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Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease
Accepted Manuscript Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease Leonardo da Silva Lara, Leonardo Andrade-Lima, Claudia Magalhães Calvet, Juliana Borsoi, Thabata Lopes Alberto Duque, Andrea Henriques-Pons, Mirian Claudia Souza Pereira, Lygia Veiga Pereira PII:
S1286-4579(18)30073-X
DOI:
10.1016/j.micinf.2018.03.002
Reference:
MICINF 4574
To appear in:
Microbes and Infection
Received Date: 14 December 2017 Revised Date:
23 February 2018
Accepted Date: 13 March 2018
Please cite this article as: L. da Silva Lara, L. Andrade-Lima, C.M. Calvet, J. Borsoi, T.L. Alberto Duque, A. Henriques-Pons, M.C. Souza Pereira, L.V. Pereira, Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease, Microbes and Infection (2018), doi: 10.1016/j.micinf.2018.03.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Trypanosoma cruzi infection of human induced pluripotent stem cell-derived
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cardiomyocytes: an in vitro model for drug screening for Chagas disease.
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Leonardo da Silva Laraaζ, Leonardo Andrade-Limabζ, Claudia Magalhães Calvetaζ, Juliana
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Borsoib, Thabata Lopes Alberto Duquec, Andrea Henriques-Ponsd, Mirian Claudia Souza
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Pereiraa*, Lygia Veiga Pereirab*
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a
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Manguinhos, RJ, 21045-900, Brazil
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b
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of São Paulo, SP 05508-090, Brazil
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Laboratório de Ultraestrutura Celular, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365,
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National Laboratory for Embryonic Stem Cells (LaNCE), Institute of Biosciences, University
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Fiocruz - Av. Brasil 4365, Manguinhos, RJ, 21045-900, Brazil
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(ζ) These authors contributed equally to the work;
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(*) These senior authors contributed equally to the work.
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Corresponding authors: Lygia V Pereira email: [email protected]; Mirian Claudia S Pereira
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email: [email protected]
Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fiocruz.
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Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo cruz,
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Abstract
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Chagas disease, caused by Trypanosoma cruzi, is an important global public health problem
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which, despite partial efficacy of benznidazole (Bz) in acute phase, urgently needs an
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effective treatment. Cardiotoxicity is a major safety concern for conduction of more accurate
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preclinical drug screening platforms. Human induced pluripotent stem cells derived
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cardiomyocytes (hiPSC-CM) are a reliable model to study genetic and infectious cardiac
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alterations and may improve drug development. Herein, we introduce hiPSC-CM as a suitable
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ACCEPTED MANUSCRIPT model to study T. cruzi heart infection and to predict the safety and efficacy of anti-T. cruzi
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drugs.
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Keywords
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hiPSC-cardiomyocytes; Chagas disease; Trypanosoma cruzi; drug screening; benznidazole
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Introduction
Chagas disease (CD), caused by Trypanosoma cruzi, is responsible for the greatest
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burden of disability adjusted life years [1]. Epidemiological surveys estimate 5-7 million
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people infected worldwide and 65 million living under risk of infection in the Americas, with
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12.000 deaths per year [2]. CD treatment is currently based on nitroheterocyclic drugs
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(benznidazole – Bz, and nifurtimox - Nif) and presents severe side effects, limited efficacy
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and low effect in the chronic phase [3]. The therapeutic failure in phase II of clinical trials of
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promising drug candidates [4] and the results of BENEFIT trails [5] lead to an improvement
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of hit and lead criteria for drug discovery [6,7]. An important recommendation is
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development of a better system for standardization and validation of new compounds prior to
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clinical trials [6].
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Drug screenings for human diseases generally use cell lineages and animal models to
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determine drug efficacy and safety. The current concern is the lack of reliable prediction tools
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for human clinical trials [8]. In this regard, the application of primary cells differentiated from
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pluripotent stem cells has been proposed as an alternative approach in screening compounds
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for cardiotoxicity and activity against human diseases, since they better model normal human
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tissues and would reduce the use of animals for research [9].
