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Title: Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits
The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell–derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes.  more » « less
Award ID(s):
1728497
NSF-PAR ID:
10119305
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Nature Genetics
ISSN:
1061-4036
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Abstract Aims

    Dissecting complex interactions among transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are central for understanding heart development and function. Although computational approaches and platforms have been described to infer relationships among regulatory factors and genes, current approaches do not adequately account for how highly diverse, interacting regulators that include noncoding RNAs (ncRNAs) control cardiac gene expression dynamics over time.

    Methods

    To overcome this limitation, we devised an integrated framework, cardiac gene regulatory modeling (CGRM) that integrates LogicTRN and regulatory component analysis bioinformatics modeling platforms to infer complex regulatory mechanisms. We then used CGRM to identify and compare the TF-ncRNA gene regulatory networks that govern early- and late-stage cardiomyocytes (CMs) generated by in vitro differentiation of human pluripotent stem cells (hPSC) and ventricular and atrial CMs isolated during in vivo human cardiac development.

    Results

    Comparisons of in vitro versus in vivo derived CMs revealed conserved regulatory networks among TFs and ncRNAs in early cells that significantly diverged in late staged cells. We report that cardiac genes (“heart targets”) expressed in early-stage hPSC-CMs are primarily regulated by MESP1, miR-1, miR-23, lncRNAs NEAT1 and MALAT1, while GATA6, HAND2, miR-200c, NEAT1 and MALAT1 are critical for late hPSC-CMs. The inferred TF-miRNA-lncRNA networks regulating heart development and contraction were similar among early-stage CMs, among individual hPSC-CM datasets and between in vitro and in vivo samples. However, genes related to apoptosis, cell cycle and proliferation, and transmembrane transport showed a high degree of divergence between in vitro and in vivo derived late-stage CMs. Overall, late-, but not early-stage CMs diverged greatly in the expression of “heart target” transcripts and their regulatory mechanisms.

    Conclusions

    In conclusion, we find that hPSC-CMs are regulated in a cell autonomous manner during early development that diverges significantly as a function of time when compared to in vivo derived CMs. These findings demonstrate the feasibility of using CGRM to reveal dynamic and complex transcriptional and posttranscriptional regulatory interactions that underlie cell directed versus environment-dependent CM development. These results with in vitro versus in vivo derived CMs thus establish this approach for detailed analyses of heart disease and for the analysis of cell regulatory systems in other biomedical fields.

