes, the membrane was incubated with anti-mouse antibody conjugated to alkaline phosphatase (1 : 10,000; Sigma). The enzyme activity was revealed with 0.17 mg/ml 5-bromo-4-chloro-3 indolyl phosphate (BCIP; Sigma) and 0.34 mg/ml nitro blue tetrazolium (NBT; Sigma) in substrate buffer (0.1 M Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5). The densitometric analysis was performed using the image-Pro Plus software. The immunoblotting experiments were performed independently at least 3 times, beginning with new cell cultures in each experiment.
Statistical analysis Student×s t-test was used to determine the significance of differences between mean values in the three Western blot assays; a p value < 0.05 was considered significant.
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Results
Disruption of costameres by T. cruzi infection 533
In our in vitro system, most cells were spread and beating after 24 hours of culture. Non differentiated cells, myoblasts and fibroblasts, displayed vinculin in focal adhesion sites located more prominently at the cell edges (Fig. 1A). Cardiac myocytes, with I bands of myofibrils detected by phalloidin-FITC labeling, displayed vinculin localized at the Z line of myofibrils, costameres, as well as anchoring myofibrils to the sarcolemma (Fig. 1B). Following the myogenesis process, the cells reached a high level of differentiation after 3 days of culture, showing well developed myofibrils and costameres (Fig. 1C). A kinetic study of the T. cruzi-cardiomyocyte interaction was performed to evaluate the effect of infection on organization of focal adhesions. At an early time of infection (24 h), vinculin
was localized at the Z line of myofibrils and at focal adhesion sites (Fig. 2), as observed in control cells. Changes in focal adhesion distribution were detected after 72 hours in T. cruziinfected cells. Confocal laser scanning microscopy revealed vinculin concentrated at the cell periphery and in the perinuclear region, while no costameric staining was visualized in infected cardiomyocytes (Fig. 3). To confirm the alteration observed in the distribution of vinculin in cardiomyocytes after 72 hours of T. cruzi infection, we evaluated the percentage of cells displaying costameres in uninfected and T. cruzi-infected cardiomyocyte cultures. Our results demonstrate an increase in the number of costamere-containing cells in uninfected cardiomyocytes during myogenesis in vitro, reaching 36% and 55% after 48 and 72 hours of culture, respectively (Fig. 4). The quantitative analysis confirmed the decrease in the costameric distribution of vinculin only after 72 hours of infection,
Fig. 1. Detection of vinculin, a focal adhesion protein, in nondifferentiated cells by imunofluorescence (A). An intense labeling for vinculin was visualized mainly in focal contacts at the cell periphery (arrows). Distribution of actin filaments and vinculin during cardiomyogenesis (B, C). Double labeling of heart muscle cell cultures with
phalloidin-FITC (green) and anti-vinculin antibody (red). After 24 h (B) and 72 h (C) in culture many cells were differentiated, showing actin organized at the myofibrils (arrowhead) and vinculin localized at costameres (long arrows). Note the presence of vinculin attaching myofibrils to the sarcolemma (short arrows).
Fig. 2. Distribution of vinculin in 24-h T. cruzi-infected cardiomyocytes. Triple labeling of infected cells with phalloidin-FITC, antivinculin antibody and DAPI. The distribution of cytoskeleton proteins was similar to that of uninfected cells, showing organized myofibrils (A;
arrowhead) and vinculin localized at the Z lines of myofibrils (long arrows), as well as at focal contacts (B; short arrows). DAPI stained the host cell nucleus and the parasite nucleus and kinetoplast (C; double arrows).
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Fig. 3. Effect of Trypanosoma cruzi infection on vinculin distribution. Confocal scanning microscopy combined with interferential contrast of uninfected (A ± C) and 72-h T. cruzi-infected cardiomyocytes (D ± F). Uninfected cardiomyocytes displayed a normal distribution pattern of vinculin, which was detected at costameres (C; long arrow) and focal
adhesion sites (A and C; short arrows). Note the concentration of vinculin at the periphery of infected cells and in the perinuclear region (short arrows), while no staining was detected in costameres (D and F). The intracellular parasites (double arrows) were visualized by interferential contrast (E and F).
displaying mean levels of 19.8%, which represents a reduction of 65.45% in the total number of costameres as compared to the control cells (Fig. 4). To address the question of whether parasite infection alters the expression of vinculin, protein extracts were obtained from uninfected and T. cruzi-infected cardiomyocyte cultures and the samples were processed for immunoblotting assay. Our
results demonstrate changes in the vinculin expression during the T. cruzi infection. The densitometric analysis revealed a significant reduction of 33% (p < 0.05) and 36.6% (p < 0.009) in the vinculin expression after 24 and 72 hours post-infection, respectively (Fig. 5). To evaluate whether vinculin distribution was also affected during the acute phase of experimental T. cruzi infection, mice
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Fig. 4. Quantitative analysis of cells containing costameres in uninfected and T. cruzi-infected cardiomyocytes. An increase in the number of cardiomyocytes displaying costameres was observed during cardiomyogenesis. However, infection with T. cruzi induced a decrease in the percentage of cells with costameres.
