DOCSLIB.ORG

The Role of Wnt/Β-Catenin Antagonists - Sclerostin and DKK1 - in Bone Homeostasis and Mechanotransduction

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Role of Wnt/Β-Catenin Antagonists - Sclerostin and DKK1 - in Bone Homeostasis and Mechanotransduction The role of Wnt/β-catenin antagonists - sclerostin and DKK1 - in bone homeostasis and mechanotransduction Alyson Ruth Morse B Biotech (Hons) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Supervisor: Prof David G Little Co-supervisors: A/Prof Aaron Schindeler Dr Michelle M McDonald 2017 The University of Sydney Sydney Medical School Statement of Originality This is to certify that to the best of my knowledge, the content of this thesis is my own work. This thesis has not been submitted for any degree or other purposes. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged. Alyson Morse ii Acknowledgments It has been a long, long journey with many people to thank. My husband, James: he is my number one supporter and inspiration. He has a drive and desire for achievement that I see unmatched. My little girl, Lyndsey: who has been the motivating force every day over the past two years to get this thesis finished! My parents and family: they have always believed in my abilities and maintained interest in my achievements - thank you for all that you have given for me. My supervisor, David: he has shown more confidence in me than I ever did in myself. I’m sure he is unaware of the full influence and ambition that he has instilled. My associate supervisors, Michelle and Aaron: each have provided invaluable guidance, experience and knowledge during different stages of my candidature - thank you! The additional experts that have helped me along the way: Prof Marjolein van der Meulen, Dr Wendy Gold, and Dr Ciara Murphy - you have been invaluable. iii Abstract Promising avenues for improving bone mass and fracture resistance include loading-based exercise and agents modulating Wnt/β-catenin signalling. This thesis examines the intersection between these. Murine models of tibial loading and unloading, genetic knockout (KO) models of Wnt antagonists sclerostin and dickkopf-1/DKK1 (encoded by Sost and Dkk1 genes), and neutralising antibodies for sclerostin (Scl-Ab) were applied. Sost KO mice underwent unloading and compressive loading of the tibiae. Sclerostin was vital for the bone response to unloading yet not for loading. Rather, loading-induced anabolism was synergistically augmented in Sost KO mice. It was hypothesised that other Wnt antagonists, such as DKK1, may up-regulate following long-term sclerostin deficiency. Similarly, Dkk1 KO mice exhibited a synergistically augmented anabolic bone response to loading compared to wild type. However, compensation by sclerostin was doubtful as the primary cause for the augmented response with similar sclerostin expression seen in non-loaded tibiae of Dkk1 KO and wild type mice, and similar down-regulation following loading. Dkk1 KO mice treated with Scl-Ab resulted in additive increases in bone volume above either individual DKK1 or sclerostin deficiency. Prominent and synergistic effects were within cancellous bone. Immunohistochemical staining did not support the hypothesis that sclerostin up- regulates to compensate for long-term DKK1 deficiency. Finally, Scl-Ab was co-administered to mice undergoing tibial compressive loading, with additional anabolic increases seen above either monotherapy. RNA sequencing provided insight into mechanisms involved in the augmented response to loading with Scl-Ab use. This thesis supports future clinical use of antibodies targeting Wnt antagonists, whereby load/resistance exercise or dual-agents may increase the efficacy of Scl-Ab or DKK1-Ab therapy. This may have a critical impact for treatment of osteoporosis and other conditions of bone-loss. iv Original Publications Over the course of this candidature, several studies have been published as articles within peer- reviewed scientific journals. Publications related to this thesis Original published articles related to the submission of this thesis: Morse A, McDonald MM, Kelly NH, Melville KM, Schindeler A, Kramer I, Kneissel M, van der Meulen MC, Little DG. Mechanical load increases in bone formation via a sclerostin-independent pathway. J Bone Miner Res, 