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Human pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are an unlimited
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source of human normal cardiomyocytes. Despite generally having an immature phenotype,
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these cells have been shown to model different cardiac diseases, including hypertrophic
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cardiomyopathy [10,11], dilated cardiomyopathy [12], and viral myocarditis caused by
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coxsackievirus infection [13]. Also, hiPSC-CM were able to predict patient specific
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susceptibility to doxy-induced cardiotoxicity [14]. Considering that cardiomyocytes are an important target of T. cruzi infection, we
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introduce the application of hiPSC-CMs as an in vitro model for anti-T. cruzi drug screening
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and toxicity studies using trypanocidal treatment with benznidazole. We demonstrate that
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hiPSC-CMs are a robust platform for studying T. cruzi infection of cardiomyocytes, and for
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assaying cardiotoxicity of new compounds with anti-T. cruzi activity.
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Materials and methods
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2.
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Detailed Materials and Methods are presented in Supplemental Material.
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3. Results
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In order to develop a strategy more easily incorporated by groups lacking expertise in
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pluripotent stem cell culture and differentiation, we used commercial cryopreserved 45 day-
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differentiated CM-hiPSCs. After thawing, four day-cultured hiPSC-CMs stained positive for
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α-actinin and cardiac troponin-T, revealing typical organized sarcomeres (Suppl. Fig. 1).
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After 7 days in culture, cells presented the characteristic contractions of CM-hiPSCs (data not
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shown).
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Before analyzing the cytotoxic and trypanocidal effect of Bz on hiPSC-CM, we
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evaluated the kinetic of infection of the human cells by T. cruzi Y strain. Seven days after
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thawing, cells were exposed to bloodstream trypomastigotes in ratios of 10:1 and 25:1
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parasite-host cell, for 90 minutes, 24 and 48 hours.
Rare intracellular parasites were
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ACCEPTED MANUSCRIPT visualized in the confluent hiPSC-CM cultures at early time of infection (90 min) regardless
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of parasite-host cell ratio (data not shown). However, the percentage of infection increased
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substantially with the course and ratio of infection. Lower level of infection was achieved
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when confluent hiPSC-CM cultures were infected at a ratio of 10:1 parasite: host cell,
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reaching 13.5±3.53 % and 26.46±2.42 % after 24 h and 48 h of infection, respectively.
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Intracellular amastigotes were typically located at the perinuclear domain. Approximately
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twice the infection rate was evidenced when the hiPSC-CM cultures were infected with
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higher multiplicity of infection (MOI = 25:1). The percentage of infection reached 23.35±4.03
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% and 50.16±1.76 % after 24 h and 48 h post-infection, respectively.
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Human iPSC-CMs were treated with a range of Bz concentration (3.9 – 500 µM), and
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no cytotoxic effect was evidenced even at the highest concentration of Bz, achieving a CC50 >
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500 µM (Fig. 1A). T. cruzi Y strain (TcII) and Dm28cLuc clone (TcI) were used to
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determine Bz sensitivity of T. cruzi-infected hiPSC-CMs. Twenty-four hours after infection,
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hiPSC-CMs monolayers were treated for 72 h with serial dilutions of Bz (0.61 – 100 µM) and
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the infection rate estimated by direct counting. High levels of infection (63%), with an
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average of 53.7±12 parasites per host cell, were evidenced after 72 h of infection with
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bloodstream trypomastigotes Y strain (25:1 parasites:host cell).
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Bz treatment of hiPSC-CM infected with T. cruzi Y strain inhibited the rate of
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infection and parasite growth in a concentration response manner. The percentage of infection
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ranged from 2% to 57% after treatment with Bz (72 h) (Fig. 2A). A significant reduction in
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the number of intracellular parasites was observed from the concentration of 1.85 µM (15.6 ±
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2.4; p≤0.05) to the highest concentration of Bz (50 µM) (1.2 ± 0.2; p=1.44E-05) (Fig. 2B).
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The inhibition of 50% in intracellular amastigotes proliferation (IC50 value) was achieved at
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1.13 ± 0.2 µM of Bz (Fig. 1B). This remarkable reduction on hiPSC-CMs infection was easily
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visualized by light microscopy (Fig. 2 C-H). Light micrographs demonstrate the susceptibility
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of hiPSC-CMs to T. cruzi infection and the efficiency of Bz treatment. Another approach towards the implementation of this hiPSC-CMs model for large
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scale screening of compounds was to analyze the effect of Bz on intracellular amastigotes
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using Dm28cLuc, a genetically modified parasite expressing luciferase, and bioluminescent
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assay, a highly sensitive method for measuring drug effect in high throughput screening. The
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luminescent-based growth inhibition revealed an IC50 value of 3.65 ± 1.24 µM for
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intracellular amastigotes (Fig. 1C).
