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
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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 ...... 84 3.1.4 Aims ...... 85 3.2 Results ...... 86
3.2.1 Body mass ...... 86 3.2.2 ECG morphology ...... 86 3.2.3 Electrophysiological parameters ...... 88 3.2.4 Arrhythmia inducibility ...... 90 3.2.5 Effect of anaesthesia ...... 92 3.3 Discussion ...... 93
3.3.1 The P-wave duration is prolonged in older sheep ...... 93 3.3.2 The corrected sinus node recovery time is prolonged in older sheep ...... 94 3.3.3 Atrial fibrillation is induced more readily and is of longer duration in older sheep ..... 95 3.3.4 Limitations ...... 96 3.4 Conclusions ...... 97
4 Action Potential Morphology ...... 98
4.1 Introduction ...... 98
4.1.1 Action potential characteristics affected by age ...... 98 4.1.2 Calcium cycling characteristics affected by age ...... 99 4.1.3 Aims ...... 99 4.2 Results ...... 101
4.2.1 Size of atrial myocytes ...... 101 4.2.2 Baseline characteristics of patch-clamped myocytes ...... 103 4.2.3 Transmembrane action potential amplitude ...... 105 P a g e | 5
4.2.4 Transmembrane action potential duration ...... 108 4.2.5 Monophasic action potential morphology ...... 110 4.2.6 Calcium transient amplitude and rate of decay ...... 112 4.3 Discussion ...... 114
4.3.1 Myocytes are larger in the right atrium than the left and hypertrophy with age ...... 114 4.3.2 Isolated atrial myocytes are depolarised at rest ...... 115 4.3.3 Action potential amplitude does not change with age...... 116 4.3.4 Action potential upstroke recorded with perforated patch does not differ with age ...... 117 4.3.5 Action potential duration is longer in isolated myocytes than intact tissue ...... 117 4.3.6 Action potential duration prolongs with age in isolated myocytes ...... 118 4.3.7 Action potential duration is longer in myocytes from the right atrium than the left at low stimulation rates ...... 120 4.3.8 Differences in calcium transient amplitude and rate of decay did not reach statistical significance ...... 120 4.3.9 Limitations ...... 121 4.4 Conclusions ...... 122
5 Alternans ...... 123
5.1 Introduction ...... 123
5.1.1 What is alternans? ...... 123 5.1.2 Historical perspective ...... 123 5.1.3 Concordant and discordant alternans ...... 124 5.1.4 Alternans in the atria ...... 127 5.1.5 Action potential restitution as a cause of alternans ...... 129 5.1.6 The interplay between calcium cycling and action potential alternans ...... 130 5.1.7 Mechanisms underlying alternans of intracellular calcium ...... 134 5.1.8 Impaired calcium reuptake as a mechanism of alternans ...... 134 5.1.9 Altered calcium release as a mechanism of alternans ...... 135 5.1.10 Subcellular alternans ...... 136 5.1.11 Modulation of alternans ...... 137 5.1.12 Higher order periodicities ...... 138 5.1.13 Alternans and ageing ...... 138 5.1.14 Aims ...... 138 5.2 Method of alternans quantification ...... 139 P a g e | 6
5.3 Results ...... 143
5.3.1 Alternans threshold in isolated myocytes ...... 143 5.3.2 Alternans magnitude in isolated myocytes ...... 145 5.3.3 Presence of higher order oscillations in action potentials from isolated myocytes ... 147 5.3.4 Alternans threshold in vivo ...... 149 5.3.5 Alternans magnitude in vivo ...... 151 5.3.6 Higher order oscillation of action potentials in vivo ...... 153 5.3.7 Relationship between in vivo alternans and arrhythmia inducibility ...... 155 5.3.8 Regions of the action potential most affected by alternans...... 157
5.3.9 Simultaneous alternans of membrane potential and [Ca]i ...... 159 2+ 5.3.10 Disruption of [Ca ]i alternans ...... 161 5.3.11 Correlations between cellular alternans and Ca handling, APD, cell size ...... 163 5.4 Discussion ...... 165
5.4.1 Alternans occurs at lower stimulation rates in isolated myocytes than in vivo ...... 165 5.4.2 Alternans threshold decreases with age ...... 166 5.4.3 Alternans magnitude increases with age ...... 167 5.4.4 Depolarisation and repolarisation alternans occur separately ...... 169 5.4.5 The relationship between alternans and atrial fibrillation ...... 170 5.4.6 Limitations ...... 170 5.5 Conclusions ...... 171
6 Atrial conduction ...... 172
6.1 Introduction ...... 172
6.1.1 Conduction velocity ...... 172 6.1.2 Cable theory ...... 172
6.1.3 INa ...... 174 6.1.4 Connexins ...... 179 6.1.5 Fibrosis ...... 181 6.1.6 Aims ...... 182 6.2 Results ...... 183
6.2.1 Conduction velocity ...... 183 6.2.2 Sodium current recordings – baseline whole cell patch clamp parameters ...... 186
6.2.3 INa amplitude and kinetics ...... 187
6.2.4 Current / voltage relationship of INa ...... 189
6.2.5 Activation and inactivation curves for INa ...... 191 P a g e | 7
6.2.6 Time-dependent recovery from inactivation and use dependence of INa ...... 193
6.2.7 Effects of action potential duration on INa ...... 195 6.2.8 Protein expression ...... 197 6.3 Discussion ...... 199
6.3.1 Right atrial conduction velocity increases with age ...... 199 6.3.2 Left atrial myocyte capacitance increases with age ...... 201
6.3.3 INa density increases and recovers faster from inactivation with age ...... 202 6.3.4 P-wave prolongation despite faster atrial conduction may reflect atrial dilatation .. 204
6.3.5 Increased action potential amplitude is due to augmented INa ...... 205 6.3.6 Cx43 expression decreases with age in the left atrium while Cx40 does not ...... 205 6.3.7 Limitations ...... 206 6.4 Conclusions ...... 207
7 General discussion ...... 208 7.1 Sheep are a valid model for human atrial ageing ...... 208 7.2 The atrial action potential increases with age in vitro but not in vivo ...... 209 7.3 Ageing promotes action potential alternans ...... 210
7.4 Right atrial conduction velocity and INa increase with age ...... 211 7.5 Conclusions ...... 212 8 References ...... 213 9 Appendix ...... Error! Bookmark not defined.
Word count 68,465 P a g e | 8
Table of Figures
Figure 1.1 – Atrial anatomy and epidemiology...... 20 Figure 1.2 – Phases of the atrial action potential and constituent ionic currents...... 24 Figure 1.3 – Calcium cycling...... 30 Figure 1.4 – Mechanisms of arrhythmia...... 39 Figure 1.5 – Dispersion of repolarisation can promote re-entry...... 42 Figure 2.1 – Ageing of sheep using dentition...... 50 Figure 2.2 – Atrial tissue specimen with nylon cannula perfusing right atrium ...... 54 Figure 2.3 – Equipment for patch clamp recording...... 59 Figure 2.4 – Calcium fluorescence schematic...... 66 Figure 2.5 - In vivo electrophysiology...... 72 Figure 3.1 – ECG differences between young and old sheep...... 87 Figure 3.2 – Electrophysiological parameters...... 89 Figure 3.3 – Arrhythmia inducibility...... 91 Figure 4.1 – Atrial myocyte size...... 102 Figure 4.2 – Atrial myocyte baseline characteristics...... 104 Figure 4.3 – Effects of stimulation rate on action potential morphology...... 106
Figure 4.4 – Transmembrane action potential amplitude, time to peak and V̇max ...... 107 Figure 4.5 – Transmembrane action potential duration in unloaded atrial myocytes...... 109 Figure 4.6 – Monophasic action potential morphology...... 111 Figure 4.7 – Calcium transient amplitude and rate of decay...... 113 Figure 5.1 – Spatially concordant and discordant alternans...... 126 Figure 5.2 – The action potential restitution curve...... 128 Figure 5.3 – Interactions between calcium cycling and the action potential...... 131 Figure 5.4 – Repolarisation alternans precipitates depolarisation alternans ...... 132 Figure 5.5 – Quantification of alternans...... 141 Figure 5.6 – Alternans threshold in isolated myocytes...... 144 Figure 5.7 – Magnitude of alternans in isolated cells...... 146 Figure 5.8 – Higher order oscillations in isolated myocytes ...... 148 Figure 5.9 – In vivo alternans thresholds...... 150 Figure 5.10 – Magnitude of in vivo right atrial alternans...... 152 Figure 5.11 – Higher order oscillations in vivo...... 154 Figure 5.12 – Relationship between alternans behaviour and arrhythmia inducibility...... 156 Figure 5.13 – Region of action potential most affected by alternans...... 158 Figure 5.14 – Simultaneous membrane potential and intracellular calcium alternans...... 160 P a g e | 9
Figure 5.15 – The effect of SERCA blockade on alternans ...... 162 Figure 5.16 – Correlations between alternans and potential mechanisms...... 164 Figure 6.1 – Sodium channel structure ...... 175 Figure 6.2 – In vivo conduction velocity recordings...... 184 Figure 6.3 – In vivo conduction velocity results...... 185
Figure 6.5 – Current / voltage (I/V) plots of peak INa...... 190
Figure 6.6 – Activation and inactivation curves for INa...... 192
Figure 6.7 – Time-dependent recovery from inactivation and use dependence of INa...... 194
Figure 6.8 – Effects of action potential duration on INa...... 196 Figure 6.9 – Expression of proteins relevant to cardiac conduction...... 198
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Table of Tables
Table 2.1 – Ca2+-free and taurine-containing solutions ...... 52 Table 2.2 – Modified Tyrode’s solution ...... 53 Table 2.3 – Pipette solution for perforated patch recordings ...... 60
Table 2.4 – Pipette solution for measurement of INa ...... 60 + Table 2.5 – Low Na extracellular solution for measurement of INa ...... 61 Table 2.6 – Lysis and resuspension buffers ...... 77 Table 2.7 – Protease inhibitors ...... 77 Table 2.8 – NuPage transfer buffer (final concentrations for use) ...... 77 Table 2.9 – TBS-T ...... 77 Table 3.1 – ECG Parameters...... 86 Table 3.2 – Electrophysiological parameters...... 88 Table 3.3 – Arrhythmia inducibility...... 90 Table 3.4 – Effect of anaesthesia on electrophysiological parameters...... 92 Table 4.1 – Atrial myocyte size...... 101 Table 4.2 – Cell size and baseline parameters...... 103
Table 4.3 – Transmembrane action potential amplitude, time to peak and V̇max ...... 105 Table 4.4 – Transmembrane action potential duration and minimum diastolic potential ..... 108 Table 4.5 – Monophasic action potential morphology at 2 Hz...... 110 Table 4.6 – Calcium transient amplitude and rate of decay...... 112 Table 5.1 – Alternans threshold in isolated myocytes...... 143 Table 5.2 – Alternans magnitude in isolated myocytes ...... 145 Table 5.3 – Prevalence of higher order oscillations in isolated myocytes...... 147 Table 5.4 – In vivo alternans thresholds...... 149 Table 5.5 – In vivo right atrial alternans magnitude ...... 151 Table 5.6 – Prevalence of higher order oscillations in vivo...... 153 Table 5.7 – Associations between arrhythmia inducibility and alternans...... 155 Table 5.8 – Region of action potential most affected by alternans...... 157 Table 5.9 – Correlations between alternans threshold and potential determinants...... 163 Table 6.1 – Conduction velocity measurements...... 183 Table 6.2 – Cell and pipette parameters for whole-cell recordings...... 186
Table 6.3 – INa amplitude and kinetics...... 187
Table 6.4 – Activation and inactivation of INa...... 191
Table 6.5 – Time dependant recovery and use dependence of INa...... 193
Table 6.6 – The effects of an action potential clamp on INa ...... 195 P a g e | 11
List of abbreviations
Abbreviation Meaning % Percent * Significant 2+ [Ca ]i Intracellular calcium concentration [K+] Potassium concentration °C Degrees Celsius µg Microgram µL Microlitre µM Micromolar µm Micrometre 4AP 4-aminopyridine A Amperes A/D Analogue to digital ADP Adenosine diphosphate AF Atrial fibrillation AM Acetoxymethyl ANOVA Analysis of variance AP Action potential APD Action potential duration
APD50 Action potential duration at 50% repolarisation
APD90 Action potential duration at 90% repolarisation ATP Adenosine triphosphate AVN Atrioventricular node BDM 2,3-butanedione monoxime BSA Bovine serum albumin Ca2+ Calcium
CaCl2 Calcium chloride CaM Calmodulin CICR Calcium-induced calcium release
Cm Membrane capacitance CRU Calcium release unit Cs+ Caesium CsCl Caesium chloride CSNRT Corrected sinus node recovery time CsOH Caesium hydroxide CV Conduction velocity Cx37 Connexin 37 Cx40 Connexin 40 Cx43 Connexin 43 Cx45 Connexin 45 DAD Delayed afterdepolarisation DMSO Dimethyl sulfoxide EAD Early afterdepolarisation EC Excitation-contraction ECG Electrocardiogram EDTA Ethylenediaminetetraacetic acid P a g e | 12
EGM Electrogram ERP Effective refractory period F Fluorescence
F0 Fluorescence during diastole
Fmax Maximum fluorescence
Fmin Minimum fluorescence G Gravitational force of earth HCl Hydrochloric acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hz Hertz I/V Current / voltage
ICa(L) L-type calcium current
ICa(T) T-type calcium current ICD Implantable cardioverter defibrillator
IK(ATP) Adenosine triphosphate-sensitive potassium current
IK1 Inward rectifier potassium current
IKr Rapid delayed rectifier potassium current
IKs Slow delayed rectifier potassium current
IKur Ultrarapid delayed rectifier potassium current
INa Sodium current
Ito Transient outward current IV Intravenous k Slope factor K+ Potassium
K2EGTA Ethylene glycol tetraacetic acid potassium salt
KCH3O3S Methane sulphonic acid potassium salt KCl Potassium chloride
Kd Dissociation constant kHz Kilohertz LA Left atrium LQT3 Long QT syndrome type III LV Left ventricle mA milliamperes MAP Monophasic action potential MDP Minimum diastolic potential MgATP Magnesium adenosine triphosphate
MgCl2 Magnesium chloride
MgSO4 Magnesium sulphate mL Millilitres MRI Magnetic resonance imaging mRNA Messenger ribonucleic acid ms Milliseconds mTWA Microvolt T-wave alternans mV Millivolts MΩ Megaohms nA Nanoamperes Na+ Sodium
Na2H2PO4 Disodium hydrogen phosphate P a g e | 13
NaCl Sodium chloride NaOH Sodium hydroxide NCX Sodium calcium exchanger NKA Sodium / potassium ATPase nm Nanometres NS Non-significant p Probability of falsely rejecting null hypothesis pA picoamperes PC Personal computer PKA Cyclic AMP-dependent protein kinase PKC Calcium-dependent protein kinase PLB Phospholamban PMSF Phenylmethanesulfonyl fluoride PMT Photomultiplier QTc Corrected QT interval RA Right atrium
Ri Intra-cellular resistance
Rm Membrane resistance RMP Resting membrane potential RV Right ventricle RyR Ryanodine receptor s Seconds SAN Sino-atrial node SCA Spatially concordant alternans SDA Spatially discordant alternans SDS Sodium dodecyl sulphate SERCA Sarco-endoplasmic reticulum calcium ATPase SNRT Sinus node recovery time SR Sarcoplasmic reticulum TBS Tris-buffered saline TEA Tetraethyl ammonium TRPV2 Transient receptor potential vanilloid-2 V Volts
V0.5 Half-maximal voltage
VAlt Alternans magnitude VBA Visual Basic for Applications
V̇max Maximum rate of rise of action potential λ Space constant π Pi
τdecay Time constant of decay Ω Ohms
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Abstract for thesis submitted for the degree of Doctor of Philosophy
Arrhythmogenesis in the ageing atria
Charles Michael Pearman, The University of Manchester, May 2015
Atrial Fibrillation (AF) is rare amongst young people whilst epidemic in the elderly. Whilst much is known about the pathophysiology of AF, the mechanisms underlying the vulnerability to AF amongst older people in incompletely understood. Young (<18 months, first quintile of life) and old (>8 years, last quintile of life) Welsh mountain sheep were used to investigate changes in atrial electrophysiology with age.
