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The Role of Microtubule-Associated Protein 1S (MAP1S) in Regulating Autophagy

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The Role of Microtubule-Associated Protein 1S (MAP1S) in Regulating Autophagy University of Manchester The role of microtubule-associated protein 1S (MAP1S) in regulating autophagy in the heart A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 Yulia Suciati Kohar School of Medical Sciences Division of Cardiovascular Sciences TABLE OF CONTENTS List of Figures ............................................................................................................... 6 List of Tables ............................................................................................................... 10 Abbreviations ............................................................................................................. 12 Abstract ...................................................................................................................... 16 Declaration ................................................................................................................. 18 Copyright statement .................................................................................................. 19 Acknowledgments ...................................................................................................... 20 1. INTRODUCTION .................................................................................................. 22 1.1. The Global Burden of Cardiovascular Disease ........................................... 22 1.2. Coronary artery disease and myocardial infarction................................... 24 1.3. Molecular mechanism of heart failure and myocardial infarction ............ 26 1.4. General overview of cardiac cell death ...................................................... 29 1.4.1. Apoptosis ............................................................................................ 30 1.4.2. Necrosis ............................................................................................... 33 1.4.3. Autophagy- dependent cell death ...................................................... 34 1.5. Autophagy ..................................................... Error! Bookmark not defined. 1.5.1. Types of autophagy ............................................................................. 34 1.5.2. Molecular mechanism of autophagy .................................................. 36 1.5.3. Autophagic flux ................................................................................... 45 1.6. The role of autophagy in cardiac homeostasis .......................................... 47 1.6.1. Autophagy in cardiomyocyte .............................................................. 47 1.6.2. Autophagy in cardiac pathological conditions .................................... 48 1.7. MAP1S ........................................................................................................ 53 1.7.1. Structure and biological function of MAP1 family of proteins ........... 53 1.7.2. Structure and biological function of MAP1S protein .......................... 56 1.7.3. The role of MAP1S in regulating autophagy and other pathologies .. 57 2 1.8. Summary of literature study ...................................................................... 60 1.9. Hypothesis .................................................................................................. 60 1.10. Aim ............................................................................................................. 61 2. MATERIALS AND METHODS ............................................................................... 63 2.1. Generation of MAP1S KO Mice .................................................................. 63 2.2. Molecular analysis ...................................................................................... 64 2.2.1. DNA Extraction .................................................................................... 64 2.2.2. PCR ...................................................................................................... 65 2.2.3. Gel electrophoresis ............................................................................. 66 2.2.4. Isolation of NRCM ............................................................................... 66 2.2.5. Isolation of MSF .................................................................................. 67 2.2.6. Protein expression analysis ................................................................. 68 2.2.7. Protein extraction ............................................................................... 68 2.2.8. Adenovirus productions ...................................................................... 72 2.2.9. siRNA Transfection .............................................................................. 78 2.2.10. pAd GFP-LC3 Transduction ................................................................. 79 2.2.11. pAdKeima, pAdParkin Transduction ................................................... 80 2.2.12. pAd/MAP1S Transduction ................................................................... 80 2.2.13. Lysotracker Analysis ............................................................................ 81 2.2.14. MitoTracker Analysis ........................................................................... 82 2.2.15. Seahorse XF Assay ............................................................................... 83 2.2.16. MTT assay ............................................................................................ 86 2.3. Animal work ............................................................................................... 86 2.3.1. Rapamycin and Chloroquine IP Injection ............................................ 87 2.3.2. TEM ..................................................................................................... 87 2.3.3. Mouse model of myocardial infarction .............................................. 88 3 2.3.4. cTnI analysis ........................................................................................ 88 2.3.5. Echocardiography ............................................................................... 89 2.4. Histological analysis ................................................................................... 91 2.4.1. Tissue fixation with formaldehyde, embedding and sectioning ......... 91 2.4.2. Masson’s Trichrome staining .............................................................. 93 2.4.3. TUNEL staining .................................................................................... 93 2.4.4. H&E staining ........................................................................................ 94 2.5. Statistical analysis ...................................................................................... 95 3. THE ROLE OF MAP1S IN MODULATING AUTOPHAGIC FLUX IN CARDIOMYOCYTES ..................................................................................................... 97 3.1. Background ................................................................................................ 97 3.2. Hypothesis .................................................................................................. 98 3.3. Aims and Objectives ................................................................................... 98 3.4. Results ........................................................................................................ 99 3.4.1. MAP1S is expressed in cardiomyocytes and in cardiac fibroblasts .... 99 3.4.2. MAP1S gene silencing in NRCM ........................................................ 100 3.4.3. Molecular cascade of LC3 activation ................................................ 101 3.4.4. Studies using MAP1S knockout (KO) mice ........................................ 111 3.4.5. The modulation effect of autophagic flux in NRCM’s lysosome ...... 120 3.5. Discussion ................................................................................................. 123 4. THE ROLE OF MAP1S IN REGULATING MITOPHAGY ........................................ 127 4.1. Background .............................................................................................. 127 4.2. Hypothesis ................................................................................................ 129 4.3. Aims and Objectives ................................................................................. 130 4.4. Results ...................................................................................................... 131 4.4.1. MAP1S gene silencing prevents binding of autophagosome with damaged mitochondria ..................................................................... 131 4 4.4.2. MAP1S gene silencing affects mitochondrial organizational network …………………………………………………………………………………………………….135 4.4.3. MAP1S gene silencing displayed reduced mitochondrial function .. 138 4.4.4. MAP1S gene silencing affects apoptotic pathway ............................ 144 4.5. Discussion ................................................................................................. 150 5. THE EFFECTS OF MAP1S GENETIC ABLATION DURING MYOCARDIAL INFARCTION ............................................................................................................. 154 5.1. Background .............................................................................................. 154 5.2. Hypothesis ................................................................................................ 155 5.3. Aims and Objectives ................................................................................
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