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The Role of Microtubule-Associated Protein 1S (MAP1S) in Regulating Pathological Cardiac Hypertrophy

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The Role of Microtubule-Associated Protein 1S (MAP1S) in Regulating Pathological Cardiac Hypertrophy The role of microtubule-associated protein 1S (MAP1S) in regulating pathological cardiac hypertrophy A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2018 Mohammed Ali Najai School of Medical Sciences Division of Cardiovascular Sciences 1 Table of Contents List of figures……………………………………………………………………….7 List of tables………………………….…………… ………..……………………..10 Abstract…………………………..……...……………… ………….……………..11 Declaration………………………..……...……………… ………………………..13 Copyright statement……………..…………...…………… …….………………..14 Acknowledgements…………...…..………………………… ……...…………..…15 Author………………………………...………………………….…………………16 Abbreviations……………………………………...………….……………………17 1 Introduction ....................................................................................................21 1.1 Heart failure ....................................................................................................21 1.2 The pathophysiological process leading to heart failure................................21 1.2.1 Cardiac hypertrophy ....................................................................................21 1.2.2 Fibrosis .........................................................................................................25 1.2.3 Inflammation ................................................................................................25 1.2.4 Cell death ......................................................................................................27 1.2.4.1 Apoptosis ...................................................................................................28 1.2.4.2 Necrosis ......................................................................................................29 1.2.5 Autophagy ....................................................................................................29 1.2.5.1 Process of autophagy .................................................................................32 1.2.5.2 Leading causes of autophagy ....................................................................35 1.2.5.2.1 Starvation................................................................................................35 1.2.5.2.2 Stress response ........................................................................................35 1.2.5.2.3 Pathogen infection ..................................................................................35 1.3.1 Cellular mechanisms leading to autophagy .................................................36 1.3.2 The role of autophagy in cardiac hypertrophy and HF ..............................36 1.4.1 Microtubule-associated proteins (MAP) .....................................................37 1.4.2 Expression and isoforms of MAP1 family ...................................................38 1.4.3 Gene organization and structure of MAP1 .................................................39 1.4.4 Interaction and function of MAP1 ...............................................................39 1.4.5 The function of MAP1 family in the heart ..................................................40 2 1.4.6 MAP1S ..........................................................................................................43 1.4.7 The role of MAP1S in suppressing tumorigenesis and other pathologies ..44 1.4.8 The role of microtubule-associated protein 1S (MAP1S) in autophagy .....45 1.5 HYPOTHESIS AND AIMS ............................................................................47 1.5.1 Hypothesis ....................................................................................................47 1.5.2 Aims ..............................................................................................................47 2. MATERIALS AND METHODS .............................................................49 2.1 MAP1S knockout mice ....................................................................................49 2.2. DNA extraction...............................................................................................51 2.3 Mice genotyping using polymerase chain reaction (PCR) .............................51 2.4 Gel electrophoresis analysis ............................................................................53 2.5 Transverse aortic constriction (TAC) and sham surgery ..............................54 2.6 Echocardiography for hypertrophy-induced mice ........................................56 2.7 RNA extraction................................................................................................58 2.8 Measurement of DNA and RNA concentration .............................................59 2.9 Gene expression quantification by quantitative PCR (qPCR) ......................59 2.10 Protein extraction from mice heart tissues ...................................................61 2.11 Measurement of protein concentration ........................................................62 2.12 Western blot ..................................................................................................62 2.13 Preparing heart cross-sections for haematoxylin and eosin and Masson’s trichrome staining .................................................................................................65 2.13.1 Haematoxylin and eosin stain for detecting cardiomyocytes morphology ...............................................................................................................................67 2.13.2 Masson’s trichrome staining for detecting fibrosis formation ..................67 2.14 PathScan Intracellular Signalling Array ......................................................68 2.15 Mitochondria structure analysis by electron transmission microscopy ......70 2.16 H9c2 cardiomyoblast cell culture .................................................................71 2.17 Protein extraction from H9C2 cells ..............................................................71 2.18 GFP-LC3 adenovirus generation ..................................................................71 2.19 Knockdown of MAP1S gene using small interfering RNA (siRNA) ...........73 2.20 Stimulation of autophagy in H9C2 cells .......................................................75 2.21 Luciferase assay.............................................................................................76 2.22 Immunofluorescence .....................................................................................76 2.23 Detection of mitochondria using MitoTracker.............................................77 3 2.24 Statistical analysis .........................................................................................77 3 The role of MAP1S in modulating cardiac hypertrophy ..................79 3.1 Introduction.....................................................................................................79 3.1.1 Cardiac hypertrophy and tumour suppressors ...........................................79 3.1.2 The tumour suppressor MAP1S and hypertrophy .....................................80 3.2 Hypothesis .......................................................................................................82 3.3 Aims .................................................................................................................82 3.4 Results .............................................................................................................83 3.4.1 Demonstrating MAP1S ablation (global knockout) in mice .......................83 3.4.2 MAP1S protein expression in mouse hearts ................................................83 3.4.3 Cardiac phenotype in MAP1S null mice after 2 weeks of TAC..................84 -/- 3.4.4 Echocardiographic analysis (ECG) of MAP1S mice after 2 weeks TAC .86 3.4.5 Cardiac function in MAP1S knockout mice after 2 weeks TAC ................87 3.4.6 Histological analysis .....................................................................................90 -/- 3.4.6.1 Cardiomyocyte cross-sectional area (CSA) in MAP1S mice after 2 weeks TAC ............................................................................................................90 3.4.6.2 Histological analysis of the fibrosis level in MAP1S-/- mice hearts after 2 weeks TAC ............................................................................................................92 3.4.7 Gene expression of cardiac hypertrophy and fibrosis markers ..................94 3.4.7.1 Gene expression of cardiac hypertrophy markers ANP and BNP ..........95 3.4.7.2 Gene expression of cardiac fibrosis marker COL1A1 and COL3A1 ......97 3.5 Discussion ...................................................................................................... 100 3.5.1 Hypertrophic response to TAC in MAP1S knockout mice ....................... 100 3.5.2 Echocardiography data of MAP1S null mice following TAC ................... 101 3.5.3 Evaluation of heart function following hypertrophy induction after 2 weeks TAC .......................................................................................................... 102 3.5.4 Cardiac structure in MAP1S-/- mice in response to hypertrophy induced by TAC ................................................................................................................ 102 3.5.5 Evaluation of the fibrosis level that accompanies hypertrophy in response to TAC ................................................................................................................. 103 3.6 Conclusion ....................................................................................................
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