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Junctional Sarcoplasmic Reticulum Protein Processing and Trafficking in Cardiac Tissue and Primary Cultured Cardiomyocytes Naama Sleiman Wayne State University

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Junctional Sarcoplasmic Reticulum Protein Processing and Trafficking in Cardiac Tissue and Primary Cultured Cardiomyocytes Naama Sleiman Wayne State University Wayne State University Wayne State University Dissertations 1-1-2014 Junctional Sarcoplasmic Reticulum Protein Processing And Trafficking In Cardiac Tissue And Primary Cultured Cardiomyocytes Naama Sleiman Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Cell Biology Commons, and the Physiology Commons Recommended Citation Sleiman, Naama, "Junctional Sarcoplasmic Reticulum Protein Processing And Trafficking In Cardiac Tissue And Primary Cultured Cardiomyocytes" (2014). Wayne State University Dissertations. Paper 1056. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. JUNCTIONAL SARCOPLASMIC RETICULUM PROTEIN PROCESSING AND TRAFFICKING IN CARDIAC TISSUE AND PRIMARY CULTURED CARDIOMYOCYTES by NA’AMA H. SLEIMAN DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2014 MAJOR: PHYSIOLOGY Approved by: ____________________________________________________ Advisor Date ____________________________________________________ ____________________________________________________ ____________________________________________________ ____________________________________________________ © COPYRIGHT BY NA’AMA H. SLEIMAN 2014 All Rights Reserved DEDICATION I dedicate this work to my dear Parents Hassan and Sakna, for their endless love and support throughout my life. To my Uncle, for his encouragement through my early college years. To my Daughter, Serena whom was and is my angel, source of happiness and inspiration as I watched her grow and learn during my Doctoral pursuit and will always encourage and wish to see accomplish success in her future endeavors. To the advancement of science and medicine. ii ACKNOWLEDGEMENTS A deep appreciation to my Dissertation advisor, Steven Cala, Ph.D. whom I admire for his passion to science and his work, for his guidance, expertise, countless hours of support and most of all his patience throughout the process. Without whom this completion would not have been possible. I would like to individually thank the members of my dissertation committee Jian-Ping Jin M.D., Ph.D., Noreen Rossi, M.D., Assia Shisheva Ph.D., Todd Leff, Ph.D for their supervision and goodwill. I extend my gratitude to the Department of Physiology Graduate Officer Douglas Yingst, Ph.D. for his support. Ms. Christine Cupps for her remarkable assistance and continuous encouragement. I am very grateful to the entire Faculty and staff in the Physiology Department, to my colleagues, friends and family. I am blessed to have crossed paths with you and wish to continue in the future. You have all contributed to this accomplishment. iii TABLE OF CONTENTS Dedication ........................................................................................................................ ii Acknowledgements ........................................................................................................ iii List of Tables .............................................................................................................................. ix List of Figures ............................................................................................................................. x List of Abbreviations ....................................................................................................... xii Preface: Introduction to Dissertation ............................................................................. xiv Chapter 1: Background ................................................................................................... 1 1.1. Cardiomyocytes ............................................................................................................. 1 1.1.1. Cardiomyocytes in the intact heart ................................................................... 1 1.1.2. Isolated and cultured cardiomyocytes .............................................................. 1 1.2. Cardiac sarcoplasmic reticulum ................................................................................... 2 1.2.1. Morphological characterization ......................................................................... 3 1.2.2. SR and ER markers ............................................................................................ 4 1.3. Proteins involved in Ca2+ homeostasis ...................................................................... 6 1.3.1. Ca2+ homeostasis in the cardiomyocyte .......................................................... 6 1.3.2. Major proteins in the Ca2+ -release complex ................................................... 7 1.4. Protein: biosynthesis and trafficking ........................................................................... 8 1.4.1. Protein synthesis and nascent polypeptide folding ........................................ 8 1.4.2. Co-translational asparagine (N)-linked glycosylation ..................................... 9 1.4.3. Post-translational modifications of the N-linked glycans ............................... 9 1.4.4. Rough ER and smooth ER .............................................................................. 11 1.4.5. ER exit sites and the intermediate compartment .......................................... 12 1.4.6. Golgi ................................................................................................................... 13 1.4.7. Protein trafficking through the cardiac secretory pathway ........................... 14 iv 1.5. Junctional sarcoplasmic reticulum proteins ............................................................. 15 1.5.1. Calsequestrin .................................................................................................... 15 1.5.1.1. Cardiac muscle calsequestrin (CSQ2) .............................................. 18 1.5.1.1.1. Ca2+ binding and polymerization ............................................. 18 1.5.1.1.2. (N)-linked glycosylation ............................................................ 19 1.5.1.1.3. Phosphorylation by protein kinase CK2 ................................. 20 1.5.1.1.4. Role of CSQ2 in Ca2+ homeostasis ........................................ 21 1.5.1.1.5. Characteristics of CSQ2-DsRed .............................................. 22 1.5.2. Triadin ................................................................................................................ 23 1.5.3. Junctin ................................................................................................................ 23 1.5.4. Junctional sarcoplasmic reticulum protein-protein interactions .................. 24 1.6. Hypertrophy ................................................................................................................. 25 1.6.1. Cardiomyopathies linked to genetic changes in protein components of the Ca2+-release complex in mice ........................................ 26 1.7. Cytoskeleton ................................................................................................................ 29 1.7.1. Cardiac cytoskeletal components ................................................................... 29 1.7.2. Microtubules and endoplasmic reticulum ...................................................... 29 1.7.2.1. Microtubules .......................................................................................... 30 1.7.2.1.1. Microtubule disrupting agents .................................................. 30 1.8. Adenoviral mediated overexpression in cardiomyocytes ....................................... 31 Chapter 2: Materials and Methods ................................................................................ 33 2.1. Animals and heart tissue ............................................................................................ 33 2.2. Purification of CSQ2 ................................................................................................... 33 2.3. Cardiomyocyte isolation ............................................................................................. 34 2.3.1. Surgery ............................................................................................................... 34 2.3.2. Isolation protocol ............................................................................................... 35 v 2.3.3. Cardiomyocyte culture ..................................................................................... 36 2.4. Cell treatments ............................................................................................................ 36 2.4.1. Adenovirus infection ......................................................................................... 36 2.4.2. Nocodazole treatment ...................................................................................... 37 2.5. Electrophoresis ............................................................................................................ 37 2.5.1. Protein extraction and concentration determination ..................................... 37 2.5.2. Gel electrophoresis ........................................................................................... 38 2.5.3. Concanavalin A binding to glycoproteins ....................................................... 38 2.6. Immunological methods
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