DOCSLIB.ORG

Modulation of Cardiac Kv Currents by Kvbeta2 and Pyridine Nucleotides. Peter Joseph Kilfoil University of Louisville

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Modulation of Cardiac Kv Currents by Kvbeta2 and Pyridine Nucleotides. Peter Joseph Kilfoil University of Louisville University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2016 Modulation of cardiac Kv currents by Kvbeta2 and pyridine nucleotides. Peter Joseph Kilfoil University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Cardiology Commons Recommended Citation Kilfoil, Peter Joseph, "Modulation of cardiac Kv currents by Kvbeta2 and pyridine nucleotides." (2016). Electronic Theses and Dissertations. Paper 2400. https://doi.org/10.18297/etd/2400 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The nivU ersity of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The nivU ersity of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. MODULATION OF CARDIAC Kv CURRENTS BY Kvβ2 AND PYRIDINE NUCLEOTIDES By Peter Joseph Kilfoil B.E., Vanderbilt University, 2001 M.S., University of Kentucky, 2007 A Dissertation Submitted to the Faculty of the University of Louisville School of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Biochemistry and Molecular Biology Department of Biochemistry and Molecular Genetics University of Louisville Louisville, KY May 2016 Copyright 2016 by Peter J Kilfoil All rights reserved MODULATION OF CARDIAC Kv CURRENTS BY Kvβ2 AND PYRIDINE NUCLEOTIDES By Peter Joseph Kilfoil B.E., Vanderbilt University, 2001 M.S., University of Kentucky, 2007 A Dissertation Approved on April 6, 2016 by the following Dissertation Committee ________________________________________________________ Dissertation Director Dr. Aruni Bhatnagar ________________________________________________________ Dr. Ronald Gregg ________________________________________________________ Dr. Daniel Conklin ________________________________________________________ Dr. Barbara Clark ________________________________________________________ Dr. Alan Cheng ii DEDICATION This dissertation is dedicated to my beloved wife Carrie and son Rowan. iii ACKNOWLEDGMENTS I would like to thank my dissertation director, Dr. Aruni Bhatnagar, for his continued support, guidance and mentorship. I thank Dr. Srinivas Tipparaju, who taught me the patch-clamp technique and remains a scientific collaborator, contributing to this work by providing his expertise in the Langendorff perfused heart model along with his post-doc Kalyan Chapalamadugu. I thank Dr. Oleg Barski, Dr. Ganapathy Jagatheesan, and Dr. Matthew Nystoriak for engaging and thoughtful scientific conversations concerning this project and others. I thank Drs. Alan Brooks and Rachel Keith for assisting me in learning the complicated procedure for the isolation of viable mouse cardiac myocytes. I thank pediatric cardiologist Dr. Frank Raucci for expert advice in cardiac electrophysiology and his help with Western blotting techniques. I thank my committee members, Drs. Daniel Conklin, Ronald Gregg, Barbara Clark and Alan Cheng for their guidance and input on this project as well as their professorship in my classes in the Department of Biochemistry and Molecular Genetics. Lastly I would like to thank all the members of the University of Louisville Diabetes and Obesity Center for either assisting in my graduate training or being my friends for the last 5 years. iv ABSTRACT MODULATION OF CARDIAC Kv CURRENTS BY Kvβ2 AND PYRIDINE NUCLEOTIDES Peter Joseph Kilfoil April 6, 2016 Myocardial voltage-gated potassium (Kv) channels regulate the resting membrane potential and the repolarization phase of the action potential. Members of the Kv1 and Kv4 family associate with ancillary subunits, such as the Kvβ proteins, that modify channel kinetics, gating and trafficking. Previous investigation into the function of cardiac β subunits demonstrated that Kvβ1 regulates Ito and IK,slow currents in the heart, but the role of Kvβ2 in the myocardium remains unknown. In heterologous expression systems, Kvβ2 increases surface expression of Kv1 channels, shifts the activation potential of Kv1 channels to more polarized voltages, and increases the inactivation of Kv1 channels. Accordingly, the electrophysiological phenotype in Kvβ2-/- mice was examined to uncover its role. To investigate the effects of the loss of Kvβ2 on cardiac repolarization, we performed whole-cell electrophysiology on primary cardiac