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The Role of MAP4K4 in Cardiac Muscle Cell Death

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The Role of MAP4K4 in Cardiac Muscle Cell Death The role of MAP4K4 in cardiac muscle cell death Micaela M. Jenkins CID: 00855768 National Heart and Lung Institute Faculty of Medicine Imperial College London A Thesis submitted to Imperial College London for Doctor of Philosophy 1 National Heart and Lung Institute Word count: 65,028 2 Acknowledgements I express my sincerest thanks to Professor Michael Schneider and Professor Sian Harding for enabling the opportunity to work on this exciting project as well as for all their help, support and advice during course of my studies. I am also very grateful to the British Heart Foundation for awarding me the studentship to undertake this research. I would like to acknowledge the extensive work carried out by the MAP4K4 team past and present, which provided the foundations for this study. I am especially grateful to Lorna Fiedler for all her scientific and moral support all the way through from my MRes days to this day. Lorna’s insight into the project has been absolutely invaluable to me as has her guidance and support. I am extremely grateful to Dr Michela Noseda for imparting to me the research and technical skills necessary to complete the work hereby presented as well as for the daily support and guidance on every respect. I am indebted to Tom Owen, Eleanor Humphrey and Carolina Pinto Ricardo not only for their generous technical and academic assistance but also for accompanying me in the happy as well as hard moments during these three years. I would also like to thank all the members of the Schneider and Harding laboratories for their support and advice and for generally making my days at work so enjoyable. I would also like to acknowledge the Carling group for having provided me with training on the Seahorse platform and kindly allowed me to use their equipment on a regular basis. Special thanks to my family, friends and particularly Quin. It was your encouragement and support that took me through these years and always made me see the light at the end of the tunnel. Finally, I would like to dedicate this thesis to my mother and grandfather, my everlasting sources of inspiration and strength. 3 Declaration of Originality I declare that the work hereby presented was conducted by the author, except where indicated by special reference in the text, and that no part of the dissertation has been submitted for any other degree. Some of the results contained in this thesis have been presented at conferences and seminars. Micaela M. Jenkins 4 Copyright Declaration ‘The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work’ 5 Table of contents Acknowledgements ................................................................................................................... 3 Declaration of Originality ........................................................................................................... 4 Copyright Declaration................................................................................................................ 5 Table of contents ...................................................................................................................... 6 List of Figures ......................................................................................................................... 10 List of Tables .......................................................................................................................... 14 List of abbreviations ................................................................................................................ 15 Abstract................................................................................................................................... 19 1. Introduction ......................................................................................................................... 20 1.1 Heart failure as a socio-economic problem ................................................................ 21 1.2 Pathobiology of heart failure ...................................................................................... 24 1.3 Cell death signalling pathways ................................................................................... 28 1.3.1 Apoptosis ............................................................................................................ 29 1.3.2 Necrosis .............................................................................................................. 33 1.3.3 Autophagy ........................................................................................................... 38 1.4 Cell death in myocardial infarction and heart failure ................................................... 39 1.4.1 Apoptosis in myocardial infarction and heart failure ........................................... 41 1.4.2 Necrosis in myocardial infarction and heart failure .............................................. 44 1.4.3 Autophagy in myocardial infarction and heart failure ........................................... 45 1.5 Metabolism ................................................................................................................ 49 1.6 Metabolism in myocardial infarction and heart failure ................................................ 51 1.7 The protein kinase complement of the human genome .............................................. 55 1.7.1 Classification and mode of action ........................................................................ 55 1.7.2 Structure ............................................................................................................. 58 1.7.3 Mitogen-activated protein kinases (MAPKs) ........................................................ 59 1.8 MAPKs in heart failure ............................................................................................... 63 1.8.1 MAP kinases in heart failure ................................................................................ 63 1.8.2 MAP3 kinases in heart failure .............................................................................. 67 1.8.3 MAP4K kinases in heart failure ........................................................................... 70 1.9 Induced pluripotent stem cells (iPSCs) as a tool for drug discovery ........................... 75 1.9.1 Characterisation of iPSC-derived cardiomyocytes ............................................... 77 1.9.2 Engineered heart tissue (EHT) as a platform for drug evaluation ........................ 78 2. The aim ....................................................................................................................... 81 2. Methods .............................................................................................................................. 84 6 2.1 hiPSC-CM culture ...................................................................................................... 85 2.2 MAP4K4 pharmacological inhibition ........................................................................... 86 2.3 Cell death induction ................................................................................................... 89 2.4 Cell death assays ...................................................................................................... 89 2.5 Immunocytostaining (2D) ........................................................................................... 90 2.6 Mitochondrial respiration analysis .............................................................................. 92 2.7 Protein quantification ................................................................................................. 93 2.8 hiPSC-CMs thawing for human engineered heart tissue (hEHTs) generation ............ 93 2.9 EHT generation ......................................................................................................... 94 2.10 EHT solutions .......................................................................................................... 94 2.11 Force calculation in EHTs and contractile assessment ............................................ 96 2.12 Immunocytochemistry .............................................................................................. 98 2.13 Calcium dynamics assessment ................................................................................ 99 2.14 Statistical Analysis ................................................................................................. 100 3. Results .............................................................................................................................. 101 Effects of MAP4K4 inhibition on cell death in 2-dimensional human iPSC-CM culture. ........ 102 3.1 Optimisation of conditions for human cardiac muscle cell death induced by H2O2 ... 104 3.2 Effect of the MAP4K4 inhibitor IC4-001 on plasma membrane disruption induced by H2O2 .............................................................................................................................. 106 3.3 Effect of the MAP4K4inhibitor IC4-001 on caspase-3 activation induced by H2O2 .... 108 3.4 Effect of the MAP4K4 inhibitor IC4-001 on BID cleavage induced by H2O2 .............. 111 3.5
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