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were inoculated with 102 trypomastigotes of T. cruzi (Y strain) and the cardiac tissue was examined. Histological analysis of healthy myocardium revealed a normal arrangement of cardiac fibers, showing regular aspect of vessels and interstitial space (Fig. 6A). At the beginning of infection up to 12 dpi, parasitemia peak, the T. cruzi-infected cardiac tissue displayed a similar pattern of myofiber structures as in control cells, despite the presence of amastigote nests in the cardiac fibers (Fig. 6B). However, a progressive enhancement of inflammatory infiltrate was noted at later stages of acute infection with the decline of parasitemia. An intense myocarditis was detected at 23 dpi, leading to an enlargement of interstitial space and local disruption of myocardium fibers (Fig. 6C). Immunolocalization of vinculin in healthy cardiac tissue cryosections showed the presence of vinculin in the Z line of myofibrils, costameres, and intercalated discs (Fig. 7A). In contrast, analysis of T. cruzi-infected myocardium demonstrated alterations in the vinculin distribution. At the 12 dpi, when amastigote nests were frequently detected, the infected cardiomyocytes displayed no vinculin staining at costameres (Fig. 7C). In addition, an irregular arrangement of cardiac fibers was observed in regions containing infected myocytes, showing disturbance in the lateral alignment of intercalated discs (Fig. 7C). Vinculin staining of intercalated discs was often detected even in areas of intense inflammatory infiltrate (Fig. 7E).
Discussion
Fig. 5. Densitometric analysis of vinculin expression during T. cruzicardiomyocyte infection. Note the reduction of 33% and 36.6% of vinculin expression after 24 h and 72 h of cardiomyocyte infection, respectively. Student t-test (*) p < 0.05.
Fig. 6. Histological analysis of uninfected and T. cruzi-infected myocardium. Cryosection of healthy mice cardiac tissue (A) revealed a normal organization of the cardiac fibers, showing regular interstitial spaces. No change was observed in the structure of cardiac fibers,
Several studies have demonstrated that the interaction between the extracellular matrix and the cytoskeleton is essential for cardiac function (Craig and Pardo, 1983; Koteliansky and Gneushev, 1983; Pardo et al., 1983a, b). As Chagas× disease may develop to severe cardiomyopathy, leading to dysfunction of cardiac tissue and heart failure, we have investigated the distribution of vinculin, a focal adhesion protein involved in the attachment of microfilament to the plasma membrane and essential for transmission of contraction force, during the Trypanosoma cruzi-cardiomyocyte interaction. Analysis of vinculin distribution in cardiomyocyte cultures revealed its localization in costameres and attaching myofibrils
despite the presence of amastigote nests at the peak of parasitemia (12 dpi; B). An intense inflammatory infiltrate (*) and enlargement of interstitial space was observed at 23 dpi (C).
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Fig. 7. Double labeling of uninfected (A) and T. cruzi-infected mice cardiac tissue (C and E) with anti-vinculin (A, C and E) and DAPI (B, D and F). Healthy cardiac tissue displayed intense vinculin labeling at intercalated discs (arrow) and costameres (A; arrowhead). An irregular lateral alignment of intercalated discs (arrow) and the lack of vinculin staining in the cardiac fibers containing intracellular parasites (*) were
observed at 12 dpi (B). Note the enlargement of interstitial space caused by inflammatory infiltrate (**), leading to a disconnection between adjacent cardiomyocytes (E). Vinculin is still visualized at costameres (arrowhead) and intercalated disc (arrow). DAPI stained the nuclei of cardiomyocytes and mononucleated cells and the nucleus and kinetoplast of the parasites (*) (B, D and F).
to the sarcolemma. It has been shown that such distribution allows the mechanical binding between the contractile apparatus and the extracellular matrix (Craig and Pardo, 1983; Koteliansky and Gneushev, 1983; Pardo et al., 1983a, b),
indicating its important role in the transmission of contraction force (Danowski et al., 1992; Imanaka-Yoshida et al., 1994). The co-localization of vinculin, extracellular matrix components and their receptors suggest that the Z line is the main
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mechanic tension site in striated muscle cells (Terracio et al., 1990; Wang and Ramirez-Mitchell, 1983). Recently, it has been demonstrated that the contraction force may be laterally transmitted between Z and M lines of adjacent cells, conferring an extensive and stable connection (Bloch and GonzalezSerratos, 2003). Changes in the cytoskeleton dynamics of the host cell induced by intracellular pathogens have been demonstrated during the parasite invasion in mammalian cells and/or in attempts of the parasites to avoid an aggressive environment during their intracellular development (Gruenheid and Finlay, 2003). Several pathogens recruit actin filaments during the invasion process through the nucleation or rearrangement of actin at the parasite adhesion site (Elliott and Clark 2000; Moon et al., 1983; Reddy et al., 2000). The mechanism of recruitment of cytoskeletal proteins seems to be dependent on both microorganism and the target cell (Barbosa, 1999). Although changes in the organization of host cell actin filaments have been demonstrated during T. cruzi invasion by endocytosis (Barbosa and Meirelles, 1995; Proco¬pio et al., 1999; Rosestolato et al., 2002; Vieira et al., 2002) or active penetration (Rodriguez et al., 1995; Schenkman and Mortara, 1992), the participation of focal adhesion components in the parasite-host cell interaction and their distribution during the intracellular development of parasites is still poorly known. The participation of vinculin, talin and a-actinin during the invasion process of amastigote and trypomastigote forms of T. cruzi into cell lineages has been reported (Proco¬pio et al., 1999). Our data demonstrate that the distribution of vinculin remains similar to that of control cells at an early time of infection (24 h), showing vinculin at focal adhesion sites, linking myofibrils