2014;29(11):2456-67. Chapter 2 p68. Morse A, Cheng TL, Schindeler A, McDonald MM, Mohanty S, Kneissel M, Kramer I, Little DG. Dkk1 KO mice treated with sclerostin antibody have additional increases in bone volume. Calcif Tissue Int. 2018 May 29; [Epub ahead of print]. Chapter 4 p113. Morse A, Schindeler A, McDonald MM, Kneissel M, Kramer I, Little DG. Sclerostin antibody augments the anabolic bone formation response in a mouse model of mechanical tibial loading. J Bone Miner Res. 2017 Nov 1; [Epub ahead of print]. Chapter 5 p146. Publications not related to this thesis Original articles published not related to the submission of this thesis: Liu R, Birke O, Morse A, Peacock L, Mikulec K, Little DG, Schindeler A. Myogenic progenitors contribute to open but not closed fracture repair. BMC Musculoskelet Disord, 2011;22(12):288. v Yang N, Schindeler A, McDonald MM, Seto JT, Houweling PJ, Lek M, Hogarth M, Morse AR, Raftery JM, Balasuriya D, MacArthur DG, Berman Y, Quinlan KG, Eisman JA, Nguyen TV, Center JR, Prince RL, Wilson SG, Zhu K, Little DG, North KN (2011). α-Actinin-3 deficiency is associated with reduced bone mass in human and mouse. Bone. 2011;49(4):790-8. McDonald MM, Morse A, Peacock L, Mikulec K, Schindeler A, Little DG. Characterization of the bone phenotype and fracture repair in osteopetrotic incisors absent rats. J Orthop Res. 2011;29(5):726-33. McDonald MM, Morse A, Mikulec K, Peacock L, Yu N, Baldock PA, Birke O, Liu M, Ke HZ, Little DG. Inhibition of sclerostin by systemic treatment with sclerostin antibody enhances healing of proximal tibial defects in ovariectomized rats. J Orthop Res. 2012;30(10):1541-8. McDonald MM, Morse A, Mikulec K, Peacock L, Baldock PA, Kostenuik PJ, Little DG. Matrix metalloproteinase-driven endochondral fracture union proceeds independently of osteoclast activity. J Bone Miner Res. 2013;28(7):1550-60. Morse A, Yu NY, Peacock L, Mikulec K, Kramer I, Kneissel M, McDonald MM, Little DG. Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength. Bone. 2015;71:155- 63. Morse A, Cheng TL, Peacock L, Mikulec K, Little DG, Schindeler A. RAP-011 augments callus formation in closed fractures in rats. J Orthop Res. 2016;34(2):320-30 McDonald MM, Reagan MR, Youlten SE, Mohanty ST, Seckinger A, Terry RL, Pettitt JA, Simic MK, Cheng TL, Morse A, Le LMT, Abi-Hanna D, Kramer I, Falank C, Fairfield H, Ghobrial IM, Baldock PA, Little DG, Kneissel M, Vanderkerken K, Bassett JHD, Williams GR, Oyajobi BO, Hose D, Phan TG, Croucher PI. Inhibiting the osteocyte specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma. Blood. 2017;129(26):3452-3464 vi Morse A, McDonald MM, Schindeler A, Peacock L, Mikulec K, Cheng TL, Liu M, Ke HZ, Little DG. Sclerostin Antibody Increases Callus Size and Strength but does not Improve Fracture Union in a Challenged Open Rat Fracture Model. Calcif Tissue Int. 2017;101(2):217-228 McDonald MM, Morse A*, Birke O, Yu NYC, Mikulec K, Peacock L, Schindeler A, Liu M, Ke HZ, Little DG. Sclerostin antibody enhances bone formation in a rat model of distraction osteogenesis. J Orthop Res. 2017; [Epub ahead of print] *equal first author McDonald MM, Morse A, Schindeler A, Mikulec K, Peacock L, Liu M, Ke HZ, Little DG. Homozygous Dkk1 knockout mice exhibit high bone mass phenotype due to increased bone formation. Calcif Tissue Int. 2017; [Epub ahead of print] vii Authorship attribution statement The work presented in this thesis was conducted during my full time candidature to fulfil the requirements of Doctor of Philosophy through the Sydney Medical School, The University of Sydney. Unless otherwise stated, all experiments were conducted, and data collected and analysed, by myself at the Orthopaedic Research and Biotechnology Unit at The Children’s Hospital at Westmead. Animal ethics was approved by the South Western Area Health Service (SWAHS) Animal Ethics Committee and/or CMRI/CHW animal ethics. Experimental conception, and manuscript and thesis editing, was conducted in conjunction with my supervisors Prof David G Little, A/Prof Aaron Schindeler and Dr Michelle M McDonald. Chapter 2 of this thesis is published as: Morse A, McDonald MM, Kelly NH, Melville KM, Schindeler A, Kramer I, Kneissel M, van der Meulen MC, Little DG. Mechanical load increases in bone formation via a sclerostin- independent pathway. J Bone