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1. OCT4-mediated inflammation induces cell reprogramming at the origin of cardiac valve development and calcification

Author

Jonathan T. Butcher, David J. Milan, Emily J. Farrar, David S. Peal, Emilye Hiriart, Ablajan Mahmut, Michel Pucéat, Bernd Jagla, Gall, Valérie, Cornell University [New York], Marseille medical genetics - Centre de génétique médicale de Marseille (MMG), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Institut Pasteur [Paris] (IP), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics, Harvard Medical School [Boston] (HMS), and We are grateful to the Leducq Foundation for funding part of this research within the MITRAL network of excellence and for awarding us for cell imaging facility [M.P.: 'Equipement de Recherche et Plateformes Technologiques' (ERPT)]. E.H. was a fellow of the Ministere de La Recherche et des technologies. We thank the NIH for funding (NIH HL128745 and HL143247 to J.T.B.) and the National Science Foundation for the Graduate Research Fellowship Program.

Subjects
Somatic cell, [SDV]Life Sciences [q-bio], Cell, Inflammation, Diseases and Disorders, [SDV.GEN] Life Sciences [q-bio]/Genetics, Biology, Cell Plasticity, medicine, Progenitor cell, [SDV.GEN]Life Sciences [q-bio]/Genetics, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Multidisciplinary, SciAdv r-articles, Embryo, medicine.disease, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, embryonic structures, Biomedicine and Life Sciences, medicine.symptom, Reprogramming, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Calcification, Research Article, Developmental Biology
Abstract

Description, Cell reprogramming is at the origin of valve calcification., Cell plasticity plays a key role in embryos by maintaining the differentiation potential of progenitors. Whether postnatal somatic cells revert to an embryonic-like naïve state regaining plasticity and redifferentiate into a cell type leading to a disease remains intriguing. Using genetic lineage tracing and single-cell RNA sequencing, we reveal that Oct4 is induced by nuclear factor κB (NFκB) at embyronic day 9.5 in a subset of mouse endocardial cells originating from the anterior heart forming field at the onset of endocardial-to-mesenchymal transition. These cells acquired a chondro-osteogenic fate. OCT4 in adult valvular aortic cells leads to calcification of mouse and human valves. These calcifying cells originate from the Oct4 embryonic lineage. Genetic deletion of Pou5f1 (Pit-Oct-Unc, OCT4) in the endocardial cell lineage prevents aortic stenosis and calcification of ApoE−/− mouse valve. We established previously unidentified self-cell reprogramming NFκB- and OCT4-mediated inflammatory pathway triggering a dose-dependent mechanism of valve calcification.

Published
2021
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2. Mutations in DCHS1 Cause Mitral Valve Prolapse

Author

Yaopan Mao, Katelynn Toomer, Charles Simpson, David J. Milan, David S. Peal, Francesca N. Delling, Roger R. Markwald, Jorge Solis, Maelle Perrocheau, Ronen Durst, Lisa A. Freed, Amanda J. Johnson, Annemarieke DeVlaming, Christopher Jett, Xavier Estivill, Leticia Fernández-Friera, Adrian H. Chester, Michel Pucéat, Matthew R. Stone, Maire Leyne, Thierry Le Tourneau, Susan A. Slaugenhaupt, Christian Dina, Jean-Jacques Schott, Monica Salani, Kenneth D. Irvine, Katherine Williams, Kimberly Sauls, Andy Wessels, Yoshikazu Tsukasaki, Daniel Trujillano, Xingju Nie, Herve LeMarec, Florie A. Charles, Patrick Bruneval, Donald R. Menick, Robert A. Levine, Stephan Ossowski, Russell A. Norris, Michael E. Talkowski, Harinath Kasiganesan, Nabila Bouatia-Naji, Ann-Marie Broome, Stacey N. Lynch, Tahirali Motiwala, Harrison Brand, Christophe Tribouilloy, Colby Chiang, Albert Hagège, Xavier Jeunemaitre, Center for humana genetic research, Harvard Medical School [Boston] (HMS), Dipartimento di Biologia Cellulare e dello Sviluppo, Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Mécanismes physiologiques et conséquences des calcifications cardiovasculaires: rôle des remodelages cardiovasculaires et osseux, Université de Picardie Jules Verne (UPJV)-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de recherche de l'institut du thorax (ITX-lab), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Laser Processing Group, Center for Genomic Regulation (CRG-UPF), CIBER de Epidemiología y Salud Pública (CIBERESP), Laboratoire de Recherche en Imagerie : Méthodes d'imagerie des Échanges transcapillaires (LRI - EA4062), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Génétique Médicale et Génomique Fonctionnelle (GMGF), Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5), Service de cardiologie [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Università degli Studi di Roma 'La Sapienza' [Rome], Université Paris Descartes - Paris 5 (UPD5)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), and Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)