to the sarcolemma and also at the Z line of myofibrils, conferring a striated pattern in differentiated cells. In contrast, a decrease in the total number of costameres was observed after 72 hours of infection, generating a severe alteration in the distribution of vinculin, which was concentrated both at the perinuclear region and the cell periphery. Additionally, our preliminary data also revealed changes in the a-actinin expression after 72 h of T. cruzi infection, suggesting a disassembly of the contraction apparatus in T. cruzi-infected cells. These results confirm previous data on alterations in the cytoskeletal organization of infected cardiomyocytes with destruction of myofibrils at the site of intracellular parasites (Pereira et al., 1993), as well as on the downregulation of acardiac actin mRNA and other sarcomeric proteins (Garg et al., 2003; Pereira et al., 2000). Furthermore, evidence of ventricular arrhythmias (reviewed by Elizari, (2002)) and reduction in the distribution of conexin-43 in T. cruzi-infected cardiomyocytes (Campos de Carvalho et al., 1992, 1993), suggest disturbance in the intercellular conductive system. Therefore, these data suggest that T. cruzi infection affects the arrangement of the contractile apparatus and the transmission of contraction force, both essential for the ventricular function, which may probably collaborate for the arrhythmias evidenced in the Chagas× disease. Although our data revealed no substantial change in vinculin distribution after 24 hours of infection, the protein expression was significantly reduced by about 33% of normal levels, suggesting that changes in gene expression probably occur at the initial stage of invasion, which may be induced by a signaling pathway during the parasite-host cell recognition. This hypothesis is supported by recent data from our group that demonstrated alterations in gene transcription by microarray proce-
Disruption of costameres by T. cruzi infection 537
dures, including focal adhesion proteins and other cytoskeletal proteins after 2 hours of T. cruzi-cardiomyocyte interaction (Pereira et al., 2002; Probst et al., 2003). Furthermore, focal adhesion alterations have been also reported in other pathogen interactions, including viral, bacterial and protozoan, acting on the disassembly of adhesion proteins via signaling pathways (Kee et al., 1999; Hintermann et al., 2002; Shifrin et al., 2002), proteins cleavage by parasite proteases (Shoeman et al., 1993, 2002) or reassembly of proteins at the host cell-parasite interface (Teoh et al., 2000). Alterations in cardiac fiber structure were also detected in murine experimental Chagas× disease. Histological analysis of cardiac tissue of T. cruzi-infected mice showed amastigote nests within cardiac fibers at parasitemia peak (12 dpi) and an intense myocarditis at late stages of acute phase of experimental infection (23 dpi). Similar effects have been reported by other investigators, using the same experimental animal lineage, parasite inoculum and T. cruzi inoculation pathway (Calvet et al., 2004; Luz et al., 1995). On the other hand, it has been demonstrated that the parasitemia and inflammatory infiltrate levels depend on the parasite inoculum, inoculation pathway and also parasite and host genotypes (Andersson et al., 2003; Soeiro et al., 2000). Analysis of vinculin distribution revealed its localization in intercalated discs and costameres in the healthy cardiac tissue, which is consistent with previous reports describing the participation of vinculin in cell-cell adherens junctions mediated by a-catenin (Gutstein et al., 2003), in cell-matrix adherens junctions and also at the Z line of sarcomeres corresponding to the costameres (Koteliansky and Gneushev, 1983; Pardo et al., 1983a, b). Although vinculin is localized at costameres in adult cardiac tissue, its costameric distribution was difficult to be visualized in some regions of the tissue section, probably due to the angle of the section. The striated pattern was only detected close to the cell surface. Pardo et al. (1983a, b) have reported that the angle of the sectioning may be determinant for the vinculin visualization, showing that transversal or longitudinal sections within the fibers do not allow the visualization of intracellular labeling. The loss of vinculin staining, including costameric distribution, in cardiomyocytes containing amastigote nests at the parasitemia peak (12 dpi) confirms the data obtained with the in vitro system, suggesting that the presence of parasites affects the vinculin distribution at the Z line of myofibrils, leading to a deficient transmission of contraction force among neighbouring cells. Although alterations in vinculin distribution may be attributed to the mechanical stress induced by the presence of the intracellular parasites, the participation of parasite proteases in this process may be relevant. Furthermore, it has been demonstrated that chicken fibroblasts transformed by Rous sarcoma virus present a decrease in vinculin expression, which may be attributed mainly to changes in half-life of this protein rather than degradation (Lee and Otto, 1996). Besides the disruption of costameres, the intense myocarditis revealed in the acute phase of disease (23 dpi) may also contribute to the instability in the propagation of the contraction force as a result of interstitial space enlargement and the disorganization of the lateral alignment of myofibrils. In summary, we have demonstrated for the first time that T. cruzi infection affects vinculin distribution, causing a disruption of costameres, suggesting that this disturbance of the cytoskeleton may alter the transmission of contraction force and contribute to the cardiac dysfunctions reported in Chagas×
538 T. G. Melo et al.
disease. Further studies will be carried out to clarify the mechanisms that promote disassembly of costameres. Acknowledgements. The authors thank Dr. Maurilio Jose¬ Soares for ¬ vila for his skillful critical reviews of the manuscript and Bruno A computer imaging support. This work was supported by grants from FundaÁaƒo Oswaldo cruz (FIOCRUZ), FundaÁaƒo de Amparo a¡ Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento CientÌfico e Tecnolo¬gico (CNPq).
