RESUMO
BACKGROUND: Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES: Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS: We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS: Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in ß-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION: The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Miócitos Cardíacos/parasitologia , Paxilina/metabolismo , Talina/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Western Blotting , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Mecanotransdução Celular/fisiologia , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The activation of signaling pathways involving protein tyrosine kinases (PTKs) has been demonstrated during Trypanosoma cruzi invasion. Herein, we describe the participation of FAK/Src in the invasion of cardiomyocytes by T. cruzi. The treatment of cardiomyocytes with genistein, a PTK inhibitor, significantly reduced T. cruzi invasion. Also, PP1, a potent Src-family protein inhibitor, and PF573228, a specific FAK inhibitor, also inhibited T. cruzi entry; maximal inhibition was achieved at concentrations of 25µM PP1 (53% inhibition) and 40µM PF573228 (50% inhibition). The suppression of FAK expression in siRNA-treated cells and tetracycline-uninduced Tet-FAK(WT)-46 cells significantly reduced T. cruzi invasion. The entry of T. cruzi is accompanied by changes in FAK and c-Src expression and phosphorylation. An enhancement of FAK activation occurs during the initial stages of T. cruzi-cardiomyocyte interaction (30 and 60min), with a concomitant increase in the level of c-Src expression and phosphorylation, suggesting that FAK/Src act as an integrated signaling pathway that coordinates parasite entry. These data provide novel insights into the signaling pathways that are involved in cardiomyocyte invasion by T. cruzi. A better understanding of the signal transduction networks involved in T. cruzi invasion may contribute to the development of more effective therapies for the treatment of Chagas' disease.
Assuntos
Quinase 1 de Adesão Focal/fisiologia , Miócitos Cardíacos/parasitologia , Transdução de Sinais/fisiologia , Trypanosoma cruzi/fisiologia , Quinases da Família src/fisiologia , Animais , Proteína Tirosina Quinase CSK , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolonas/farmacologia , RNA Interferente Pequeno/fisiologia , Sulfonas/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
The treatment of Chagas disease (CD), a neglected parasitic condition caused by Trypanosoma cruzi, is still based on only two drugs, nifurtimox (Nif) and benznidazole (Bz), both of which have limited efficacy in the late chronic phase and induce severe side effects. This scenario justifies the continuous search for alternative drugs, and in this context, the natural naphthoquinone ß-lapachone (ß-Lap) and its derivatives have demonstrated important trypanocidal activities. Unfortunately, the decrease in trypanocidal activity in the blood, high toxicity to mammalian cells and low water solubility of ß-Lap limit its systemic administration and, consequently, clinical applications. For this reason, carriers as drug delivery systems can strategically maximize the therapeutic effects of this drug, overcoming the above mentioned restrictions. Accordingly, the aim of this study is to investigate the in vitro anti-T. cruzi effects of ß-Lap encapsulated in2-hydroxypropyl-ß-cyclodextrin (2HP-ß-CD) and its potential toxicity to mammalian cells.
Assuntos
Portadores de Fármacos/química , Naftoquinonas/química , Naftoquinonas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , SolubilidadeRESUMO
Chagas disease is responsible for more than 10,000 deaths per year and about 6 to 7 million infected people worldwide. In its chronic stage, patients can develop mega-colon, mega-esophagus, and cardiomyopathy. Differences in clinical outcomes may be determined, in part, by the genetic background of the parasite that causes Chagas disease. Trypanosoma cruzi has a high genetic diversity, and each group of strains may elicit specific pathological responses in the host. Conflicting results have been reported in studies using various combinations of mammalian host-T. cruzi strains. We previously profiled the transcriptomic signatures resulting from infection of L6E9 rat myoblasts with four reference strains of T. cruzi (Brazil, CL, Y, and Tulahuen). The four strains induced similar overall gene expression alterations in the myoblasts, although only 21 genes were equally affected by all strains. Cardiotrophin-like cytokine factor 1 (Clcf1) was one of the genes found to be consistently upregulated by the infection with all four strains of T. cruzi. This cytokine is a member of the interleukin-6 family that binds to glycoprotein 130 receptor and activates the JAK/STAT signaling pathway, which may lead to muscle cell hypertrophy. Another commonly upregulated gene was tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (Ywhaq, 14-3-3 protein Θ), present in the Cell Cycle Pathway. In the present work, we reanalyzed our previous microarray dataset, aiming at understanding in more details the transcriptomic impact that each strain has on JAK/STAT signaling and Cell Cycle pathways. Using Pearson correlation analysis between the expression levels of gene pairs in biological replicas from each pathway, we determined the coordination between such pairs in each experimental condition and the predicted protein interactions between the significantly altered genes by each strain. We found that although these highlighted genes were similarly affected by all four strains, the downstream genes or their interaction partners were not necessarily equally affected, thus reinforcing the idea of the role of parasite background on host cell transcriptome. These new analyses provide further evidence to the mechanistic understanding of how distinct T. cruzi strains lead to diverse remodeling of host cell transcriptome.
