1Department of Clinical Biochemistry, Faculty of Chemical Sciences, National University of Córdoba, Córdoba 5000, Argentina; and 2Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
Submitted 7 July 2003 ; accepted in final form 14 September 2003
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ABSTRACT |
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apoptosis; Bcl-2; Bcl-xL; nitric oxide synthase; nitric oxide
Many pathogenic mechanisms have been postulated to explain the cardiomyopathy. These mechanisms include death of cardiac cells by apoptosis or necrosis (5), cellular hyperplasia and hypertrophy (3), immunity to parasite antigens persisting in the tissue (13), and autoimmunity (25), among others. In this sense, cruzipain, a major T. cruzi antigen, has been detected in heart specimens from patients with chronic Chagas disease (28), suggesting a potential role in the pathogenesis. Moreover, we have previously demonstrated that mice immunization with cruzipain in absence of infection elicited humoral and cellular autoreactive responses against myosin from skeletal and heart muscles, leading to functional and structural alterations in target tissues (14, 15, 16).
We have also reported that macrophages stimulated with cruzipain exhibited an enhanced arginase activity (16, 32), which favors parasite spreading. This metabolic pathway has been poorly investigated in cardiac cells. In mammals, two arginase isoforms, the cytosolic arginase-1 and the mitochondrial arginase-2, are expressed. T helper (Th)-2 cytokines IL-4, IL-10, and IL-13 induce arginase-1 in macrophages (29). Both isoenzymes catalyze the same reaction; they hydrolyze L-arginine to urea and L-ornithine. Ornithine decarboxylase metabolizes L-ornithine to the polyamine putrescine, which is the precursor of the polyamines spermine and spermidine. These molecules play an integral role in cell homeostasis and tissue repair (19, 21). In addition, it has been described that putrescine bursts parasite growth within host cells (11).
In contrast, resistance to T. cruzi infection is associated with the capacity of lymphocytes to generate interferon- (IFN-
), which, in turn, can activate macrophages to produce nitric oxide (NO), the main effector molecule. In addition to macrophages, many other cells are able to produce NO. Murine myocardial cells produce NO in vitro by cytokines such as IFN-
and IL-1
or by IL-6 and tumor necrosis factor-
(TNF-
) in the presence of lipopolysaccharide (LPS) (26).
The aim of the present work was to study the effect of cruzipain, devoid of enzymatic activity, on cell death and survival of cardiomyocytes employing neonatal BALB/c mouse cardiomyocyte cultures. We further investigated the influence of cruzipain on the L-arginine metabolic pathways, evaluating the arginase and NOS activities and their protein levels. We found that cruzipain, as well as T. cruzi infection, increased cardiomyocyte survival, diminishing the apoptotic rate induced by serum deprivation. In addition, this glycoprotein was able to enhance the arginase activity and the expression of arginase-2 isoform. Moreover, the inhibition of arginase activity abrogated the antiapoptotic action of cruzipain. Altogether, results show that cruzipain may be a cardioprotective factor for cardiomyocytes because it stimulated the arginase-2, which is involved in the antiapoptotic effect exerted by cruzipain.
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MATERIALS AND METHODS |
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Neonatal mouse primary cardiomyocyte culture. The cardiomyocyte culture was performed following a procedure previously described (35). One- to three-day-old neonatal mice were euthanized by cervical dislocation. The hearts from the mice were removed aseptically and kept in Hanks' balanced salt solution without Ca2+ and Mg2+ (HBSS) (in g/l: 0.4 KCl, 0.06 KH2PO4, 8.0 NaCl, and 0.05 Na2HPO4, pH 7.4) on ice. The tissues were washed three times and minced into small fragments. The cells were dissociated with trypsin (0.25% wt/vol in HBSS) at 37°C. The cells released after the first digestion were discarded, whereas the cells from subsequent digestion were added to an equal volume of cold HBSS with Ca2+ and Mg2+ (in g/l: HBSS plus 0.14 CaCl2, 0.047 MgCl2, 0.049 MgSO4, 0.35 NaHCO3, and 1.0 D-glucose, pH 7.4) until all cardiac cells were isolated. The resulting cell suspension was centrifuged at 200 g for 8 min, and the cells were resuspended in DMEM supplemented with 10% FBS (vol/vol). To exclude nonmuscle cells, the isolated cells were first plated in tissue culture dishes at 37°C for 2 h under a water-saturated atmosphere of 5% CO2.