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Trypanosoma cruzi infection disrupts vinculin costameres in cardiomyocytes Tatiana G. Melo, Danielle S. Almeida, Maria de Nazareth S. L. de Meirelles, Mirian Claudia S. Pereira1) Departamento de Ultra-estrutura e Biologia Celular, Laborato¬rio de Ultra-estrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil Received May 28, 2004 Received in revised version August 11, 2004 Accepted August 19, 2004
Cardiomyocytes; Trypanosoma cruzi; Focal adhesion protein; Vinculin
Introduction
Chagas× disease cardiomyopathy is an important manifestation of Trypanosoma cruzi infection, leading to cardiac dysfunction and serious arrhythmias. We have here investigated by indirect immunofluorescence assay the distribution of vinculin, a focal adhesion protein with a major role in the transmission of contraction force, during the T. cruzi-cardiomyocyte infection in vitro and in vivo. No change in vinculin distribution was observed after 24 h of infection, where control and T. cruziinfected cardiomyocytes displayed vinculin localized at costameres and intercalated discs. On the other hand, a clear disruption of vinculin costameric distribution was noted after 72 h of infection. A significant reduction in the levels of vinculin expression was observed at all times of infection. In murine experimental Chagas× disease, alteration in the vinculin distribution was also detected in the infected myocardium, with no costameric staining in infected myocytes and irregular alignment of intercalated discs in cardiac fibers. These data suggest that the disruption of costameric vinculin distribution and the enlargement of interstitial space due to inflammatory infiltration may contribute to the reduction of transmission of cardiac contraction force, leading to alterations in the heart function in Chagas× disease.
Trypanosoma cruzi, the causative agent of Chagas× disease, needs to invade vertebrate host cells to survive and replicate. Therefore, its ability to enter non-professional phagocytic cells is a critical factor to enhance its subsistence. Although many cell types are infected by T. cruzi in vitro (Piras et al., 1983), a defined tropism to muscle cells has been reported (Brener, 1973; Cabrine-Santos et al., 2001; Vera-Cruz et al., 2003), leading to progressive cardiomyopathy and megaesophagus in the chronic phase of Chagas× disease (reviewed by Higuchi et al. (2003), Kirchhoff (1996), Lages-Silva et al. (2001)). Thus, a detailed investigation of the T. cruzi-muscle cell interaction may contribute to the knowledge of biological and molecular events that occur during the infectious process. Changes in the cytoskeleton of host cells have been reported to facilitate several microbe-host cell interactions (reviewed by Gruenheid and Finlay (2003), Smith and Enquist (2002)). Rearrangement of actin is required for many pathogens to invade the host cell (Elliot and Clark, 2000; Mortara et al., 1991; Tan and Andrews, 2002). Once within the host cell, the pathogens may modulate the cytoskeleton dynamics of the host cell to their advantage by remodeling the parasitophorous vacuole (Guerin and De Chastellier, 2000; Meresse et al., 2001) or facilitating their intracellular motility (Frischknecht and Way, 2001; Goldberg, 2001). Focal adhesion proteins have been also reported to participate in many parasite-host cell processes (Shifrin et al., 2002; Teoh et al., 2000; Yilmaz et al., 2003). Vinculin, talin and a-actinin have been identified in association with microfilaments at the parasite-host cell interface (Finlay et al., 1991, 1992; Freeman et al., 2000; Tran Van Nhieu et al., 1997). In addition, vinculin has been reported to play an important role in generating force to Shigella motility within and between adjacent host cells, leading to a systemic disease (Laine et al., 1997). Vinculin, a major protein of adherens-type junctions (Geiger and Ginsberg, 1991), displays several binding sites for many
Abbreviations. Abbreviations. BSA Bovine serum albumin. ± DABCO 1,4Diazabicyclo-(2.2.2)-octane. ± DAPI 4',6-Diamidino-2-phenylindole. ± DMEM Dulbecco×s modified Eagle×s medium. ± dpi Days post infection. ± FBS Fetal bovine serum. ± PIP2 Phosphatidylinositol 4,5-bisphosphate. ± PBS Phosphate-buffered saline. 1)
Corresponding author: Dr. Mirian Claudia S. Pereira, Departamento de Ultra-estrutura e Biologia Celular, Laborato¬rio de Ultra-estrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, RJ, 21045 ± 900, Brazil, e-mail: [email protected], Fax: 5521 2260 4434.
0171-9335/04/083/10-531 $30.00/0
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cytoskeletal proteins. The vinculin primary structure consists of a globular head connected by a proline-rich neck to a long flexible tail (Milam, 1985; Molony and Burridge, 1985; Winkler et al., 1996; Zamir and Geiger, 2001). Once activated by exposure to phosphatidylinositol 4,5-bisphosphate (Gilmore and Burridge 1995, 1996; H¸ttelmaier et al., 1997), which binds to the protein tail (Johnson et al., 1998; Sechi and Wehland, 2000), vinculin exposes its multidomains, allowing the interaction with a-actinin (Wachsstock et al., 1987; Kroemker et al., 1994) and talin (Burridge and Mangeat, 1984; Johnson and Craig, 1994) at the head region, while tail domains bind to Factin, paxillin, VASP, ponsin, vinexin, lipid bilayer and also to the vinculin head in its inactive conformation (Critchley, 2000; Geiger et al., 2001; Jockusch et al., 1995; Johnson and Craig, 1995). Vinculin plays an important role in anchoring microfilaments to the plasma membrane at focal adhesion sites. In muscle cells, this protein is also located at the Z line of myofibrils, termed costameres, in association with the plasma membrane (Pardo et al., 1983a, b; Craig and Pardo, 1983; Koteliansky and Gneushev, 1983), suggesting its participation in the mechanical force transmission during the contractionrelaxation process (Danowski et al., 1992; Imanaka-Yoshida et al., 1994). Chagas× disease cardiomyopathy leads to cardiac damage and dysfunction with ventricular and atrial arrhythmias (Elizari, 1999, 2002). However, the mechanism leading to the evolution of the disease is still unknown. Many studies have reported that mutations in cytoskeletal protein genes are involved in the hypertrophic and dilated cardiomyopathy (Daehmlow et al., 2002; Marian and Roberts 1998, 2001). Although previous data revealed that T. cruzi affects organization of the cytoskeleton and regulation of the actin gene in cardiomyocytes (Pereira et al., 1993, 2000), little is known about actin-binding proteins, such as focal adhesion proteins, during the T. cruzi-host cell interaction. In the present study we report for the first time changes in the expression and distribution of vinculin in cardiomyocytes induced by T. cruzi infection.
