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Investigating the Role of Mitochondrial Dynamic Proteins Mid49 and Mid51, As Novel Targets of Cardioprotection

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Investigating the Role of Mitochondrial Dynamic Proteins Mid49 and Mid51, As Novel Targets of Cardioprotection INVESTIGATING THE ROLE OF MITOCHONDRIAL DYNAMIC PROTEINS MID49 AND MID51, AS NOVEL TARGETS OF CARDIOPROTECTION Thesis submitted by Parisa Samangouei BSc, MSc (First class with Honours) For the degree of Doctor of Philosophy University College London, UK Primary supervisor: Professor Derek Hausenloy Secondary supervisor: Professor Derek Yellon Tertiary supervisor : Dr Andrew Hall Institute of Cardiovascular Science Hatter Cardiovascular institute, University College London, 67 Chenies Mews, London WC1E 6HX. March 2018 DECLARATION I, Parisa Samangouei confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Data gathered from pilot studies conducted by other investigators, within our research group, presented in the introduction sections, has been acknowledged and noted. Any collaborations and assistance provided by other investigators in aiding to generate experimental results, presented in this thesis, has been indicated in the respective methods and results sections. Parisa Samangouei 1 ABSTRACT Background Acute myocardial infarction and the heart failure that often ensues are the leading causes of death and disability worldwide. As such, novel therapeutic strategies are required to protect the heart from acute ischaemia-reperfusion injury (IRI). Mitochondria play a central role in determining the fate of cardiomyocytes, during acute myocardial IRI. Genetic and pharmacological inhibition of Drp1-mediated mitochondrial fission has been shown to reduce cardiomyocyte death, during acute IRI. Accordingly, we investigated the role of the newly described mitochondrial- specific Drp1 receptors, MiD49 and MiD51, as novel targets for cardioprotection. We hypothesised that inhibition of MiD49 and MiD51 would render the heart more resistant to acute IRI, and provide novel therapeutic targets for cardioprotection. Methods and Results 1. In cardiac cell-lines, the genetic knockdown (KD) of both MiD49 and MiD51 promoted mitochondrial elongation, inhibited mitochondrial permeability transition pore opening, reduced cell death and prevented mitochondrial calcium overload during simulated IRI, when compared to control cells. 2. In adult mice, MiD49 deficiency had no significant effect on mitochondrial morphology, cardiac size and function, or myocardial infarct size, when compared to wild-type littermates. The effect of dual cardiac-specific genetic ablation of MiD49 and MiD51 in the heart on the susceptibility to acute IRI is currently being investigated. 3. We have developed a biophysical assay to high-throughput screen for novel small molecule inhibitors of the interaction between Drp1 and MiD49 and MiD51, as a therapeutic strategy for inhibiting mitochondrial fission. Conclusions and Further studies For the first time, we provide evidence for the role of MiD49 and MiD51 as novel therapeutic targets for cardioprotection. Studies are ongoing to validate their roles in the adult heart. In order to provide a novel therapeutic strategy for inhibiting 2 mitochondrial fission as a cardioprotective strategy, we aim to identify novel small molecule inhibitors of the interaction between Drp1 and MiD49/MiD51. 3 CONTENTS DECLARATION ....................................................................................................... 1 ABSTRACT ............................................................................................................. 2 CONTENTS ............................................................................................................. 4 ACKNOWLEDGEMENTS ........................................................................................ 9 PUBLICATIONS .................................................................................................... 10 FIGURES............................................................................................................... 11 TABLES ................................................................................................................. 14 ABBREVIATIONS .................................................................................................. 15 CHAPTER 1: General introduction ................................................................... 19 1.1 Epidemiology of ischaemic heart disease .........................................................19 1.1.1 Atherosclerosis .............................................................................................20 1.1.2 Myocardial infarction .....................................................................................21 1.2 Cardiac metabolism ..........................................................................................23 1.2.1 Oxidative phosphorylation ............................................................................23 1.2.2 Cardiac Ischaemia ........................................................................................25 1.2.3 Cardiac Reperfusion .....................................................................................27 1.3 Mitochondrial Dynamics and Cardioprotection ..................................................30 1.3.1 Mitochondrial Fusion ....................................................................................30 1.3.2 Mitochondrial fission .....................................................................................32 1.4 Mitochondrial arrangement and dynamics in the heart .....................................35 1.4.1 Observing mitochondrial dynamics in cardiomyocytes ..................................36 1.4.2 Inhibition of mitochondrial fission in the heart during IRI ...............................37 1.5 Mitochondrial fission proteins MiD49 and MiD51 as targets of cardioprotection ...............................................................................................................39 1.5.1 Discovery .....................................................................................................39 1.5.2 Cellular expression of MiD49 and MiD51 ......................................................41 1.5.3 MiD49 and MiD51 are Drp1 adaptors involved in the mitochondrial fission machinery .....................................................................................................42 1.5.4 The molecular structure of MiD49 and MiD51 in relation to their function .....47 1.5.5 MiD-ER interaction .......................................................................................52 1.5.6 Regulation of MiD49 and MiD51 activity during physiological and stressed conditions ..................................................................................................54 CHAPTER 2: Research Objectives ................................................................... 56 4 2.1 Overall objective ...............................................................................................56 2.2 Overall hypothesis ............................................................................................56 2.3 Aims .................................................................................................................56 CHAPTER 3: Investigating the role of MiD49 and MiD51, as targets of cardioprotection: in vitro studies ............................................................................. 58 3.1 Introduction ......................................................................................................58 3.2 Research objectives .........................................................................................59 3.2.1 Aim1: Investigating the effect of MiD49 and MiD51 overexpression: in vitro studies ..............................................................................................................59 3.2.2 Aim 2: Investigating the effect of MiD49 and MiD51 genetic ablation: in vitro studies ..............................................................................................................60 3.3 Materials and Methods .....................................................................................60 3.3.2 The general method of cell passage and seeding .........................................61 3.3.3 Freezing cell stocks ......................................................................................62 3.3.4 Thawing of frozen cell lines ..........................................................................63 3.3.5 Transfection of cell lines ...............................................................................63 3.3.6 Cell fixation ...................................................................................................65 3.3.7 Fluorescence activated cell sorting ...............................................................65 3.3.8 Fluorescence microscopy .............................................................................66 3.3.9 Confocal microscopy ....................................................................................66 3.3.10 MPTP opening ..............................................................................................67 3.3.11 Determining cell survival after SIRI ...............................................................69 3.3.12 Real-time simulated ischaemia-reperfusion injury confocal set-up ................70 3.3.13 Statistical analysis ........................................................................................71 Aim 1: Investigating the effect of MiD49 and MiD51 overexpression: in vitro studies .............................................................................................................................
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