The suspended cells were then collected and plated at a density of 1.0 x 105 cells/cm2 in 10% FBS-DMEM. More than 90% of cells were cardiomyocytes as detected by immunostaining with antibody to mAchR M2. After 24 h, the culture medium was changed to 0.1% FBS-DMEM containing different stimuli: cruzipain (10 µg/ml), IL-4 (10 U/ml), LPS/IFN- (20 µg/ml-10 U/ml), and control cells were maintained in 0.1% FBS-DMEM. In some assays, cells were pretreated with NOHA (50 µmol/l) for 30 min and incubated in a medium containing NOHA in the presence or absence of cruzipain (10 µg/ml) in 0.1% FBS-DMEM during 48 h for the determination of cell viability.
Cruzipain purification. Cruzipain was purified following the method previously described (14). Briefly, epimastigote forms of T. cruzi Tulahuen strain were grown at 28°C in brain heart infusion supplemented with 0.5% tryptose, 10% FBS, 200 mg/ml hemin, 100 U/ml penicillin, and 100 mg/ml streptomycin. Parasites were harvested at the exponential growth phase, centrifuged at 5,000 g at 4°C for 10 min, and washed with PBS. Parasites were resuspended in 3 vol of 0.25 M sucrose, 5 mM KCl, the irreversible protease inhibitor TLCK (1 mM), and PMSF (1 mM). Epimastigotes were disrupted by three cycles of freezing (20°C) and thawing (4°C). The homogenate was centrifuged at 7,000 g for 15 min at 4°C, and the pellet was discarded. Saturated ammonium sulfate solution, adjusted to pH 7 with NH4OH, was added to the supernatant at 50% saturation. The precipitate obtained after centrifugation of this suspension was carefully dissolved and dialyzed against 50 mM Tris·HCl and 150 mM NaCl, pH 7.4. CaCl2, MgCl2, and MnCl2 were added to a final concentration of 5 mM each. Subsequently, the enzyme was further purified by affinity chromatography using ConA Sepharose as previously described (24). After the electrophoresis performed at 120 V, the gel was incubated with 50 mM sodium phosphate buffer, pH 5.7, at 37°C overnight, and it was then stained with Coomassie brilliant blue R250. As previously described by Giordanengo et al. (14), the purity of the cruzipain used was demonstrated by SDS-PAGE analyses and staining with silver. In our study, protease activity of cruzipain was inhibited excluding the action of the enzymatic activity on cardiac myocytes. The samples were neither reduced nor boiled (6). The lack of enzymatic activity was tested in SDS-PAGE (10% gel) containing 0.1% of copolymerized gelatin as substrate.
T. cruzi infection. Cardiomyocyte monolayers were cultured in medium with 10% FBS for 24 h and then infected with 1.5 x 105, and 5 x 105 T. cruzi trypomastigotes, Tulahuen strain, per milliliter. After 24 h, unattached parasites were removed by washing and the cells were switched to 0.1% FBS-DMEM for 48 h. To assess the effect of cruzipain on cardiomyocytes survival, other cultures were previously incubated with cruzipain (10 µg/ml) for 24 h and then infected with 5 x 105 T. cruzi trypomastigotes. Intracellular parasites were identified by indirect immunofluorescence with chagasic IgG as primary antibody and FITC-labeled anti human IgG as secondary antibody.