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Treatment of hiPSC-CM with Bz revealed no changes in the cardiac cell phenotype
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(Fig. 3A). Ultrastructural analysis showed a large number of mitochondria (M), myofibrils
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(Mf), sarcoplasmic reticulum cisternae (SR), glycogen granules (Gly) and junction complex
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(J). Nevertheless, additional eletromechanical parameters should be analyzed to rigorously
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exclude cardiotoxicity [14]. Parasites were visualized within the cytoplasm of untreated
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hiPSC-CM in close association with host cell organelles (Fig. 3B). Treatment of T. cruzi-
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infected hiSPC-CM with Bz induced morphological changes in the intracellular parasites.
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Swelling of kinetoplast-mitochondria was observed even at the lowest concentration of Bz
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(IC25) (Fig. 3C). Damage of kinetoplast-mitochondria was more evident at the highest
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concentration of Bz (IC50) (Fig. 3D).
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3. Discussion
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Heart diseases, including cardiovascular diseases and cardiomyopathies, are
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responsible for high rates of heart failures and death worldwide [15]. Human induced
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pluripotent stem cells emerged as a potential study model in scientific research and
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revolutionized the understanding of numerous pathologies, with great contributions to
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inherited cardiomyopathies [16]. Advances in the detailing of pathological features have been
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ACCEPTED MANUSCRIPT also achieved with the hiPSC disease model [17], which recapitulate disease phenotype,
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improving the understanding of the intrinsic mechanism of the disease and also becoming a
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valuable tool for drug discovery. However, cardiomyopathies caused by infectious diseases
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lack insights from hiPSC-based studies. Here we show that hiPSC-CM, a relevant target of T.
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cruzi human infection, reproduces the intracellular cycle of the parasite and, therefore, is a
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potential in vitro model for the study of T. cruzi infection in human heart and for the
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development of new drugs for CD. To our knowledge, this is the first report showing T. cruzi
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infection of human cardiomyocytes derived from pluripotent stem cells.
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Cardiotoxic effect is a serious problem in many therapeutic regimens [18].
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Ultrastructural and biochemical alterations have been reported in heart tissue after treatment
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with nifurtimox, highlighting a serious problem in CD therapy [19]. Our findings
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demonstrated no cytotoxicity effect in hiPSC-CM induced by Bz, reinforcing previous data of
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lower risk of heart function after Bz treatment [20].
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candidate for treatment of CD [21], failed in antifungal clinical trials due to cardiotoxic
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effects by inhibiting the hERG channel [22]. Thus, the application of the hiPSC-CM model in
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CD preclinical screenings may contribute for the demonstration of cardiotoxicity thereby
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increasing safety of clinical trials.
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In contrast, D0870, an antifungal
Human iPSC-CM may also be used as an in vitro platform to determine the efficacy of
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any potential anti-T. cruzi drugs. Bz treatment of T. cruzi-infected hiPSC-CM lead to
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intracellular amastigotes reduction, thus validating hiPSC-CM as a suitable model for
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screening of novel bioactive compounds against T. cruzi. Damage of kinetoplast-mitochondria
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was an evident effect of Bz treatment. Swelling of mitochondria without alteration in the
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topology of kDNA has been previously reported [23], suggesting that incorporation of
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oxidized nucleotides, an important mechanism of action of Bz, occurs more slowly in kDNA.
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Nevertheless, the current high cost of hiPSC-CMs hampers their use in large-scale screens for
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new drugs. Their use may be more cost effective in secondary screening of promising
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candidate compounds and in cardiotoxicity tests prior to clinical trials. Human iPSC technology is an emerging tool for the area of infectious disease
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research, providing primary human target tissue to study parasite infections and their response
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to therapy in vitro [13,24,25]. Thus, further exploration of hiPSC-derived in vitro models will
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contribute substantially to the understanding of parasitic infections of medical importance.
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Acknowledgements
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The authors thank Renata Soares Dias de Souza for technical support and the Rudolf
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Barth Electron Microscopy and the Bioassay Platform at Institute Oswaldo Cruz. This work
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was supported by Fundação Oswaldo Cruz, Fundação de Amparo à Pesquisa do Estado do
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Rio de Janeiro, Fundação de Amparo à Pesquisa do Estado de São Paulo (CEPID
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2013/08135-2) and Conselho Nacional de Desenvolvimento Científico e Tecnológico.
