University of Manchester The role of PMCA1 in post- myocardial infarction remodelling A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2017 Vera Stankovikj School of Medical Sciences Division of Cardiovascular Sciences Table of contents List of figures ....................................................................................................................... 10 List of tables ........................................................................................................................ 14 List of abbreviations ............................................................................................................. 16 Declaration .......................................................................................................................... 20 Copyright statement ............................................................................................................ 21 Abstract ............................................................................................................................... 22 Acknowledgements ............................................................................................................. 24 1. Introduction ................................................................................................................. 26 1.1 Heart failure ........................................................................................................... 26 1.2 Coronary artery disease-induced myocardial infarction .......................................... 27 1.2.1 Epidemiology ......................................................................................................... 27 1.2.2 Pathophysiology .................................................................................................... 27 1.2.3 Diagnosis of MI and current treatment strategies ................................................. 31 1.3 Post-myocardial infarction remodelling .................................................................. 32 1.3.1 Infarct expansion ................................................................................................... 34 1.3.2 Non-ischaemic infarct extension ........................................................................... 35 1.3.3 Cardiac hypertrophy .............................................................................................. 35 1.4 Mechanisms of cardiomyocyte death after MI ........................................................ 37 1.4.1 Necrosis ................................................................................................................. 37 1.4.2 Autophagy post-MI ................................................................................................ 39 1.4.3 Apoptosis post-MI .................................................................................................. 41 1.4.4 Oxidative stress after MI ........................................................................................ 45 1.5 Mechanisms of cardiac repair after MI ................................................................... 46 2 1.5.1 Cellular pathways of cardiomyocyte survival after MI - ERK1/2 signalling pathway 48 1.6 Genome-wide association studies elucidated a potential link between CAD, MI and atp2b1 ............................................................................................................................. 50 1.7 The plasma membrane calcium ATPase (PMCA) ..................................................... 54 1.7.1 Structure of PMCA and regulation of PMCA activity ............................................. 55 1.7.2 Expression of PMCA and links of PMCA isoforms with human disease ................. 57 1.7.2.1 PMCA1 ............................................................................................................. 57 1.7.2.2 PMCA2 ............................................................................................................. 57 1.7.2.3 PMCA3 ............................................................................................................. 58 1.7.2.4 PMCA4 ............................................................................................................. 58 1.7.3 Role of PMCA in cellular proliferation and death .................................................. 59 1.7.4 Role of PMCA in the heart ..................................................................................... 60 1.7.5 PMCA1 and cardiovascular disease ....................................................................... 63 1.7.5.1 PMCA1 is involved in development of hypertension ...................................... 64 1.8 Hypothesis ............................................................................................................. 65 1.9 Aims ....................................................................................................................... 65 2 Methods ........................................................................................................................ 67 2.1 Characterisation of a mouse model of myocardial infarction .................................. 67 2.1.1 Animal subjects ...................................................................................................... 67 2.1.2 Genotype confirmation by PCR .............................................................................. 67 2.1.3 Evaluation of the anatomy of the left coronary artery in both wild type and PMCA1Ht mice ..................................................................................................................... 69 2.1.4 Mouse model of myocardial infarction ................................................................. 70 2.1.5 Conscious electrocardiography (ECG) .................................................................... 70 2.1.6 Assessment of plasma levels of mouse cardiac Troponin I .................................... 71 2.1.6.1 Blood sampling and plasma preparation ......................................................... 71 3 2.1.6.2 Determination of plasma levels of mouse cardiac troponin I (cTnI) ............... 72 2.2 Characterisation of the post-MI remodelling process .............................................. 73 2.2.1 Experiment timeline .............................................................................................. 73 2.2.2 In vivo cardiac investigations ................................................................................. 74 2.2.2.1 Transthoracic two-dimensional echocardiography (TTE) ................................ 74 2.2.2.2 Unconscious ECG ............................................................................................. 75 2.2.2.3 Haemodynamic assessment of cardiac function ............................................. 76 2.2.3 Histological investigations ..................................................................................... 77 2.2.3.1 Tissue processing, paraffin embedding and sectioning of mouse hearts ........ 77 2.2.3.2 Masson’s Trichrome staining ........................................................................... 78 2.2.3.3 Picrosirius red staining .................................................................................... 81 2.2.3.4 Haematoxylin and Eosin staining ..................................................................... 82 2.2.3.5 Dihydroethidium (DHE) staining ...................................................................... 83 2.2.3.6 Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) Nick-End Labeling (TUNEL) assay ....................................................................... 84 2.2.4 Molecular analysis ................................................................................................. 86 2.2.4.1 Tissue collection for molecular analysis .......................................................... 86 2.2.4.2 Gene expression analysis ................................................................................. 86 2.2.4.3 Protein expression analysis ............................................................................. 91 2.3 Statistical analysis .................................................................................................. 95 3 Characterisation of a mouse model of MI ...................................................................... 97 3.1 Introduction ........................................................................................................... 97 3.2 Aims ....................................................................................................................... 98 3.3 Results ................................................................................................................... 98 3.3.1 Expression of PMCA1 in a mouse model of MI ...................................................... 98 3.3.2 Expression of PMCA4 in a mouse model of MI ...................................................... 99 4 3.3.3 PMCA1Ht mouse model .......................................................................................... 99 3.3.3.1 Generation of PMCA1Ht mice ........................................................................... 99 3.3.3.2 Genotype confirmation ................................................................................. 100 3.3.3.3 Expression of atp2b1 in PMCA1Ht mice ......................................................... 101 3.3.3.4 Expression of