Miner Res. 2014;29(11):2456-67. KNH and KMM performed strain gauging experiments. IK, MK and MCVDM were involved with experimental design and the review of the manuscript and data. Guy Smith assisted with some DXA data collection. Chapter 3 of this thesis is drafted for submission for review with Bone. Morse A, Ko F, McDonald MM, Schindeler A, van der Meulen MC, Lee L, Little DG. A Dkk1 KO mouse shows an increased anabolic bone response following compressive tibial loading. FK assisted in strain gauging experiments. MCVDM was involved in experimental design and review of manuscript and data. LL, Kathy Mikulec-Langton and Matthew Summers assisted with some specimen collection/homogenisation and/or RNA extractions. Kathy Mikulec-Langton and Lauren Peacock assisted with some mechanical loading and DXA scans. viii Chapter 4 of this thesis is published as: Morse A, Cheng TL, Schindeler A, McDonald MM, Mohanty S, Kneissel M, Kramer I, Little DG. Dkk1 KO mice treated with sclerostin antibody have additional increases in bone volume. Calcif Tissue Int. 2018 May 29 [Epub ahead of print]. With my assistance, TLC performed and analysed mechanical testing experiments. SM performed immunohistochemistry. IK and MK were involved with experimental design and the review of the manuscript and data.
Load more

Recommended publications
  • FLNC Missense Variants in Familial Noncompaction Cardiomyopathy
    Cardiogenetics 2019; volume 9:8181 FLNC missense variants than 2 according to current echocardio- in familial noncompaction graphic criteria, or 2.3 on CMR.1,2 Correspondence: Jaap I. van Waning, Approximately 10% of patients diagnosed Department of Clinical Genetics, EE 2038, cardiomyopathy with NCCM have concurrent congenital Erasmus MC, POB 2040, 3000CA Rotterdam, heart defects (CHD).3,4 the Netherlands. Tel.: +3107038388 - Fax: +3107043072. Jaap I. van Waning,1 In 30-40% of cases diagnosed with E-mail: [email protected] Yvonne M. Hoedemaekers,2 NCCM a pathogenic variant can be identi- 2,3 4 Wouter P. te Rijdt, Arne I. Jpma, fied. Around 80% of these pathogenic vari- Acknowledgements: JVW was supported by a Daphne Heijsman,4 Kadir Caliskan,5 ants involve the same sarcomere genes, that grant from the Jaap Schouten Foundation. Elke S. Hoendermis,6 are the major causes for hypertrophic car- WPTR was supported by a Young Talent Program (CVON PREDICT) grant 2017T001 Tineke P. Willems,7 diomyopathy (HCM) and dilated cardiomy- - Dutch Heart Foundation. 8 opathy (DCM), in particular MYH7, Arthur van den Wijngaard, 5,6 3 MYBPC3 and TTN. Filamin C (FLNC) Albert Suurmeijer, Conflict of interest: the authors declare no plays a central role in muscle functioning Marjon A. van Slegtenhorst,1 potential conflict of interest. by maintaining the structural integrity of the Jan D.H. Jongbloed,2 muscle fibers. Pathogenic variants in FLNC Received for publication: 20 March 2019. Danielle F. Majoor-Krakauer,1 2 were found to be associated with a wide Revision received: 29 July 2019. Paul A.
    [Show full text]
  • The Cytoskeleton in Cell-Autonomous Immunity: Structural Determinants of Host Defence
    Mostowy & Shenoy, Nat Rev Immunol, doi:10.1038/nri3877 The cytoskeleton in cell-autonomous immunity: structural determinants of host defence Serge Mostowy and Avinash R. Shenoy Medical Research Council Centre of Molecular Bacteriology and Infection (CMBI), Imperial College London, Armstrong Road, London SW7 2AZ, UK. e‑mails: [email protected] ; [email protected] doi:10.1038/nri3877 Published online 21 August 2015 Abstract Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. 1 Mostowy & Shenoy, Nat Rev Immunol, doi:10.1038/nri3877 Cell-autonomous immunity, which is defined as the ability of a host cell to eliminate an invasive infectious agent, is a first line of defence against microbial pathogens 1 . It relies on antimicrobial proteins, specialized degradative compartments and programmed host cell death 1–3 . Cell- autonomous immunity is mediated by tiered innate immune signalling networks that sense microbial pathogens and stimulate downstream pathogen elimination programmes. Recent studies on host– microorganism interactions show that components of the host cell cytoskeleton are integral to the detection of bacterial pathogens as well as to the mobilization of antibacterial responses (FIG.