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The authors declare no conflict of interest.
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Figure legends
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Fig. 1. Benznidazole cytotoxicity and Trypanocidal effect in hiPSC-CM. (A) Bz did not
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elicit any toxic effect in hiPSC-CM even at high concentrations (CC50 > 500 µM). (B-C) Bz
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Dm28cLuc clone, reaching a IC50 value of 1.1 µM and 3.65 µM, respectively.
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Fig. 2. Profile of T. cruzi infection of hiPSC-CM treated with benznidazole (Bz). (A) A
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concentration response effect of Bz treatment was evident. A significant reduction was
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observed in the percentage of T. cruzi (Y strain) infection. (B) Note the drastic reduction in
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the number of intracellular parasites after Bz treatment (*) p<0.05. (C-H) Light microscopy of
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hiPSC-CM infected by T. cruzi Y strain and treated with Bz. (C) Untreated cells presented
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high level of infection. (D-H) Treatment of T. cruzi-infected hiPSC-CM with Bz markedly
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reduced the level of infection. Few amastigotes (arrows) were visualized at (D) 33 µM, (E) 11
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µM and (F) 3.3 µM of Bz. Amastigote nests were mostly visualized at low concentrations of
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Bz, (G) 1.2 µM and (H) 0.43 µM.
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Fig. 3. Transmission electron micrographs of uninfected and T. cruzi-infected hiPSC-
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CM treated with Bz. (A) No cardiotoxic effect was observed on uninfected hiPSC-CM after
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treatment with Bz at IC50 value for the parasite. Human iPSC-CM shows organized myofibrils
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(Mf), abundance of mitochondria (Mt), glycogen granules (Gly) and endoplasmic reticulum
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(ER). (B) Untreated T. cruzi-infected hiPSC-CM showing well preserved intracellular
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parasites. (C-D) Treatment of hiPSC-CM infected by T. cruzi Y strain with (C) IC25 and (D)
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IC50 of Bz concentrations showing alterations in the intracellular parasites. Swelling of
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kinetoplast-mitochondria (asterisk) was noticed in both Bz concentrations but it was more
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pronounced after treatment with IC50. N-nucleus; J-cell junction; K-kinetoplast and F-
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flagellum.
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Supplemental Fig. 1. Characterization of of hiPSC-CM. Immunoflorescence of hiPSC-CM
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stained with (A) anti-α-actinin (green), and (B) anti-cardiac troponin T (TNNT2) (green), and
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showing high efficiency of differentiation (98% of cells positive for cardiac troponin-T).
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S1286-4579(18)30073-X
DOI:
10.1016/j.micinf.2018.03.002
Reference:
MICINF 4574
To appear in:
Microbes and Infection
Received Date: 14 December 2017 Revised Date:
23 February 2018
Accepted Date: 13 March 2018
Please cite this article as: L. da Silva Lara, L. Andrade-Lima, C.M. Calvet, J. Borsoi, T.L. Alberto Duque, A. Henriques-Pons, M.C. Souza Pereira, L.V. Pereira, Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease, Microbes and Infection (2018), doi: 10.1016/j.micinf.2018.03.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Trypanosoma cruzi infection of human induced pluripotent stem cell-derived
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cardiomyocytes: an in vitro model for drug screening for Chagas disease.
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Leonardo da Silva Laraaζ, Leonardo Andrade-Limabζ, Claudia Magalhães Calvetaζ, Juliana
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Borsoib, Thabata Lopes Alberto Duquec, Andrea Henriques-Ponsd, Mirian Claudia Souza
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Pereiraa*, Lygia Veiga Pereirab*
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a
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Manguinhos, RJ, 21045-900, Brazil
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b
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of São Paulo, SP 05508-090, Brazil
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Laboratório de Ultraestrutura Celular, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365,
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National Laboratory for Embryonic Stem Cells (LaNCE), Institute of Biosciences, University
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Fiocruz - Av. Brasil 4365, Manguinhos, RJ, 21045-900, Brazil
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(ζ) These authors contributed equally to the work;
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(*) These senior authors contributed equally to the work.
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Corresponding authors: Lygia V Pereira email: [email protected]; Mirian Claudia S Pereira
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email: [email protected]
Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fiocruz.