Assuntos
Trypanosoma cruzi , Animais , Brasil , Ciclo Celular , Humanos , Mioblastos , Ratos , Transdução de Sinais , Transcriptoma , Trypanosoma cruzi/genéticaRESUMO
Herein, we present the design, synthesis and trypanocidal evaluation of sixteen new 1,3,4-thiadiazole derivatives from N-aminobenzyl or N-arylhydrazone series. All derivatives were assayed against the trypomastigote form of Trypanosoma cruzi, showing IC50 values ranging from 3 to 226⯵M, and a better trypanocidal profile was demonstrated for the 1,3,4-thiadiazole-N-arylhydrazones (3a-g). In this series, the 2-pyridinyl fragment bound to the imine subunit of the hydrazine moiety presented pharmacophoric behavior for trypanocidal activity. Compounds 2a, 11a and 3e presented remarkable activity and excellent selectivity indexes. Compound 2a was also active against the intracellular amastigote form of T. cruzi. Moreover, its corresponding hydrochloride, compound 11a, showed the most promising profile, producing phenotypic changes similar to those caused by posaconazole, a well-known inhibitor of sterol biosynthesis. Thus, 1,3,4-thiadiazole derivative 11a could be considered a good prototype for the development of new drug candidates for Chagas disease therapy.
Assuntos
Doença de Chagas/tratamento farmacológico , Tiadiazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/parasitologia , Concentração Inibidora 50 , Camundongos , Relação Estrutura-Atividade , Tiadiazóis/síntese química , Tiadiazóis/química , Tripanossomicidas/síntese química , Tripanossomicidas/químicaRESUMO
We analyzed the distribution and expression of cadherin and beta-catenin during Trypanosoma cruzi-cardiomyocyte interaction. Confocal microscopy revealed cadherin associated with beta-catenin at the cell-cell contacts. After 24h of infection, the spatial distribution and expression of both adherens junction (AJ) proteins remained unaltered. In contrast, loss of N-cadherin-catenin complex was visualized in highly infected cardiomyocytes. Immunoblotting assays corroborated the spatial disorder, showing a 46% reduction in both N-cadherin and beta-catenin expression at later infection (72h of infection). Our data demonstrate that T. cruzi infection disturbs AJs, which can result in loss of cardiac tension and may contribute to the cardiac dysfunctions present in T. cruzi infection.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Miócitos Cardíacos/metabolismo , Trypanosoma cruzi/patogenicidade , beta Catenina/metabolismo , Animais , Células Cultivadas , Camundongos , Microscopia Confocal , Miócitos Cardíacos/parasitologia , Miócitos Cardíacos/ultraestrutura , Trypanosoma cruzi/metabolismoRESUMO
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. cruzi-infected 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.
Assuntos
Cardiomiopatia Chagásica/patologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Miofibrilas/ultraestrutura , Trypanosoma cruzi , Vinculina/metabolismo , Animais , Membrana Celular/ultraestrutura , Células Cultivadas , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Coração/parasitologia , Interações Hospedeiro-Parasita , Immunoblotting , Masculino , Camundongos , Microscopia de Fluorescência , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Miofibrilas/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
BACKGROUND Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in β-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.
Assuntos
Animais , Camundongos , Trypanosoma cruzi/fisiologia , Cardiomiopatia Chagásica/metabolismo , Miócitos Cardíacos/parasitologia , Mecanotransdução Celular/genética , Western Blotting , Reação em Cadeia da Polimerase , Imunofluorescência , Paxilina/metabolismoRESUMO
Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits multiple strategies to ensure its establishment and persistence in the host. Although this parasite has the ability to infect different organs, heart impairment is the most frequent clinical manifestation of the disease. Advances in knowledge of T. cruzi-cardiomyocyte interactions have contributed to a better understanding of the biological events involved in the pathogenesis of Chagas disease. This brief review focuses on the current understanding of molecules involved in T. cruzi-cardiomyocyte recognition, the mechanism of invasion, and on the effect of intracellular development of T. cruzi on the structural organization and molecular response of the target cell.