Materials and methods Animals Swiss Webster male mice weighing 18 ± 20 g were obtained from the animal facilities of the Oswaldo Cruz Institute ± FIOCRUZ, Rio de Janeiro, Brazil. A total of 60 mice, divided in 3 different experimental groups, were intraperitoneally inoculated with 102 bloodstream trypomastigote forms of Trypanosoma cruzi, Y strain. Parasitemia was individually checked by counting the number of parasites/5 ml of blood at different days post-infection (dpi) (Brener, 1962; Luz et al., 1995). Cardiac tissue was obtained from 3 uninfected and 3 infected animals sacrificed at different stages of the acute infection: at negative parasitemia, at the peak of parasitemia, and after parasitemia decline. The hearts were embedded in Tissue-Tek (Sakura Finetek, CA, USA) and frozen in liquid nitrogen.
Cell culture Primary cultures of cardiomyocytes were obtained from 18-day-old mouse embryos as previously described (Meirelles et al., 1986). Briefly, cardiac fragments were dissociated in phosphate-buffered saline (PBS, pH 7.2) containing 0.025% trypsin (Sigma; St. Louis, MO, USA) plus 0.01% collagenase (Worthington, New Jersey, USA) and plated (1 105 cells/ml) into 24-well culture dishes containing glass coverslips previously coated with 0.01% gelatin (Sigma). For biochemical analysis,
the isolated cells were plated at a density of 2.5 106 cells in 60-mm culture dishes. The cells were cultivated in Dulbeccos× modified Eagle medium (DMEM; Sigma) supplemented with 10% horse serum (Sigma), 5% fetal bovine serum (FBS; Sigma), 2.5 mM CaCl2, 1 mM L-glutamine (Sigma), 2% chicken embryo extract, and maintained at 37 8C in a 5% CO2 atmosphere.
Parasites and infection of cultured cells Bloodstream trypomastigote forms of Trypanosoma cruzi, Y strain, were obtained from Swiss mice at the peak of parasitemia as described (Meirelles et al., 1984). Cardiac cell cultures were infected at a multiplicity of 10 parasites per host cell. After 24 h of interaction, free trypomastigotes were removed by washing the cultures with Ringer×s solution (154 mM NaCl, 56 mM KCl, 17 mM Na2HPO4, pH 7.0). The time course of infection was interrupted after 24 and 72 h.
Indirect immunofluorescence Uninfected and T. cruzi-infected cardiomyocytes were fixed for 20 min at 4 8C with 4% paraformaldehyde (Sigma) in PBS. Thereafter, the cells were washed and permeabilized with PBS containing 0.5% Triton X-100 (Sigma). The cultures were then washed with PBS containing 4% bovine serum albumin (BSA; Sigma) to block unspecific reactions and incubated overnight at 4 8C with anti-vinculin antibody (Sigma) diluted 1 : 300 in PBS. The antigen-antibody complex was revealed by incubation for 1 h at 37 8C with TRITC-conjugated anti-mouse IgG (Sigma). Controls were performed by incubation with mouse serum or by omission of the primary antibody. Actin filaments and DNA were detected with 4 mg/ml phalloidin-FITC (Sigma) and 10 mg/ml 4,6diamidino-2-phenylindole (DAPI; Sigma), respectively. The cells were mounted in 2.5% 1,4-diazabicyclo-(2,2,2)-octane (DABCO; Sigma) in PBS containing 50% glycerol, pH 7.2. The percentage of cells (n 500 cells, from 3 different experiments) displaying a costameric distribution of vinculin was evaluated in controls and T. cruzi-infected cells after 24 and 72 hours of infection. The fluorescence images were obtained using either an Axioplan 2 microscope equipped with epifluorescence or a confocal laser scanning microscope (BX51 Olympus). The cardiac tissue cryosections were fixed for 10 min at 20 8C in acetone and processed as described above.
Protein extraction and immunoblotting assay Total proteins were extracted using a lysis solution (50 mM Tris-HCl containing 1% Triton X-100 plus 10 mM E-64 (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma) and 1 mM pepstatin (Sigma), pH 8.0). The amount of protein was determined by the Bradford method (Bradford, 1976) and a total of 20 mg of protein was eletrophoretically separated in a 12% polyacrylamide gel, by SDS-PAGE. The proteins were then transferred at 30 V (60 mA) overnight at 4 8C to a Polyvinylidene difluoride (PVDF) membrane (Bio-Rad, California, USA) in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol, pH 8.3). After protein transfer, the membrane was blocked with 0.5% I-Block (Sigma), 0.1% Tween 20 (Sigma) in PBS and incubated overnight at room temperature with anti-vinculin antibody diluted 1 : 2000 in blocking solution. After several washes, the membrane was incubated with anti-mouse antibody conjugated to alkaline phosphatase (1 : 10,000; Sigma). The enzyme activity was revealed with 0.17 mg/ml 5-bromo-4-chloro-3 indolyl phosphate (BCIP; Sigma) and 0.34 mg/ml nitro blue tetrazolium (NBT; Sigma) in substrate buffer (0.1 M Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5). The densitometric analysis was performed using the image-Pro Plus software. The immunoblotting experiments were performed independently at least 3 times, beginning with new cell cultures in each experiment.