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Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo cruz,
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Abstract
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Chagas disease, caused by Trypanosoma cruzi, is an important global public health problem
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which, despite partial efficacy of benznidazole (Bz) in acute phase, urgently needs an
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effective treatment. Cardiotoxicity is a major safety concern for conduction of more accurate
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preclinical drug screening platforms. Human induced pluripotent stem cells derived
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cardiomyocytes (hiPSC-CM) are a reliable model to study genetic and infectious cardiac
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alterations and may improve drug development. Herein, we introduce hiPSC-CM as a suitable
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drugs.
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Keywords
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hiPSC-cardiomyocytes; Chagas disease; Trypanosoma cruzi; drug screening; benznidazole
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Introduction
Chagas disease (CD), caused by Trypanosoma cruzi, is responsible for the greatest
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burden of disability adjusted life years [1]. Epidemiological surveys estimate 5-7 million
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people infected worldwide and 65 million living under risk of infection in the Americas, with
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12.000 deaths per year [2]. CD treatment is currently based on nitroheterocyclic drugs
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(benznidazole – Bz, and nifurtimox - Nif) and presents severe side effects, limited efficacy
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and low effect in the chronic phase [3]. The therapeutic failure in phase II of clinical trials of
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promising drug candidates [4] and the results of BENEFIT trails [5] lead to an improvement
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of hit and lead criteria for drug discovery [6,7]. An important recommendation is
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development of a better system for standardization and validation of new compounds prior to
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clinical trials [6].
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Drug screenings for human diseases generally use cell lineages and animal models to
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determine drug efficacy and safety. The current concern is the lack of reliable prediction tools
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for human clinical trials [8]. In this regard, the application of primary cells differentiated from
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pluripotent stem cells has been proposed as an alternative approach in screening compounds
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for cardiotoxicity and activity against human diseases, since they better model normal human
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tissues and would reduce the use of animals for research [9].
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Human pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are an unlimited
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source of human normal cardiomyocytes. Despite generally having an immature phenotype,
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these cells have been shown to model different cardiac diseases, including hypertrophic
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cardiomyopathy [10,11], dilated cardiomyopathy [12], and viral myocarditis caused by
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coxsackievirus infection [13]. Also, hiPSC-CM were able to predict patient specific
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susceptibility to doxy-induced cardiotoxicity [14]. Considering that cardiomyocytes are an important target of T. cruzi infection, we
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introduce the application of hiPSC-CMs as an in vitro model for anti-T. cruzi drug screening
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and toxicity studies using trypanocidal treatment with benznidazole. We demonstrate that
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hiPSC-CMs are a robust platform for studying T. cruzi infection of cardiomyocytes, and for
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assaying cardiotoxicity of new compounds with anti-T. cruzi activity.
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Materials and methods
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Detailed Materials and Methods are presented in Supplemental Material.
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3. Results
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In order to develop a strategy more easily incorporated by groups lacking expertise in
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pluripotent stem cell culture and differentiation, we used commercial cryopreserved 45 day-
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differentiated CM-hiPSCs. After thawing, four day-cultured hiPSC-CMs stained positive for
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α-actinin and cardiac troponin-T, revealing typical organized sarcomeres (Suppl. Fig. 1).
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After 7 days in culture, cells presented the characteristic contractions of CM-hiPSCs (data not
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shown).
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Before analyzing the cytotoxic and trypanocidal effect of Bz on hiPSC-CM, we
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evaluated the kinetic of infection of the human cells by T. cruzi Y strain. Seven days after
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thawing, cells were exposed to bloodstream trypomastigotes in ratios of 10:1 and 25:1
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parasite-host cell, for 90 minutes, 24 and 48 hours.
Rare intracellular parasites were
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ACCEPTED MANUSCRIPT visualized in the confluent hiPSC-CM cultures at early time of infection (90 min) regardless
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of parasite-host cell ratio (data not shown). However, the percentage of infection increased
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substantially with the course and ratio of infection. Lower level of infection was achieved
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when confluent hiPSC-CM cultures were infected at a ratio of 10:1 parasite: host cell,
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reaching 13.5±3.53 % and 26.46±2.42 % after 24 h and 48 h of infection, respectively.
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Intracellular amastigotes were typically located at the perinuclear domain. Approximately
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twice the infection rate was evidenced when the hiPSC-CM cultures were infected with
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higher multiplicity of infection (MOI = 25:1). The percentage of infection reached 23.35±4.03
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% and 50.16±1.76 % after 24 h and 48 h post-infection, respectively.
