Trypanosoma cruzi infection and endothelin-1 cooperatively activate pathogenic inflammatory pathways in cardiomyocytes

Abstract

Trypanosoma cruzi, the causative agent of Chagas' disease, induces multiple responses in the heart, a critical organ of infection and pathology in the host. Among diverse factors, eicosanoids and the vasoactive peptide endothelin-1 (ET-1) have been implicated in the pathogenesis of chronic chagasic cardiomyopathy. In the present study, we found that T. cruzi infection in mice induces myocardial gene expression of cyclooxygenase-2 (Cox2) and thromboxane synthase (Tbxas1) as well as endothelin-1 (Edn1) and atrial natriuretic peptide (Nppa). T. cruzi infection and ET-1 cooperatively activated the Ca(2+)/calcineurin (Cn)/nuclear factor of activated T cells (NFAT) signaling pathway in atrial myocytes, leading to COX-2 protein expression and increased eicosanoid (prostaglandins E(2) and F(2α), thromboxane A(2)) release. Moreover, T. cruzi infection of ET-1-stimulated cardiomyocytes resulted in significantly enhanced production of atrial natriuretic peptide (ANP), a prognostic marker for impairment in cardiac function of chagasic patients. Our findings support an important role for the Ca(2+)/Cn/NFAT cascade in T. cruzi-mediated myocardial production of inflammatory mediators and may help define novel therapeutic targets.

Figure 1. Trypanosoma cruzi infection induces Cox2, Tbxas1, Edn1 and Nppa in infected heart tissue.

(A and B) C57BL/6 (black circles) and BALB/c (white circles) mice were infected with 2×10³ blood-trypomastigote forms of the Y strain. (A) Parasitemia expressed as the mean ± standard error of the mean (s.e.m.) of the number of parasites per 5 µl of blood. (B) Percent of mice survival. Results are representative of 2 independent experiments, each performed with 6 mice per group. (C) Tissue inflammation, parasitism and COX-2 expression in heart from uninfected (left panels) and T. cruzi-infected (21 days post-infection, right panels) mice. Representative results of histological analysis (Mason's trichrome staining) of cardiac tissue specimens from BALB/c and C57BL/6 mice (top and center panels, respectively) are shown. Bars = 100 µm. Bottom panels display representative results of COX-2 immunostaining (IS) in the hearts from C57BL/6 mice. Original magnification for microphotographs ×400. (D) Cox2 (COX-2), Tbxas1 (TXS), Edn1 (ET-1) and Nppa (ANP) gene expression in the heart during the acute phase of infection in C57BL/6 and BALB/c mice. RNA from heart tissue at different days post-infection was used to perform RT-PCR with specific probes, and normalized to ribosomal 18S RNA as described in ‘Materials and Methods’. Values are expressed as means ± s.e.m. from 3 independent infections, each performed with 3 mice per group. *P<0.05. (E) Levels of circulating peptides (ET-1 and ANP) and eicosanoids (PGF_2α and TxB₂) in the sera of uninfected (black bars) and T. cruzi-infected (grey bars) C57BL/6 mice. Mouse sera were collected before and after 21 days of infection, and were assayed in triplicate by capture ELISA for ANP (top panel), ET-1 (central panel), PGF_2α and TxB₂ (bottom panel). Each bar represents the mean values for groups of 6 mice ± s.e.m. Similar results were obtained in two additional experiments. ^* P<0.05; ^** P<0.01.

Figure 2. Trypanosoma cruzi infection of endothelin-1-pre-treated HL-1 cardiomyocytes induces cyclooxygenase-2 expression.

(A) COX-2 protein expression in primary BALB/c cardiac myocytes infected with T. cruzi. Neonatal mouse heart cells were isolated and ex vivo infected with Y strain trypomastigotes (cell∶parasite ratio 1∶5) for 24 h. To obtain a positive control, the cells were incubated with 25 U/ml recombinant IFN-γ plus 1 µg/ml LPS. Uninfected cells (Mock) were used as controls. The levels of COX-2 and β-actin proteins were analysed by immunoblotting as described under ‘Materials and methods’. (B) Effects of ET-1 pre-treatment and T. cruzi infection of HL-1 cardiomyocytes on Cox2 mRNA expression. HL-1 atrial muscle cells were stimulated with 0.3 nM ET-1 for 2 h, and/or infected with T. cruzi trypomastigotes (cell∶parasite ratio 1∶5) for 3 h, and the levels of Cox2 mRNA were assessed by reverse transcription and PCR; Actb (β-actin) was used as a loading marker. (C) Effects of ET-1 pre-treatment and T. cruzi infection of HL-1 cardiomyocytes on COX-2 protein expression. HL-1 atrial muscle cells were stimulated with 0.3 nM ET-1 for 2 h, and/or infected with T. cruzi trypomastigotes for 3 h, and the levels of COX-2 and α-tubulin proteins were analysed by immunoblotting. (D) Effects of ET-1 pre-treatment and T. cruzi infection of HL-1 cardiomyocytes on the inducibility of the Cox2 promoter. Cells were transiently transfected with the P2-1900-Cox-2-LUC reporter construct, and then stimulated with 0.3 nM ET-1 for 2 h, and/or infected with trypomastigotes for 3 h. For some experiments, FK506 (100 ng/ml) was added to [T. cruzi+ET-1]-activated cardiomyocytes. PMA+Ion was used as a standard stimulus. Luciferase activity is expressed as fold induction relative to the transfection with empty expression vector. Data are the means ± s.e.m. of three independent experiments, each performed in triplicate. *P<0.05. (E) Involvement of NFAT in Cox2 induction by T. cruzi plus ET-1. HL-1 cells were transiently transfected with the P2-1900-Cox-2-LUC reporter construct, with the P2-274-Cox-2 promoter construct, or with the same construct containing distal and/or proximal NFAT sites (dNFAT and pNFAT, respectively), and/or actvated protein-1 (AP-1) site mutated (indicated by X). For some experiments, the cells were transiently co-transfected with the P2-274-Cox-2-LUC reporter plasmid along with a dominant-negative version of NFAT (dn-NFAT). Three hours later, the cells were stimulated with ET-1 (0.3 nM) for 2 h and infected with T. cruzi parasites for 3 h. Luciferase activity is expressed as percentage of induction (mean ± s.e.m.) relative to that achieved in P2-1900-Cox-2-LUC transfected cells. One out of three separate experiments performed is shown. *P<0.05; ** P<0.001 (respect to the P2-274 construct).