Statistical analysis Student×s t-test was used to determine the significance of differences between mean values in the three Western blot assays; a p value < 0.05 was considered significant.
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Results
Disruption of costameres by T. cruzi infection 533
In our in vitro system, most cells were spread and beating after 24 hours of culture. Non differentiated cells, myoblasts and fibroblasts, displayed vinculin in focal adhesion sites located more prominently at the cell edges (Fig. 1A). Cardiac myocytes, with I bands of myofibrils detected by phalloidin-FITC labeling, displayed vinculin localized at the Z line of myofibrils, costameres, as well as anchoring myofibrils to the sarcolemma (Fig. 1B). Following the myogenesis process, the cells reached a high level of differentiation after 3 days of culture, showing well developed myofibrils and costameres (Fig. 1C). A kinetic study of the T. cruzi-cardiomyocyte interaction was performed to evaluate the effect of infection on organization of focal adhesions. At an early time of infection (24 h), vinculin
was localized at the Z line of myofibrils and at focal adhesion sites (Fig. 2), as observed in control cells. Changes in focal adhesion distribution were detected after 72 hours in T. cruziinfected cells. Confocal laser scanning microscopy revealed vinculin concentrated at the cell periphery and in the perinuclear region, while no costameric staining was visualized in infected cardiomyocytes (Fig. 3). To confirm the alteration observed in the distribution of vinculin in cardiomyocytes after 72 hours of T. cruzi infection, we evaluated the percentage of cells displaying costameres in uninfected and T. cruzi-infected cardiomyocyte cultures. Our results demonstrate an increase in the number of costamere-containing cells in uninfected cardiomyocytes during myogenesis in vitro, reaching 36% and 55% after 48 and 72 hours of culture, respectively (Fig. 4). The quantitative analysis confirmed the decrease in the costameric distribution of vinculin only after 72 hours of infection,
Fig. 1. Detection of vinculin, a focal adhesion protein, in nondifferentiated cells by imunofluorescence (A). An intense labeling for vinculin was visualized mainly in focal contacts at the cell periphery (arrows). Distribution of actin filaments and vinculin during cardiomyogenesis (B, C). Double labeling of heart muscle cell cultures with
phalloidin-FITC (green) and anti-vinculin antibody (red). After 24 h (B) and 72 h (C) in culture many cells were differentiated, showing actin organized at the myofibrils (arrowhead) and vinculin localized at costameres (long arrows). Note the presence of vinculin attaching myofibrils to the sarcolemma (short arrows).
Fig. 2. Distribution of vinculin in 24-h T. cruzi-infected cardiomyocytes. Triple labeling of infected cells with phalloidin-FITC, antivinculin antibody and DAPI. The distribution of cytoskeleton proteins was similar to that of uninfected cells, showing organized myofibrils (A;
arrowhead) and vinculin localized at the Z lines of myofibrils (long arrows), as well as at focal contacts (B; short arrows). DAPI stained the host cell nucleus and the parasite nucleus and kinetoplast (C; double arrows).
534 T. G. Melo et al.
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Fig. 3. Effect of Trypanosoma cruzi infection on vinculin distribution. Confocal scanning microscopy combined with interferential contrast of uninfected (A ± C) and 72-h T. cruzi-infected cardiomyocytes (D ± F). Uninfected cardiomyocytes displayed a normal distribution pattern of vinculin, which was detected at costameres (C; long arrow) and focal
adhesion sites (A and C; short arrows). Note the concentration of vinculin at the periphery of infected cells and in the perinuclear region (short arrows), while no staining was detected in costameres (D and F). The intracellular parasites (double arrows) were visualized by interferential contrast (E and F).
displaying mean levels of 19.8%, which represents a reduction of 65.45% in the total number of costameres as compared to the control cells (Fig. 4). To address the question of whether parasite infection alters the expression of vinculin, protein extracts were obtained from uninfected and T. cruzi-infected cardiomyocyte cultures and the samples were processed for immunoblotting assay. Our
results demonstrate changes in the vinculin expression during the T. cruzi infection. The densitometric analysis revealed a significant reduction of 33% (p < 0.05) and 36.6% (p < 0.009) in the vinculin expression after 24 and 72 hours post-infection, respectively (Fig. 5). To evaluate whether vinculin distribution was also affected during the acute phase of experimental T. cruzi infection, mice
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Fig. 4. Quantitative analysis of cells containing costameres in uninfected and T. cruzi-infected cardiomyocytes. An increase in the number of cardiomyocytes displaying costameres was observed during cardiomyogenesis. However, infection with T. cruzi induced a decrease in the percentage of cells with costameres.