Old sheep were more vulnerable to induced AF than young sheep. On the surface ECG, p-wave duration increased with age suggesting increasing atrial size. The corrected sinus node recovery time increased with age, suggesting deteriorating sinus node function. These findings confirmed the validity of sheep as a model for human ageing.
In isolated atrial myocytes, action potentials (APs) were recorded using the perforated patch clamp technique. AP duration increased with age, and an increase in AP amplitude was also seen at the lowest stimulation rates. Right atrial AP durations were prolonged compared to those from left atrial myocytes, and the inter-atrial difference was similar between old and young. However, when right atrial monophasic APs were recorded from anaesthetised sheep in vivo, no difference in AP duration was seen between age groups.
Alternans occurred at lower stimulation rates in old compared to young myocytes and was of greater magnitude. These age-related differences were present in isolated myocytes and in vivo. Alternans mechanisms were explored by simultaneously recording APs and intracellular calcium concentration. Atrial alternans was driven by alternans of Ca2+ cycling at low stimulation rates. However, despite disabling Ca2+ cycling using thapsigargin, alternans could still be elicited from myocytes during rapid stimulation.
Right atrial conduction velocity (CV) was assessed in vivo and found to increase with + age. A key determinant of CV, the Na current INa was investigated using the whole cell patch clamp technique. INa increased with age in left atrial myocytes and recovered faster from inactivation. Protein expression was investigated using Western blotting. Expression of the Na+ channel α-subunit did not change with age. The gap junction protein Cx43 was expressed less in older subjects, but Cx40 expression was similar.
This work has cast light on several aspects of atrial electrophysiology in which the effects of age have not been thoroughly investigated. The longer cellular APs seen with age decrease the wavelength of potential re-entrant circuits which could be seen as protective against AF. However, AP prolongation is also associated with afterdepolarisations which could serve to trigger AF. The increase in alternans behaviour may set the stage for wavebreak, leading to re-entrant circuit formation. The increase in CV was surprising and might be seen as protective against AF as it increases arrhythmia wavelength, and is likely to be caused by the increased INa. P a g e | 15
Declaration
The Western blots for Connexin 40 and Connexin 43 presented in Chapter 6 were performed with Mr George Madders and this data has also supported his application for an undergraduate Degree in Pharmacology at The University of Manchester.
No other portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.
Charles Michael Pearman School of Medicine Faculty of Medical and Human Sciences
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Copyright statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Theses
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Acknowledgements
I would like to thank the British Heart Foundation for funding this work.
I would also like to thank my supervisors, Dr Katharine Dibb and Professor David Eisner for their patient teaching and mentoring. Professor Andy Trafford has also provided great supervision in all but formal title, and Dr. Luigi Venetucci has been a great sounding board with whom to talk through ideas. Thanks are also due to my advisor, Professor George Hart.
I must thank all members of the laboratory - everyone from team atrium, especially Dr. Jess Clarke for teaching me how to isolate cells and patch, Dr. Mark Richards, Dr. Dan Wrigley and Dr. Aiste Adomaviciene. Thanks to everyone from team ventricle, especially those who uncomplainingly held sheep and administered anaesthesia for many hours - Ms. Emma Radcliffe and her motivation for running, Dr. Mike Lawless whose cooking inspires me to try harder, Ms. Charlotte Smith for her commitment to tea and Ms. Amy Watkins. Special thanks to Dr. Sarah Briston for providing example traces of afterdepolarisations and Mr. George Madders for contributing to the western blotting data in Chapter 6. Thanks to team biochem, Ms. Jess Caldwell, Dr. Margaux Horn and Dr. Becky Taylor for teaching me how to blot and not moaning too much about my gradually expanding share of the workbenches. Special thanks to Dr. Graeme Kirkwood and Dr. Dave Greensmith for showing what is possible with VBA, introducing me to XKCD and solidly reaffirming socialist values.
Finally, and most importantly, I want to thank my family – my parents, my wonderful wife Annabelle for all her support, encouragement, and hugs; my daughter Lily for making me smile; and the bump now known as Poppy for staying put just long enough. P a g e | 18
Biography
Charles Michael Pearman obtained his degree of Bachelor of Medicine and Surgery from the University of Liverpool in 2005. As part of his undergraduate studies, he undertook an intercalated MSc under Professor George Hart, studying the effects of the PKA inhibitor H-89 on the transient outward potassium current in isolated rat ventricular myocytes. Following his undergraduate studies, he completed foundation and core medical training in Liverpool, before being awarded a national training number and commencing training in clinical cardiology in the Mersey Deanery. After three years of cardiology training, he was appointed as a British Heart Foundation clinical research fellow at the University of Manchester.
Charles has experience in cellular electrophysiology, isolating cardiac myocytes and using the patch clamp technique. He has used Western blotting for protein quantification. In his clinical and research work he has an understanding and practical experience of electrocardiography; echocardiography; invasive angiography; electro-anatomical mapping; and the implantation of cardiac devices such as pacemakers, implantable cardioverter-defibrillators and vagal nerve stimulators.
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1 INTRODUCTION
You, along with anyone else reading this, have a roughly one in five chance of suffering a stroke at some time in your life1. A quarter of these potentially life-changing or life-ending strokes can be directly attributed to a disorder affecting up to one in ten older people, atrial fibrillation (AF)2. Yet still, our understanding of how AF develops in the old is incomplete.
AF, the commonest arrhythmia seen in clinical practice3, is a disturbance of the normal rhythm of electrical conduction in the cardiac atria. In healthy sinus rhythm, regular waves of depolarisation spread from the sino-atrial node (Figure 1.1A) to the remainder of the atria in a coordinated fashion, moving on in turn to the ventricles via the atrio-ventricular node. In contrast, AF is characterised by rapid meandering wavelets that, while organised in some regions, by and large traverse the atria in a chaotic manner. This lack of coordination abolishes any meaningful atrial propulsion and severely impairs the heart’s ability to effectively control the rate of ventricular contraction. Consequently patients may experience palpitations, fatigue or dyspnoea3. More seriously, atria that no longer effectively contract promote stagnation of blood in some regions of their complex geometry, specifically the pouch known as the left atrial appendage. This stagnant blood is prone to clot. The resulting thrombus carries a significant risk of floating away with the stream of blood, only to lodge in critical organs such as the brain (embolisation), obstructing blood flow and causing stroke4.
While AF is rarely seen in the young, it is endemic in the elderly. Both the incidence (the number of new cases in a period of time) and the prevalence (the total number of cases) rise steeply with age (Figure 1.1B). In North America, the prevalence of AF increases with age from less than 1% in those aged less than 50 to greater than 9% in those aged over 805). In Europe, the situation may be even worse, as prevalence rates of AF as high as 17.8 % in those aged over 85 have been described 6. We are faced with an ageing population, leading to an epidemic of AF. Despite this real and growing problem, we still do not understand the mechanisms underpinning why the old develop AF.
In this chapter, the natural history of AF will be explored. To understand how this occurs, we will examine how individual myocytes function in terms of the electrical signals that coordinate them, and the central role of calcium (Ca2+) in atrial contraction. We will need to understand how networks of cells communicate to enable them to work in unison. Armed with this knowledge, we will be well placed to see not only how abnormal heart rhythms can arise from single cells, but how this can develop into self-perpetuating arrhythmias in cardiac tissue. Finally, these fundamental mechanisms common to all arrhythmias will be put into the context of AF. P a g e | 20
Figure 1.1 – Atrial anatomy and epidemiology. A Cartoon of cardiac anatomy highlighting the atria. B Incidence and prevalence of atrial fibrillation stratified by age. Adapted from Heeringa (2006).
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1.1 The natural history and classification of AF
AF may be a temporary arrhythmia, and the reversion of AF to sinus rhythm is known as cardioversion. This may occur spontaneously or can be induced by pharmacological means or via passage of an electrical current through the atria known as ‘DC cardioversion’. The development of AF is dependent on initiating impulses, most commonly from the pulmonary vein sleeves7 (the trigger), interacting with atrial tissue that is susceptible to fibrillation (the substrate). When a healthy atrial substrate is exposed to triggering impulses, these will either fail to initiate AF or lead to merely a brief paroxysm of AF before cardioverting to sinus rhythm. If all episodes of AF spontaneously cardiovert within 7 days the condition is referred to as ‘paroxysmal AF’. A more vulnerable atrial substrate develops sustained episodes of AF in response to triggering stimuli. If the atria are not overwhelmingly diseased, these episodes may still spontaneously cardiovert, but restoration of sinus rhythm may require assistance. Episodes of AF lasting more than seven days or requiring chemical or electrical cardioversion are known as ‘persistent AF’3. A severely diseased substrate may develop ‘permanent AF’ that resists spontaneous or induced cardioversion.
A vulnerable atrial substrate can be due to extrinsic factors such as electrolyte disturbances such as abnormally low blood potassium (K+) concentrations (hypokalaemia), hormonal imbalances such as elevated thyroid hormones (thyrotoxicosis), or local inflammation such as pericarditis3. However, atrial vulnerability is more commonly due to factors intrinsic to the atrial myocardium.
Following an episode of AF, the atrial substrate remodels both structurally and electrically leading to an increased propensity to further AF, neatly summarised by Wjiffels as ‘AF begets AF’ 8. This leads to the natural history of AF often seen as initially brief self-terminating paroxysms of AF evolving to progressively longer episodes of persistent AF, and eventually permanent AF9. Once permanent AF ensues, all hope of returning to sinus rhythm is lost. If this occurs, the only treatment options remaining are to control ventricular rate and reduce the blood’s ability to clot, thereby reducing the risk of stroke. The changes in atrial structure and electrophysiology as a result of AF are known as ‘atrial tachycardia remodelling’.
To better understand what AF is, and what makes one person’s atria more susceptible to AF than another’s, we will start by looking at how the electrical activity in a single cell is brought about. P a g e | 22
1.2 The cardiac action potential
1.2.1 Phases of the action potential and their underlying ionic currents
The pattern of depolarisation and repolarisation of a cardiac myocyte known as the action potential (AP) is governed by the passage of ions across the cell membrane (Figure 1.2A and C). Although there are some key differences between the APs of ventricular and atrial myocytes which will be discussed later, the fundamental processes remain broadly similar between chambers of the heart.
The time course of the AP can be divided into five phases (Figure 1.2B). In the resting state, the sodium (Na+) / K+ ATPase (NKA) produces a large transmembrane concentration gradient of
+ + both these ions, augmenting intracellular K concentrations ([K ]i) while diminishing the + + intracellular concentration of Na ([Na ]i). While this causes a small polarisation of the cell membrane, the resting membrane potential (RMP) is for the most part set by the permeability
+ + of the cell to K . This is in turn controlled by the inward rectifying K current IK1 leading to an RMP of ~-80 mV. At low stimulation rates, ample time between stimuli is present for full repolarisation to occur. Conversely, at high stimulation rates the interval between stimuli may be insufficient for the repolarising currents discussed below to return the cell to its resting polarisation. In this case the most negative potential seen is termed the minimum diastolic potential (MDP).
From the RMP, a triggering stimulus causes a partial depolarisation of the cell membrane. In healthy intact myocardium, the trigger arises firstly from the specialised conducting tissue of the SAN, located in right atrium. The depolarisation of one myocyte leads to a passive spread of charge through gap junctions, causing a partial depolarisation of neighbouring cells. This partial depolarisation is enough to activate voltage gated Na+ channels, giving rise to the Na+
+ current INa. The activation of a minority of Na channels leads to further depolarisation, in turn triggering a cascade of Na+ channels. This positive feedback loop rapidly creates a torrential influx of Na+. The abrupt shift in membrane potential from ~-80 mV to ~+30 mV over the course of ~10 ms is termed phase 0. The maximum rate of rise of the AP (V̇max), time to peak, and the AP amplitude are all determined by INa.
The brisk flow of Na+ is, however, short lived. Na+ channels undergo time-dependent
10 inactivation, shutting off INa within two milliseconds under physiological conditions . The depolarisation also initiates repolarising currents. The first to appear is the transient outward
+ current Ito comprised of K flowing out of the cell (efflux) and, in some species although not humans Cl- flowing into the cell (influx)11, 12, both of which cause a net decrease in positive P a g e | 23 charge from the cytosol. These currents create a sharp initial repolarisation of the AP termed phase 1.
2+ The Ca currents ICa(L) and ICa(T), which have remained dormant until the cell membrane has significantly depolarised during phase 0, activate slowly and only attain their maximum current by the time Na+ channels have inactivated. These additional depolarising inward currents are counterbalanced by the repolarising efflux of K+ through the delayed rectifier K+ currents IKur, IKr, and IKs. For a time, the inward and outward currents are nearly equal, causing a period of little net change in membrane potential. This has the appearance of a plateau and enables a sustained contraction of the myocyte. The plateau is referred to as phase 2 of the AP and can be easily identified in the ventricular AP in Figure 1.2Bi but is much less pronounced in the atrial AP in Figure 1.2Bii.
As the plateau continues, repolarising K+ currents increase while depolarising Ca2+ currents decrease. This creates an acceleration of repolarisation back towards the RMP. However, offsetting this is the extrusion of Ca2+ ions in exchange for Na+ ions via the Na+ / Ca2+ exchanger (NCX). The exchanger permits the entry of three Na+ for every Ca2+, thereby causing a net inward current. As a passive transporter, this current is driven by the electrochemical gradient and therefore is predominantly active when the cell has largely repolarised13. The region of the AP demonstrating pronounced repolarisation is termed phase 3 of the action potential. The action potential duration (APD) is the time taken from AP onset until repolarisation. This is commonly specified as repolarisation to 50% of MDP (APD50) or 90% repolarisation (APD90), and its calculation can be seen in Figure 4.4. The final phase of the AP is phase 4 – a return to the RMP. P a g e | 24
Figure 1.2 – Phases of the atrial action potential and constituent ionic currents. A Ionic currents responsible for the atrial action potential. Note differences in scaling on y-axis. B Phases of the (i) ventricular action potential and (ii) atrial action potential. C Superimposed ionic currents to highlight relative amplitudes. Adapted from Grandi et al. (2011). P a g e | 25
1.2.2 Regional differences in action potential morphology
This description of cardiomyocyte APs contrasts with that seen in cardiac conduction tissue, which is characterised by spontaneous diastolic depolarisations in phase 4 and a slower phase 0 upstroke14. The slow phase 4 depolarisation, caused by the hyperpolarisation activated current If, eventually reaches threshold and triggers another AP. Faster phase 4 depolarisation reaches threshold sooner, therefore elevating the heart rate. The rate of phase 4 depolarisation is faster in the SAN than in the AV node, which is again faster than that seen in the Purkinje fibres. This means that in health, the dominant pacemaker is the SAN. If myocytes from another region of the heart depolarise faster during phase 4, as might be seen if the SAN is abnormally slow or another region is abnormally fast, the faster region becomes the dominant pacemaker.