Measurements of cell viability. The amount of live cells was assessed by staining trypsinized cells after treatment with trypan blue, and the fractions of blue cells (dead) and nonstained cells (live) were quantified by counting in a Neubauer camera. The percentage of live cells was calculated as the ratio between the number of dead cells and total cell number x 100. The analyzer was blinded toward the treatment of cells. To include cells that already detached from the plate during the process of cell death, cells from the supernatant of each dish were also counted. In each dish, a total number of at least 500 cells were counted.
Measurement of inducible nitric oxide synthase and arginase activities. Cardiomyocytes were treated with different stimuli or maintained in 0.1% FBS-DMEM during 24 or 48 h. Nitric oxide synthase (NOS) activity was evaluated indirectly by assaying nitrites in culture supernatants (9). Briefly, 100 µl of culture supernatant was mixed with an equal volume of Griess reagent for 15 min. Absorbance was measured at 540 nm and nitrite concentration was calculated using a standard curve of sodium nitrite.
Arginase activity was measured in cell lysates as previously described (8). Briefly, 1 x 104 cells were stimulated with different stimuli during 48 h. Cells were lysated with 50 µl of 0.1% Triton X-100 and protease inhibitors. The mixture was then stirred at room temperature for 30 min. Then 50 µl of 10 mM MnCl2 and 50 mM Tris·HCl, pH 7.5, were added to activate the enzyme by heating at 55°C for 10 min. Arginine hydrolysis was carried out by the addition of 25 µl of 0.5 M arginine, pH 9.7, to a 25 µl of the lysate. Incubation was performed at 37°C for 60 min, and the reaction was stopped by the addition of 400 µl of an acid mixture containing H2SO4 (96%), H3PO4 (85%), and H2O (1:3:7, vol/vol/vol). After adding 25 µl of 9% ISPF (-isonitrosopropiophenone) and heating at 100°C for 45 min, the formed urea was colorimetrically quantified at 540 nm. A calibration curve was prepared with increasing amounts of urea solution.
Identification of apoptotic nuclei. Cells were fixed in 4% neutral buffered formalin for 40 min at room temperature. Apoptotic nuclei were ascertained by two assays: staining with PI (1 µg/ml in PBS) and terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) with the In Situ Cell Death Detection kit (Roche Diagnostics) according to the manufacturer's protocol. For TUNEL technique, myocyte identification was indicated by staining with anti-mAchR M2 by use of rhodamine-labeled secondary antibody. For each treatment, more than 500 cells were counted within random fields and the apoptotic index was calculated as the ratio between the number of cells with apoptotic features (condensed and/or fragmented nuclei for PI staining or TUNEL-positive cells) and total cell number x 100 in triplicate samples from three separate experiments.
Flow cytometric analysis. The method was based on the use of PI as previously described (34). Briefly, 1.2 x 106 cells were washed and resuspended in fluorochrome solution (50 µg of PI/ml). Fluorescence of individual nuclei was acquired with the Research program in a flow cytometer (Cytoron Absolute Count-Ortho). The number of apoptotic cells was determined by evaluating the percentage of hypodiploid nuclei in the <2N DNA peak. A minimum of 104 events per sample was analyzed, and all measurements were made at the same instrument setting.
Immunoblot analysis. Cardiomyocytes were lysated with a buffer containing 50 mmol/l Tris·HCl, pH 7.5, 150 mmol/l NaCl, 1% NP-40, 10 mmol/l EDTA, and a protease inhibitor cocktail. After centrifugation, different samples with equal amount of protein were fractionated in a 12% SDS-PAGE and electrotransferred to nitrocellulose membranes. After being blocked, the membranes were sequentially incubated with anti-arginase-1 antibody and HRP-conjugated anti-mouse IgG or anti-arginase-2 and HRP-conjugated anti-rabbit IgG. To detect Bcl-2 and Bcl-xL, the cell lysates were subjected to Western blot, as above except for the use of a hamster anti-mouse Bcl-2, biotinylated anti-hamster IgG, and HRP-conjugated streptavidin or rabbit anti-mouse Bcl-xL following HRP-conjugated anti-rabbit IgG. Reaction was visualized by enhanced chemiluminiscence (Amersham Pharmacia Biotech). Immunoblots were quantified by densitometric analysis of the films (Scion Image Program).