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Human iPSC-CMs were treated with a range of Bz concentration (3.9 – 500 µM), and
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no cytotoxic effect was evidenced even at the highest concentration of Bz, achieving a CC50 >
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500 µM (Fig. 1A). T. cruzi Y strain (TcII) and Dm28cLuc clone (TcI) were used to
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determine Bz sensitivity of T. cruzi-infected hiPSC-CMs. Twenty-four hours after infection,
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hiPSC-CMs monolayers were treated for 72 h with serial dilutions of Bz (0.61 – 100 µM) and
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the infection rate estimated by direct counting. High levels of infection (63%), with an
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average of 53.7±12 parasites per host cell, were evidenced after 72 h of infection with
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bloodstream trypomastigotes Y strain (25:1 parasites:host cell).
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Bz treatment of hiPSC-CM infected with T. cruzi Y strain inhibited the rate of
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infection and parasite growth in a concentration response manner. The percentage of infection
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ranged from 2% to 57% after treatment with Bz (72 h) (Fig. 2A). A significant reduction in
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the number of intracellular parasites was observed from the concentration of 1.85 µM (15.6 ±
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2.4; p≤0.05) to the highest concentration of Bz (50 µM) (1.2 ± 0.2; p=1.44E-05) (Fig. 2B).
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The inhibition of 50% in intracellular amastigotes proliferation (IC50 value) was achieved at
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1.13 ± 0.2 µM of Bz (Fig. 1B). This remarkable reduction on hiPSC-CMs infection was easily
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visualized by light microscopy (Fig. 2 C-H). Light micrographs demonstrate the susceptibility
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of hiPSC-CMs to T. cruzi infection and the efficiency of Bz treatment. Another approach towards the implementation of this hiPSC-CMs model for large
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scale screening of compounds was to analyze the effect of Bz on intracellular amastigotes
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using Dm28cLuc, a genetically modified parasite expressing luciferase, and bioluminescent
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assay, a highly sensitive method for measuring drug effect in high throughput screening. The
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luminescent-based growth inhibition revealed an IC50 value of 3.65 ± 1.24 µM for
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intracellular amastigotes (Fig. 1C).
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Treatment of hiPSC-CM with Bz revealed no changes in the cardiac cell phenotype
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(Fig. 3A). Ultrastructural analysis showed a large number of mitochondria (M), myofibrils
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(Mf), sarcoplasmic reticulum cisternae (SR), glycogen granules (Gly) and junction complex
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(J). Nevertheless, additional eletromechanical parameters should be analyzed to rigorously
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exclude cardiotoxicity [14]. Parasites were visualized within the cytoplasm of untreated
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hiPSC-CM in close association with host cell organelles (Fig. 3B). Treatment of T. cruzi-
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infected hiSPC-CM with Bz induced morphological changes in the intracellular parasites.
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Swelling of kinetoplast-mitochondria was observed even at the lowest concentration of Bz
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(IC25) (Fig. 3C). Damage of kinetoplast-mitochondria was more evident at the highest
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concentration of Bz (IC50) (Fig. 3D).
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3. Discussion
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Heart diseases, including cardiovascular diseases and cardiomyopathies, are
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responsible for high rates of heart failures and death worldwide [15]. Human induced
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pluripotent stem cells emerged as a potential study model in scientific research and
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revolutionized the understanding of numerous pathologies, with great contributions to
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inherited cardiomyopathies [16]. Advances in the detailing of pathological features have been
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improving the understanding of the intrinsic mechanism of the disease and also becoming a
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valuable tool for drug discovery. However, cardiomyopathies caused by infectious diseases
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lack insights from hiPSC-based studies. Here we show that hiPSC-CM, a relevant target of T.
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cruzi human infection, reproduces the intracellular cycle of the parasite and, therefore, is a
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potential in vitro model for the study of T. cruzi infection in human heart and for the
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development of new drugs for CD. To our knowledge, this is the first report showing T. cruzi
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infection of human cardiomyocytes derived from pluripotent stem cells.
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Cardiotoxic effect is a serious problem in many therapeutic regimens [18].
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Ultrastructural and biochemical alterations have been reported in heart tissue after treatment
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with nifurtimox, highlighting a serious problem in CD therapy [19]. Our findings
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demonstrated no cytotoxicity effect in hiPSC-CM induced by Bz, reinforcing previous data of
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lower risk of heart function after Bz treatment [20].