Important differences also exist between ventricular and atrial APs. The RMP of atrial myocytes is 5-10 mV less negative than that seen in ventricular myocytes, due to a six-fold
15 reduction in IK1 . Despite this reduction in one repolarising current, a shorter APD is seen in the atria than the ventricles. This is due in part to an increase in the ultrarapid delayed
+ + rectifying K current IKur and the acetylcholine sensitive K current IK(Ach), which are absent or only present at low levels in the ventricle16,17. Differences in repolarizing currents are also present between the left and right atria, as the right atrium exhibits less IKr than the left atrium, leading to the shorter left atrial APD18.
There are also atrioventricular differences in depolarizing currents. Less Ca2+ influx is seen in
19 2+ atrial than ventricular myocytes due to faster decay of ICa(L) , mirrored by lower Ca efflux and 20 lower NCX expression . Conversely, the contribution of ICa(T) is negligible to the AP of healthy ventricular myocytes but is of greater significance in the atria13.
1.2.3 Modulation of action potential morphology
Far from being an unchanging motif, the shape of the AP adapts to changing circumstances. A physiological modulator of AP morphology is the autonomic nervous system. Independently of changes in heart rate, β-adrenergic stimulation increases ICa(L) thereby elevating the atrial AP 21 plateau and prolonging APD50 . In contrast, the effects of adrenergic tone on the terminal portion of the atrial AP are less clear. There are conflicting reports of longer22, shorter23 or
21 unchanging APD90 in response to the β-adrenergic agonist isoprenaline. As isoprenaline enhances many repolarising currents as well as ICa(L), the variety of effects seen may represent 24 differences in the balance of currents expressed between species . INa is increased by 25 26 27 adrenergic stimulation , resulting in an increase in atrial AP amplitude and V̇max . P a g e | 26
27 Parasympathetic tone forces the MDP to more negative potentials by increasing IK(ACh) . This indirectly increases AP amplitude and V̇max by enhancing INa recovery from inactivation. APD50 27, 28 and APD90 both decrease in response to parasympathetic stimulation . It has been suggested that the effect of vagal stimulation on MDP and APD may be enhanced in older atria28, 29.
In addition to autonomic modulation, AP morphology is influenced by the metabolic status of the myocyte. At times of cardiac stress such as ischaemia or increased myocardial energy use, the concentration of adenosine triphosphate (ATP) decreases while the concentration of
+ adenosine diphosphate (ADP) increases. The ATP-sensitive K current IK(ATP) is blocked by ATP 30 and opened by ADP . Activation of IK(ATP) has the effect of shortening AP duration and hyperpolarizing the cell membrane. It has been suggested that the APD shortening caused by
30 IK(ATP) may offer some form of myocardial protection at times of stress , as a shortening of the 31 AP plateau reduces myocyte contractility . However, activation of IK(ATP), while shortening APD, does not appear to influence the Ca2+ transient32, a decrease of which could reduce myocardial energy demands. Instead, the AP shortening produced by IK(ATP) can lead to early after-depolarisations33, contributing to the risk of arrhythmias in the setting of acute ischaemia.
Mechanical changes affecting the atrium as a whole also influence the AP. Atrial stretch may occur due to changes in circulating volume, ventricular function or valvular heart disease, which may in turn promote atrial fibrillation34. In a Langendorff-perfused rabbit heart model, acute atrial stretch by volume overload led to a shortening of the monophasic AP with an associated increase in vulnerability to AF35. It is thought that these changes are due to the stretch-activated K+ current possessed by atrial myocytes36.
2+ [Ca ]i influences various ion currents responsible for AP morphology, achieving this via two mechanisms: firstly, Ca2+ interacts with calmodulin (CaM), an important second messenger protein that directly regulates ion channels, pumps and transporters. Secondly, Ca2+ bound to CaM controls Ca2+ - calmodulin kinase II (CaMKII), an enzyme that phosphorylates many
37 cellular constituents . CaMKII suppresses Ito, which contributes to AP shortening in the 2+ 38 presence of persistently elevated [Ca ]i . In addition, there is some evidence for specific Ca2+-activated K+ currents39. In response to Ca2+ overload caused, for example, by sustained rapid stimulation as seen in AF, Ca2+-activated K+ currents open, hyperpolarising the cell membrane and shortening the AP duration.
Finally, the interactions between myocytes and other cell types may influence the shape of the AP. These ‘electrotonic effects’ between myocytes and fibroblasts have been shown P a g e | 27
40 experimentally to partially depolarise the RMP . This leads to a reduction in INa due to impaired recovery from inactivation, thereby slowing V̇max and reducing AP amplitude. Coupling of myocytes to electrically active fibroblasts has been suggested by modeling studies to shorten AP duration41, whereas coupling to electrically inactive cells may prolong AP duration42.
1.2.4 Action potential characteristics affected by AF remodelling
AP duration decreases following induction of AF in animal studies43. Similarly, patients suffering from longstanding persistent AF44, 45 but not paroxysmal AF46 exhibit a shorter atrial AP duration than those in sinus rhythm, and this has been postulated to be a major cause for the vicious circle of ‘AF begets AF’8. The decreased AP duration is largely due to a reduction in
44 2+ ICa(L) . Calpains, a family of enzymes known to degrade Ca channels, may be responsible for 43 the decrease in ICa(L) seen in response to AF . Increases in repolarizing currents such as IK1 and 44, IK(Ach) but potentially decreases in IKur and Ito may also contribute to the shortening of the AP 47, 48. The Ca2+ overload caused by continuous rapid atrial stimulation activates CaMKII, which
38 2+ increases Ito and therefore contributes to APD shortening . The increase in Ca may also potentiate Ca2+-activated K+ currents which also shorten the AP duration. However, any effect on Ca2+-activated K+ channels must occur solely in the early stages of the remodeling process as these channels are downregulated in patients with chronic AF39.
The effects of AF remodelling upon AP amplitude are unclear, with some groups reporting that
43, 49 50 amplitude increases , while others suggest that it decreases . INa, a key determinant of AP amplitude, has consistently been shown to decrease following rapid atrial pacing51, 52 and therefore another explanation is needed to bring these disparate findings together. Some reports showing an increased AP amplitude following AF can be explained by concomitant
49 hyperpolarisation of the RMP which would enhance INa recovery from inactivation, although this idea cannot support the findings from all studies43.
Again, the effects of AF on the RMP are uncertain, as studies have shown a more negative49,
43 50 less negative or unchanged RMP after rapid atrial pacing in dogs. IK1, which sets the RMP, does not differ between dogs in sinus rhythm and those in AF50, although patients with AF
44 have a greater IK1 than those in sinus rhythm . These discrepancies may be explained by the duration of tachycardia remodelling. In atrial myocytes from patients with chronic AF, hyperpolarisation of the RMP was seen47. However, a short period of tachypacing applied to myocytes from patients in sinus rhythm led to depolarisation of the RMP47. The full cellular electrophysiological changes of AF may therefore take place over an extended timescale. P a g e | 28
1.3 Excitation-contraction coupling
1.3.1 Calcium cycling
The AP is in part a messenger, synchronising the excitation of many cells to provide a purposeful atrial contraction. Within each individual cell, this message is carried from the cell
2+ 2+ membrane to the contractile elements by Ca . The rise and fall of [Ca ]i during a contraction, known as a Ca2+ transient, is governed not only by the flow of Ca2+ into and out of the cell but also the release and reuptake of Ca2+ from intracellular stores.
Some Ca2+ exists in a freely ionised state, but more than 99% of total intracellular Ca2+ is bound to proteins such as troponin C and SERCA. To flow across cell membranes, between cellular compartments, or activate myofilaments contraction, Ca2+ needs to be in an unbound, freely ionised state. The majority of the Ca2+ entering the cytoplasm will become bound to proteins,
2+ 2+ 2+ meaning that free [Ca ]i will rise less than the total Ca entry. The same is seen when Ca is 2+ released from intracellular stores – a lot is released to achieve a small increase in free [Ca ]i. This is referred to as Ca2+ buffering, and an increase in buffering means that more Ca2+ entry or
2+ 2+ Ca release from the SR is required to achieve the same increase in free [Ca ]i.
The membrane depolarisation of the AP causes Ca2+ to enter the cell via the L-type Ca2+ channel, which as described above leads to the current ICa(L) (Figure 1.3B(i)). This small influx is insufficient to cause substantial cellular contraction by itself. Instead, a contraction requires a
2+ 2+ large increase in [Ca ]i which can only be provided by the major intracellular Ca store, the sarcoplasmic reticulum (SR)13. Specialised Ca2+ release units on the SR known as ryanodine receptors (RyRs) exhibit the curious positive feedback property of discharging a large amount
2+ 2+ of Ca in response to exposure to a small amount of Ca . This is referred to as ‘calcium- induced calcium release’ (CICR) (Figure 1.3B(ii)).
Deep invaginations of the cell membrane known as T-tubules bring the L-type Ca2+ channel close to the RyRs on the SR membrane, thus enabling rapid synchronous CICR. The size of the Ca2+ transient is governed firstly by how much Ca2+ is present within the SR, secondly by how
2+ 2+ much of this SR Ca content is released, and thirdly by how much Ca enters the cell on ICa(L). 2+ In this respect ICa(L) contributes twice, as increasing ICa(L) augments the proportion of SR Ca content that is released through the RyR.
That which rises must fall. Ca2+ released from the SR must be returned, and Ca2+ that enters the cell must leave it, allowing the cell to relax. Ca2+ reuptake is achieved via the sarco- endoplasmic Ca2+ ATPase (SERCA) (Figure 1.3B(iii)), while Ca2+ extrusion occurs for the most P a g e | 29 part passively via the Na+ / Ca2+ exchanger (NCX) (Figure 1.3B(iv)), but ~1% is extruded actively using the sarcolemmal Ca2+ ATPase. During steady contractions, the flow back and forth across the cell membrane must balance otherwise the cell would gradually gain or lose Ca2+. Similarly, the flow to and from the SR must also balance53.
In addition to flow between the cytoplasm and the SR, other cellular compartments may influence Ca2+ cycling. The mitochondria make up ~30% of the cardiomyocyte volume and Ca2+ is integral to mitochondrial function. However, the flow into and out of the mitochondria takes place over a longer timescale than that seen during a typical cardiac Ca2+ transient, meaning that changes in mitochondrial [Ca2+] are almost undetectable during a single cardiac cycle13.
P a g e | 30
Figure 1.3 – Calcium cycling. A Key proteins involved in Ca2+cycling. B Ca2+ enters the cell via the L-type Ca2+ channel. C Ca2+ entry stimulates Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. D Ca2+ is taken back into the SR by SERCA. E Ca2+ is removed from the cytosol by the Na+ / Ca2+ exchanger. LTCC - L-type Ca2+ channel, NCX - Na+ / Ca2+ exchanger, NKA - Na+ / K+ ATPase, PLB - phospholamban, SERCA - Sarco-endoplasmic reticulum Ca2+ ATPase. Adapted from Bers (2001). P a g e | 31
1.3.2 Modulation of calcium cycling
AP morphology is determined in part by ICa(L). Conversely, ICa(L) is determined in part by AP morphology. Any ionic current is governed by both the electromotive driving force and also the activation / inactivation state of the channel. A longer AP means that the membrane is held at a less negative potential for longer. This provides less of a driving force for Ca2+ entry, but equally keeps more Ca2+ channels in an activated state. The net effect of these opposing
2+ 54 influences is that a longer AP leads to a greater total Ca influx . The increase in ICa(L) leads to greater CICR, and it has been shown that prolongation of the AP plateau increases Ca2+ transient amplitude, although prolongation of the terminal phase of AP repolarisation alone
31 (when ICa(L) is inactive) does not .
Ca2+ exerts a negative feedback effect on voltage gated Ca2+ channels via CaM. This is seen as
2+ Ca -dependent inactivation of ICa(L) whereby the current inactivates faster in the presence of Ca2+ than when Ca2+ is chelated and barium is used as a charge carrier13. On the other hand, a positive feedback can also be seen, modulated by CaMKII. In response to an increase in pacing
2+ rate, the Ca influx increases. This effect is blocked by inhibitors of CaMKII, and is thought to 2+ 55 represent facilitation of ICa(L) by CaMKII in response to increasing [Ca ]i . Facilitation of 2+ ICa(L),combined with increasing SR Ca content contribute to the ‘positive staircase’ effect whereby myocytes contract more forcefully at faster stimulation rates.
The autonomic nervous system modulates ICa(L). β-adrenergic stimulation increases ICa(L) via protein kinase A (PKA) 56. This, in turn, potentiates an increase in Ca2+ transient amplitude via CICR.
2+ 2+ RyR function can be modulated, and is strongly influenced by [Ca ]i. An increase in SR Ca content can be expected to increase the flow through the RyR simply as a function of an increased concentration gradient. However, in addition to this, elevated SR Ca2+ content increases the sensitivity of the RyR to cytoplasmic Ca2+ 57. The effects of CaMKII on RyR function are uncertain, with some lines of evidence pointing to CaMKII enhancement58 while others suggest CaMKII suppression of RyR function59. The RyR can also be phosphorylated by PKA, leading to both an increase in RyR peak opening probability but also more rapid inactivation60.
SERCA function is modulated predominantly by the protein phospholamban (PLB) 61. PLB inhibits SERCA, slowing Ca2+ reuptake. PLB can be phosphorylated, but phosphorylation by different enzymes occurs in separate locations and has very different effects. When PLB is phosphorylated by PKA, the inhibitory effects of PLB on SERCA are reversed (i.e. increases P a g e | 32
SERCA pumping activity). Conversely, phosphorylation by PKC potentiates PLB (i.e. decreases SERCA pumping activity)13. While PLB is responsible for the vast majority of SERCA modulation in the ventricle, in the atrium PLB is complemented by a similar protein called sarcolipin62, 63. Sarcolipin has a similar role to PLB, acting as a brake on SERCA activity thereby slowing Ca2+ reuptake, and in the presence of β-adrenergic stimulation the inhibitory effect of sarcolipin is diminished, accelerating Ca2+ reuptake.
To summarise the effects of β-adrenergic stimulation on Ca2+ cycling: β-adrenoceptors activate
PKA which directly increases ICa(L); the increased ICa(L) may be potentiated by the positive feedback effect of CaMKII; SERCA function is enhanced by withdrawal of PLB inhibition; enhanced SERCA elevates SR Ca2+ content; the elevated SR Ca2+ content increases the responsiveness of the RyR; the RyR is also directly stimulated by PKA. The net result is a larger and more rapidly decaying Ca2+ transient.
1.3.3 Calcium cycling characteristics affected by AF remodelling
The entry of Ca2+ into the cell is altered by atrial tachycardia remodeling. In keeping with the reduction in ICa(L) seen in patients with AF, tachycardia remodeling in sheep leads to a reduction 64 2+ in ICa(L) . Even though SR Ca content does not change with AF, the reduced ICa(L) leads to a decrease in fractional SR Ca2+ release and therefore reduced Ca2+ transient amplitude64. The
65 change in ICa(L) occurs rapidly and can be seen after only three minutes of atrial tachycardia . This decrease is due to Ca2+ overload, and is initially reversible65 but becomes permanent after long-term stimulation64. This timecourse may represent the acute effect of CaMKII on channel function but a chronic effect of CaMKII on channel synthesis.