myocytes. We found Kv current density was reduced and action potential duration prolonged in v myocytes lacking Kvβ2. To isolate the molecular interactions by which Kvβ2 was affecting Kv currents, we show that Kvβ2 co-immunoprecipitates with Kv1.4 and Kv1.5 in heart lysates. To measure if surface expression of these Kv channels was reduced with the loss of Kvβ2, we performed immunofluorescent confocal microscopy of isolated cardiac myocytes. We found that the surface expression of Kv1.5 was reduced in Kvβ2-/- myocytes. We also performed a membrane fractionation technique to demonstrate that the proportion of total cellular Kv1.5 at the membrane was reduced in Kvβ2-/-. Together, these findings support our hypothesis that Kvβ2 plays a role in the generation of functional Kv currents in the myocardium by interacting with members of the Kv family. The pyridine nucleotides, NAD[P](H), are ubiquitous cofactors utilized as electron donors and acceptors by over 250 cellular oxidoreductases. Work out of our laboratory has shown that the Kvβ proteins are functional enzymes of the aldo- keto reductase family, that utilize NAD[P]H to catalyze the reduction of substrates. Furthermore, follow up work has shown that the redox status of bound pyridine nucleotide (PN) modifies the gating of Kvα-Kvβ channel complexes in heterologous expression systems. To examine a physiological role for PN in cardiac repolarization, whole-cell and single channel cardiac myocyte currents were recorded under the exposure to various PN redox states. We found that the inactivation rates and open probabilities of Kv currents in isolated myocytes are sensitive to the redox status of PN, and that surface action potentials in an isolated heart model are prolonged by treatment with factors that increase intracellular NADH concentration. vi TABLE OF CONTENTS PAGE DEDICATION..………………………………………………………………….…........iii ACKNOWLEDGEMENTS…………………………………………………….…..……iv ABSTRACT…………………………………………………………………..…………..v LIST OF FIGURES………………………………………………………………..…….x LIST OF TABLES…………………………………………………………………..…..xii LIST OF SCHEMES………………………………………………...………….….….xiii CHAPTER I. INTRODUCTION a. Background………………………………………..……………..….1 b. The Action Potential……………………………………….…......…5 c. Voltage-gated K+ channels……………………………………....…7 d. Kv channel structure and function…………………….…….....….9 e. Kv channel ancillary subunits…………………………….…….…14 i. KChIPs………………………………………………….......15 ii. DPPLs………………………………………………………16 iii. KCNEs……………………………………………………...16 f. The Kvβ subunits vii i. Structure of the Kvβ proteins…………………......………17 ii. Phosphorylation………………………………………....…20 iii. The Kvβ proteins are functional enzymes…………...….21 g. Kv1 currents in the myocardium and the action potential……..24 h. The molecular identities of mouse cardiac Kv currents i. The transient outwad current Ito………………….....……26 ii. The outwardly rectifying currents IK,slow1 and IK,slow2……29 II. Detailed Methods and Materials a. Immunoprecipitation and Western blot analysis………........….37 b. Electrophysiological recordings…………………………..………39 c. Analysis of electrophysiological recordings……………………..40 d. Mice…………………………………………………………………41 e. Isolation of adult mouse cardiac myocytes………………......…41 f. Cell immunofluorescence……………………………………....…44 g. Echocardiography……………………………………………....…45 h. Cardiac histology…………………………………………...…...…46 i. Quantitative RT-PCR………………………………………...……47 j. Statistics…………………………………………………….…...…48 III. Kvβ2 MODULATES CARDIAC Kv CURRENTS AND REPOLARIZATION a. Introduction……………………………………………….…...…...49 b. Results…………………………………………………….……..…50 c. Discussion……………………………………………………...…..55 viii IV. REGULATION OF CARDIAC Kv CURRENTS AND REPOLARIZATION BY PYRIDINE NUCLEOTIDES a. Introduction…………………………………………………...…….74 b. Results…………………………………………………………...…76 c. Discussion……………………………………………………...…..79 REFERENCES………………………………………………………………………...91 APPENDIX.......…………………………………………………..………………......115 CURRICULUM VITAE…………………………………………………………….…142 ix LIST OF FIGURES FIGURE PAGE 1.1 Overall structure of the Kv1.2 tetramer………………………………….......…31 1.2 Models of N-type and C-type inactivation of K+ channels…………….......….32 1.3 C-type and N-type inactivating currents……………………………….....….…33 1.4 Kvβ1 imparts rapid N-type inactivation………………………………….......…34 1.5 Model of physiological regulation of voltage-gated potassium (Kv) channel by pyridine nucleotides……………………………………………………………....35 1.6 Action potentials and underlying ionic currents………………………........….36 3.1 Expression of Kvα and Kvβ proteins in the mouse ventricle…………........…59 3.2 Quantification of Western blots………………………………………….....……60 3.3 Detection of Kvβ2 by immunofluorescence…………….…………..….....……61 3.4 Immunoprecipitation of Kv1.4 and Kv1.5 by Kvβ2………………….........……62 3.5 The ratio of Kv1.5 in the sarcolemma to cytosol is reduced in Kvβ2-/- hearts………………………………………..……………………………………...…..63 3.6 Cellular localization of Kv1.5……………………………….…………….......…64 3.7 Isolated ventricular cardiac myocyte recorded