Figure 3. Activation of the Ca²⁺/Calcineurin/NFAT intracellular signaling pathway in endothelin-1-stimulated and Trypanosoma cruzi-infected cardiomyocytes.

(A) HL-1 cells, exposed or not to 0.3 nM ET-1, were loaded with the Ca²⁺ indicator Fura-2/M and changes in [Ca²⁺]_i upon T. cruzi infection were recorded. Uninfected cells were used as a control. Arrows indicate the time (min) when either culture medium (M) or T. cruzi trypomastigotes (T) was added. The results presented are representative of three independent experiments. (B) ET-1 stimulated and T. cruzi-infected HL-1 cardiomyocytes were disrupted and the protein expression of the four NFAT isoforms (c1 to c4) was analysed by immunoblotting. Alpha-tubulin protein levels were determined as a control of loading. (C) HL-1 cells were incubated for 2 h with ET-1 (0.3 nM) and subsequently infected with T. cruzi trypomastigotes for 3 h. For some experiments, FK506 (100 ng/ml) was added 1 h before stimulation. Fractionated extracts from both untreated and treated cells were analysed by immunoblotting with an antiserum to NFATc4. The phosphorylated cytosolic (P-NFATc4) or dephosphorylated nuclear (NFATc4) forms of the factor are indicated. Cyto, cytosolic extracts; Nucl, nuclear extracts. (D) Electrophoretic mobility shift assay (EMSA) analysis to determine NFATc4 binding to the NFAT sites of the Cox2 gene (Cox-2 NFAT). HL-1 myocytes were stimulated with 0.3 nM ET-1 for 2 h and/or infected with T. cruzi trypomastigotes for 3 h. For some experiments, FK506 (100 ng/ml) was added 1 h before stimulation. Mock-treated cells were considered as controls. PMA (15 ng/ml) supplemented with 1 µM Ion was used as a standard stimulus. Nuclear extracts were analysed by EMSA using a Cox-2 NFAT radiolabeled probe. A 50-fold molar excess of unlabeled Cox-2 NFAT oligonucleotide (T. cruzi+ET-1+Cox-2 NFAT) was added to determine specific binding. NFATc4 antibody or normal rabbit IgG was added to the extracts before incubation with the probe. Arrows indicate specific supershifted complexes. This is representative of at least three independent experiments.

Figure 4. Production of eicosanoids and atrial natriuretic peptide by endothelin-1-stimulated and Trypanosoma cruzi-infected HL-1 cardiac cells.

(A) Cardiomyocytes were serum-starved for 12 h, then incubated for 1 h in the presence of cyclooxygenase inhibitors (10 µM indomethacin -Indo- or 10 µM NS-398) and further stimulated with 0.3 nM ET-1 for 2 h. After treatment, the cells were infected with T. cruzi trypomastigotes for 24 h. HL-1 myocytes infected with the parasite or stimulated with ET-1 alone were included in the assay. Culture supernatants were collected and analysed for PGE₂, PGF_2α and TxB₂ (TxA₂ stable metabolite) by ELISA (Cayman). The results represent means ± s.e.m. of three individual experiments assayed in triplicate. *P<0.05 and **P<0.001 compared with mock-treated cells; ^# P<0.05 and ^## P<0.001 compared with NS-398- and Indo-treated cells, respectively. (B) Effects of T. cruzi infection and ET-1 stimulation on the expression of prostanoid terminal synthases in HL-1 cardiac cells. Myocytes were incubated with 0.3 nM ET-1 for 2 h and/or infected with T. cruzi trypomastigotes for 3 h. Uninfected and mock-treated cells were used as controls. Immunoblot analysis of the protein expression of prostanoid terminal synthases (mPGES-2, PGFS and TXS) after exposure to stimulus and/or parasite is shown. Alpha-tubulin was used as loading control. The results presented are representative of three independent experiments with similar outcome. (C) HL-1 cells were treated with ET-1 (0.3 nM) for 2 h and/or T. cruzi-infected for 24 h and supernatants were collected. Uninfected and mock-treated cells were used as controls. ANP release (mean ± s.e.m.) was analyzed using an ELISA kit (Kamiya Biomedical) following the manufacturer's instructions. The results are representative of three independent experiments performed in quadruplicate. Statistically significant differences are indicated (*P<0.05, compared with mock; ^# P<0.05, [T. cruzi+ET-1]-activated cells versus T. cruzi-infected myocytes).