Subjects
Male, Pathology, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Messenger, 030204 cardiovascular system & hematology, medicine.disease_cause, Mice, 0302 clinical medicine, Cell Movement, Mitral valve, Missense mutation, Mitral valve prolapse, Atrioventricular canal defect, Pair 11, Zebrafish, Genetics, 0303 health sciences, Mutation, Multidisciplinary, Mitral Valve Prolapse, Protein Stability, Cadherins, 3. Good health, Pedigree, medicine.anatomical_structure, Phenotype, cardiovascular system, Mitral Valve, Female, Human, medicine.medical_specialty, Cadherin Related Proteins, Biology, Chromosomes, Article, 03 medical and health sciences, medicine, Animals, Humans, RNA, Messenger, 030304 developmental biology, Body Patterning, Mitral regurgitation, Mitral valve repair, DCHS1, Chromosomes, Human, Pair 11, Zebrafish Proteins, medicine.disease, RNA
Abstract

International audience; Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Published
2015
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3. miR-21 represses Pdcd4 during cardiac valvulogenesis

Author

Russell A. Norris, Heather J. Kolpa, David S. Peal, Roger R. Markwald, Andrea C. Giokas, Calum A. MacRae, Joyce Bischoff, David J. Milan, Patrick T. Ellinor, Shibnath Ghatak, Stacey N. Lynch, and Suniti Misra

Subjects
Time Factors, Endothelium, Biology, Mice, Downregulation and upregulation, Cell Movement, microRNA, medicine, Animals, Humans, Molecular Biology, Zebrafish, Crosses, Genetic, Research Articles, Regulation of gene expression, Gene knockdown, Atrioventricular valve, Endothelial Cells, Gene Expression Regulation, Developmental, RNA-Binding Proteins, Zebrafish Proteins, biology.organism_classification, Heart Valves, Molecular biology, Cell biology, Endothelial stem cell, MicroRNAs, medicine.anatomical_structure, Apoptosis Regulatory Proteins, Developmental Biology
Abstract

The discovery of small non-coding microRNAs has revealed novel mechanisms of post-translational regulation of gene expression, the implications of which are still incompletely understood. We focused on microRNA 21 (miR-21), which is expressed in cardiac valve endothelium during development, in order to better understand its mechanistic role in cardiac valve development. Using a combination of in vivo gene knockdown in zebrafish and in vitro assays in human cells, we show that miR-21 is necessary for proper development of the atrioventricular valve (AV). We identify pdcd4b as a relevant in vivo target of miR-21 and show that protection of pdcd4b from miR-21 binding results in failure of AV development. In vitro experiments using human pulmonic valve endothelial cells demonstrate that miR-21 overexpression augments endothelial cell migration. PDCD4 knockdown alone was sufficient to enhance endothelial cell migration. These results demonstrate that miR-21 plays a necessary role in cardiac valvulogenesis, in large part due to an obligatory downregulation of PDCD4.

Published
2013
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4. Patterning and Development of the Atrioventricular Canal in Zebrafish

Author

Stacey N. Lynch, David S. Peal, and David J. Milan

Subjects
animal structures, Body Patterning, Morphogenesis, Danio, Pharmaceutical Science, Model system, Early death, Article, Genetics, Animals, Atrioventricular cushions, Zebrafish, Genetics (clinical), biology, fungi, Gene Expression Regulation, Developmental, Anatomy, biology.organism_classification, Heart Valves, Molecular Medicine, Atrioventricular canal, Endocardial Cushions, sense organs, Cardiology and Cardiovascular Medicine, Neuroscience, Signal Transduction
Abstract

Proper atrioventricular canal (AVC) patterning and subsequent valvulogenesis is a complex process, and defects can result in disease or early death. The zebrafish Danio rerio has become a useful model system for studying AVC development, and much progress has been made in dissecting out the critical steps. Here, we review the recent advances in the field and highlight the cellular and molecular changes observed during zebrafish AVC development.