Likewise, the release of Ca2+ from the SR is affected by atrial tachycardia remodeling. A study of dogs exposed to atrial tachypacing suggested that total RyR mRNA and protein were decreased by AF66, although this finding has not been repeated67, 68. The RyR becomes hyperphosphorylated by PKA during AF in atrial tachypaced dogs, and similar changes are seen when comparing atrial tissue from patients in AF vs. sinus rhythm69. The phosphorylated status conveys an increased open probability under diastolic conditions, predisposing to spontaneous Ca2+ release, afterdepolarisations, ectopic beats and therefore reinitiation of arrhythmia. Increased phosphorylation of the RyR by CaMKII has also been seen in atrial tachypaced goats67. The changes in RyR phosphorylation may take some time to be realised. One week of tachypacing reduces Ca2+ transient amplitude without affecting RyR expression or phosphorylation in dogs68 and rabbits70. This is reflected in a similar rate of spontaneous Ca2+ sparks between control and one-week tachypaced rabbits70. P a g e | 33
Furthermore, AF may affect the reuptake of Ca2+ into the SR. Inconsistent results have been reported, potentially relating to the duration of AF. Patients with longstanding AF show slowed Ca2+ reuptake71, although other groups have shown similar expression of SERCA and PLB between these groups72. In contrast, in human studies examining short-term (paroxysmal) AF, accelerated Ca2+ reuptake was seen. Surprisingly, this corresponded with a decrease in SERCA expression but could be explained by increased phosphorylation of PLB46. In animal studies examining the short term effects of atrial tachycardia remodeling, no difference in Ca2+ reuptake was seen between 6-week atrial tachypaced dogs and control73. In atrial tachypaced goats, the proportion of PLB that had been phosphorylated by PKA was reduced while total PLB was unaffected67.
In summary, AF leads to reduced Ca2+ entry and smaller Ca2+ transients, although SR Ca2+ content is unaffected. RyRs are hyperphosphorylated, potentially leading to leak and spontaneous Ca2+ release. Ca2+ reuptake may be slowed in chronic AF, but this does not seem to be the case in AF of shorter duration.
P a g e | 34
1.4 Cardiac conduction
We have seen above how an electrical impulse acts on a single cell, opening channels to flood the cell with positively charged ions, triggering the release of Ca2+ from intracellular stores, only for the cascade to reverse and restore the equilibrium present before the impulse arrived. A single cell contracting independently is of little use, and to achieve anything meaningful myocardial cells need to contract together. How does one cell communicate with the next to coordinate their efforts?
Within tissue, the region of membrane belonging to one myocyte that touches another contains specialised proteins that link one cell to the next. If we picture a line of cells as a train, some proteins physically hold the cells together like coupling linkages between the carriages, but other proteins called gap junctions connect the cytoplasm of cells, like the rubberised tunnels that passengers can walk through. Charged particles can flow through the gap junctions connecting one cell to another. When one cell becomes excited and an AP fires, some of the large depolarisation can be transmitted through gap junctions to partially depolarise the inactive neighbouring cell. This brings the neighbouring cell’s membrane potential to the threshold needed for Na+ channel activation and triggers an action potential.
Instead of a single train, myocardial tissue is arranged more like many parallel trains side by side forming sheets, with several sheets stacked vertically. While connections exist between parallel trains, it is much easier to walk along a train than clambering from one to another. Myocardial tissue is much the same, with many gap junctions at the ends of cells and fewer along the sides. It is therefore easier to propagate an impulse along a fibre than it is for the impulse to travel perpendicular to the fibres. This disparity in how fast an impulse travels in one direction compared to another is called anisotropic conduction74. Some have suggested tissue that is overly anisotropic, that has very poor side-to-side conduction, is more prone to arrhythmias75. However, the changes in distribution of gap junctions as a result of atrial tachycardia remodelling tend to decrease anisotropy76, which does not immediately fit with our understanding of how AF begets AF.
The excitation of one cell spreading out to activate the next, and the next, forms a wave of depolarisation. The wave normally travels in only one direction. Excitation is prevented from travelling back in the direction it has come by the slow repolarisation of cells that have just been excited. A second AP cannot be triggered from the upstream cells immediately, as the Na+ channels needed for the AP upstroke need to recover from inactivation, which only occurs once the cell has largely repolarised. By the time the upstream cell is ready to fire again, the P a g e | 35 wave of depolarisation has passed. The time period during which a second AP cannot be elicited is called the effective refractory period (ERP), an important parameter in arrhythmogenesis.
The speed with which a wave propagates can be measured, and is referred to as the conduction velocity (CV). The factors that determine CV will be explored in detail in Chapter 6.
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1.5 Mechanisms of arrhythmia
We have seen how in health, starting from the SAN, electrical impulses conduct through sheets of myocardial tissue as individual cells depolarise sequentially, conveying the impulse to adjacent myocytes through gap junctions. The wave of depolarisation formed is followed by repolarisation, before which no further excitation can be produced. However, this order can break down. The initiation of arrhythmias often begins with a premature impulse arising from somewhere other than the SAN (an ectopic focus). Regions of the heart may spontaneously depolarise faster than the SAN (enhanced automaticity), and secondary depolarisations can be generated immediately following an AP (triggered activity). After an initiating premature impulse, an arrhythmia can be maintained by the breakdown of an orderly wavefront, instead forming self-perpetuating circuits around an obstacle (re-entry). These can form a vortex, becoming spiral or scroll waves that may disintegrate at their edges into chaotic, seemingly random wavelets.
1.5.1 Enhanced automaticity
In health, only specialised conduction tissue shows automaticity, spontaneously depolarising to produce APs with the SAN leading the way. Under pathological conditions, contractile myocardium can also become automatic, and the resulting spontaneous APs are often produced at a much faster rate than normal. This enhanced automaticity is seen when the
77 + RMP becomes less negative . As the RMP is set by K currents, predominantly IK1, a less negative RMP can be caused by mishandling of K+. Excess extracellular [K+] (hyperkalaemia) reduces the driving force for K+ efflux and depolarises the RMP13. Genetic conditions that inhibit IK1 such as Andersen-Tawil syndrome achieve a similar effect, depolarising the RMP and generating abnormal automaticity78. Conversely, abnormally low extracellular [K+] (hypokalaemia) hyperpolarises the RMP, but can instead promotes arrhythmias through other mechanisms79. Hyperpolorisation of the RMP can slow automaticity, as can be brought about by rapidly stimulating or ‘overdrive pacing’ spontaneously depolarising tissue80. This phenomenon can be exploited to test sinus node function, as will be seen in Chapter 3.
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1.5.2 Triggered activity
A second source of ectopic discharges comes from ‘triggered activity’, so called because the abnormal discharges occur after, or are triggered by, a normal AP. These discharges take place within a single cell, although may require simultaneous occurrence in a number of cells within a tight region to generate an impulse that propagates through tissue81.
Early afterdepolarisations (EADs) refer to a second excitation that occurs before the initial AP has fully repolarised, and are usually seen in the context of abnormally long APs (Figure 1.4A). These long APs are commonly caused by genetic conditions such as long QT syndrome, which promotes EADs and the malignant arrhythmia Torsades de Pointes78. A similar phenotype is seen in clinical practice secondary to drugs that prolong the AP such as the IKr antagonist sotalol. Although the precise mechanism of EADs is still debated and may take different forms, the prolonged APs may allow L-type Ca2+ channels to recover from inactivation whilst still providing a sufficiently depolarised membrane to permit re-activation82. This theory is
+ supported by pharmacological evidence that increasing ICa(L) or inhibiting K currents promotes EADS.
Triggered activity also occurs after the AP has completed. These are known as delayed afterdepolarisations (DADs), and are produced by a different mechanism to EADs (Figure 1.4B). RyRs can spontaneously release Ca2+ during diastole. If this opening leads to a sufficiently large Ca2+ flux, neighboring RyRs open via CICR. This progresses as a slowly propagating ‘Ca2+ wave’
2+ + along the SR. The resulting increase in [Ca ]i is extruded via NCX in exchange for Na , producing a net inward current. If large enough, this inward current brings the membrane to threshold, triggering an AP. The clinical exemplar is catecholaminergic polymorphic ventricular tachycardia, a genetic condition causing defective, leaky RyRs. During additional SR Ca2+ loading such as during sympathetic stimulation, Ca2+ waves develop, causing DADs and a bidirectional ventricular tachycardia on the surface ECG78. DADs occur more frequently in atrial myocytes from patients with a history of AF71. These have been suggested to be due to enhanced leak from CaMKII phosphorylated RyRs, increasing the chance of Ca2+ waves. Furthermore, elevated NCX expression in these cells creates a larger inward current in response to waves, increasing the chance that depolarisation will reach threshold and trigger a premature AP71.
A third type of triggered activity has been described which may have particular relevance to AF. ‘Late phase 3 EADS’ combine features of EADs and DADs, occurring before the end of the AP similar to the EAD, provoked by the enhanced SR Ca2+ loading similar to the DAD but crucially occurring when the AP is shortened , rather than the prolongation that produces P a g e | 38
EADs78. These circumstances can be produced by combined sympathetic and parasympathetic activation, with sympathetic tone enhancing SR Ca2+ content and parasympathetic tone augmenting IK(ACh), shortening the AP duration. These conditions have been shown to induce AF associated with phase 3 EADs in dog atria83.
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Figure 1.4 – Mechanisms of arrhythmia. A (From left to right) Action potential from sheep ventricular myocyte, small EADs, large EADs. B (From left to right) small EADs and sub-threshold DAD, EAD and DAD triggering a premature AP. Note artefact from stimulation present in second action potential. C Cartoon of re-entrant circuit formation. EAD - early afterdepolarisation, DAD - delayed afterdepolarisation, Stim - stimulation artefact. Traces of action potentials from ventricular myocytes have been filtered for illustrative clarity and are courtesy of Dr. Sarah Briston.
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1.5.3 Re-entry
Triggered activity is a powerful mechanism by which an arrhythmia can be initiated. Re-entry is a powerful mechanism by which it can be maintained. Rather than following a linear path from, for example, the top of the atrium to the bottom, a re-entrant circuit is one by which the wave of depolarisation circles around an obstacle, persistently chasing its own tail. The obstacle can be structural, such as the mitral valve or a scar from a myocardial infarction; but may be functional, being only a region that is temporarily refractory to stimulation.
Re-entry is often initiated by a premature impulse caused by, for example, triggered activity. In a situation where an obstacle to propagation is encountered by the wave of depolarisation, several things may happen. If the tissues on either side of the obstacle share a similar CV and ERP, the wavefront will split around the obstacle, conduct similarly on either side, and reform distally (Figure 1.4C(i)). However, if the tissue on one side of the obstacle repolarises slowly, giving a long ERP, a premature impulse may block on only one side of the obstacle (unidirectional block, Figure 1.4C(ii)). If conduction proceeds slowly enough down the excitable side of the obstacle, the wavefront can proceed down and back up the other side, by which time the once refractory limb has now regained excitability (Figure 1.4C(iii)). The wavefront then continues to proceed around the obstacle, exciting tissue rapidly on either side but maintaining its own circuit.
Three conditions are needed for a premature impulse to generate re-entry : an obstacle, unidirectional block (a long enough ERP to block initially, but short enough to recover in time) and a zone of slow conduction. The minimum distance a re-entrant circuit can successfully accommodate is determined by the product of the ERP and the CV, and is called the wavelength. A shorter wavelength can successfully re-enter around a smaller obstacle and allow more simultaneous re-entrant circuits to coexist in a fixed volume of tissue. This suggests that AF may be facilitated by shorter wavelengths. Supporting this view, in animal84 and human85 studies the duration of induced AF increases as atrial wavelength decreases. Changes in wavelength also contribute to the electrical aspect of atrial tachycardia remodelling, as the atrial ERP and CV decrease in response to sustained AF86. Wavelength cannot fully explain atrial vulnerability, as others have not found a significant relationship between the two87, 88.
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1.5.4 Factors promoting wavebreak
Re-entrant circuits commonly form around anatomical obstacles, with a classical example being typical atrial flutter whereby the depolarising wave moves from top to bottom and back again around the right atrium, circumnavigating the inferior vena cava and the tricuspid valve. Re-entrant circuits also occur where no structural barriers are found, and the core of the circuit is formed from tissue that is merely functionally refractory at a critical point in time.
The conditions needed to set this up rely upon variability in how rapidly small regions of tissue can repolarise, referred to as the dispersion or repolarisation. All myocytes naturally show some variability, but these differences are smoothed out when cells are connected electrically, known as electrotonic effects. Unsurprisingly, deficient intercellular coupling is associated with a greater dispersion of repolarisation89. If the differences persist then one area of tissue may be refractory to stimulation while adjacent tissue has regained excitability. This is demonstrated in Figure 1.5. If tissue repolarises at the same rate throughout, a premature impulse will either conduct everywhere or be blocked everywhere. (Figure 1.5A). When significant dispersion of repolarisation is present (Figure 1.5B), a premature impulse blocks in some regions that are still refractory but conducts in others that have regained excitability. The wavefront curves around towards excitable cells (Figure 1.5C,D), and can form a complete re-entrant circuit (Figure 1.5E).
Rather than being a fixed property, dispersion is influenced by the rate of stimulation. Tissue that is homogenous when stimulated slowly can show marked dispersion when stimulated rapidly. This is particularly seen when, rather than every AP in a sequence looking similar, an oscillation occurs whereby short APs are followed by long APs, repeating in a long-short-long-short pattern known as alternans. Alternans, generating dispersion of repolarisation, is heavily implicated in the onset of arrhythmias90, and will be discussed at length in Chapter 5.
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Figure 1.5 – Dispersion of repolarisation can promote re-entry. A When repolarisation is homogenous throughout tissue, waves of depolarisation (glowing cells) proceed in an orderly fashion. Any premature stimulus is uniformly blocked and does not proceed. B If repolarisation is delayed in some regions (dark cells) but not others (light cells), a premature impulse can be conducted along only one side of a functional obstacle. C The wave front curves towards excitable tissue. D Completion of repolarisation in the refractory cells allow the wavefront to curve back in the direction in came from. E The circuit completes and re- entry continues. P a g e | 43
1.6 Arrhythmic mechanisms applied to atrial fibrillation
The general arrhythmia mechanisms described above of enhanced automaticity and triggered activity generating premature impulses from ectopic foci, interacting with tissue that has a dispersion of repolarisation causing wavebreak and re-entry, are common to many arrhythmias. How does AF fit into this conceptual framework?
1.6.1 Pulmonary vein ectopy
The ectopic foci generating the impulses that initiate AF are often found within sleeves of myocardium that extend into the pulmonary veins91. These myocytes can develop enhanced automaticity or may experience early or delayed afterdepolarisations92. Some of these myocytes may express pacemaker currents, and combined sympathetic and parasympathetic activation often prompts their automaticity. Despite being frequently found as a source for ectopic foci, some investigators have found no difference between the Ca2+ handling characteristics of pulmonary vein sleeve myocytes with those from the left atrium93. The frequent contribution of the pulmonary veins s to AF initiation can be testified to by the effects of electrically disconnecting them from the body of the left atrium, a maneuver which often prevents AF occurrence in the short term94.
Some have suggested that in addition to initiating AF, continuous rapid discharges from the pulmonary veins may maintain AF in some cases. Discharges from the pulmonary veins occurring at a faster rate than left atrial activation have been seen in tachypaced dogs95, but the relevance of this to human AF has been questioned96.
In response to premature stimuli, the atria may continue to conduct normally, but in a vulnerable atrial substrate wavebreak occurs. The significance of dispersion to wavebreak is borne out by experimental work showing that atria displaying greater dispersion are more vulnerable to AF84, as are atria that are prone to alternans97. Equally, atrial tachycardia remodelling increases atrial dispersion87 and alternans98.
