{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T12:36:39Z","timestamp":1787315799214,"version":"build-2736575974"},"posted":{"date-parts":[[2024,9,23]]},"group-title":"Developmental Biology","reference-count":52,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2024,9,23]],"date-time":"2024-09-23T00:00:00Z","timestamp":1727049600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,9,28]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as\n                  <jats:italic>SCN5A, HCN4 and ID2,<\/jats:italic>\n                  and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing\n                  <jats:italic>in silico<\/jats:italic>\n                  models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings\n                  <jats:italic>in vitro<\/jats:italic>\n                  , demonstrating that our new cell-specific\n                  <jats:italic>in vitro<\/jats:italic>\n                  model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.\n                <\/jats:p>","DOI":"10.1101\/2024.09.22.614353","type":"posted-content","created":{"date-parts":[[2024,9,23]],"date-time":"2024-09-23T11:45:12Z","timestamp":1727091912000},"source":"Crossref","is-referenced-by-count":2,"title":["Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes Towards a Purkinje Fiber Fate"],"prefix":"10.64898","author":[{"given":"David M.","family":"Gonzalez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafael","family":"Dariolli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Moyett","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bhavana","family":"Shewale","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacqueline","family":"Bliley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Clarke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avi","family":"Ma\u2019ayan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stacey","family":"Rentschler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3338-5456","authenticated-orcid":false,"given":"Adam","family":"Feinberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric","family":"Sobie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8467-3004","authenticated-orcid":false,"given":"Nicole C.","family":"Dubois","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2024100111100981000_2024.09.22.614353v2.1","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1139\/cjpp-2020-0234","article-title":"Different effects of amiodarone and dofetilide on the dispersion of repolarization between well-coupled ventricular and Purkinje fibers1","volume":"99","year":"2021","journal-title":"Can. J. Physiol. Pharmacol"},{"key":"2024100111100981000_2024.09.22.614353v2.2","doi-asserted-by":"publisher","DOI":"10.1097\/HCO.0000000000000134"},{"key":"2024100111100981000_2024.09.22.614353v2.3","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3519"},{"key":"2024100111100981000_2024.09.22.614353v2.4","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.113.303060"},{"key":"2024100111100981000_2024.09.22.614353v2.5","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2999"},{"key":"2024100111100981000_2024.09.22.614353v2.6","doi-asserted-by":"crossref","first-page":"1130","DOI":"10.1002\/dvdy.24441","article-title":"Probing early heart development to instruct stem cell differentiation strategies","volume":"245","year":"2016","journal-title":"Dev Dyn"},{"key":"2024100111100981000_2024.09.22.614353v2.7","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2003-03-0832"},{"key":"2024100111100981000_2024.09.22.614353v2.8","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3280"},{"key":"2024100111100981000_2024.09.22.614353v2.9","doi-asserted-by":"crossref","first-page":"4223","DOI":"10.1111\/febs.13773","article-title":"Notch signalling in ventricular chamber development and cardiomyopathy","volume":"283","year":"2016","journal-title":"FEBS J"},{"key":"2024100111100981000_2024.09.22.614353v2.10","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3161"},{"key":"2024100111100981000_2024.09.22.614353v2.11","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2005"},{"key":"2024100111100981000_2024.09.22.614353v2.12","first-page":"122","article-title":"Endocardial Regulation of Cardiac Development","volume":"9","year":"2022","journal-title":"J Cardiovasc Dev Dis"},{"key":"2024100111100981000_2024.09.22.614353v2.13","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1161\/CIRCRESAHA.118.314578","article-title":"Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution","volume":"125","year":"2019","journal-title":"Circ Res"},{"key":"2024100111100981000_2024.09.22.614353v2.