DISSERTATION CHARACTERIZATION OF ZEBRAFISH MODELS OF FILAMIN C RELATED CARDIOMYOPATHY Submitted by Rasha M. Alnefaie Graduate Degree Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Spring 2019 Doctoral Committee: Advisor: Deborah Garrity ASN Reddy Paul Lybourn Racheal Muller Copyright by Rasha M. Alnefaie 2019 All Rights Reserved ABSTRACT CHARACTERIZATION OF ZEBRAFISH MODELS OF FILAMIN C RELATED CARDIOMYOPATHY Cardiomyopathies are a group of cardiac muscle diseases characterized by abnormal function and/or structure of the myocardium which cause arrhythmia, heart failure or sudden death. In many cases, cardiomyopathy is a genetic disease and the majority of inherited cases are caused by mutations in genes that encode cardiac costameric and sarcomeric proteins. Cardiomyopathies include different types, such as dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), and arrythmogenic cardiomyopathy (ARCM). These groups of disorders can have common cellular phenotypes and mechanisms. To date, few studies have described the roles of filamin C in cardiac development or explained how mutations in filamin C cause cardiomyopathic disease. Due to the lack of a suitable animal model, the pathological mechanisms underlying this disease and the role of filaminC in cardiac development remain unclear. Here, we created a zebrafish loss-of-function model for two flnc paralogous genes. We investigated several genetic lines bearing mutations in filamin C that target different sites of the gene. As for humans, zebrafish mutants exhibited variable penetrance and variable expressivity. Double flnca and flncb mutant hearts exhibited more pronounced cardiac morphological defects compared to single mutants, leading to the conclusion that these paralogs play redundant roles in zebrafish heart development. The cardiac morphological phenotype of double flnc mutant embryos is characterized by a decrease in cardiac output and stroke volume as is also observed in patients who suffer from cardiomyopathies. Using a transgenic line expressing GFP in cardiac z-discs, we find that knockdown of flnca and flncb via morpholinos and double flnca and flncb mutant hearts ii exhibited irregular z-discs. In support of this finding, ultrastructural analysis by transmission electronic microscopy for flncb morphant embryos and double flnca and flncb mutants indicated disorganized myofibrils with fewer consecutive sarcomeres. Particularly, z-discs were irregular or apparently absent, and numerous small vacuoles and potentially autophagous vesicles were observed. Additionally, through double flnca and flncb mutant, we demonstrated that filamin C is required for normal cardiomyocyte morphology and microfilament arrangement. In summary, the zebrafish model demonstrates an essential requirement for filamin C function during heart and skeletal muscle development. Depletion of filamin C impairs both sarcomerogenesis and alters the cytoskeletal architecture of cardiomyocytes. iii ACKNOWLEDGMENTS I would like to thank God for providing me with the ability to complete the PhD program. My special thanks to my advisor Dr. Deborah Garrity; without her guidance and mentoring this project would not have been completed. I benefitted a lot from her supervision. I’m grateful to my committee members for all their helpful suggestions to improve my project. In addition, a thank you to Dr. David Bark for providing the high speed camera and all his professional guidance. I also wish to thank Alex Gendernalik for his help in using the high speed camera and Matlab. I would like to thank all the Garrity lab members (graduate and undergraduate students) for creating an awesome environment. Special thanks for my undergraduate student Max Hostetter for all his help to complete this project. I would like to give thanks to Suzanne Royer for performing TEM. I would also like to thank Alissa Williams for helping in editing my dissertation. I am extremely grateful for the opportunity granted by Albaha University for financial support for the PhD program. I’m also thankful to my family who have supported me as I accomplished my project. Lastly, I would like to thank all those who helped me in any way in my PhD journey. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGMENTS ............................................................................................................. iv Chapter1: Introduction .................................................................................................................... 1 Summary.................................................................................................................................. 1 Zebrafish and Developmental Biology .................................................................................................... 3 Taxonomic classification ......................................................................................................... 3 Natural habitat of zebrafish ..................................................................................................... 4 Embryonic development of zebrafish ...................................................................................... 4 Zebrafish as a model organism ................................................................................................................. 9 Overview ................................................................................................................................. 9 Advantages of zebrafish as a cardiovascular model .............................................................. 11 A brief description of embryonic zebrafish heart development ............................................ 12 The zebrafish genome............................................................................................................ 17 Aquarium and laboratory strains: .......................................................................................... 19 Cardiomyopathy ....................................................................................................................................... 19 History of the concept of Cardiomyopathies ......................................................................... 19 Cardiomyopathy definition .................................................................................................... 20 Classification of Cardiomyopathies ...................................................................................... 20 Dilated Cardiomyopathy (DCM) ........................................................................................... 24 Hypertrophic Cardiomyopathy (HCM) ................................................................................. 27 Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) ............................................. 28 Filamin C and myopathies ...................................................................................................................... 30 Filamin (FLN) ....................................................................................................................... 30 Cellular localization of filamin isoforms ............................................................................... 33 Biological function of filamin family proteins ...................................................................... 34 Myofibrillar myopathy (MFM) related to FLNC .................................................................. 37 Animal Models for FLNC function: ...................................................................................... 39 Dissertation Aims ..................................................................................................................................... 41 Chapter 2: flnca and flncb are required for normal heart development ........................................ 44 Introduction ............................................................................................................................................... 44 Structure ................................................................................................................................ 44 Functions ............................................................................................................................... 45 FLNC ..................................................................................................................................... 45 Myopathy diseases................................................................................................................. 47 Animal models used to study FLNC ..................................................................................... 48 Zebrafish as a model organism .............................................................................................. 48 Results ........................................................................................................................................................ 50 Genetic conservation: ............................................................................................................ 50 FLNC morpholino knockdown and phenotypic analysis: ..................................................... 50 Zebrafish flncb is required for sarcomeric arrangement