1.6.2 Multiple wavelets
Following wavebreak, AF has classically been described as devolving into a chaotic pattern of activation with no discernible order. In 1964, Moe et al. proposed the multiple wavelet hypothesis to explain this99. They envisaged many tiny functional re-entrant circuits that continually extinguished and reformed. Their simulations suggested that a critical number of wavelets were required to maintain AF, therefore needing a minimum volume of atrial tissue. This idea ties into observations that dilated atria are more susceptible to AF100. Furthermore, P a g e | 44
AF can be suppressed by creating electrical barriers within the atria to reduce any contiguous areas below the minimum required to sustain the arrhythmia101. Their model also predicted the proarrhythmic potential of shortening the ERP and increasing dispersion.
Meandering wavefronts showing breakdown and reformation were seen in vitro in large mammalian102 and human tissue103. Functional re-entry that drifted through the tissue before extinguishing at tissue boundaries could be stabilised and converted to an organised regular tachycardia if structural obstacles were introduced104.
The arrhythmia wavelength paradigm complemented this hypothesis, as more re-entrant circuits can be accommodated with a shorter wavelength105. Compared to goats undergoing short-term atrial remodelling, goats subjected to six months of atrial tachycardia remodelling demonstrated more simultaneous wavelets in conjunction with slower atrial conduction106.
1.6.3 Rotors
An alternative theory is that of a dominant re-entrant circuit or mother rotor. If chaotic wavelets were the sole driver of AF, it would be expected that the frequencies of activation measured at any point in the atria would be random. Instead, in the isolated sheep heart it was shown that one or a small number of dominant frequencies of activation occurred, an order shining from the chaos107. The dominant frequency varied from region to region, and was explained as a hierarchy whereby the points exhibiting the highest frequencies were the ‘masters’ while other regions were the ‘slaves’. The regions with the fastest dominant frequency, proposed as the drivers of AF, were suggested to represent the core of a rapidly rotating re-entrant circuit108.
Rotors can be seen as a spiral wave in two dimensions, or a ‘scroll wave’ when visualised in three dimensions. The core of the rotor is not excited, and may meander. The electrotonic influences from the non-excited core mean that these cells can contribute to the repolarisation of excited cells near the core, shortening their AP durations. Furthermore, a curved rotating wavefront accelerates the usual CV. These combine to enable a very high stimulation frequency at the centre. However, further from the core, the spiral has slower conduction and little electronic influence from non-excited tissue, decreasing the frequency of activation109. Further out still, the rotor may break down into wavelets.
Rotors have been demonstrated for some time in animal models of AF110, but have only more recently been shown in man111. It is still debated to what extent rotors play a role in human AF. Some suggest that rotors are seen in the majority of cases, and targeting them with P a g e | 45 ablation may be a powerful tool to prevent AF recurrence112, while others have found little evidence of rotors in their patients113. P a g e | 46
1.7 Atrial structural remodelling
As the rhythm of the heart is an electrical process, when trying to understand AF it is natural to focus on the electrical properties of cells and tissues. However, properties of the atria that do not directly relate to electrical function are also important such as atrial size and the glue binding myocytes that is the extracellular matrix.
The atria are elastic structures. When exposed to a short term increase in pressure, the atria dilate, and then recoil when the load is decreased114. However, in response to long term changes such as a leaking mitral valve, dilatation occurs that is only partially reversible once the valve has been repaired115. Similar changes occur in response to AF. AF is associated with atrial dilatation116, and while this certainly reflects larger atria being more vulnerable to AF, a role for AF generating dilatation is suggested by the decrease in atrial size seen once sinus rhythm is restored by electrical cardioversion of AF117. The powerful impact atrial size has on vulnerability to AF is clear, as dilatation and age were found to be the strongest predictors of AF following cardiac surgery118. The susceptibility to AF of dilated atria could lie within the multiple wavelet theory described above, where a greater tissue mass permits more concurrent re-entrant circuits, reducing the chance of them simultaneously extinguishing. Additionally, stretching the atria incurs changes in the extracellular matrix which may promote arrhythmias by other means.
Myocytes are held within a framework of protein fibres, comprised mainly of collagen and elastin. In between the fibres are a ‘ground substance’ full of materials that interact with cells and the fibrous component of the extracellular matrix. These matrix components are maintained by specific cells, fibroblasts. In response to a diverse array of insults, an increase in extracellular matrix volume occurs, termed fibrosis. In the context of AF, the insult can represent the extremely high activation rates of atrial myocytes, leading to the accumulation of Ca2+ and cellular damage. Fibrosis may replace damaged myocytes (replacement fibrosis), but may represent an expansion of the matrix that normally separates myocytes (reactive fibrosis)119. Disease not only increases the volume of fibrosis but also alters its composition, affecting the ratio of collagen subtypes120. As well as manipulating the matrix, fibroblasts themselves change in response to injury, becoming myofibroblasts and developing the ability to contract. While this is a helpful property if the injury is a physical gap that needs filling, in diffuse atrial injury it may be counterproductive.
Atrial fibrosis has well-established links with atrial fibrillation, with AF promoting fibrosis and fibrosis in turn increasing atrial vulnerability121. Fibrosis leads to conduction slowing, often in a P a g e | 47 heterogeneous pattern, predisposing to excitation re-entry. The promotion of atrial fibrosis by left ventricular failure leads to discrete areas of conduction slowing within the atrium122. As well as the overall collagen content of tissue, the distribution of fibrosis may have an effect on CV123, and a network of thin fibrotic strands may be more likely to cause conduction slowing and arrhythmogenesis than larger volume but organised linear fibrotic bands.
Atrial fibrosis is the final common pathway for many cardiac pathologies that predispose to AF119 and has been associated in autopsy studies with AF persistence120. AF remodelling leads to further fibrosis, although these changes take place over an extended timescale. Despite alterations within the myocytes, no changes in atrial connective tissue were observed after 16 weeks of rapid atrial pacing in a goat model124. The extent of atrial fibrosis can be assessed on a macroscopic scale in vivo use Gadolinium-enhanced MRI 125, and has been used as a marker of disease progression to identify those least likely to benefit from catheter ablation126.
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1.8 Atrial fibrillation and ageing
Given the complexities in maintaining a normal sinus rhythm, it is unsurprising that the system can fail at many points. Many aspects of atrial electrophysiology can malfunction, thereby predisposing an individual to AF. These can be seen from the single cell such as abnormal Ca2+ release promoting afterdepolarisations, to those affecting tissue such as repolarisation dispersion promoting wavebreak, to whole organ traits such as atrial size permitting many re- entrant circuits.
It is already well-established how some of these parameters change with age. Heart rate127 and blood pressure128 increase with age, associated with an increase in sympathetic tone and decrease in vagal tone127, 129. The atria enlarge with age130, prolonging the total atrial conduction time measured on the ECG as the p-wave131. The atrial effective refractory period, too, is longer in older subjects132. However, many factors remain under- or unexplored. This thesis will investigate some of the gaps in our knowledge, using sheep as a model of ageing.
Firstly, electrophysiology of the ageing sheep atria will be explored in areas that are well established in human ageing – the vulnerability to AF, changes on the surface ECG and invasive measurements of sinus node and AV node function to confirm the relevance of this model to human ageing. Secondly, the action potentials and Ca2+ handling characteristics of individual myocytes will be compared between old and young sheep. This will be complemented by recording atrial action potentials in vivo from anaesthetised sheep. Thirdly, the alternans behavior of the atrium will be examined, both in vivo and in isolated cells. Finally, atrial conduction, the Na+ current, and gap junction expression will be assessed.
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2 METHODS
2.1 Determining the age of sheep
This body of work aims to explore changes in the cardiac atria that occur with age. In order to answer these questions, we first required a method for determining the age of experimental subjects.
Welsh mountain sheep were used for all experiments. The age of individual sheep was established by physical examination. The principle physical feature was dentition. Sheep have a fully developed set of narrow temporary incisors by the age of three months133. The first incisors or pinchers are replaced by broad permanent teeth at the age of 12-15 months. From then on, one pair of intermediate incisors is replaced annually by broad permanent teeth. A ‘full mouth’ or complete set of permanent teeth has usually developed by four years of age. Excluding traumatic loss, senile tooth loss occurs after 7-8 years.
In line with this, sheep were classed as young (<18 months) if they had a complete set of short teeth and the first pair of incisors were broad but the remaining teeth were all narrow (Figure 2.1A). Sheep were classed as old (>8 years) if all teeth present were long and at least 25% were absent, reducing the chance of mistaking traumatic loss for senile loss (Figure 2.1B and C). Old sheep frequently exhibited other signs of frailty such as a reduction in adiposity but this was not a universal finding.
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Figure 2.1 – Ageing of sheep using dentition. A Young sheep aged <18 months. 1st pair of incisors are broad and adult, the remainder are small temporary teeth. B Old sheep aged > 8 years. >25% of teeth are absent. Remaining teeth are broad and elongated. C Edentulous old sheep aged > 8 years.
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2.2 Cell isolation
Much of the experimentation in this body of work was performed on isolated myocytes. The first step in the experimental process, therefore, requires the separation of an intact heart into individual atrial myocytes. Following a brief background to cell isolation, the solutions used will be listed. The details of the isolation procedure itself will then be described.
2.2.1 Background
The ability to work with individual cardiac myocytes has several advantages. From an electrophysiologist’s perspective, membrane currents can be better controlled and studied in greater depth in isolated cells than intact tissue. Viable cardiac myocytes were first isolated from rat ventricle in 1972 by Gould and Powell134. Early protocols generated myocytes that were intolerant of Ca2+ and produced poor yields of viable cells. The synergistic use of mechanical, chemical and enzymatic methods has been iteratively improved since then135. Solution composition, particularly with respect to the addition of excitation-contraction (EC) uncoupling agents and taurine has improved the consistency of myocyte isolation. The choice of tissue dispersing enzyme is often seen as a crucial step and is largely based on trial and error.
2.1.1 Solutions
A Ca2+-free solution has been shown by many investigators to be useful for isolating myocytes. It has been reported that a period of Ca2+-free perfusion leads to separation of myocytes at the intercalated disks136. The Ca2+-free solution used here (Table 2.1) is similar to Tyrode’s original solution137. As well as being nominally Ca2+-free (even double distilled water contains micromolar traces of Ca2+), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) has been added to aid the pH buffering already provided by disodium hydrogen phosphate
(Na2H2PO4). Bovine serum albumin (BSA) is used to increase oncotic pressure and thereby prevent myocyte rupture by uncontrolled water influx during electrolyte shifts138. 2,3-butanedione monoxime (BDM) helps to mitigate the effects of the “Ca2+paradox”139, whereby myocytes exposed to a Ca2+ free solution undergo damage and expire upon reintroduction of Ca2+140. BDM blocks EC coupling and prevents the uncontrolled contractions that can be seen on Ca2+ reintroduction that lead to sarcolemmal trauma141. However, BDM is also a non-specific phosphatase inhibitor. As a result, BDM affects cardiac electrophysiology
142 143 by, for example, inhibiting NCX , ICa(L), and Ito . P a g e | 52
A taurine-containing solution (Table 2.1) is applied during the second stage of the isolation procedure. This differs from the Ca2+-free solution in that it contains taurine, a β-amino sulfonic acid144 found in healthy myocytes145. Supplemental taurine has been found to increase viable cell yield during myocyte isolation145. A possible mechanism for this is that additional taurine, once within the cytosol, is extruded via a Na+ / taurine symporter, preventing Na+ overload. If Na+ loading is allowed to proceed unchecked, Ca2+ reintroduction causes Ca2+ overload via reverse mode Na+ / Ca2+ exchange146.
Substance Ca2+-free solution (mM) Taurine solution (mM) NaCl 134 113 HEPES 10 10 Glucose 11 11 KCl 4 4
MgSO4 1.2 1.2
Na2H2PO4 1.2 1.2 BSA 0.5mg/mL 0.5mg/mL BDM 10 10 Taurine - 50
CaCl2 - 0.1 pH 7.34 with NaOH
Table 2.1 – Ca2+-free and taurine-containing solutions
Following cell isolation, myocytes were stored in a solution composed of equal measures of taurine-containing solution and modified Tyrode’s solution (Table 2.2). This facilitated a gradual elevation of Ca2+ to physiological levels and allowed washout of BDM. The original description of Tyrode’s solution 137 was an enhancement to earlier physiological salines such as Ringer’s saline147. Magnesium, a vital modulator of ion channel and intracellular enzyme function148, was added, and pH buffering augmented using disodium hydrogen phosphate and bicarbonate. In the modified Tyrode’s solution used here, the buffering function is fulfilled by 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Probenecid, a blocker of transmembrane pannexin channels that connect the cytosol to the extracellular space149, is also present and serves to prevent efflux of small molecules such as fluorescent indicators from isolated myocytes. However, probenecid is also an agonist of transient receptor potential vanilloid-2 (TRPV2) channels which are stretch- and osmotically-activated Ca2+- selective channels. Whilst predominantly found in neural and renal tissue, TRPV2 channels have also been found in cardiac tissue and TRPV2 activation is positively inotropic150.
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Substance Concentration (mM) NaCl 140 HEPES 10 Glucose 10 KCl 4
MgCl2 1
CaCl2 1.8 Probenecid 2 pH 7.34 with NaOH
Table 2.2 – Modified Tyrode’s solution
Isolation solutions were made on the day of experimentation and warmed to 37°C. Modified Tyrode’s solution was made up to two weeks in advance and used within one week of glucose and Ca2+ addition.
2.2.2 Euthanasia and coronary cannulation
20,000 IU of heparin sodium (Wockhardt, Wrexham, UK) was administered via the cephalic vein two minutes prior to euthanasia in order to reduce the chances of thrombus formation in the coronary circulation. Sheep were euthanised using 40mL of 20% sodium pentobarbital (AnimalCare, York, UK) and death was confirmed by the absence of corneal reflexes. The heart was rapidly removed and washed free from blood using Ca2+-free isolation solution (Table 2.1).
The cardiac atria and aortic root were dissected away from the ventricles below the atrioventricular groove. The ostia of the left mainstem and right coronary arteries were identified. If left atrial isolation was planned, cannulation of the first branch from the left mainstem (LMS) leading to the left atrial appendage was attempted. Failing this, the circumflex artery was cannulated and the non-appendage branches ligated. If right atrial isolation was planned, the ostium of the right coronary artery (RCA) was cannulated and the non-appendage branches ligated. The cannula, once correctly sited, was sutured in place (Figure 2.2A). Perfusion was tested using a 50mL syringe connected to the cannula. Once satisfactory perfusion had been confirmed, the tissue was transferred to the Langendorff setup (Figure 2.2B).
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Figure 2.2 – A Atrial tissue specimen with nylon cannula perfusing right atrium via right coronary artery. Note clips on cut edge of ventricle to prevent excessive leakage of solutions. B Isolation setup for perfusion method of myocytes from atria of large mammals. LA – left atrium, LMS – left main stem, LV – left ventricle, RA – right atrium, RCA – right coronary artery, RV – right ventricle.
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The tissue was perfused via a Langendorff apparatus with Ca2+-free solution warmed to 37°C (Figure 2.2B). A flow rate was selected that caused blanching and turgidity of the tissue, typically 20-30mL/min. Exposed tissue was washed with Ca2+-free isolation solution. The opposing coronary circulation (RCA if LMS cannulated) was flushed with Ca2+-free solution using a Pasteur pipette. Expelled solution was discarded. These measures were performed to minimise any residual blood which could affect enzyme activity.
Although the coronary anatomy supplying the left atrial appendage was quite consistent, considerable variation was seen in the arterial supply to the right atrial appendage. Whilst in the majority of cases the right coronary artery supplied the right atrial appendage, in other cases the supply was derived from the circumflex system.