Disruption of costameres by T. cruzi infection 535
were inoculated with 102 trypomastigotes of T. cruzi (Y strain) and the cardiac tissue was examined. Histological analysis of healthy myocardium revealed a normal arrangement of cardiac fibers, showing regular aspect of vessels and interstitial space (Fig. 6A). At the beginning of infection up to 12 dpi, parasitemia peak, the T. cruzi-infected cardiac tissue displayed a similar pattern of myofiber structures as in control cells, despite the presence of amastigote nests in the cardiac fibers (Fig. 6B). However, a progressive enhancement of inflammatory infiltrate was noted at later stages of acute infection with the decline of parasitemia. An intense myocarditis was detected at 23 dpi, leading to an enlargement of interstitial space and local disruption of myocardium fibers (Fig. 6C). Immunolocalization of vinculin in healthy cardiac tissue cryosections showed the presence of vinculin in the Z line of myofibrils, costameres, and intercalated discs (Fig. 7A). In contrast, analysis of T. cruzi-infected myocardium demonstrated alterations in the vinculin distribution. At the 12 dpi, when amastigote nests were frequently detected, the infected cardiomyocytes displayed no vinculin staining at costameres (Fig. 7C). In addition, an irregular arrangement of cardiac fibers was observed in regions containing infected myocytes, showing disturbance in the lateral alignment of intercalated discs (Fig. 7C). Vinculin staining of intercalated discs was often detected even in areas of intense inflammatory infiltrate (Fig. 7E).
Discussion
Fig. 5. Densitometric analysis of vinculin expression during T. cruzicardiomyocyte infection. Note the reduction of 33% and 36.6% of vinculin expression after 24 h and 72 h of cardiomyocyte infection, respectively. Student t-test (*) p < 0.05.
Fig. 6. Histological analysis of uninfected and T. cruzi-infected myocardium. Cryosection of healthy mice cardiac tissue (A) revealed a normal organization of the cardiac fibers, showing regular interstitial spaces. No change was observed in the structure of cardiac fibers,
Several studies have demonstrated that the interaction between the extracellular matrix and the cytoskeleton is essential for cardiac function (Craig and Pardo, 1983; Koteliansky and Gneushev, 1983; Pardo et al., 1983a, b). As Chagas× disease may develop to severe cardiomyopathy, leading to dysfunction of cardiac tissue and heart failure, we have investigated the distribution of vinculin, a focal adhesion protein involved in the attachment of microfilament to the plasma membrane and essential for transmission of contraction force, during the Trypanosoma cruzi-cardiomyocyte interaction. Analysis of vinculin distribution in cardiomyocyte cultures revealed its localization in costameres and attaching myofibrils
despite the presence of amastigote nests at the peak of parasitemia (12 dpi; B). An intense inflammatory infiltrate (*) and enlargement of interstitial space was observed at 23 dpi (C).
536 T. G. Melo et al.
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Fig. 7. Double labeling of uninfected (A) and T. cruzi-infected mice cardiac tissue (C and E) with anti-vinculin (A, C and E) and DAPI (B, D and F). Healthy cardiac tissue displayed intense vinculin labeling at intercalated discs (arrow) and costameres (A; arrowhead). An irregular lateral alignment of intercalated discs (arrow) and the lack of vinculin staining in the cardiac fibers containing intracellular parasites (*) were
observed at 12 dpi (B). Note the enlargement of interstitial space caused by inflammatory infiltrate (**), leading to a disconnection between adjacent cardiomyocytes (E). Vinculin is still visualized at costameres (arrowhead) and intercalated disc (arrow). DAPI stained the nuclei of cardiomyocytes and mononucleated cells and the nucleus and kinetoplast of the parasites (*) (B, D and F).
to the sarcolemma. It has been shown that such distribution allows the mechanical binding between the contractile apparatus and the extracellular matrix (Craig and Pardo, 1983; Koteliansky and Gneushev, 1983; Pardo et al., 1983a, b),
indicating its important role in the transmission of contraction force (Danowski et al., 1992; Imanaka-Yoshida et al., 1994). The co-localization of vinculin, extracellular matrix components and their receptors suggest that the Z line is the main
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mechanic tension site in striated muscle cells (Terracio et al., 1990; Wang and Ramirez-Mitchell, 1983). Recently, it has been demonstrated that the contraction force may be laterally transmitted between Z and M lines of adjacent cells, conferring an extensive and stable connection (Bloch and GonzalezSerratos, 2003). Changes in the cytoskeleton dynamics of the host cell induced by intracellular pathogens have been demonstrated during the parasite invasion in mammalian cells and/or in attempts of the parasites to avoid an aggressive environment during their intracellular development (Gruenheid and Finlay, 2003). Several pathogens recruit actin filaments during the invasion process through the nucleation or rearrangement of actin at the parasite adhesion site (Elliott and Clark 2000; Moon et al., 1983; Reddy et al., 2000). The mechanism of recruitment of cytoskeletal proteins seems to be dependent on both microorganism and the target cell (Barbosa, 1999). Although changes in the organization of host cell actin filaments have been demonstrated during T. cruzi invasion by endocytosis (Barbosa and Meirelles, 1995; Proco¬pio et al., 1999; Rosestolato et al., 2002; Vieira et al., 2002) or active penetration (Rodriguez et al., 1995; Schenkman and Mortara, 1992), the participation of focal adhesion components in the parasite-host cell interaction and their distribution during the intracellular development of parasites is still poorly known. The participation of vinculin, talin and a-actinin during the invasion process of amastigote and trypomastigote forms of T. cruzi into cell lineages has been reported (Proco¬pio et al., 1999). Our data demonstrate that the distribution of vinculin remains similar to that of control cells at an early time of infection (24 h), showing vinculin at focal adhesion sites, linking myofibrils to the sarcolemma