Published
2011
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5. Chondroitin sulfate expression is required for cardiac atrioventricular canal formation

Author

Calum A. MacRae, David S. Peal, C. Geoffrey Burns, and David J. Milan

Subjects
Cardiac Jelly, biology, Cell migration, biology.organism_classification, Cell biology, Extracellular matrix, chemistry.chemical_compound, chemistry, Immunology, cardiovascular system, Atrioventricular canal, Chondroitin, Chondroitin sulfate, Zebrafish, Endocardium, Developmental Biology
Abstract

Defects in cardiac valvulogenesis are a common cause of congenital heart disease, and the study of this process promises to provide mechanistic insights and lead to novel therapeutics. Normal valve development involves multiple signaling pathways, and recently roles have been identified for extracellular matrix components, including glycosaminoglycans. We, therefore, explored the role of the glycosaminoglycan chondroitin sulfate during zebrafish cardiac development. Beginning at 33 hr, there is a distinct zone of chondroitin sulfate expression in the atrioventricular (AV) boundary, in the cardiac jelly between the endocardium and myocardium. This expression is both spatially and temporally restricted, and is undetectable after 48 hr. Chemical as well as genetic inhibition of chondroitin synthesis results in AV canal (AVC) defects, including loss of the atrioventricular constriction, blood regurgitation, and failure of circulation. Lack of chondroitin disrupts a marker of cell migration, results in a loss of myocardial and endothelial markers of valvulogenesis, and misregulates bone morphogenetic protein expression, supporting an early role in AVC development. In summary, we have defined a requirement for chondroitin sulfate expression in the normal patterning of the AV boundary, suggesting that this component of the cardiac jelly provides a necessary signal in this critical transition in vertebrate cardiogenesis. Developmental Dynamics 238:3103–3110, 2009. © 2009 Wiley-Liss, Inc.

Published
2009
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6. Mitral valve disease−morphology and mechanisms

Author

Mark D. Handschumacher, David P. Milan, Patrick Bruneval, Magdi H. Yacoub, Harry C. Dietz, Hervé Le Marec, Robert A. Levine, Jean Mérot, Maelle Perrocheau, David S. Peal, Francesca N. Delling, Nadia Rosenthal, Vincent Probst, Albert Hagège, Jonathan Beaudoin, Joyce Bischoff, Russell A. Norris, Leticia Fernández-Friera, Jacob P. Dal-Bianco, Miguel Chaput, Catherine Clusel, Morten O. Jensen, Thierry Le Tourneau, Muralidhar Padala, Xavier Jeunemaitre, Emmanuel Messas, Susan A. Slaugenhaupt, Roger R. Markwald, Jorge Solis, Jae-Kwan Song, Christian Dina, Jonathan T. Butcher, Jean-Jacques Schott, Tui Neri, Alain Carpentier, Daniel P. Judge, Nabila Bouatia-Naji, Ronen Durst, Adrian H. Chester, Elena Aikawa, Michael Pucéat, Ehud Schwammenthal, Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Medicine, The Johns Hopkins University School of Medicine, Laboratoire de Recherche en Imagerie : Méthodes d'imagerie des Échanges transcapillaires (LRI - EA4062), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), UMR 1302 Structures et Marchés Agricoles, Ressources et Territoires, Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST-Structures et Marchés Agricoles, Ressources et Territoires (SMART), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Mouse Biology Unit, EMBL, Monterotondo (EMBL), EMBL Mouse Biology Unit, Laser Processing Group, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Structures et Marché Agricoles, Ressources et Territoires (SMART-LERECO), Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST, Unité de recherche de l'institut du thorax (ITX-lab), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Structures et Marchés Agricoles, Ressources et Territoires (SMART), Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-Université d'Évry-Val-d'Essonne (UEVE), Université Paris Descartes - Paris 5 (UPD5)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5), and Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)

Subjects
medicine.medical_specialty, Mitral regurgitation, business.industry, [SDV]Life Sciences [q-bio], Hypertrophic cardiomyopathy, Ventricular outflow tract obstruction, Mitral Valve Insufficiency, Degeneration (medical), Disease, medicine.disease, Article, 3. Good health, Pathogenesis, medicine.anatomical_structure, Internal medicine, Mitral valve, Heart failure, Cardiology, cardiovascular system, Medicine, cardiovascular diseases, medicine.symptom, Cardiology and Cardiovascular Medicine, business, humans
Abstract

International audience; Mitral valve disease is a frequent cause of heart failure and death. Emerging evidence indicates that the mitral valve is not a passive structure, but−even in adult life−remains dynamic and accessible for treatment. This concept motivates efforts to reduce the clinical progression of mitral valve disease through early detection and modification of underlying mechanisms. Discoveries of genetic mutations causing mitral valve elongation and prolapse have revealed that growth factor signalling and cell migration pathways are regulated by structural molecules in ways that can be modified to limit progression from developmental defects to valve degeneration with clinical complications. Mitral valve enlargement can determine left ventricular outflow tract obstruction in hypertrophic cardiomyopathy, and might be stimulated by potentially modifiable biological valvular-ventricular interactions. Mitral valve plasticity also allows adaptive growth in response to ventricular remodelling. However, adverse cellular and mechanobiological processes create relative leaflet deficiency in the ischaemic setting, leading to mitral regurgitation with increased heart failure and mortality. Our approach, which bridges clinicians and basic scientists, enables the correlation of observed disease with cellular and molecular mechanisms, leading to the discovery of new opportunities for improving the natural history of mitral valve disease.