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.devcel.2006.12.011"},{"key":"2024100111100981000_2024.09.22.614353v2.15","doi-asserted-by":"publisher","DOI":"10.1038\/nrcardio.2015.193"},{"key":"2024100111100981000_2024.09.22.614353v2.16","doi-asserted-by":"crossref","first-page":"H466","DOI":"10.1152\/ajpheart.2000.279.2.H466","article-title":"A comparison of transient outward currents in canine cardiac Purkinje cells and ventricular myocytes","volume":"279","year":"2000","journal-title":"American Journal of Physiology-Heart and Circulatory Physiology"},{"key":"2024100111100981000_2024.09.22.614353v2.17","doi-asserted-by":"publisher","DOI":"10.1242\/dev.119271"},{"key":"2024100111100981000_2024.09.22.614353v2.18","doi-asserted-by":"crossref","first-page":"13287","DOI":"10.1038\/srep13287","article-title":"Purkinje Cells as Sources of Arrhythmias in Long QT Syndrome Type 3","volume":"5","year":"2015","journal-title":"Sci Rep"},{"key":"2024100111100981000_2024.09.22.614353v2.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2010.12.008"},{"key":"2024100111100981000_2024.09.22.614353v2.20","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.191362598"},{"key":"2024100111100981000_2024.09.22.614353v2.21","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.116.309877"},{"key":"2024100111100981000_2024.09.22.614353v2.22","doi-asserted-by":"crossref","first-page":"e53255","DOI":"10.1371\/journal.pone.0053255","article-title":"Diclofenac Prolongs Repolarization in Ventricular Muscle with Impaired Repolarization Reserve","volume":"7","year":"2012","journal-title":"PloS one"},{"key":"2024100111100981000_2024.09.22.614353v2.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2017.07.003"},{"key":"2024100111100981000_2024.09.22.614353v2.24","doi-asserted-by":"crossref","first-page":"2382","DOI":"10.1038\/s41598-018-20829-9","article-title":"Genetic targeting of Purkinje fibres by Sema3a-CreERT2","volume":"8","year":"2018","journal-title":"Sci Rep"},{"key":"2024100111100981000_2024.09.22.614353v2.25","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1200250109"},{"key":"2024100111100981000_2024.09.22.614353v2.26","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1038\/nprot.2012.150","article-title":"Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt\/\u03b2-catenin signaling under fully defined conditions","volume":"8","year":"2013","journal-title":"Nature protocols"},{"key":"2024100111100981000_2024.09.22.614353v2.27","doi-asserted-by":"crossref","first-page":"1042","DOI":"10.1007\/s00246-018-1859-y","article-title":"Zinc and Zinc Transporters: Novel Regulators of Ventricular Myocardial Development","volume":"39","year":"2018","journal-title":"Pediatr Cardiol"},{"key":"2024100111100981000_2024.09.22.614353v2.28","doi-asserted-by":"publisher","DOI":"10.1172\/JCI96993"},{"key":"2024100111100981000_2024.09.22.614353v2.29","unstructured":"Lux\u00e1n, G. , D\u2019Amato, G. and de la Pompa, J. L. (2016). Intercellular Signaling in Cardiac Development and Disease: The NOTCH pathway. In Etiology and Morphogenesis of Congenital Heart Disease: From Gene Function and Cellular Interaction to Morphology (ed. Nakanishi, T .), Markwald, R. R .), Baldwin, H. S .), Keller, B. B .), Srivastava, D .), and Yamagishi, H .), p. Tokyo: Springer."},{"key":"2024100111100981000_2024.09.22.614353v2.30","doi-asserted-by":"crossref","first-page":"1102","DOI":"10.1002\/stem.1921","article-title":"Isolation and characterization of embryonic stem cell-derived cardiac Purkinje cells","volume":"33","year":"2015","journal-title":"Stem Cells"},{"key":"2024100111100981000_2024.09.22.614353v2.31","doi-asserted-by":"publisher","DOI":"10.1016\/S0070-2153(10)92011-5"},{"key":"2024100111100981000_2024.09.22.614353v2.32","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.stem.2020.10.003","article-title":"BMP10 Signaling Promotes the Development of Endocardial Cells from Human Pluripotent Stem Cell-Derived Cardiovascular Progenitors","volume":"28","year":"2021","journal-title":"Cell Stem Cell"},{"key":"2024100111100981000_2024.09.22.614353v2.33","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.110.218156"},{"key":"2024100111100981000_2024.09.22.614353v2.34","doi-asserted-by":"publisher","DOI":"10.1007\/s00246-018-1906-8"},{"key":"2024100111100981000_2024.09.22.614353v2.