Project description:ABSTRACT Background: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing mutant of the coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs. Viral proliferation on hiPSC-CMs was subsequently quantified using bioluminescence imaging. For drug screening, select antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate (PDTC), and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of some of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with the reported drug effects in previous studies. Finally, mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways within these hiPSC-CMs after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to confirm antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that could be used to screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion. For this experiment, human induced pluripotent stem cell derived cardiomyocytes were infected with coxsackievirus at multiplicity of infection (MOI) of 5 for 8 hours. Cells were treated with and without interferon beta 1 in order to determine if treatment activates antiviral response genes and/or viral clearance pathways. 4 total samples (2 for each condition) were analyzed

Project description:Cardiac involvement is an important determinant of mortality amongst sarcoidosis patients. While granulomatous inflammation is a hallmark finding in cardiac sarcoidosis, the precise immune cell populations that comprise the granuloma remain unresolved. Furthermore, it is unclear how the cellular and transcriptomic landscape of cardiac sarcoidosis differs from other inflammatory heart diseases. We leveraged spatial transcriptomics (GeoMx DSP) and single nucleus RNA sequencing (snRNAseq) to elucidate the cellular and transcriptional landscape of cardiac sarcoidosis. Using GeoMX DSP technology, we compared the transcriptomal profile of CD68+ rich immune cell infiltrates in human cardiac sarcoidosis, giant cell myocarditis, and lymphocytic myocarditis. We performed snRNAseq of human cardiac sarcoidosis to identify immune cell types and examined their transcriptomic landscape and regulation. Using multi-channel immunofluorescence staining, we validated immune cell populations identified by snRNAseq, determined their spatial relationship, and devised an immunostaining approach to distinguish cardiac sarcoidosis from other inflammatory heart diseases. Despite overlapping histological features, spatial transcriptomics identified transcriptional signatures and associated pathways that robustly differentiated cardiac sarcoidosis from giant cell myocarditis and lymphocytic myocarditis. snRNAseq revealed the presence of diverse populations of myeloid cells in cardiac sarcoidosis with distinct molecular features. We identified GPNMB as a novel marker of multinucleated giant cells and predicted that the MITF family of transcription factors regulated this cell type. We also detected additional macrophage populations in cardiac sarcoidosis including HLA-DR+ macrophages, SYTL3+ macrophages and CD163+ resident macrophages. HLA-DR+ macrophages were found immediately adjacent to GPMMB+ giant cells, a distinct feature compared with other inflammatory cardiac diseases. SYTL3+ macrophages were located scattered throughout the granuloma and CD163+ macrophages, CD1c+ dendritic cells, non-classical monocytes, and T-cells were located at the periphery and outside of the granuloma. Finally, we demonstrate mTOR pathway activation is associated with proliferation and is selectively found in HLA-DR+ and SYLT3+ macrophages. In this study, we identified diverse populations of immune cells with distinct molecular signatures that comprise the sarcoid granuloma. These findings provide new insights into the pathology of cardiac sarcoidosis and highlight opportunities to improve diagnostic testing.

Project description:ABSTRACT Background: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing mutant of the coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs. Viral proliferation on hiPSC-CMs was subsequently quantified using bioluminescence imaging. For drug screening, select antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate (PDTC), and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of some of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with the reported drug effects in previous studies. Finally, mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways within these hiPSC-CMs after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to confirm antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that could be used to screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion. For this experiment, human induced pluripotent stem cell derived cardiomyocytes were infected with coxsackievirus at multiplicity of infection (MOI) of 5 for 8 hours. Cells were treated with and without interferon beta 1 in order to determine if treatment activates antiviral response genes and/or viral clearance pathways.

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Dissecting the Cellular Landscape and Transcriptome Network in Viral Myocarditis by Single-Cell RNA Sequencing

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