    [Show full text]
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals
    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
    [Show full text]
  • Genetic Mutations and Mechanisms in Dilated Cardiomyopathy
    Genetic mutations and mechanisms in dilated cardiomyopathy Elizabeth M. McNally, … , Jessica R. Golbus, Megan J. Puckelwartz J Clin Invest. 2013;123(1):19-26. https://doi.org/10.1172/JCI62862. Review Series Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of congestive heart failure. In hypertrophic cardiomyopathy (HCM), cardiac output is limited by the thickened myocardium through impaired filling and outflow. Mutations in the genes encoding the thick filament components myosin heavy chain and myosin binding protein C (MYH7 and MYBPC3) together explain 75% of inherited HCMs, leading to the observation that HCM is a disease of the sarcomere. Many mutations are “private” or rare variants, often unique to families. In contrast, dilated cardiomyopathy (DCM) is far more genetically heterogeneous, with mutations in genes encoding cytoskeletal, nucleoskeletal, mitochondrial, and calcium-handling proteins. DCM is characterized by enlarged ventricular dimensions and impaired systolic and diastolic function. Private mutations account for most DCMs, with few hotspots or recurring mutations. More than 50 single genes are linked to inherited DCM, including many genes that also link to HCM. Relatively few clinical clues guide the diagnosis of inherited DCM, but emerging evidence supports the use of genetic testing to identify those patients at risk for faster disease progression, congestive heart failure, and arrhythmia. Find the latest version: https://jci.me/62862/pdf Review series Genetic mutations and mechanisms in dilated cardiomyopathy Elizabeth M. McNally, Jessica R. Golbus, and Megan J. Puckelwartz Department of Human Genetics, University of Chicago, Chicago, Illinois, USA. Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of conges- tive heart failure.
    [Show full text]
  • Inhibition of Β-Catenin Signaling Respecifies Anterior-Like Endothelium Into Beating Human Cardiomyocytes Nathan J
    © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 3198-3209 doi:10.1242/dev.117010 RESEARCH ARTICLE STEM CELLS AND REGENERATION Inhibition of β-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes Nathan J. Palpant1,2,3,*, Lil Pabon1,2,3,*, Meredith Roberts2,3,4, Brandon Hadland5,6, Daniel Jones7, Christina Jones3,8,9, Randall T. Moon3,8,9, Walter L. Ruzzo7, Irwin Bernstein5,6, Ying Zheng2,3,4 and Charles E. Murry1,2,3,4,10,‡ ABSTRACT lineages. Anterior mesoderm (mid-streak) gives rise to cardiac and endocardial endothelium, whereas posterior mesoderm (posterior During vertebrate development, mesodermal fate choices are regulated streak) gives rise to the blood-forming endothelium and vasculature by interactions between morphogens such as activin/nodal, BMPs and (Murry and Keller, 2008). Wnt/β-catenin that define anterior-posterior patterning and specify Well-described anterior-posterior morphogen gradients, downstream derivatives including cardiomyocyte, endothelial and including those of activin A/nodal and BMP4, are thought to hematopoietic cells. We used human embryonic stem cells to explore pattern mesoderm subtypes (Nostro et al., 2008; Sumi et al., 2008; how these pathways control mesodermal fate choices in vitro. Varying Kattman et al., 2011). Such gradients are proposed to specify doses of activin A and BMP4 to mimic cytokine gradient polarization anterior mesodermal fates like cardiomyocytes versus posterior in the anterior-posterior axis of the embryo led to differential activity mesodermal fates like blood. Remarkably, a recent study showed of Wnt/β-catenin signaling and specified distinct anterior-like (high activin/ that ectopic induction of a nodal/BMP gradient in zebrafish embryos low BMP) and posterior-like (low activin/high BMP) mesodermal was sufficient to create an entirely new embryonic axis that could populations.