Load more

Recommended publications
  • The Role of Transient Receptor Potential Cation Channels in Ca2þ Signaling
    Downloaded from http://cshperspectives.cshlp.org/ on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press The Role of Transient Receptor Potential Cation Channels in Ca2þ Signaling Maarten Gees, Barbara Colsoul, and Bernd Nilius KU Leuven, Department of Molecular Cell Biology, Laboratory Ion Channel Research, Campus Gasthuisberg, Herestraat 49, bus 802, Leuven, Belgium Correspondence: [email protected] The 28 mammalian members of the super-family of transient receptor potential (TRP) channels are cation channels, mostly permeable to both monovalent and divalent cations, and can be subdivided into six main subfamilies: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and the TRPA (ankyrin) groups. TRP channels are widely expressed in a large number of different tissues and cell types, and their biological roles appear to be equally diverse. In general, considered as poly- modal cell sensors, they play a much more diverse role than anticipated. Functionally, TRP channels, when activated, cause cell depolarization, which may trigger a plethora of voltage-dependent ion channels. Upon stimulation, Ca2þ permeable TRP channels 2þ 2þ 2þ generate changes in the intracellular Ca concentration, [Ca ]i,byCa entry via the plasma membrane. However, more and more evidence is arising that TRP channels are also located in intracellular organelles and serve as intracellular Ca2þ release channels. This review focuses on three major tasks of TRP channels: (1) the function of TRP channels as Ca2þ entry channels; (2) the electrogenic actions of TRPs; and (3) TRPs as Ca2þ release channels in intracellular organelles. ransient receptor potential (TRP) channels choanoflagellates, yeast, and fungi are primary Tconstitute a large and functionally versatile chemo-, thermo-, or mechanosensors (Cai 2008; family of cation-conducting channel proteins, Wheeler and Brownlee 2008; Chang et al.
    [Show full text]
  • The Two-Pore Channel TPCN2 Mediates NAADP-Dependent Ca2+-Release from Lysosomal Stores
    Pflugers Arch - Eur J Physiol (2009) 458:891–899 DOI 10.1007/s00424-009-0690-y ION CHANNELS, RECEPTORS AND TRANSPORTERS The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores Xiangang Zong & Michael Schieder & Hartmut Cuny & Stefanie Fenske & Christian Gruner & Katrin Rötzer & Oliver Griesbeck & Hartmann Harz & Martin Biel & Christian Wahl-Schott Received: 30 May 2009 /Accepted: 2 June 2009 /Published online: 26 June 2009 # The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Second messenger-induced Ca2+-release from show that TPCN2, a novel member of the two-pore cation intracellular stores plays a key role in a multitude channel family, displays the basic properties of native of physiological processes. In addition to 1,4,5-inositol NAADP-dependent Ca2+-release channels. TPCN2 tran- 2+ trisphosphate (IP3), Ca , and cyclic ADP ribose (cADPR) scripts are widely expressed in the body and encode a that trigger Ca2+-release from the endoplasmatic reticulum lysosomal protein forming homomers. TPCN2 mediates (ER), nicotinic acid adenine dinucleotide phosphate intracellular Ca2+-release after activation with low- (NAADP) has been identified as a cellular metabolite that nanomolar concentrations of NAADP while it is desensitized mediates Ca2+-release from lysosomal stores. While by micromolar concentrations of this second messenger and NAADP-induced Ca2+-release has been found in many is insensitive to the NAADP analog nicotinamide adenine tissues and cell types, the molecular identity of the channel dinucleotide phosphate (NADP). Furthermore, TPCN2- (s) conferring this release remained elusive so far. Here, we mediated Ca2+-release is almost completely abolished when the capacity of lysosomes for storing Ca2+ is pharmacolog- 2+ Xiangang Zong and Michael Schieder contributed equally to this work.