     
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  2. Abstract

    Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

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Although the development of novel calcium reporters enables assays of CRISPR/Cas-9 genome-edited induced pluripotent stem cell–derived cardiomyocytes and primary adult cardiomyocytes, existing calcium-detection technologies are often proprietary and require specialist equipment, whereas open-source workflows necessitate considerable user expertise and manual input. Objective: We aimed to develop an easy to use open-source, adaptable, and automated analysis pipeline for measuring cellular calcium transients, from image stack to data output, inclusive of cellular identification, background subtraction, photobleaching correction, calcium transient averaging, calcium transient fitting, data collation, and aberrant behavior recognition. Methods and Results: We developed CalTrack, a MatLab-based algorithm, to monitor fluorescent calcium transients in living cardiomyocytes, including isolated single cells or those contained in 3-dimensional tissues or organoids, and to analyze data acquired using photomultiplier tubes or employing line scans. CalTrack uses masks to segment cells allowing multiple cardiomyocyte transients to be measured from a single field of view. After automatically correcting for photobleaching, CalTrack averages and fits a string of transients and provides parameters that measure time to peak, time of decay, tau, peak fluorescence/baseline fluorescence (F max /F 0 ), and others. We demonstrate the utility of CalTrack in primary and induced pluripotent stem cell–derived cell lines in response to pharmacological compounds and in phenotyping cells carrying hypertrophic cardiomyopathy variants. Conclusions: CalTrack, an open-source tool that runs on a local computer, provides automated high-throughput analysis of calcium transients in response to development, genetic or pharmacological manipulations, and pathological conditions. 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Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC −/− ), or haploinsufficiency ( FLNC +/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC +/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC −/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. 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However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current ( I k1 ). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking I k1 -like currents with BaCl 2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with I k1 ’s role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting. NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized. </div> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> </div><div class="clearfix"></div> </div> </li> </ol> <div class="push_top"></div> </div> </div> <div class="col-md-3"> <div id="citation-sidebar"> <ul class="nav nav-list" id="citation-fulltext-sidebar" style="font-size: 14px; font-family: Georgia Regular;"> <li style="font-weight: bold; margin-bottom: 5px; font-size:13px;">Free Publicly Accessible Full Text</li> <li> <span class="text-muted pull-right"><small id="citation-fulltext-sidebar-sizetext"></small></span><small><a class="misc" href="https://par.nsf.gov/servlets/purl/10119305" target="_blank">Accepted Manuscript1.0<img src="https://par.nsf.gov/img/ui/page_white_acrobat.png" alt="Accepted Manuscript" id="citation-fulltext-sidebar-size" class="ft_icon"/></a></small> </li> <li class="divider"></li> <li style="font-weight: bold;font-size:13px;">Journal Article:</li> <li style="word-break:break-all" class="small"> <a href="https://doi.org/10.1038/s41588-019-0499-3" target="_blank" rel="noopener noreferrer" title="Document DOI URL" class="external-link" data-ostiid="10119305" style="word-wrap: break-word;">https://doi.org/10.1038/s41588-019-0499-3  <span class="fas fa-external-link-alt"></span></a></li> </ul> <div class="hidden-print"> <ul class="nav nav-list clearfix" id="sidebar-feedback" style="margin-top: 20px; margin-bottom: 20px; clear: both;"> <li style="position: relative;"> <div class="feedback-container"> <div style="font-family: Georgia Regular; font-size: 14px; color: #313b52; padding:20px;"> Have feedback or suggestions for a way to improve these results?<br/> <span style="text-decoration: underline;"> <script type="text/javascript" defer>/* <![CDATA[ */ user = "feedback"; site = "research.gov"; subject = "?subject=Comments or Suggestions"; content = "<span class='far fa-envelope'></span><span class='span-link' style='padding-left:5px'>Let us know</span>"; id = ""; document.write('<a itemprop="'+ id +'" href="mailto:' + user + '@' + site + subject + '">' + (content != '' ? content : (user + '@' + site)) + '</a>'); /* ]]> */</script> <noscript></noscript>!</span> </div> </li> </ul> <ul class="nav nav-list" style="font-size: 14px; font-family: Arial Regular;"> <li class="nav-header header-format">Citation Formats</li> <li class="links-format"><a href="#cite-mla" data-toggle="modal">MLA</a> <div id="cite-mla" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-mla_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-mla_label">Cite: MLA Format</strong> </div> <div class="modal-body" >Benaglio, Paola, D’Antonio-Chronowska, Agnieszka, Ma, Wubin, Yang, Feng, Young Greenwald, William W., Donovan, Margaret K., DeBoever, Christopher, Li, He, Drees, Frauke, Singhal, Sanghamitra, Matsui, Hiroko, van Setten, Jessica, Sotoodehnia, Nona, Gaulton, Kyle J., Smith, Erin N., D’Antonio, Matteo, Rosenfeld, Michael G., and Frazer, Kelly A. <em>Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits</em>. Retrieved from https://par.nsf.gov/biblio/10119305. <em>Nature Genetics</em> . Web. doi:10.1038/s41588-019-0499-3. </div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="links-format"><a href="#cite-apa" data-toggle="modal">APA</a> <div id="cite-apa" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-apa_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-apa_label">Cite: APA Format</strong> </div> <div class="modal-body">Benaglio, Paola, D’Antonio-Chronowska, Agnieszka, Ma, Wubin, Yang, Feng, Young Greenwald, William W., Donovan, Margaret K., DeBoever, Christopher, Li, He, Drees, Frauke, Singhal, Sanghamitra, Matsui, Hiroko, van Setten, Jessica, Sotoodehnia, Nona, Gaulton, Kyle J., Smith, Erin N., D’Antonio, Matteo, Rosenfeld, Michael G., & Frazer, Kelly A. <em>Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits</em>. <em>Nature Genetics</em>, <em></em> (). Retrieved from https://par.nsf.gov/biblio/10119305. <a href="https://doi.org/10.1038/s41588-019-0499-3">https://doi.org/10.1038/s41588-019-0499-3</a> </div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="links-format"><a href="#cite-chi" data-toggle="modal">Chicago</a> <div id="cite-chi" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-chi_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-chi_label">Cite: Chicago Format</strong> </div> <div class="modal-body">Benaglio, Paola, D’Antonio-Chronowska, Agnieszka, Ma, Wubin, Yang, Feng, Young Greenwald, William W., Donovan, Margaret K., DeBoever, Christopher, Li, He, Drees, Frauke, Singhal, Sanghamitra, Matsui, Hiroko, van Setten, Jessica, Sotoodehnia, Nona, Gaulton, Kyle J., Smith, Erin N., D’Antonio, Matteo, Rosenfeld, Michael G., and Frazer, Kelly A. "Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits". <em>Nature Genetics</em> (). Country unknown/Code not available. <a href="https://doi.org/10.1038/s41588-019-0499-3">https://doi.org/10.1038/s41588-019-0499-3.</a> <a href="https://par.nsf.gov/biblio/10119305">https://par.nsf.gov/biblio/10119305</a>. </div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="links-format"><a href="#cite-bib" data-toggle="modal">BibTeX</a> <div id="cite-bib" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-bib_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-bib_label">Cite: BibTeX Format</strong> </div> <div class="modal-body"> @article{osti_10119305,<br/> place = {Country unknown/Code not available}, title = {Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits}, url = {https://par.nsf.gov/biblio/10119305}, DOI = {10.1038/s41588-019-0499-3}, abstractNote = {The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell–derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes.}, journal = {Nature Genetics}, author = {Benaglio, Paola and D’Antonio-Chronowska, Agnieszka and Ma, Wubin and Yang, Feng and Young Greenwald, William W. and Donovan, Margaret K. and DeBoever, Christopher and Li, He and Drees, Frauke and Singhal, Sanghamitra and Matsui, Hiroko and van Setten, Jessica and Sotoodehnia, Nona and Gaulton, Kyle J. and Smith, Erin N. and D’Antonio, Matteo and Rosenfeld, Michael G. and Frazer, Kelly A.}, }</div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="divider"></li> </ul> <ul class="nav nav-list" style="font-size: 14px; font-family: Arial Regular;"> <li class="nav-header header-format">Export Metadata</li> <li class="links-format"><a href="https://par.nsf.gov/endnote?osti_id=10119305">EndNote</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:excel/osti-id:10119305">Excel</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:csv/osti-id:10119305">CSV</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:xml/osti-id:10119305">XML</a></li> <li class="divider"></li> </ul> <ul class="nav nav-list" style="font-size: 14px; 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