    [Show full text]
  • Cardiomyopathy
    UNIVERSITY OF MINNESOTA PHYSICIANS OUTREACH LABS Submit this form along with the appropriate MOLECULAR DIAGNOSTICS (612) 273-8445 Molecular requisition (Molecular Diagnostics or DATE: TIME COLLECTED: PCU/CLINIC: Molecular NGS Oncology). AM PM PATIENT IDENTIFICATION DIAGNOSIS (Dx) / DIAGNOSIS CODES (ICD-9) - OUTPATIENTS ONLY SPECIMEN TYPE: o Blood (1) (2) (3) (4) PLEASE COLLECT 5-10CC IN ACD-A OR EDTA TUBE ORDERING PHYSICIAN NAME AND PHONE NUMBER: Tests can be ordered as a full panel, or by individual gene(s). Please contact the genetic counselor with any questions at 612-624-8948 or by pager at 612-899-3291. _______________________________________________ Test Ordered- EPIC: Next generation sequencing(Next Gen) Sunquest: NGS Brugada syndrome DMD Aortopathy Full panel DNAJC19 SCN5A DSP Full panel GPD1L Cardiomyopathy, familial MYH11 CACNA1C hypertrophic ACTA2 SCN1B Full panel MYLK KCNE3 ANKRD1 FBN2 SCN3B JPH2 SLC2A10 HCN4 PRKAG2 COL5A2 MYH7 COL5A1 MYL2 COL3A1 Cardiomyopathy ACTC1 CBS Cardiomyopathy, dilated CSRP3 SMAD3 Full panel TNNC1 TGFBR1 LDB3 MYH6 TGFBR2 LMNA VCL FBN1 ACTN2 MYOZ2 Arrhythmogenic right ventricular DSG2 PLN dysplasia NEXN CALR3 TNNT2 TNNT2 NEXN Full panel RBM20 TPM1 TGFB3 SCN5A MYBPC3 DSG2 MYH6 TNNI3 DSC2 TNNI3 MYL3 JUP TTN TTN RYR2 BAG3 MYLK2 TMEM43 DES Cardiomyopathy, familial DSP CRYAB EYA4 restrictive PKP2 Full panel Atrial fibrillation LAMA4 MYPN TNNI3 SGCD MYPN TNNT2 Full panel CSRP3 SCN5A MYBPC3 GJA5 TCAP ABCC9 ABCC9 SCN1B PLN SCN2B ACTC1 KCNQ1 MYH7 KCNE2 TMPO NPPA VCL NPPA TPM1 KCNA5 TNNC1 KCNJ2 GATAD1 4/1/2014
    [Show full text]
  • Novel Pathogenic Variants in Filamin C Identified in Pediatric Restrictive Cardiomyopathy
    Novel pathogenic variants in filamin C identified in pediatric restrictive cardiomyopathy Jeffrey Schubert1, 2, Muhammad Tariq3, Gabrielle Geddes4, Steven Kindel4, Erin M. Miller5, and Stephanie M. Ware2. 1 Department of Molecular Genetics, Microbiology, and Biochemistry, University of Cincinnati College of Medicine, Cincinnati, OH; 2 Departments of Pediatrics and Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN; 3 Faculty of Applied Medical Science, University of Tabuk, Tabuk, Kingdom of Saudi Arabia; 4Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI; 5Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Correspondence: Stephanie M. Ware, MD, PhD Department of Pediatrics Indiana University School of Medicine 1044 W. Walnut Street Indianapolis, IN 46202 Telephone: 317 274-8939 Email: [email protected] Grant Sponsor: The project was supported by the Children’s Cardiomyopathy Foundation (S.M.W.), an American Heart Association Established Investigator Award 13EIA13460001 (S.M.W.) and an AHA Postdoctoral Fellowship Award 12POST10370002 (M.T.). ___________________________________________________________________ This is the author's manuscript of the article published in final edited form as: Schubert, J., Tariq, M., Geddes, G., Kindel, S., Miller, E. M., & Ware, S. M. (2018). Novel pathogenic variants in filamin C identified in pediatric restrictive cardiomyopathy. Human Mutation, 0(ja). https://doi.org/10.1002/humu.23661 Abstract Restrictive cardiomyopathy (RCM) is a rare and distinct form of cardiomyopathy characterized by normal ventricular chamber dimensions, normal myocardial wall thickness, and preserved systolic function. The abnormal myocardium, however, demonstrates impaired relaxation. To date, dominant variants causing RCM have been reported in a small number of sarcomeric or cytoskeletal genes, but the genetic causes in a majority of cases remain unexplained especially in early childhood.