Cytokine assay. Culture supernatants were assayed by a capture ELISA for granulocyte-macrophage colony stimulating factor (GM-CSF) as previously described (16). ELISA plates (Corning) were coated with anti-cytokine antibody at 4°C overnight. Plates were washed and blocked with 10% FBS at room temperature for 2 h. Supernatants (100 µl) from different cultures were added to the plates and incubated at 4°C overnight. Plates were washed and incubated with biotinylated anti-cytokine antibody for 1 h at room temperature. After being washed, extravidin-peroxidase was added to the wells and incubated for an additional 30-min period. Plates were washed and developed using O-phenylendiamine and H2O2 substrate, and the absorbance was determined at 490 nm in an ELISA plate reader (Bio-Rad). Standard curves were generated using recombinant cytokine.
Statistical analysis. Pairwise comparison between groups was evaluated with a two-tailed Student's t-test. Percentages of live and apoptotic cells were compared by using Fisher's exact test. A value of P < 0.05 was considered significant. Values are given as means ± SD of triplicate determinations from a minimum of three separate experiments.
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RESULTS |
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In the absence of cruzipain, cardiac myocyte cultures displayed cell viability about 60%, whereas, in the presence of cruzipain, the viability increased about 93% (Fig. 1).
Interestingly, these results were correlated with neonatal mouse cardiomyocyte monolayers infected with T. cruzi and subjected to serum starvation, which were also more viable than noninfected cultures. These findings were confirmed by counting the cells with condensed and fragmented nuclei stained with PI to reveal apoptotic nuclei and with chagasic IgG to discern intracellular infection. The proportion of cardiac cells with pycknotic nuclei was correlated inversely with the rate of cardiomyocytes bearing T. cruzi amastigotes (Fig. 2, A and B). Virtually, none of the infected cells exhibited apoptotic nuclei despite being starved for 48 h in serum-minimum medium (Fig. 2A). Remarkably, infected cardiomyocyte cultures remained beating after 48 h of serum starvation in contrast to uninfected control cultures, which did not beat at this time. When cardiomyocytes were previously incubated with cruzipain before parasite infection, the apoptotic death decreased significantly compared with cultures infected but not stimulated (P < 0.02) (Fig. 2B).
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To further document the cytoprotective effect of cruzipain under the conditions of our assays, we employed the TUNEL technique in cultures treated with cruzipain for 48 h. We also tested LPS/IFN-, a treatment mediating the apoptotic death of cardiac cells (20). Cultures maintained in 0.1% FBS-DMEM were used as controls. Apoptotic cardiac cells (Fig. 3A) were counted within random fields, and the results are depicted in Fig. 3B. We found that the proportion of apoptotic nuclei was significantly lower (19%) in cultures treated with cruzipain compared with controls maintained in minimum-serum conditions (35%, P < 0.05). In contrast, LPS/IFN-
produced 62% of apoptotic cells (P < 0.001).
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To obtain a more complete picture of the role of cruzipain on cell death, we also performed flow cytometric analysis assaying cardiomyocytes stained with PI. Cultures treated during 48 h with cruzipain were compared with control ones. As shown in Fig. 3C, cruzipain was able to decrease the apoptotic rate compared with nontreated cultures.
Apoptosis is a genetically controlled process, regulated by the interplay of pro- and antiapoptotic factors. Because the antiapoptotic family members, Bcl-2 and Bcl-xL, have been involved in blocking apoptosis of cardiac cells (30, 38), we evaluated the effect of cruzipain on the expression of both protein by Western blot. Cruzipain increased Bcl-2 expression in cultures treated during 20 h and 48 h. Additionally, Bcl-xL was detected, but this molecule did not show changes compared with cruzipain-treated or control cultures (Fig. 3D).