2.2.3 Enzymatic digestion
After 10 minutes washout the tissue was perfused with 100 mL of enzyme-containing solution comprising the Ca2+-free isolation solution with the addition of 5.5-6.5 mg of Type XIV Protease (Worthington, New Jersey, USA) and 55-65 mg of Type 2 Collagenase (Worthington, New Jersey, USA). After two to three minutes depending on solution flow rate, recirculation of this solution was commenced (Figure 2.2B).
The extent of digestion was estimated by the viscosity of the exudate, the development of translucent areas in the atrial appendage and a reduction in tissue rigidity. Sufficient digestion was obtained after typically eight to fifteen minutes. Following this, perfusion was switched to a taurine-containing solution (Table 2.1) for 20 minutes of washout and recirculation was discontinued.
Well-digested atrial tissue was excised from the left or right atrial appendages or the right atrial free wall. Tissue was dissected into approximately 1mm3 fragments. Chunks were agitated in taurine-containing solution using a Pasteur pipette. 10 mL of supernatant was removed every three minutes and filtered through 200 µm nylon mesh and replaced with 10 mL of fresh taurine-containing solution. The process was repeated until 10 tubes of supernatant had been filtered.
The cell-containing solution was left to settle for 20-30 minutes to allow pellets of cells to develop. The cells were then re-suspended in a cell storage solution consisting of 50% taurine-containing solution and 50% Tyrode’s solution (Table 2.2). The tubes were gently centrifuged by hand and a second re-suspension was performed to ensure that no residual enzyme was present and to facilitate removal of dead cells. P a g e | 56
The majority of experiments used cells that had not been loaded with fluorescent indicators. In those experiments where fluorescence was recorded, 50 µg aliquots of Fluo-5F acetoxymethyl (AM) ester (Life technologies, Paisley, UK) were reconstituted with 50 µL of 20% pluronic dimethyl sulphoxide (DMSO) (Life technologies, Paisley, UK). 10 µL of reconstituted Fluo-5F AM was added to 2 mL of cell suspension and incubated at room temperature for 10 minutes. Following this, 12 mL of cell storage solution was added to the suspension and incubated for a further 60 minutes at room temperature to allow de-esterification of the indicator. Finally, 12 mL of supernatant was removed leaving a cell pellet in 2 mL of cell storage solution.
Experimentation on myocytes was completed within 12 hours of cell isolation.
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2.3 Patch Clamp
The electrophysiological properties of isolated cells were examined using the patch clamp technique. This was used to study AP morphology as well as how a series of APs changed from a uniform to an alternating state with increasing pacing rate. The background to the patch clamp technique will be described before detailing the solutions and experimental methods used here. The specific voltage protocols used will be referred to in the corresponding results chapters.
2.3.1 Background
The measurement of electrical activity from single cells began with the AP recordings of the giant squid axon by Hodgkin and Huxley in 1939151. By virtue of the large diameter of giant squid neurons they were able to pass an electrode into the cytoplasm via the cut end of the axon. Electrophysiological measurements subsequently evolved, via the gap method, to sharp microelectrode techniques152. While this enabled AP recordings from small cells, the very fine electrode tips prohibited operator control of cellular potentials.
Patch clamping, a technique used to record ionic currents from cells or tissues, was first described by Neher and Sakman in 1976153. This approach was initially applied to the recording of single ion channels from an area of membrane removed from the cell of interest. In an improvement to their technique154, the same group reported the formation of gigaohm seals and the whole cell method of recording currents from intact cells.
The whole cell technique involves firstly the fabrication of a hollow glass micropipette. Formed from glass capillary tubes, the proximal end has the diameter of the original material while the distal is simultaneously heated and stretched to form a tapered orifice with a diameter of 1-3 µm155. A solution that resembles the cytosolic fluid is used to fill the micropipette and an electrical conductor, usually a silver wire with a silver chloride coating, is inserted into the filled pipette lumen (Figure 2.3A). A second, ‘bath’ electrode, usually made from a pellet of silver chloride, is placed in the fluid surrounding the cell.
The micropipette is brought into contact with the cell of interest and gentle suction is applied. This causes the cell membrane to adhere firmly to the tip of the micropipette. The quality of attachment between cell and pipette, or seal, can be quantified by measuring the electrical resistance between the pipette and the extracellular fluid using a small square wave voltage pulse. A high quality seal has a resistance in the gigaohm range i.e a large impedance to the flow of charged particles between the pipette and the bath. At this point, further suction is P a g e | 58 applied, rupturing the area of membrane directly beneath the pipette tip whilst maintaining the integrity of the seal and surrounding membrane (Figure 2.3Bi). Electrical access to the cytosol is now achieved. An electrical circuit can be created from patch pipette to cytosol, through the cell membrane, to the extracellular solution and then the bath electrode (Figure 2.3A).
An electrical amplifier is used to control and measure electrical activity (Figure 2.3C). In ‘current clamp’ mode, a fixed current is applied across the membrane and the amplifier records the membrane potential required to create this current. In ‘voltage clamp’ mode, a fixed membrane potential is applied across the membrane while the amplifier records the resulting currents.
A disadvantage to the whole cell approach is that the cytosol is effectively replaced by the pipette solution – an event known as cell dialysis. While this may in some instances be a useful tool in itself, it creates a non-physiological state in that at best only a crude approximation to the cytosol can be used. Many constituents important to intracellular signalling are lost in the process of dialysis.
The problem of intracellular dialysis was solved to a large extent following the introduction of the perforated patch technique by Horn and Marty156. This method makes use of an ionophore within the pipette solution to create pores permeable to monovalent ions exclusively in the area of membrane under the pipette tip (Figure 2.3Bii). These pores are too small to allow passage of larger substances and therefore serve to preserve the cytoplasmic environment. The initial description used nystatin as a pore-forming agent but other agents including amphotericin B157, β-escin158 and gramicidin159 have also been used. A disadvantage of ionophores is that the access resistance between pipette and cytosol is significantly greater than when membrane rupture is used. For many applications this is not of great consequence. However, the study of some events which take place over a very brief timescale, such as the recording of Na+ currents, is not possible using perforated patch. P a g e | 59
Figure 2.3 – Equipment for patch clamp recording. A Schematic of a patched myocyte. B (i) Whole cell recording (ii) Perforated patch recording. C Schematic of equipment used for patch clamp experiments
P a g e | 60
2.3.2 Solutions
The pipette solution used for current clamp experiments was designed to mimic the high K+
2+ intracellular solution. Ethylene glycol tetraacetic acid (K2EGTA) was added to buffer Ca . HEPES was used as a pH buffer. Amphotericin B was used as the pore-forming agent to achieve an electrical connection between micropipette and cell (see above).
Substance Concentration (mM)
KCH3O3S 125 KCl 20 NaCl 10 HEPES 10
MgCl2 5
K2EGTA 0.1 Amphotericin-B (dissolved in DMSO) 0.26 pH 7.2 with KOH
Table 2.3 – Pipette solution for perforated patch recordings
160 The pipette solution used for recording INa was similar to that used by Baba et al . They included adenosine triphosphate (MgATP) and phosphocreatine to reduce run-down during experimentation by supplementing cellular energy reserves. 10 mM EGTA was used to strongly buffer Ca2+.
Substance Concentration (mM) CsOH 125 Aspartic acid 125 TEA 20 HEPES 10 MgATP 5 EGTA 10 Phosphocreatine 3.6 pH 7.3 with CsOH
Table 2.4 – Pipette solution for measurement of INa
+ + The low Na extracellular solution used for the recording of INa replaces extracellular Na with tetraethyl ammonium (TEA), which maintains the osmolarity of the solution whilst also blocking K+ channels161. 4-aminopyrdidine (4AP) is included to further block K+ currents162, 163.
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Substance Concentration (mM) NaCl 3
MgCl2 1.2
CaCl2 1.8 TEA 127 CsCl 5 HEPES 20 Glucose 11 4AP 3
MnCl2 2 pH 7.3 with CsOH
+ Table 2.5 – Low Na extracellular solution for measurement of INa
2.3.3 General setup
Isolated ovine atrial myocytes were patch clamped using the perforated patch and whole cell patch clamp techniques. Cells were pipetted into a bath made from a block of Perspex with a central well. The base of the well was formed from a glass cover-slip and attached using vacuum grease. The bath was suspended over a Nikon TE200 inverted microscope.
The bath was superfused with solutions warmed to 37˚C and delivered via a gravity fed solution changer and heated tip inflow controlled by a TC2 temperature controller (Cell Microcontrols, Virginia, USA). Solution was removed via a metal outflow fashioned from a 25 gauge needle attached to a vacuum source. An Ag/AgCl reference electrode was suspended in the bath (Figure 2.3).
Glass micropipettes were pulled on a PC-10 pipette puller (Narishige Int., London, UK) using 1.5mm borosilicate glass capillary tubes (Harvard, Edenbridge, UK).
Recordings were digitised at 2.5 kHz and low pass filtered at 1 kHz. The pipette was manoeuvred directly over a suitable cell using MHW-3 coarse and fine manipulators (Narishige Int., London, UK) under continuous visualisation from an MyoCam video camera (IonOptix, Milton, USA) connected to a SSM125CE monitor (Sony Europe, Weybridge, UK). Input offset was nullified before sealing. Seals were obtained using oral suction. Cell dimensions were measured on the video display and subsequently scaled using a graticule (Graticules Ltd, Tonbridge, UK)
2.3.4 Perforated patch current clamp
For current clamp recordings, micropipettes were attached to an HS-2A headstage (Axon Instruments, California, USA), connected to an Axoclamp 2B amplifier (Axon Instruments, California, USA), digitised using a Digidata (Axon Instruments, California, USA) and P a g e | 62 simultaneously controlled and recorded by a personal computer (PC) running Clampex v9 (Axon Instruments, California, USA). Recordings were made at 37°C. Micropipette resistances were 5.0-6.5MΩ when filled with pipette solution (Table 2.3).
Myocytes were perfused with modified Tyrode’s solution (Table 2.2). Once a seal had been formed, a holding potential of -80 mV was set. The progress of perforation was monitored using a -80 mV to -40 mV depolarising step at a frequency of 0.5Hz. Complete perforation, marked by the appearance of an inward current stable in amplitude and decay time, typically took 10-20 minutes.
Once perforation was complete, the amplifier was switched to the current clamp configuration. Resting membrane potential was recorded. Cells were almost uniformly depolarised with a typical resting membrane potential of -40 mV. Current was applied to force the maximum diastolic potential to -80 mV when stimulated at 1Hz. The same holding current was used for all stimulation rates. Once current clamp was initialised, cells were stimulated at 1Hz for 3 minutes before recording was commenced. Cells were only accepted if the seal was >500MΩ, the current required to force the resting membrane potential to -80 mV was <0.06nA, and the peak of the AP was ≥0 mV when stimulated at 0.25Hz.
For steady state pacing, myocytes were stimulated with 0.8 ms pulses at 150% of the threshold amplitude at frequencies from 0.25 to 8Hz. At least 50 cycles were recorded after each change in frequency.
The final 10 representative APs at each stimulation frequency were averaged in Clampfit v10.3. This data was then transferred to Microsoft Excel 2010 and AP characteristics were automatically analysed using a programme custom-written in Visual Basic for Applications (VBA).
Maximum diastolic potential was defined as the average of the 10 samples immediately prior to stimulation. AP amplitude was defined as the peak of the AP minus the maximum diastolic potential. AP duration was defined as time from peak to 50% (APD50) and 90% (APD90) repolarisation. The maximum rate of rise of AP (V̇max) was defined as the maximum first derivative of the AP after the pulse artefact had decayed.
2.3.5 Voltage clamp
For voltage clamp recordings, micropipettes were attached to a CV 203BU headstage (Axon Instruments, California, USA), connected to an Axopatch 200B amplifier (Axon Instruments, California, USA), digitised using a Digidata (Axon Instruments, California, USA) and P a g e | 63 simultaneously controlled and recorded by a PC running Clampex v9 (Axon Instruments, California, USA). Recordings were made at room temperature. Micropipette resistances were 1.5-2.5MΩ when filled with pipette solution (Table 2.4).
Once a seal had been obtained, pipette compensation was applied. Gentle oral suction was used to rupture the membrane under the pipette tip. Cell capacitance was calculated automatically by the acquisition software using a 10 mV hyperpolarising step from 40 mV. The resulting current was integrated using equation 2.1 to calculate membrane capacitance.
퐼. 푇 퐶 = 푉
Equation 2.1 where C is membrane capacitance I is current T is time V is the voltage step
Cell capacitance and access resistance were noted. If the access resistance was above 10 MΩ attempts were made to improve this using pressure applied orally. Cell capacitance and access resistance were compensated using the amplifier before series resistance compensation was applied. The perfusing solution was changed to the low Na+ solution in Table 2.5. A minimum of five minutes was allowed to elapse to allow for cellular dialysis before recordings began.
The voltage protocols used will be described alongside their corresponding results. P a g e | 64
2.4 Calcium fluorescence measurements
In a subset of experiments, patch clamp recordings were complemented by simultaneous measurement of intracellular Ca2+ concentrations.
2.4.1 Background
Intracellular Ca2+ concentrations can be measured using fluorescent indicators. These probes emit light with a specific spectral peak when light at a different wavelength is applied. The intensity of light emitted varies with the concentration of Ca2+ bound to the indicator in accordance with equation 2.2163.
2+ (퐹 – 퐹푚푖푛) [Ca ]푖 = 퐾푑 (퐹푚푎푥 − F)
Equation 2.2
where Kd is the dissociation constant of the indicator F is fluorescence intensity 2+ Fmin is fluorescence intensity in the absence of Ca 2+ Fmax is fluorescence intensity in the presence of saturating concentrations of Ca
Fluo-5F AM is an esterified Ca2+ fluorescence indicator. At 37°C this indicator has a dissociation
2+ constant (Kd) of 1.0 µM, making it suitable for measuring intracellular Ca in the 1µM to 1mM range. This fits with the typical concentrations of unbound intracellular Ca2+ which range from ~100 µM during diastole to ~1 mM during systole13. The probe traverses the cell membrane as an uncharged ester that does not possess Ca2+-dependent fluorescence properties. Once within the myocyte, non-specific cellular esterases separate the acetoxymethyl group from the carboxylic acid residue of the indicator, restoring Ca2+-dependent fluorescence. This lysis returns the indicator to a charged state, thereby slowing extrusion of the indicator from the cytoplasm164. The peak light absorption of Fluo-5F occurs at 494 nm while peak emission occurs at 516 nm164 .
2.4.2 Experimental equipment
Light emitted from a Xenon arc lamp (Cairn Research, Faversham, UK) was passed via heat, neutral density, and interference filters to a shutter (Cairn Research, Faversham, UK) (Figure 2.4). When the shutter was opened, light passed to a 500 nm dichroic mirror. Dichroic mirrors split incoming light into a reflected and transmitted portion based on the wavelength of the light. The first dichroic mirror passed light with a wavelength of <500 nm to the P a g e | 65 microscope objective and thence to the myocytes, maximising the light within the absorption spectrum of Fluo-5F while minimising wavelengths that lead to cell damage without providing useful information. The fluorophore within the myocytes absorbed light at a peak wavelength of 494nm and in the presence of Ca2+ emitted light at a peak wavelength of 516 nm. The
2+ intensity of fluorescence was proportional to [Ca ]i. Simultaneously, the cell bath was illuminated with light from the microscope lamp that had been passed through a 750 nm high pass filter, thereby allowing cell visualisation without further exciting the fluorophore.