    [Show full text]
  • Expression Profiling of Ion Channel Genes Predicts Clinical Outcome in Breast Cancer
    UCSF UC San Francisco Previously Published Works Title Expression profiling of ion channel genes predicts clinical outcome in breast cancer Permalink https://escholarship.org/uc/item/1zq9j4nw Journal Molecular Cancer, 12(1) ISSN 1476-4598 Authors Ko, Jae-Hong Ko, Eun A Gu, Wanjun et al. Publication Date 2013-09-22 DOI http://dx.doi.org/10.1186/1476-4598-12-106 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Ko et al. Molecular Cancer 2013, 12:106 http://www.molecular-cancer.com/content/12/1/106 RESEARCH Open Access Expression profiling of ion channel genes predicts clinical outcome in breast cancer Jae-Hong Ko1, Eun A Ko2, Wanjun Gu3, Inja Lim1, Hyoweon Bang1* and Tong Zhou4,5* Abstract Background: Ion channels play a critical role in a wide variety of biological processes, including the development of human cancer. However, the overall impact of ion channels on tumorigenicity in breast cancer remains controversial. Methods: We conduct microarray meta-analysis on 280 ion channel genes. We identify candidate ion channels that are implicated in breast cancer based on gene expression profiling. We test the relationship between the expression of ion channel genes and p53 mutation status, ER status, and histological tumor grade in the discovery cohort. A molecular signature consisting of ion channel genes (IC30) is identified by Spearman’s rank correlation test conducted between tumor grade and gene expression. A risk scoring system is developed based on IC30. We test the prognostic power of IC30 in the discovery and seven validation cohorts by both Cox proportional hazard regression and log-rank test.
    [Show full text]
  • A Two-Pore Channel Protein Required for Regulating Mtorc1 Activity On
    Chang et al. BMC Biology (2020) 18:8 https://doi.org/10.1186/s12915-019-0735-4 RESEARCH ARTICLE Open Access A two-pore channel protein required for regulating mTORC1 activity on starvation Fu-Sheng Chang1, Yuntao Wang1, Phillip Dmitriev1, Julian Gross2, Antony Galione2 and Catherine Pears1* Abstract Background: Two-pore channels (TPCs) release Ca2+ from acidic intracellular stores and are implicated in a number of diseases, but their role in development is unclear. The social amoeba Dictyostelium discoideum proliferates as single cells that aggregate to form a multicellular organism on starvation. Starvation is sensed by the mTORC1 complex which, like TPC proteins, is found on acidic vesicles. Here, we address the role of TPCs in development and under starvation. Results: We report that disruption of the gene encoding the single Dictyostelium TPC protein, TPC2, leads to a delay in early development and prolonged growth in culture with delayed expression of early developmental genes, although a rapid starvation-induced increase in autophagy is still apparent. Ca2+ signals induced by extracellular cAMP are delayed in developing tpc2− cells, and aggregation shows increased sensitivity to weak bases, consistent with reduced acidity of the vesicles. In mammalian cells, the mTORC1 protein kinase has been proposed to suppress TPC channel opening. Here, we show a reciprocal effect as tpc2− cells show an increased level of phosphorylation of an mTORC1 substrate, 4E-BP1. mTORC1 inhibition reverses the prolonged growth and increases the efficiency of aggregation of tpc2− cells. Conclusion: TPC2 is required for efficient growth development transition in Dictyostelium and acts through modulation of mTORC1 activity revealing a novel mode of regulation.