Because the cytokine network is involved in the induction and regulation of apoptotic cell death and GM-CSF is a potent cytokine that may affect the function, growth, and apoptosis in the heart, we investigated whether cardiomyocyte cultures could release this inflammatory cytokine. We found that cruzipain was unable to generate the production of GM-CSF, whereas LPS/IFN- induced high levels of this cytokine in cardiomyocyte cultures (Fig. 4).
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Cruzipain modulates L-arginine metabolic pathways mediated by NOS and arginase in neonatal cardiomyocytes. It is known that L-arginine is metabolized to important regulatory molecules involved in the normal cell homeostasis modulation (19, 21). NOS converts L-arginine into citrulline plus NO. Alternatively, L-arginine is metabolized to L-ornithine and urea by arginase. Although the NOS pathway has been previously evaluated in cardiomyocytes, arginase pathway has been poorly investigated. Taking into account that cruzipain is able to stimulate arginase activity in macrophages (16, 32), we have decided to explore the effects of this T. cruzi glycoprotein on L-arginine metabolic pathways in cardiomyocyte cultures.
We first analyzed whether treatment of confluent and serum-starved mouse neonatal cardiomyocytes with cruzipain was able to induce NOS activity. Thus we evaluated nitrite production in cardiomyocytes cultured with different stimuli during 24 or 48 h (Fig. 5). Nitrite concentrations in culture supernatants of cells stimulated with cruzipain or IL-4 were low at 24 h, as well as 48 h, similar to controls maintained in medium. Conversely, high nitrite levels were detected in supernatants of cardiomyocytes incubated with LPS/IFN- for 24 h (P < 0.05), and the increase was higher after 48 h of treatment (P < 0.005). In addition, a strong iNOS expression was only observed by immunocytochemistry in cells treated with LPS/IFN-
. In contrast, iNOS stain was weaker in cultures treated with cruzipain, IL-4, or in controls (data not shown).
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Arginase activity was evaluated by measuring the urea levels produced by cell lysates of cardiomyocytes cultured with cruzipain. Treatments with IL-4 or LPS/IFN- were used as positive control for arginase and NOS activities, respectively. Although a basal level of urea was detected in unstimulated cells, cruzipain increased the urea level about twofold, whereas IL-4 was able to stimulate the urea production about fourfold compared with controls maintained in 0.1% FBS medium. In addition, we observed that LPS/IFN-
also increased the urea production (Fig. 5B).
To study the expression of arginase isoforms (1 and 2) induced by cruzipain, we analyzed their expression by Western blot (Fig. 5C). We found that arginase-2 isoform but not arginase-1 was strongly increased after treatment with cruzipain, whereas a basal expression of arginase-1 and 2 was present in cardiomyocytes cultured with medium alone (Fig. 5C). These results were confirmed by immunocytochemistry (data not shown).
In addition, when cultures were preincubated with NOHA, a specific arginase inhibitor, the survival action of cruzipain was inhibited as was assessed by trypan blue exclusion. In fact, the cell viability decreased from 93% without NOHA to 57% with the arginase inhibitor (P < 0.01). The assay was performed in quadruplicate in two independent experiments.
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DISCUSSION |
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Apoptosis is one type of active cell death (1, 2), modulated by multiple positive and negative regulators. Regarding the mechanisms associated with the antiapoptotic effects and cell survival signals, we focused on Bcl-2 and Bcl-xL expressions, because these factors have critical roles counteracting apoptotic signals in a variety of cells, including cardiomyocytes (30, 38). We found that Bcl-2 expression in cardiomyocytes may be involved in the antiapoptotic effect achieved by cruzipain. However, it should also be stressed that these results were obtained with mouse neonatal cardiomyocytes and that the effects on adult cardiomyocytes remain to be assessed.