    [Show full text]
  • Sarcomeres Regulate Murine Cardiomyocyte Maturation Through MRTF-SRF Signaling
    Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling Yuxuan Guoa,1,2,3, Yangpo Caoa,1, Blake D. Jardina,1, Isha Sethia,b, Qing Maa, Behzad Moghadaszadehc, Emily C. Troianoc, Neil Mazumdara, Michael A. Trembleya, Eric M. Smalld, Guo-Cheng Yuanb, Alan H. Beggsc, and William T. Pua,e,2 aDepartment of Cardiology, Boston Children’s Hospital, Boston, MA 02115; bDepartment of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02215; cDivision of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115; dAab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; and eHarvard Stem Cell Institute, Harvard University, Cambridge, MA 02138 Edited by Janet Rossant, The Gairdner Foundation, Toronto, ON, Canada, and approved November 24, 2020 (received for review May 6, 2020) The paucity of knowledge about cardiomyocyte maturation is a Mechanisms that orchestrate ultrastructural and transcrip- major bottleneck in cardiac regenerative medicine. In develop- tional changes in cardiomyocyte maturation are beginning to ment, cardiomyocyte maturation is characterized by orchestrated emerge. Serum response factor (SRF) is a transcription factor that structural, transcriptional, and functional specializations that occur is essential for cardiomyocyte maturation (3). SRF directly acti- mainly at the perinatal stage. Sarcomeres are the key cytoskeletal vates key genes regulating sarcomere assembly, electrophysiology, structures that regulate the ultrastructural maturation of other and mitochondrial metabolism. This transcriptional regulation organelles, but whether sarcomeres modulate the signal trans- subsequently drives the proper morphogenesis of mature ultra- duction pathways that are essential for cardiomyocyte maturation structural features of myofibrils, T-tubules, and mitochondria.
    [Show full text]
  • Transcriptomic Uniqueness and Commonality of the Ion Channels and Transporters in the Four Heart Chambers Sanda Iacobas1, Bogdan Amuzescu2 & Dumitru A
    www.nature.com/scientificreports OPEN Transcriptomic uniqueness and commonality of the ion channels and transporters in the four heart chambers Sanda Iacobas1, Bogdan Amuzescu2 & Dumitru A. Iacobas3,4* Myocardium transcriptomes of left and right atria and ventricles from four adult male C57Bl/6j mice were profled with Agilent microarrays to identify the diferences responsible for the distinct functional roles of the four heart chambers. Female mice were not investigated owing to their transcriptome dependence on the estrous cycle phase. Out of the quantifed 16,886 unigenes, 15.76% on the left side and 16.5% on the right side exhibited diferential expression between the atrium and the ventricle, while 5.8% of genes were diferently expressed between the two atria and only 1.2% between the two ventricles. The study revealed also chamber diferences in gene expression control and coordination. We analyzed ion channels and transporters, and genes within the cardiac muscle contraction, oxidative phosphorylation, glycolysis/gluconeogenesis, calcium and adrenergic signaling pathways. Interestingly, while expression of Ank2 oscillates in phase with all 27 quantifed binding partners in the left ventricle, the percentage of in-phase oscillating partners of Ank2 is 15% and 37% in the left and right atria and 74% in the right ventricle. The analysis indicated high interventricular synchrony of the ion channels expressions and the substantially lower synchrony between the two atria and between the atrium and the ventricle from the same side. Starting with crocodilians, the heart pumps the blood through the pulmonary circulation and the systemic cir- culation by the coordinated rhythmic contractions of its upper lef and right atria (LA, RA) and lower lef and right ventricles (LV, RV).