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2008.02.008"},{"key":"2024100111100981000_2024.09.22.614353v2.36","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1016\/j.bbrc.2012.10.078","article-title":"PARM-1 promotes cardiomyogenic differentiation through regulating the BMP\/Smad signaling pathway","volume":"428","year":"2012","journal-title":"Biochem Biophys Res Commun"},{"key":"2024100111100981000_2024.09.22.614353v2.37","doi-asserted-by":"crossref","first-page":"709","DOI":"10.3389\/fphys.2018.00709","article-title":"Automatic Optimization of an in Silico Model of Human iPSC Derived Cardiomyocytes Recapitulating Calcium Handling Abnormalities","volume":"9","year":"2018","journal-title":"Front Physiol"},{"key":"2024100111100981000_2024.09.22.614353v2.38","first-page":"7","article-title":"Development and Function of the Cardiac Conduction System in Health and Disease","volume":"4","year":"2017","journal-title":"J Cardiovasc Dev Dis"},{"key":"2024100111100981000_2024.09.22.614353v2.39","doi-asserted-by":"crossref","first-page":"105402","DOI":"10.1016\/j.isci.2022.105402","article-title":"Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells","volume":"25","year":"2022","journal-title":"iScience"},{"key":"2024100111100981000_2024.09.22.614353v2.40","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3745"},{"key":"2024100111100981000_2024.09.22.614353v2.41","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCULATIONAHA.112.103390"},{"key":"2024100111100981000_2024.09.22.614353v2.42","doi-asserted-by":"crossref","first-page":"3036","DOI":"10.1016\/j.stemcr.2021.10.005","article-title":"A library of induced pluripotent stem cells from clinically well-characterized, diverse healthy human individuals","volume":"16","year":"2021","journal-title":"Stem Cell Reports"},{"key":"2024100111100981000_2024.09.22.614353v2.43","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2019"},{"key":"2024100111100981000_2024.09.22.614353v2.44","doi-asserted-by":"publisher","DOI":"10.1016\/j.devcel.2007.11.018"},{"key":"2024100111100981000_2024.09.22.614353v2.45","doi-asserted-by":"publisher","DOI":"10.1016\/j.vascn.2007.04.005"},{"key":"2024100111100981000_2024.09.22.614353v2.46","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.48890"},{"key":"2024100111100981000_2024.09.22.614353v2.47","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.yjmcc.2020.04.001","article-title":"Human Purkinje in silico model enables mechanistic investigations into automaticity and pro-arrhythmic abnormalities","volume":"142","year":"2020","journal-title":"J Mol Cell Cardiol"},{"key":"2024100111100981000_2024.09.22.614353v2.48","doi-asserted-by":"crossref","first-page":"1089","DOI":"10.1016\/j.stemcr.2015.04.015","article-title":"Efficient Generation of Cardiac Purkinje Cells from ESCs by Activating cAMP Signaling","volume":"4","year":"2015","journal-title":"Stem Cell Reports"},{"key":"2024100111100981000_2024.09.22.614353v2.49","doi-asserted-by":"publisher","DOI":"10.1038\/s41569-018-0031-y"},{"key":"2024100111100981000_2024.09.22.614353v2.50","unstructured":"van Weerd, J. H. and Christoffels, V. M. (2016). Regulation of Vertebrate Conduction System Development. In Etiology and Morphogenesis of Congenital Heart Disease: From Gene Function and Cellular Interaction to Morphology (ed. Nakanishi, T .), Markwald, R. R .), Baldwin, H. S .), Keller, B. B .), Srivastava, D .), and Yamagishi, H .), p. Tokyo: Springer."},{"key":"2024100111100981000_2024.09.22.614353v2.51","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0147155"},{"key":"2024100111100981000_2024.09.22.614353v2.52","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2018.12.048"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2024.09.22.614353","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T07:06:29Z","timestamp":1768460789000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2024.09.22.614353"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,23]]},"references-count":52,"URL":"https:\/\/doi.org\/10.1101\/2024.09.22.614353","relation":{},"subject":[],"published":{"date-parts":[[2024,9,23]]},"subtype":"preprint"}}