In the present study, we additionally found that cruzipain strongly stimulates the L-arginine metabolic pathway mediated by arginase. In mammals, two arginase isoforms are expressed: the cytosolic arginase-1 and the mitochondrial arginase-2. The isoforms catalyze the same reaction but are encoded by different genes and differ in their tissue distribution (22). Arginase-1 is highly expressed in the liver, where it serves as the fifth and final enzyme of the urea cycle and constitutes the majority of total body arginase activity (21). Conversely to arginase-1, arginase-2 is expressed at low levels in many tissues (18, 27). Unlike arginase-1, the physiological role of arginase-2 has not been well established. In this study, we demonstrated for the first time that arginase-2 isoform is upregulated in cardiomyocytes primary cultures after cruzipain treatment. Although a basal level of arginase activity was found in cardiomyocyte cultures corresponding to both arginase isoforms, only arginase-2 expression was increased by cruzipain.
The ability of cruzipain to increase arginase activity has also been demonstrated on spleen macrophages from BALB/c mice stimulated with cruzipain (16, 32). Interestingly, we observed a strong arginase-2 expression in amastigote pseudocysts in hearts from T. cruzi infected BALB/c mice (Aoki MP, unpublished data). In this way, cruzipain generates an adequate environment for the parasite growth and its dissemination in the host heart. Interestingly, we found that IL-4 also increases arginase activity in myocardial cells. This is in agreement with previous studies with other cell types such as macrophages or smooth muscle cells in which IL-4 treatment significantly increased the arginase activity (29, 37).
In contrast, cruzipain was unable to stimulate the L-arginine metabolic pathway mediated by NOS. In fact, cardiomyocytes cultures maintained in medium alone have a basal NOS level that is not enhanced by cruzipain or IL-4 treatment. However, cultures stimulated with LPS/IFN- resulted in a significant increase of NO levels, similar to previously shown results (4). Therefore, it is probable that the strong NOS induction elicited by this treatment leads cardiomyocytes to apoptosis (20).
Esch et al. (10) demonstrated and confirmed the antiapoptotic activity of arginase in a pure preparation of recombinant arginase. Arginase ability to inhibit apoptosis resulting in cell survival was reported in multiple paradigms. The authors postulated that this mechanism is independent of NOS inhibition. Rather, arginase-induced depletion of arginine leads to inhibition of protein synthesis, resulting in cell survival. Interestingly, Gotoh and Mori (17) demonstrated that the induction of arginase-2 prevents NO-mediated apoptosis of RAW cells treated with LPS/IFN. In the present work, we found that cruzipain significantly enhanced the expression of arginase-2 and, in turn, decreased the apoptotic fate induced by serum starvation. Moreover, the inhibition of arginase activity by NOHA abrogated the antiapoptotic action exerted by cruzipain, suggesting that arginase activity is required for the survival effect of cruzipain. Our findings provide new insights into the complex regulation of this still rather unknown family of enzymes involved in the L-arginine metabolic pathways and should help to clarify the functional importance of arginase within the cardiovascular physiology.
Altogether, these results strongly suggest that cruzipain could act as a cardioprotective factor promoting survival signals that rescue the cells from death by apoptosis. Schnapp et al. (31) suggested that cruzipain may be a T. cruzi vaccine candidate. In this sense, recombinant cruzipain developed a strong cruzipain memory Th-1 response associated with parasite protective immunity. However, they suggested that it will be important to complete the studies with biochemical and functional analyses to ensure that the vaccine does not trigger immunopathological mechanisms.
Recently, it has been reported that another T. cruzi antigen, a trans-sialidase, is a potent and specific survival factor for Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling (7). The study of the signal transduction pathways involved in the effect of cruzipain on cardiomyocyte cultures will be the subject of future investigations.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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