    [Show full text]
  • Genetic Cardiomyopathies: the Lesson Learned from Hipscs
    Journal of Clinical Medicine Review Genetic Cardiomyopathies: The Lesson Learned from hiPSCs Ilaria My 1,2 and Elisa Di Pasquale 2,3,* 1 Department of Biomedical Sciences, Humanitas University, Pieve Emanuele, 20090 Milan, Italy; [email protected] 2 Humanitas Clinical and Research Center—IRCCS, Rozzano, 20089 Milan, Italy 3 Institute of Genetic and Biomedical Research (IRGB)—UOS of Milan, National Research Council (CNR), 20138 Milan, Italy * Correspondence: [email protected] Abstract: Genetic cardiomyopathies represent a wide spectrum of inherited diseases and constitute an important cause of morbidity and mortality among young people, which can manifest with heart failure, arrhythmias, and/or sudden cardiac death. Multiple underlying genetic variants and molecular pathways have been discovered in recent years; however, assessing the pathogenicity of new variants often needs in-depth characterization in order to ascertain a causal role in the disease. The application of human induced pluripotent stem cells has greatly helped to advance our knowledge in this field and enabled to obtain numerous in vitro patient-specific cellular models useful to study the underlying molecular mechanisms and test new therapeutic strategies. A milestone in the research of genetically determined heart disease was the introduction of genomic technologies that provided unparalleled opportunities to explore the genetic architecture of cardiomyopathies, thanks to the generation of isogenic pairs. The aim of this review is to provide an overview of the main research that helped elucidate the pathophysiology of the most common genetic cardiomyopathies: hypertrophic, dilated, arrhythmogenic, and left ventricular noncompaction cardiomyopathies. A special focus is provided on the application of gene-editing techniques in understanding key disease characteristics and on the therapeutic approaches that have been tested.
    [Show full text]
  • In Vitro Generation of Functional Murine Heart Organoids Via FGF4
    ARTICLE https://doi.org/10.1038/s41467-020-18031-5 OPEN In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix ✉ Jiyoung Lee 1 , Akito Sutani1,2, Rin Kaneko1, Jun Takeuchi3, Tetsuo Sasano4, Takashi Kohda1,5, Kensuke Ihara3, ✉ Kentaro Takahashi3, Masahiro Yamazoe3, Tomohiro Morio2, Tetsushi Furukawa3 & Fumitoshi Ishino 1 Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a 1234567890():,; method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation pro- tocol, but also represents a promising research tool with a broad range of applications, including drug testing. 1 Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan. 2 Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan. 3 Department of Bio-Informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan.
    [Show full text]
  • Perturbation of the Titin/MURF1 Signaling Complex Is Associated With
    © 2019. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2019) 12, dmm041103. doi:10.1242/dmm.041103 RESEARCH ARTICLE Perturbation of the titin/MURF1 signaling complex is associated with hypertrophic cardiomyopathy in a fish model and in human patients Yuta Higashikuse1,2, Nishant Mittal1, Takuro Arimura3,*, Sung Han Yoon4, Mayumi Oda1, Hirokazu Enomoto1, Ruri Kaneda1, Fumiyuki Hattori1, Takeshi Suzuki2, Atsushi Kawakami5, Alexander Gasch6, Tetsushi Furukawa7, Siegfried Labeit6, Keiichi Fukuda1, Akinori Kimura3 and Shinji Makino1,8,‡ ABSTRACT INTRODUCTION Hypertrophic cardiomyopathy (HCM) is a hereditary disease Zebrafish is well established as a model vertebrate in gene function characterized by cardiac hypertrophy with diastolic dysfunction. studies (Seeley et al., 2007; Xu et al., 2002). The medaka fish, Gene mutations causing HCM have been found in about half of Oryzias latipes, recently emerged as another important such model HCM patients, while the genetic etiology and pathogenesis remain system because of its smaller genome size and larger genome unknown for many cases of HCM. To identify novel mechanisms diversity (Kasahara et al., 2007). In particular, medaka is an excellent underlying HCM pathogenesis, we generated a cardiovascular- model for studying the cardiovascular system because the embryos mutant medaka fish, non-spring heart (nsh), which showed are transparent and able to tolerate an absence of blood flow under diastolic dysfunction and hypertrophic myocardium. The nsh sufficient oxygen delivery by diffusion (Burggren and Pinder, 1991). homozygotes had fewer myofibrils, disrupted sarcomeres and It is therefore possible to attribute cardiac phenotype directly to expressed pathologically stiffer titin isoforms. In addition, the nsh particular genes by ruling out the secondary effect of hypoxia.
    [Show full text]