The combination of light from the microscope lamp and fluorescence passed back through the objective to the dichroic mirror. Light with wavelengths >500 nm now passed through an adjustable diaphragm used to block light from everything except the myocyte of interest. A second dichroic mirror with a threshold of 620 nm further split the light beam. Light of wavelengths >620 nm passed to a video camera for real time cell visualisation. Light <620 nm passed to an Integra photomultiplier (PMT) tube (Cairn Research, Faversham, UK), thereby excluding light originating from the microscope lamp from reaching the PMT. The PMT converted an input of light intensity to an output of voltage. The voltage was digitised using a Digidata (Molecular Devices, California, USA) and recorded using Clampex v9 (Molecular Devices, California, USA).
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Figure 2.4 – Calcium fluorescence schematic. Coloured arrows represent light path.
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2.4.3 Fluorescence recording
Myocytes were patch clamped using the perforated patch clamp technique as described in section 2.3.4. A rectangular diaphragm was used to ensure that only that myocyte of interest was visible. Once adequate perforation had been achieved, the arc lamp shutter was opened and the PMT activated. Fluorescence was recorded simultaneously alongside membrane potential data, with 2.5 kHz acquisition and low pass filtering at 1 kHz. At completion of each experiment, the myocyte was killed by impalement and the maximum fluorescence Fmax was recorded. The bath was then manoeuvred so that no myocytes were in the field of view and minimum fluorescence Fmin was recorded.
Accurate measurements of Fmax were not obtained in all cells. To ensure data consistency, 2+ [Ca ]i has therefore been expressed as fractional fluorescence (F/F0) using equation 2.3
(퐹 – 퐹푚푖푛) 퐹/퐹0 = (퐹0 − 퐹푚푖푛)
Equation 2.3 where F is fluorescence intensity at a given timepoint
F0 is diastolic fluorescence Fmin is the background fluorescence intensity without a cell in the field of view
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2.5 In vivo electrophysiology
Recordings from isolated cells were complemented by in vivo measurements. These took the form of non-invasive ECGs, invasive electrophysiological studies and the use of permanent pacemakers to assess arrhythmia inducibility. Again, a background to these procedures will be described, followed by details of their implementation.
2.5.1 Background
The surface ECG represents a summation of the several billion APs occurring throughout the heart. The net movement of electrical charge within the myocardium can be recorded from the vantage point of two or more electrodes attached to the skin. Careful electrode placement enables a series of electrical traces to be tracked, recording electrical activity from several directions simultaneously165. A wave of depolarisation moving towards an electrode is shown as a positive, upward deflection on the ECG trace while depolarisation away from an electrode leads to a downward, negative deflection. The first recordings of human cardiac electrical activity were made by Waller in 1887 using a Lipmann capillary electrometer166. These crude recordings only permitted the detection of two waves corresponding to ventricular depolarisation and repolarisation. Einthoven improved on the technique, and after mathematically correcting for instrumental artefacts, provided the nomenclature that is still in use today167. From a flat or isoelectric line, the initial deflection, or P-wave, corresponds to atrial depolarisation. This is followed by a short pause (the PR interval) before ventricular depolarisation causes a triphasic wave referred to as the QRS complex. There is another pause (the ST segment) before a final wave known as the T wave occurs, signifying ventricular repolarisation. An illustration of the ECG can be found in Figure 3.1. The surface ECG provides useful information for our purposes about conduction times, as well as diagnostic clues towards specific pathologies.
While recordings of cardiac electrical activity are more commonly performed from outside the heart, recordings can also be made from inside the heart, or endocardially. The advantage of an endocardial approach is that the heart can be stimulated as well as observed.
Although electrical stimulation of the human heart via a needle had been reported 30 years earlier, the first internal cardiac pacemaker was implanted in 1958168 . The original epicardial leads were superseded by transvenous endocardial leads, and the atria and ventricles could be paced independently by the 1970s. P a g e | 69
Transvenous endocardial pacing opened to the door to invasive in vivo electrophysiology. Measurement of the sinus node recovery time (SNRT) highlighting depressed automaticity of the sinus node was first described by Rosen et al. in 1971169. Arrhythmic mechanisms at an organ level began to be elucidated over the following decades170. The electrograms (EGMs) generated take the form brief voltage spikes corresponding with the passage of a wave of depolarisation near to a recording electrode that has been gently opposed to the tissue under study. Recording in a bipolar configuration, whereby the difference in potential between two closely spaced electrodes is measured, allows good localisation in time. Recording in a unipolar configuration whereby the potential at a local electrode is compared against a much more distant electrode allows better location in space, while suffering the disadvantage of potential contamination by external electrical noise.
In addition to unipolar and bipolar EGMs, electrical activity that approximates the cellular action potential can be recorded from the endocardial surface of the intact heart. These monophasic action potentials (MAPs) are a method of recording extracellular waveforms that bear distinctive relationships to transmembrane APs. Being, by their nature, recorded from tissue rather than individual cells, the recordings represent the aggregation of APs from a collection of cells directly underneath the recording electrode. The first MAP recordings predate the surface ECG by several years171.
A bipolar electrode is oriented perpendicular to a region of tissue and gentle pressure is applied. The pressure causes the cells immediately underneath the electrode to partially depolarise. The tissue that surrounds but is not directly beneath the electrode remains polarised. This creates a flow of current from the partially depolarised myocytes beneath the electrode towards the adjacent muscle, which can be recorded as a negative voltage in resting myocardium. During systole, the adjacent muscle fully depolarises while the nearfield remains in a partially depolarised state. At this time, current flow has now reversed so it now flows towards the electrode, recorded as a positive voltage. The voltage pattern generated approximates the timecourse of a cellular AP172.
2.5.2 ECG recording
6-lead electrocardiograms (ECGs) were recorded using an AmpliPower BO2 amplifier and ECGAuto software (Emka Technologies, Paris, France). Data was digitised at 1 kHz. Electrodes were attached using crocodile clips to each limb on shaven and cleaned skin that was not overlying muscle. P a g e | 70
The ECGs used for feature extraction and morphological analysis were performed in the anaesthetised state with the sheep lying in the right lateral position, as these were the traces least contaminated with electrical noise and movement artefact. Conscious ECGs were performed during electrophysiological studies with the subject gently restrained in a seated position (Figure 2.5A). Noise was reduced as far as possible by removing sources of electrical interference and wetting clips to reduce electrode to skin impedance.
Following conversion of data files within ECGAuto, recordings were analysed in LabChart (ADInstruments Europe, Oxford, UK). In each subject, the ECG lead displaying the cleanest and most clearly defined p-wave was used for analysis. QRS complexes were identified automatically. Empirically determined parameters found to reliably detect QRS complexes were a QRS duration of 40 ms, minimum RR interval of 400 ms and QRS polarity determined by trace inspection. Detection was verified manually before averaging 50 consecutive beats to create a composite. Morphological characteristics were analysed automatically and adjusted manually. P-wave onset and offset were defined as the timepoint of deviation from and return to the isoelectric line after completion of a uni- or biphasic response. T wave offset was defined as the intersection of a tangent from the steepest portion of the terminal T-wave with the isoelectric line. 1-5 composite beats were analysed and then the means of each parameters used for each subject. All ECG morphology data was obtained from anaesthetised subjects as these provided the cleanest recordings.
2.5.3 Electroanatomical mapping
Prior to surgery, surgical equipment was sterilised using steam, while electrophysiological equipment was sterilised using ethylene oxide (Andersen products, Clacton-on-Sea, UK).
Welsh mountain sheep were anaesthetised with isofluorane (Abbott laboratories, Maidenhead, UK) following induction with nitrous oxide. An endotracheal tube was inserted following application of lidocaine spray (AstraZeneca, London, UK) to the pharynx. Ventilation was maintained using a Zoovent ventilator (Universal lung ventilators, Milton Keynes, UK) and Ohmeda 7810 anaesthetic machine (GE Healthcare, Hatfield, UK). Physiological parameters were continuously monitored using 6 lead ECG (Emka, Paris, France), pulse oximeter and automated blood pressure recording (Midmark, Oklahoma, USA). Depth of anaesthesia was assessed clinically using ocular reflexes. Prophylactic antibiotics were administered in the form of oxytetracycline 20mg/kg (Norbrook, Corby, UK). Meloxicam 0.5 mg/kg (Boehringer Ingelheim UK, Bracknell, UK) was used for post-operative analgesia. P a g e | 71
Following clipping and cleaning of the overlying skin with Povidone (Ecolab, Garforth, UK), an incision was made over the right jugular vein. The jugular was dissected free using blunt dissection and 2-0 MerSilk (Ethicon Europe, Norstedt, Germany) sutures were placed tightly around the proximal end and loosely around the distal end. Two small venotomies were made and guidewires advanced into the jugular lumen, followed by haemostatic sheaths. At the end of the procedure the distal jugular was tied with MerSilk and the wound closed in two layers with 2-0 Vicryl (Ethicon Europe, Norstedt, Germany). The electroanatomical (EA) mapping comprised three steps – recording monophasic APs, measuring conduction velocity (CV) and implanting a permanent pacemaker for subsequent arrhythmia inducibility assessment. Subjects had any one, two or all three of these steps performed.
2.5.4 Monophasic action potentials
Electrodes were advanced under fluoroscopic guidance to the right atrium (RA). A Blazer (Boston Scientific) steerable catheter was manipulated until a clean MAP signal was recorded from the posterior wall of the right atrium (Figure 2.5B). The tip position was visualised orthogonally in antero-posterior and lateral fluoroscopic projections. The heart was stimulated from the RA appendage using a passive fixation J-tipped permanent pacing lead (Medtronic, Minnesota, USA) at intervals of 500 ms to 250 ms. At the end of recording the catheter and passive fixation lead were removed.
Signals were recorded using LabChart (ADInstruments, Oxford, UK) and processed with 50 Hz notch, 2 Hz high pass, and 100 Hz low pass filters. MAPs were transferred to custom written software in Microsoft Excel to calculate AP durations. These were defined as time from the peak of the dome to 50% and 90% repolarisation.
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Figure 2.5 - In vivo electrophysiology. A Conscious ECG recording from gently restrained sheep. B Fluoroscopic image showing passive fixation pacing lead in right atrial appendage with Blazer catheter tip against right atrial posterior wall. C Fluoroscopic image used to measure expansion of Constellation catheter in-situ. D Schematic showing position of Constellation catheter relative to heart. E En-face view of Constellation catheter demonstrating spline arrangement.
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2.5.5 Conduction Velocity
In order to record RA conduction velocity (CV), a 48mm Constellation (Boston Scientific, Massachusetts, USA) basket mapping catheter was advanced under fluoroscopic guidance (Figure 2.5C) into the RA through a FlexCath (Medtronic, Minnesota, USA) steerable sheath (Figure 2.5D). Initial attempts using a 64 unipolar electrode configuration proved unusable due to an unacceptable signal to noise ratio. Usable results were obtained after reconfiguring to a 32 channel bipolar pair configuration. Signals were amplified using a USB-6211 (National Instruments, Texas, USA) A/D converter and recorded using custom software written in Labview (National Instruments, Texas, USA). Signals were first recorded during normal sinus rhythm. The RA was then stimulated through proximal and mid-spline electrode pairs using a Carelink (Medtronic, Minnesota, USA) pacemaker analyser.
In order to calculate CV, knowledge of the distances between electrode pairs was required. The inter-electrode spacing along each spine corresponding to axial atrial conduction was easily measured. The spacing between splines was estimated using fluoroscopy. Fluoroscopic images showing the deployed catheter and sheath were acquired (Figure 2.5C). The diameter of the deployed catheter was measured in ImageJ (National Institutes of Health, USA) using the diameter of the sheath as a known distance for calibration. Assuming a circular cross section (Figure 2.4E), the inter-spline electrode spacing was calculated as
휋d 푠푝푎푐푖푛푔 = 8
Equation 2.4 where d is the deployed diameter at the level of the electrode pair in question.
Recordings were transferred to LabChart (ADInstruments UK, Oxford, UK) and signals were band-pass filtered at 30Hz to 300Hz. Recordings were then transferred to software that was custom written in Microsoft Excel. Typically 20-50% of electrode pairs provided usable data. The onset of each electrogram and of the farfield stimulation artefact were marked allowing calculation of the time taken for an electrical impulse to travel from one electrode pair to another.
CV was calculated as distance / time. Axial CV was calculated by stimulating from the proximal electrode pair of a spline and measuring the time to reach the 2nd, 3rd and 4th electrode pairs by using the time-invariant farfield stimulation artefact as a reference point. Circumferential CV was calculated by stimulating from a mid-spline electrode pair and measuring the time to P a g e | 74 reach the corresponding pair in every other spline. In each case, CV was calculated by performing a linear regression through all measurement points including the origin.
2.5.6 Pacemaker implantation
In some subjects, an implantable cardioverter defibrillator (ICD, various models, Medtronic, Minnesota, USA) was placed to enable assessment of atrial vulnerability to fibrillation. An active fixation pacing lead (various models, Medtronic, Minnesota, USA) was advanced to the RA appendage and screwed into place. A satisfactory position was confirmed by correct placement on fluoroscopy, pacing threshold <2V at 0.4 ms pulse duration, impedance of <1200 Ω and a p-wave of >2 mV. Some subjects also received a pacing lead (various models, Medtronic, Minnesota, USA) to the apex of the right ventricle (RV). These sheep went on to have further experimentation under a tachypacing heart failure protocol after completion of all experiments listed here.
The pacing lead(s) were secured proximally with 2-0 MerSilk (Ethicon Europe, Norstedt, Germany) before being connected to the ICD which was inserted into a subcutaneous pocket. The wound was closed with 2-0 Vicryl (Ethicon Europe, Norstedt, Germany) in three layers.
2.5.7 Conscious electrophysiological studies
A minimum of one week was allowed to pass for healing and inflammation to settle following pacemaker implantation. Conscious electrophysiological studies were performed while the subject was gently restrained in a seated position. Each study followed the same protocol. Data was used solely from subjects with a pacing threshold of ≤2V as a pace threshold above 2V suggests poor electrical contact between pacing lead and myocardium, potentially influencing arrhythmia inducibility. Steady state pacing was performed from 500 ms, decrementing until either the Wenckebach point or 200 ms was reached, whichever occurred first. SNRT was assessed, and correction for the underlying sinus rate was performed using the sinus cycle length immediately following pacing. The ERP was measured using a drivetrain of 8 beats at an S1S1 interval of 400 ms. The ERP was defined as the longest S1S2 interval that failed to generate a low frequency atrial capture signal on the EGM. High frequency pacing artefacts were ignored. Data from subjects where signals were not clear enough to reliably determine ERP was discarded.
2.5.8 Arrhythmia inducibility
Arrhythmia inducibility was performed immediately following electrophysiological study. An incremental voltage protocol was used first whereby bursts of 50 Hz stimulation at 5 V were P a g e | 75 applied for 1, 2, 5, and 10 s. This was followed by a protocol of incremental voltage. Here, 5 s bursts of 50 Hz stimulation were applied from 1 V to 8 V. This protocol was followed twice. If AF persisted for >30 minutes the protocol was terminated early for the sake of subject comfort. AF voltage threshold was determined as the minimum voltage of 50 Hz burst required to induce ≥2s AF. Threshold was deemed to be 10 V if AF was non-inducible despite delivering the maximal voltage of 8 V. AF duration was assessed as the mean duration of AF induced from 5 V, 5 s bursts, as this combination occurred three times during the two protocols. To minimise skewing of data from a small number of episodes of AF of great length, during analysis the AF duration was capped at three minutes. Traces were analysed in LabChart v7 (ADInstruments Europe, Oxford, UK). P a g e | 76
2.6 Western Blotting
Protein expression was quantified to understand if any observed changes in membrane currents or in vivo findings were associated with changes in membrane proteins. Once more, a background to the technique will be described, followed by the solutions used and details of the experimental protocols.
