Arrhythmogenesis in the ageing atria A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences 2015 Charles Michael Pearman School of Medicine P a g e | 2 TABLE OF CONTENTS Table of Figures ......................................................................................................................... 8 Table of Tables ........................................................................................................................ 10 List of abbreviations ................................................................................................................ 11 Abstract for thesis submitted for the degree of Doctor of Philosophy .................................. 14 Declaration .............................................................................................................................. 15 Copyright statement ............................................................................................................... 16 Acknowledgements ................................................................................................................. 17 Biography ................................................................................................................................ 18 1 Introduction ........................................................................................................................ 19 1.1 The natural history and classification of AF ................................................................ 21 1.2 The cardiac action potential ....................................................................................... 22 1.2.1 Phases of the action potential and their underlying ionic currents ............................. 22 1.2.2 Regional differences in action potential morphology................................................... 25 1.2.3 Modulation of action potential morphology ................................................................ 25 1.2.4 Action potential characteristics affected by AF remodelling ........................................ 27 1.3 Excitation-contraction coupling .................................................................................. 28 1.3.1 Calcium cycling .............................................................................................................. 28 1.3.2 Modulation of calcium cycling ...................................................................................... 31 1.3.3 Calcium cycling characteristics affected by AF remodelling ......................................... 32 1.4 Cardiac conduction ..................................................................................................... 34 1.5 Mechanisms of arrhythmia ......................................................................................... 36 1.5.1 Enhanced automaticity ................................................................................................. 36 1.5.2 Triggered activity .......................................................................................................... 37 1.5.3 Re-entry ......................................................................................................................... 40 1.5.4 Factors promoting wavebreak ...................................................................................... 41 1.6 Arrhythmic mechanisms applied to atrial fibrillation ................................................. 43 1.6.1 Pulmonary vein ectopy ................................................................................................. 43 1.6.2 Multiple wavelets .......................................................................................................... 43 1.6.3 Rotors ............................................................................................................................ 44 P a g e | 3 1.7 Atrial structural remodelling ....................................................................................... 46 1.8 Atrial fibrillation and ageing ........................................................................................ 48 2 Methods .............................................................................................................................. 49 2.1 Determining the age of sheep .................................................................................... 49 2.2 Cell isolation ................................................................................................................ 51 2.2.1 Background ................................................................................................................... 51 2.1.1 Solutions ........................................................................................................................ 51 2.2.2 Euthanasia and coronary cannulation .......................................................................... 53 2.2.3 Enzymatic digestion ...................................................................................................... 55 2.3 Patch Clamp ................................................................................................................ 57 2.3.1 Background ................................................................................................................... 57 2.3.2 Solutions ........................................................................................................................ 60 2.3.3 General setup ................................................................................................................ 61 2.3.4 Perforated patch current clamp ................................................................................... 61 2.3.5 Voltage clamp ............................................................................................................... 62 2.4 Calcium fluorescence measurements ......................................................................... 64 2.4.1 Background ................................................................................................................... 64 2.4.2 Experimental equipment .............................................................................................. 64 2.4.3 Fluorescence recording ................................................................................................. 67 2.5 In vivo electrophysiology ............................................................................................ 68 2.5.1 Background ................................................................................................................... 68 2.5.2 ECG recording................................................................................................................ 69 2.5.3 Electroanatomical mapping .......................................................................................... 70 2.5.4 Monophasic action potentials ...................................................................................... 71 2.5.5 Conduction Velocity ...................................................................................................... 73 2.5.6 Pacemaker implantation ............................................................................................... 74 2.5.7 Conscious electrophysiological studies ......................................................................... 74 2.5.8 Arrhythmia inducibility ................................................................................................. 74 2.6 Western Blotting ......................................................................................................... 76 2.6.1 Background ................................................................................................................... 76 2.6.2 Solutions ........................................................................................................................ 77 2.6.3 Protein extraction and quantification ........................................................................... 78 2.6.4 Gel electrophoresis and transfer .................................................................................. 78 P a g e | 4 2.6.5 Protein detection .......................................................................................................... 79 2.6.6 Analysis ......................................................................................................................... 79 2.7 Statistical and analytical methods .............................................................................. 80 2.7.1 Data presentation ......................................................................................................... 80 2.7.2 Tests of statistical significance ...................................................................................... 80 2.1.2 Curve fitting................................................................................................................... 81 3 In vivo characterisation of sheep ageing model ................................................................. 82 3.1 Introduction ................................................................................................................ 82 3.1.1 The surface ECG ............................................................................................................ 82 3.1.2 Non-invasive electrophysiological testing .................................................................... 83 3.1.3 Arrhythmia inducibility ................................................................................................