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candidate for treatment of CD [21], failed in antifungal clinical trials due to cardiotoxic
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effects by inhibiting the hERG channel [22]. Thus, the application of the hiPSC-CM model in
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CD preclinical screenings may contribute for the demonstration of cardiotoxicity thereby
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increasing safety of clinical trials.
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Human iPSC-CM may also be used as an in vitro platform to determine the efficacy of
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any potential anti-T. cruzi drugs. Bz treatment of T. cruzi-infected hiPSC-CM lead to
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intracellular amastigotes reduction, thus validating hiPSC-CM as a suitable model for
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screening of novel bioactive compounds against T. cruzi. Damage of kinetoplast-mitochondria
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was an evident effect of Bz treatment. Swelling of mitochondria without alteration in the
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topology of kDNA has been previously reported [23], suggesting that incorporation of
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oxidized nucleotides, an important mechanism of action of Bz, occurs more slowly in kDNA.
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Nevertheless, the current high cost of hiPSC-CMs hampers their use in large-scale screens for
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new drugs. Their use may be more cost effective in secondary screening of promising
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candidate compounds and in cardiotoxicity tests prior to clinical trials. Human iPSC technology is an emerging tool for the area of infectious disease
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research, providing primary human target tissue to study parasite infections and their response
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to therapy in vitro [13,24,25]. Thus, further exploration of hiPSC-derived in vitro models will
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contribute substantially to the understanding of parasitic infections of medical importance.
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Acknowledgements
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The authors thank Renata Soares Dias de Souza for technical support and the Rudolf
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Barth Electron Microscopy and the Bioassay Platform at Institute Oswaldo Cruz. This work
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was supported by Fundação Oswaldo Cruz, Fundação de Amparo à Pesquisa do Estado do
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Rio de Janeiro, Fundação de Amparo à Pesquisa do Estado de São Paulo (CEPID
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2013/08135-2) and Conselho Nacional de Desenvolvimento Científico e Tecnológico.
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The authors declare no conflict of interest.
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Figure legends
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Fig. 1. Benznidazole cytotoxicity and Trypanocidal effect in hiPSC-CM. (A) Bz did not
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elicit any toxic effect in hiPSC-CM even at high concentrations (CC50 > 500 µM). (B-C) Bz
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Dm28cLuc clone, reaching a IC50 value of 1.1 µM and 3.65 µM, respectively.
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Fig. 2. Profile of T. cruzi infection of hiPSC-CM treated with benznidazole (Bz). (A) A
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concentration response effect of Bz treatment was evident. A significant reduction was
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observed in the percentage of T. cruzi (Y strain) infection. (B) Note the drastic reduction in
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the number of intracellular parasites after Bz treatment (*) p<0.05. (C-H) Light microscopy of
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hiPSC-CM infected by T. cruzi Y strain and treated with Bz. (C) Untreated cells presented
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high level of infection. (D-H) Treatment of T. cruzi-infected hiPSC-CM with Bz markedly
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reduced the level of infection. Few amastigotes (arrows) were visualized at (D) 33 µM, (E) 11
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µM and (F) 3.3 µM of Bz. Amastigote nests were mostly visualized at low concentrations of
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Bz, (G) 1.2 µM and (H) 0.43 µM.
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Fig. 3. Transmission electron micrographs of uninfected and T. cruzi-infected hiPSC-
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CM treated with Bz. (A) No cardiotoxic effect was observed on uninfected hiPSC-CM after
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treatment with Bz at IC50 value for the parasite. Human iPSC-CM shows organized myofibrils
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(Mf), abundance of mitochondria (Mt), glycogen granules (Gly) and endoplasmic reticulum
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(ER). (B) Untreated T. cruzi-infected hiPSC-CM showing well preserved intracellular
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parasites. (C-D) Treatment of hiPSC-CM infected by T. cruzi Y strain with (C) IC25 and (D)
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IC50 of Bz concentrations showing alterations in the intracellular parasites. Swelling of
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kinetoplast-mitochondria (asterisk) was noticed in both Bz concentrations but it was more
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pronounced after treatment with IC50. N-nucleus; J-cell junction; K-kinetoplast and F-
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flagellum.
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Supplemental Fig. 1. Characterization of of hiPSC-CM. Immunoflorescence of hiPSC-CM
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stained with (A) anti-α-actinin (green), and (B) anti-cardiac troponin T (TNNT2) (green), and
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showing high efficiency of differentiation (98% of cells positive for cardiac troponin-T).
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