    [Show full text]
  • Methylome and Transcriptome Maps of Human Visceral and Subcutaneous
    www.nature.com/scientificreports OPEN Methylome and transcriptome maps of human visceral and subcutaneous adipocytes reveal Received: 9 April 2019 Accepted: 11 June 2019 key epigenetic diferences at Published: xx xx xxxx developmental genes Stephen T. Bradford1,2,3, Shalima S. Nair1,3, Aaron L. Statham1, Susan J. van Dijk2, Timothy J. Peters 1,3,4, Firoz Anwar 2, Hugh J. French 1, Julius Z. H. von Martels1, Brodie Sutclife2, Madhavi P. Maddugoda1, Michelle Peranec1, Hilal Varinli1,2,5, Rosanna Arnoldy1, Michael Buckley1,4, Jason P. Ross2, Elena Zotenko1,3, Jenny Z. Song1, Clare Stirzaker1,3, Denis C. Bauer2, Wenjia Qu1, Michael M. Swarbrick6, Helen L. Lutgers1,7, Reginald V. Lord8, Katherine Samaras9,10, Peter L. Molloy 2 & Susan J. Clark 1,3 Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional diferences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic diferences and their contribution to cell type and depot-specifc function. We found that DNA methylomes were notably distinct between diferent adipocyte depots and were associated with diferential gene expression within pathways fundamental to adipocyte function. Most striking diferential methylation was found at transcription factor and developmental genes. Our fndings highlight the importance of developmental origins in the function of diferent fat depots.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • (253), Re15. [DOI: 10.1126/Stke.2532004Re15] 2004
    The VGL-Chanome: A Protein Superfamily Specialized for Electrical Signaling and Ionic Homeostasis Frank H. Yu and William A. Catterall (5 October 2004) Sci. STKE 2004 (253), re15. [DOI: 10.1126/stke.2532004re15] The following resources related to this article are available online at http://stke.sciencemag.org. This information is current as of 7 July 2009. Article Tools Visit the online version of this article to access the personalization and article tools: http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/253/re15 Supplemental "Supplementary Table 1" Materials http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/253/re15/DC1 Related Content The editors suggest related resources on Science's sites: http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2006/360/tw376 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2006/350/pe33 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2006/333/tw149 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2005/307/pe50 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2005/302/pe46 Downloaded from http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2005/270/tw55 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2004/233/pe22 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2004/233/pe23 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2004/227/pe16 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2004/219/re4 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2003/194/pe32 http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2003/188/re10
    [Show full text]
  • Stem Cells and Ion Channels
    Stem Cells International Stem Cells and Ion Channels Guest Editors: Stefan Liebau, Alexander Kleger, Michael Levin, and Shan Ping Yu Stem Cells and Ion Channels Stem Cells International Stem Cells and Ion Channels Guest Editors: Stefan Liebau, Alexander Kleger, Michael Levin, and Shan Ping Yu Copyright © 2013 Hindawi Publishing Corporation. All rights reserved. This is a special issue published in “Stem Cells International.” All articles are open access articles distributed under the Creative Com- mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board Nadire N. Ali, UK Joseph Itskovitz-Eldor, Israel Pranela Rameshwar, USA Anthony Atala, USA Pavla Jendelova, Czech Republic Hannele T. Ruohola-Baker, USA Nissim Benvenisty, Israel Arne Jensen, Germany D. S. Sakaguchi, USA Kenneth Boheler, USA Sue Kimber, UK Paul R. Sanberg, USA Dominique Bonnet, UK Mark D. Kirk, USA Paul T. Sharpe, UK B. Bunnell, USA Gary E. Lyons, USA Ashok Shetty, USA Kevin D. Bunting, USA Athanasios Mantalaris, UK Igor Slukvin, USA Richard K. Burt, USA Pilar Martin-Duque, Spain Ann Steele, USA Gerald A. Colvin, USA EvaMezey,USA Alexander Storch, Germany Stephen Dalton, USA Karim Nayernia, UK Marc Turner, UK Leonard M. Eisenberg, USA K. Sue O’Shea, USA Su-Chun Zhang, USA Marina Emborg, USA J. Parent, USA Weian Zhao, USA Josef Fulka, Czech Republic Bruno Peault, USA Joel C. Glover, Norway Stefan Przyborski, UK Contents Stem Cells and Ion Channels, Stefan Liebau,
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • Fig1-13Tab1-5.Pdf
    Supplementary Information Promoter hypomethylation of EpCAM-regulated bone morphogenetic protein genes in advanced endometrial cancer Ya-Ting Hsu, Fei Gu, Yi-Wen Huang, Joseph Liu, Jianhua Ruan, Rui-Lan Huang, Chiou-Miin Wang, Chun-Liang Chen, Rohit R. Jadhav, Hung-Cheng Lai, David G. Mutch, Paul J. Goodfellow, Ian M. Thompson, Nameer B. Kirma, and Tim Hui-Ming Huang Tables of contents Page Table of contents 2 Supplementary Methods 4 Supplementary Figure S1. Summarized sequencing reads and coverage of MBDCap-seq 8 Supplementary Figure S2. Reproducibility test of MBDCap-seq 10 Supplementary Figure S3. Validation of MBDCap-seq by MassARRAY analysis 11 Supplementary Figure S4. Distribution of differentially methylated regions (DMRs) in endometrial tumors relative to normal control 12 Supplementary Figure S5. Network analysis of differential methylation loci by using Steiner-tree analysis 13 Supplementary Figure S6. DNA methylation distribution in early and late stage of the TCGA endometrial cancer cohort 14 Supplementary Figure S7. Relative expression of BMP genes with EGF treatment in the presence or absence of PI3K/AKT and Raf (MAPK) inhibitors in endometrial cancer cells 15 Supplementary Figure S8. Induction of invasion by EGF in AN3CA and HEC1A cell lines 16 Supplementary Figure S9. Knockdown expression of BMP4 and BMP7 in RL95-2 cells 17 Supplementary Figure S10. Relative expression of BMPs and BMPRs in normal endometrial cell and endometrial cancer cell lines 18 Supplementary Figure S11. Microfluidics-based PCR analysis of EMT gene panel in RL95-2 cells with or without EGF treatment 19 Supplementary Figure S12. Knockdown expression of EpCAM by different shRNA sequences in RL95-2 cells 20 Supplementary Figure S13.