and also at the Z line of myofibrils, conferring a striated pattern in differentiated cells. In contrast, a decrease in the total number of costameres was observed after 72 hours of infection, generating a severe alteration in the distribution of vinculin, which was concentrated both at the perinuclear region and the cell periphery. Additionally, our preliminary data also revealed changes in the a-actinin expression after 72 h of T. cruzi infection, suggesting a disassembly of the contraction apparatus in T. cruzi-infected cells. These results confirm previous data on alterations in the cytoskeletal organization of infected cardiomyocytes with destruction of myofibrils at the site of intracellular parasites (Pereira et al., 1993), as well as on the downregulation of acardiac actin mRNA and other sarcomeric proteins (Garg et al., 2003; Pereira et al., 2000). Furthermore, evidence of ventricular arrhythmias (reviewed by Elizari, (2002)) and reduction in the distribution of conexin-43 in T. cruzi-infected cardiomyocytes (Campos de Carvalho et al., 1992, 1993), suggest disturbance in the intercellular conductive system. Therefore, these data suggest that T. cruzi infection affects the arrangement of the contractile apparatus and the transmission of contraction force, both essential for the ventricular function, which may probably collaborate for the arrhythmias evidenced in the Chagas× disease. Although our data revealed no substantial change in vinculin distribution after 24 hours of infection, the protein expression was significantly reduced by about 33% of normal levels, suggesting that changes in gene expression probably occur at the initial stage of invasion, which may be induced by a signaling pathway during the parasite-host cell recognition. This hypothesis is supported by recent data from our group that demonstrated alterations in gene transcription by microarray proce-
Disruption of costameres by T. cruzi infection 537
dures, including focal adhesion proteins and other cytoskeletal proteins after 2 hours of T. cruzi-cardiomyocyte interaction (Pereira et al., 2002; Probst et al., 2003). Furthermore, focal adhesion alterations have been also reported in other pathogen interactions, including viral, bacterial and protozoan, acting on the disassembly of adhesion proteins via signaling pathways (Kee et al., 1999; Hintermann et al., 2002; Shifrin et al., 2002), proteins cleavage by parasite proteases (Shoeman et al., 1993, 2002) or reassembly of proteins at the host cell-parasite interface (Teoh et al., 2000). Alterations in cardiac fiber structure were also detected in murine experimental Chagas× disease. Histological analysis of cardiac tissue of T. cruzi-infected mice showed amastigote nests within cardiac fibers at parasitemia peak (12 dpi) and an intense myocarditis at late stages of acute phase of experimental infection (23 dpi). Similar effects have been reported by other investigators, using the same experimental animal lineage, parasite inoculum and T. cruzi inoculation pathway (Calvet et al., 2004; Luz et al., 1995). On the other hand, it has been demonstrated that the parasitemia and inflammatory infiltrate levels depend on the parasite inoculum, inoculation pathway and also parasite and host genotypes (Andersson et al., 2003; Soeiro et al., 2000). Analysis of vinculin distribution revealed its localization in intercalated discs and costameres in the healthy cardiac tissue, which is consistent with previous reports describing the participation of vinculin in cell-cell adherens junctions mediated by a-catenin (Gutstein et al., 2003), in cell-matrix adherens junctions and also at the Z line of sarcomeres corresponding to the costameres (Koteliansky and Gneushev, 1983; Pardo et al., 1983a, b). Although vinculin is localized at costameres in adult cardiac tissue, its costameric distribution was difficult to be visualized in some regions of the tissue section, probably due to the angle of the section. The striated pattern was only detected close to the cell surface. Pardo et al. (1983a, b) have reported that the angle of the sectioning may be determinant for the vinculin visualization, showing that transversal or longitudinal sections within the fibers do not allow the visualization of intracellular labeling. The loss of vinculin staining, including costameric distribution, in cardiomyocytes containing amastigote nests at the parasitemia peak (12 dpi) confirms the data obtained with the in vitro system, suggesting that the presence of parasites affects the vinculin distribution at the Z line of myofibrils, leading to a deficient transmission of contraction force among neighbouring cells. Although alterations in vinculin distribution may be attributed to the mechanical stress induced by the presence of the intracellular parasites, the participation of parasite proteases in this process may be relevant. Furthermore, it has been demonstrated that chicken fibroblasts transformed by Rous sarcoma virus present a decrease in vinculin expression, which may be attributed mainly to changes in half-life of this protein rather than degradation (Lee and Otto, 1996). Besides the disruption of costameres, the intense myocarditis revealed in the acute phase of disease (23 dpi) may also contribute to the instability in the propagation of the contraction force as a result of interstitial space enlargement and the disorganization of the lateral alignment of myofibrils. In summary, we have demonstrated for the first time that T. cruzi infection affects vinculin distribution, causing a disruption of costameres, suggesting that this disturbance of the cytoskeleton may alter the transmission of contraction force and contribute to the cardiac dysfunctions reported in Chagas×
538 T. G. Melo et al.
disease. Further studies will be carried out to clarify the mechanisms that promote disassembly of costameres. Acknowledgements. The authors thank Dr. Maurilio Jose¬ Soares for ¬ vila for his skillful critical reviews of the manuscript and Bruno A computer imaging support. This work was supported by grants from FundaÁaƒo Oswaldo cruz (FIOCRUZ), FundaÁaƒo de Amparo a¡ Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento CientÌfico e Tecnolo¬gico (CNPq).
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