Published
2015
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7. Identification of heart rate-associated loci and genes

Author

R. J. F. Loos, Ruth J. F. Loos, Ody C. M. Sibon, Toby Johnson, David S. Peal, Harm-Jan Westra, Marcel den Hoed, Robert E. Handsaker, Alicia Lundby, Tõnu Esko, Peter M. Visscher, Cristen J. Willer, Lude Franke, Ayellet V. Segrè, David J. Milan, Mark Eijgelsheim, Nilesh J. Samani, Harold Snieder, and Jian Yang

Subjects
Genetics, Heart rate, Expression quantitative trait loci, General Earth and Planetary Sciences, Identification (biology), Biology, Gene, General Environmental Science
Published
2013
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8. Novel Chemical Suppressors of Long QT Syndrome Identified by an in vivo Functional Screen

Author

Janet M. Mosley, David S. Peal, Robert W. Mills, Evi Lim, David J. Milan, Craig T. January, Randall T. Peterson, Stacey N. Lynch, and Patrick T. Ellinor

Subjects
medicine.medical_specialty, ERG1 Potassium Channel, Heart block, Long QT syndrome, Action Potentials, QT interval, Article, Afterdepolarization, Ventricular action potential, Animals, Genetically Modified, Physiology (medical), Internal medicine, Chlorocebus aethiops, medicine, Animals, Humans, Zebrafish, biology, Zebrafish Proteins, biology.organism_classification, medicine.disease, Phenotype, Ether-A-Go-Go Potassium Channels, Cell biology, High-Throughput Screening Assays, Long QT Syndrome, Endocrinology, HEK293 Cells, Gene Knockdown Techniques, COS Cells, Flurandrenolone, Mutation, Cardiology and Cardiovascular Medicine, Atrioventricular block
Abstract

Background— Genetic long QT (LQT) syndrome is a life-threatening disorder caused by mutations that result in prolongation of cardiac repolarization. Recent work has demonstrated that a zebrafish model of LQT syndrome faithfully recapitulates several features of human disease, including prolongation of ventricular action potential duration, spontaneous early afterdepolarizations, and 2:1 atrioventricular block in early stages of development. Because of their transparency, small size, and absorption of small molecules from their environment, zebrafish are amenable to high-throughput chemical screens. We describe a small-molecule screen using the zebrafish KCNH2 mutant breakdance to identify compounds that can rescue the LQT type 2 phenotype. Methods and Results— Zebrafish breakdance embryos were exposed to test compounds at 48 hours of development and scored for rescue of 2:1 atrioventricular block at 72 hours in a 96-well format. Only compounds that suppressed the LQT phenotype in 3 of 3 fish were considered hits. Screen compounds were obtained from commercially available small-molecule libraries (Prestwick and Chembridge). Initial hits were confirmed with dose-response testing and time-course studies. Optical mapping with the voltage-sensitive dye di-4 ANEPPS was performed to measure compound effects on cardiac action potential durations. Screening of 1200 small molecules resulted in the identification of flurandrenolide and 2-methoxy-N-(4-methylphenyl) benzamide (2-MMB) as compounds that reproducibly suppressed the LQT phenotype. Optical mapping confirmed that treatment with each compound caused shortening of ventricular action potential durations. Structure activity studies and steroid receptor knockdown suggest that flurandrenolide functions via the glucocorticoid signaling pathway. Conclusions— Using a zebrafish model of LQT type 2 syndrome in a high-throughput chemical screen, we have identified 2 compounds, flurandrenolide and the novel compound 2-MMB, as small molecules that rescue the zebrafish LQT type 2 syndrome by shortening the ventricular action potential duration. We provide evidence that flurandrenolide functions via the glucocorticoid receptor–mediated pathway. These 2 molecules and future discoveries from this screen should yield novel tools for the study of cardiac electrophysiology and may lead to novel therapeutics for human LQT patients.

Published
2010

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