    [Show full text]
  • Pflugers Final
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Serveur académique lausannois A comprehensive analysis of gene expression profiles in distal parts of the mouse renal tubule. Sylvain Pradervand2, Annie Mercier Zuber1, Gabriel Centeno1, Olivier Bonny1,3,4 and Dmitri Firsov1,4 1 - Department of Pharmacology and Toxicology, University of Lausanne, 1005 Lausanne, Switzerland 2 - DNA Array Facility, University of Lausanne, 1015 Lausanne, Switzerland 3 - Service of Nephrology, Lausanne University Hospital, 1005 Lausanne, Switzerland 4 – these two authors have equally contributed to the study to whom correspondence should be addressed: Dmitri FIRSOV Department of Pharmacology and Toxicology, University of Lausanne, 27 rue du Bugnon, 1005 Lausanne, Switzerland Phone: ++ 41-216925406 Fax: ++ 41-216925355 e-mail: [email protected] and Olivier BONNY Department of Pharmacology and Toxicology, University of Lausanne, 27 rue du Bugnon, 1005 Lausanne, Switzerland Phone: ++ 41-216925417 Fax: ++ 41-216925355 e-mail: [email protected] 1 Abstract The distal parts of the renal tubule play a critical role in maintaining homeostasis of extracellular fluids. In this review, we present an in-depth analysis of microarray-based gene expression profiles available for microdissected mouse distal nephron segments, i.e., the distal convoluted tubule (DCT) and the connecting tubule (CNT), and for the cortical portion of the collecting duct (CCD) (Zuber et al., 2009). Classification of expressed transcripts in 14 major functional gene categories demonstrated that all principal proteins involved in maintaining of salt and water balance are represented by highly abundant transcripts. However, a significant number of transcripts belonging, for instance, to categories of G protein-coupled receptors (GPCR) or serine-threonine kinases exhibit high expression levels but remain unassigned to a specific renal function.
    [Show full text]
  • 1 1 2 3 Cell Type-Specific Transcriptomics of Hypothalamic
    1 2 3 4 Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to 5 weight-loss 6 7 Fredrick E. Henry1,†, Ken Sugino1,†, Adam Tozer2, Tiago Branco2, Scott M. Sternson1,* 8 9 1Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 10 20147, USA. 11 2Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, 12 Cambridge CB2 0QH, UK 13 14 †Co-first author 15 *Correspondence to: [email protected] 16 Phone: 571-209-4103 17 18 Authors have no competing interests 19 1 20 Abstract 21 Molecular and cellular processes in neurons are critical for sensing and responding to energy 22 deficit states, such as during weight-loss. AGRP neurons are a key hypothalamic population 23 that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific 24 transcriptomics can be used to identify pathways that counteract weight-loss, and here we 25 report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived 26 young adult mice. For comparison, we also analyzed POMC neurons, an intermingled 27 population that suppresses appetite and body weight. We find that AGRP neurons are 28 considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell 29 type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion 30 channels, neuropeptides, and receptors. Combined with methods to validate and manipulate 31 these pathways, this resource greatly expands molecular insight into neuronal regulation of 32 body weight, and may be useful for devising therapeutic strategies for obesity and eating 33 disorders.
    [Show full text]