2.6.1 Background
The technique known as ‘Western Blotting’ is a method of identification and semi-quantitative analysis of specific proteins. The process by which proteins could be separated by electrophoresis before being transferred to a membrane was first described by Towbin in 1979173, although the commonly used name was not coined until 1981174.
A sample is separated into its constituent proteins by means of gel electrophoresis. The sample is incubated with a sample buffer such as sodium dodecyl sulphate (SDS) that adds charged residues to the proteins. A reducing agent together with heat is applied to reduce covalent disulphide bonds, facilitating denaturing of the protein and binding with SDS. The samples are loaded at one end of a polyacrylamide gel and an electrical current applied along the gel. The resulting electrical field pulls the proteins to the far end of the gel. Smaller proteins travel faster through the gel and therefore the individual proteins become distributed according to molecular weight175.
For further processing, the proteins need to be transferred to a material more robust than a polyacrylamide gel that allows access to the proteins of interest from additional reagents. Commonly used materials are nitrocellulose or polyvinylidene difluoride (PVDF) membranes176. An electrical current is applied to a stack comprising gel, membrane, filter paper and padding. This pulls the proteins from the gel and onto the membrane. The transfer can be performed with the gel and membrane either immersed in a solution or in a dry state.
Once the proteins are located within the membrane they can be identified using antibodies that are ideally specific to the protein of interest. Antibodies may react non-specifically with the membrane. A blocking step, involving a reagent which attaches to non-specific binding sites on the nitrocellulose, is therefore performed. A two-step antibody protocol is often preferred. The primary antibody binds to the protein of interest. The secondary antibody, containing a residue that catalyses a luminescent reaction, binds to an epitope on the primary antibody. Finally, luminescent reagents are applied that emit light only when in the presence P a g e | 77 of the secondary antibody. This light can be detected as one or more bands on the membrane corresponding to the location of the protein of interest.
2.6.2 Solutions
Lysis and resuspension buffers were used in the extraction of protein from atrial tissue. In order to prevent degradation of the proteins of interest by endogenous proteases, protease inhibitors were added.
Substance Lysis buffer (mM) Resuspension buffer (mM) Tris HCl 20 20 EDTA 1 1 Sucrose 250 - SDS - 69.4 (2%)
Table 2.6 – Lysis and resuspension buffers
Substance Concentration Aprotinin 1mg/mL Leupeptin 1mg/mL Sodium orthovanadate 100mM PMSF 0.1mg/mL
Table 2.7 – Protease inhibitors
Transfer buffer was used during the transfer of protein from gel to nitrocellulose membrane. Tris buffered saline (TBS) had a detergent, Tween 20, added to facilitate the washing steps.
Substance Concentration (mM) Bicine 1.25 Bis-Tris 1.25 EDTA 0.05
Table 2.8 – NuPage transfer buffer (final concentrations for use)
Substance Concentration Tris 20mM NaCl 150mM Tween 20 0.1% pH 7.6 with HCl
Table 2.9 – TBS-T
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2.6.3 Protein extraction and quantification
The proteins of interest to this work are normally found in the cell membrane and expressed at comparatively low levels with respect to other cellular proteins. Therefore, protein was extracted using a membrane fractionation protocol to concentrate the protein of interest. Tissue samples from the left and right atrial appendages were obtained immediately after euthanasia with sodium pentobarbital (AnimalCare, York, UK) and snap frozen in liquid nitrogen. Samples were stored in liquid nitrogen until required. 200-400 µg of tissue was thawed and homogenised using an Ultra-turrax T25 (IKA, Staufen, Germany) on ice in 1.5 mL of Lysis buffer (Table 2.6). Protease inhibitors (Table 2.7) were added to reduce protein degradation by cellular proteases. The suspension was spun in a Biofuge (Heraeus, Hanau, Germany) centrifuge at 10,000 G for 10 minutes at 4°C to remove non-homogenised tissue. The supernatant was removed and centrifuged again at 10,000 G for 10 minutes at 4°C. The remaining supernatant was spun for a 3rd time in an Optima Max Ultracentrifuge (Beckman Coulter, California, USA) at 100,000 G for two hours at 4°C to extract membrane components. The supernatant was discarded and the precipitate was reconstituted with 200 µL of resuspension buffer (Table 2.6) with protease inhibitors added (Table 2.9).
5 µL of protein suspension was added to 25µL of Reagent A’ made by combining 1mL alkaline copper tartrate Reagent A (BioRad, California, USA) and 20µL of surfactant solution Reagent S (BioRad, California, USA), and then adding 200 µL of Folin Reagent B (BioRad, California, USA). The copper tartrate reacts with the Folin reagent in the presence of protein to produce a colour change, detectable by photospectroscopy177. The spectrum of the protein sample was compared to that of six protein standards with known concentrations of BSA. An ELX 800 microplate absorbance microplate reader (BioTek, Vermont, USA) and Gen5 software (BioTek, Vermont, USA) were used to quantify total sample protein. All measurements were repeated in triplicate. Protein samples were stored at -80°C.
2.6.4 Gel electrophoresis and transfer
Protein samples were prepared by combining 20 µg protein, 5 µL of sample buffer (Life technologies, Paisley, UK) and 2 µL of NuPage sample reducing agent (Life technologies, Paisley, UK) with resuspension buffer (Table 2.6) to make a total volume of 20 µL per lane. Samples were heated at 70°C for 10 minutes before being loaded into NuPage 3-8% Tris-acetate gels (Life technologies, Paisley, UK). Precision Blue (BioRad, California, USA), a pre-stained marker of known molecular weight, was loaded into the first lane to aid band identification. Gels were assembled into XCell Surelock Mini-Cell tanks (Life technologies, Paisley, UK) before filling with Tris-acetate SDS running buffer (Life Technologies, Paisley, UK) P a g e | 79 with 0.25% Antioxidant (Life technologies, Paisley, UK). Gels were run at 200 V for 90 minutes using a PowerPac 200 (BioRad, California, USA).
Protein was transferred to nitrocellulose membrane (GE healthcare, Hatfield, UK) using an XCell II Blot module (Life technologies, Paisley, UK). The module was filled with NuPage transfer buffer (Life technologies, Paisley, UK, constituents in Table 2.8) with 20% methanol and 0.1% antioxidant. Gel transfer was performed at 100 mA at 4°C for 22 hours.
2.6.5 Protein detection
Following transfer, the membrane was stained with 0.1% Ponceau-S (AppliChem, Gatersleben, Germany). The membrane was imaged using the Syngene Chemi Genius Bioimaging system (Syngene Europe, Cambridge, UK). Ponceau-S was washed off using TBS-T (Table 2.9). The membrane was blocked with a solution of 5% milk powder dissolved in TBS-T for one hour at room temperature and subsequently washed with TBS-T.
After blocking, the membrane was incubated with the primary antibody diluted with TBS-T overnight at 4°C on a rotary shaker. The membrane was washed again with TBS-T before the secondary antibody diluted with TBS-T was applied. The secondary antibody was incubated at room temperature for one hour on an orbital shaker. A final wash with TBS-T was performed before protein was detected.
1mL of Amersham ECL Western Blotting Reagent 1 and 1mL of Amersham ECL Western Blotting Reagent 2 (GE Healthcare, Hatfield, UK) were simultaneously applied to the membrane for three minutes. A 10 minute exposure was used to detect luminescence with the Syngene Chemi Genius Bioimaging system (Syngene Europe, Cambridge, UK).
Details of the antibodies used will be provided alongside their corresponding results.
2.6.6 Analysis
The GeneTools programme (Syngene Europe, Cambridge, UK) was used to quantify protein bands. Densitometry was compared against a single internal control of ovine left ventricular tissue that was used for all blots. Blots were performed in triplicate to reduce loading errors and the results expressed as the mean normalised value. Other groups have used housekeeping loading controls to minimise errors. However, as has been shown in other models178, we have shown that the ovine model of ageing leads to changes in expression of the proteins commonly used for this purpose GAPDH and β-actin179, rendering this approach inapplicable for the work described here. P a g e | 80
2.7 Statistical and analytical methods
2.7.1 Data presentation
Data are presented as mean ± standard error of the mean in tables and graphs unless stated otherwise in the accompanying text. Number of subjects is presented as [n = number of cells, number of animals].
2.7.2 Tests of statistical significance
Statistical tests were performed in SigmaPlot (Systat Software, California, USA).
Data was tested for normality using the Shapiro-Wilk test. Non-normally distributed data was transformed using log10 or square root if the dataset contained values of zero. If data was found to be persistently non-normally distributed despite transformation, the Mann – Whitney rank sum test was used.
Continuous data in which only two groups were present was tested for significance using a two-tailed Student’s t-test. Where more than two groups were present, comparisons were made using one-way, two-way or two-way repeated measures ANOVA. Significance was taken as an overall difference between groups. Post-hoc tests were applied to assess for interactions between groups and if found to be significant have been described in the text.
Categorical data was compared using the Chi-squared test. In cases where expected values were less than 5, Fisher’s exact test was used instead as this has greater accuracy under these conditions.
Statistical significance was nominally taken as p<0.05. Bonferroni’s correction was used for multiple comparisons.
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2.1.2 Curve fitting
It was necessary to fit mathematical curves to some datasets in order to extract key parameters. In each case, macros were written in Microsoft Excel making use of the ‘Solver’ function. The least-squares method was used, employing Solver as an optimiser.
The single exponential (Equation 2.5) was used to fit curves to the decay phase and recovery
2+ from inactivation of INa, and the decay phase of Ca transients.
−푥 ⁄푡 푦 = 푦0 + 퐴1푒 1
Equation 2.5
Where y0 is the Y offset A1 is the amplitude t1 is the time constant of decay
The Boltzmann curve (Equation 2.6) was used to fit the activation and inactivation kinetics of
INa.
퐴1 − 퐴2 푦 = + 퐴2 1 + 푒(푥−푥0)⁄푘
Equation 2.6
Where A1 is the upper bound A2 is the lower bound x0 is the centre point at which y is halfway between the upper and lower bounds k is the slope factor, representing the width of the central steep region of the curve
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3 IN VIVO CHARACTERISATION OF SHEEP AGEING MODEL
3.1 Introduction If the objective of this work is to shine new light onto why older persons develop AF, we must first ensure that the experimental model that we are using is relevant to humans. To this end, the first set of experiments performed assess parameters that are well known to change with age in man, where possible using techniques commonly performed in clinical practice.
3.1.1 The surface ECG
As described in Chapter 2.5, the surface ECG records electrical activity from the whole heart using electrodes attached to the chest wall and limbs. As regions of the heart activate in sequence, the activity of specific cardiac structures can be identified within the ECG trace as characteristic waves – the P-wave representing atrial depolarisation, the QRS complex caused by ventricular depolarisation, and the T wave due to ventricular repolarisation (Figure 3.1A).
The duration of the P-wave measured on the surface ECG reflects atrial size and both inter- and intra- atrial conduction velocity. P-wave duration is associated with the future risk of developing AF131, 180 and is also associated with all-cause mortality in patients prescribed AV- node modulating medication131. Advancing age has been shown to lead to prolongation of the P-wave131, 132. Interestingly, an age-related reduction in P-wave amplitude has been reported in single channel ECG recordings131. While this could represent decreasing atrial mass, single channel recordings cannot exclude a shift in P-wave axis that could have shown a corresponding increase in amplitude in other leads.
The PR interval reflects the time taken for a wave of depolarisation to pass from the sinus node, through the right atrium, atrioventricular (AV) node and proximal bundle of His before activating the ventricular myocardium. Any change in PR interval can therefore reflect disease at any or all of these locations. An abnormally long PR interval has been shown to predict the likelihood of developing AF180. The PR interval increases with age in man131, explained at least in part by age-related fibrosis of the AVN181.
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3.1.2 Non-invasive electrophysiological testing
Invasive electrophysiological testing can be performed by cannulating the heart with electrodes that are then used to both stimulate and record the activity of the myocardium. Although a slight misnomer as an invasive intervention is required at some point, non-invasive electrophysiological testing refers to stimulation and recording performed through a pre- implanted cardiac pacemaker. This can be advantageous as the procedure causes little discomfort to experimental animals and therefore can be performed while the subjects are awake.
The sinus node recovery time (SNRT) is a marker of sino-atrial node (SAN) function and is used clinically as supportive evidence for the need for a pacemaker182. Following a period of rapid atrial pacing, a delay is observed before sinus rhythm resumes. This pause is due to hyperpolarisation of the SAN myocytes mediated by acetylcholine80, 183. The hyperpolarisation could occur due to intracellular Na+ accumulation which is then removed from the cell via the
+ + 78 Na / K pump, facilitating IK1 . The SNRT may be expressed as a raw value of time from last pacing stimulus to first sinus beat. Alternatively, it may be corrected for the underlying sinus rate (CSNRT) by subtracting the preceding sinus cycle length182. The SNRT has been shown to increase with age in rodents184, dogs185 and humans186.
The Wenckebach phenomenon is a characteristic pattern of loss of 1:1 atrioventricular conduction due to electrical conduction block occurring within the atrioventricular node (AVN). It is seen as a progressive lengthening of the PR interval until block occurs. The subsequent beat shows resetting of the PR interval to a shorter value before progressive lengthening begins again187. The occurrence of the Wenckebach phenomenon is universally seen during rapid atrial pacing even in health, but may reflect pathology if seen at resting heart rates. The longest cycle length at which the Wenckebach phenomenon occurs, referred to as the Wenckebach point, is an indicator of AVN function. The utility of Wenckebach point assessment for clinical decision making is hampered by poor sensitivity to significant AVN disease188 as a considerable proportion of patients requiring a pacemaker have normal Wenckebach points. Senescence is associated with a deterioration of AVN function. The Wenckebach point has been positively correlated with age in rodent189 and human studies186, 190, potentially caused in part by nodal fibrosis181.
The ERP is defined as the longest interval between two consecutive stimulations that fails to propagate on the second impulse191. As discussed in Chapter 1.4, the ERP is a consequence of action potential duration and Na+ channel recovery from inactivation. AF remodelling leads to shortening of the atrial ERP192, and some but not all reports have suggested that a short atrial P a g e | 84
ERP may be pro-fibrillatory84. However, despite the vulnerability to arrhythmias of senescent atria, age is generally accepted as leading to an increase in atrial ERP in humans132 and large mammals193 but not rodents194.
3.1.3 Arrhythmia inducibility
If atrial fibrillation is to be studied, the most important parameter to measure is atrial vulnerability itself. The least artificial means of assessing vulnerability to AF is long-term non-invasive measurement of spontaneous cardiac rhythm. This is difficult to achieve. Clinical research can approximate this with repeated ECG recordings or short to medium term continuous ambulatory ECG recording195. A more invasive approach involves using implantable devices capable of recording cardiac rhythm such as pacemakers196 or implantable loop recorders197.
A disadvantage of these approaches is the low event frequency in healthy experimental subjects and therefore the requirement for unacceptably long monitoring periods to detect a single arrhythmic episode. A less physiological but more pragmatic approach involves the artificial induction of AF. Several methods have been used to induce AF experimentally. The two most commonly used approaches to stimulation involve an extrastimulus approach or burst pacing. The extrastimulus method used a train of paced beats (S1) separated by a constant gap (S1S1 interval), followed by two or more extrastimuli (S2, S3 etc.) separated by a brief interval that gets progressively shorter as the test continues (S1S2 interval, S2S3 interval etc.). Burst pacing involves stimulating the tissue at very high frequency, typically 50-100 Hz. Stimulation may be delivered to the left198 or right199 atrium via an endocardial200, epicardial201, or transoesophageal202 approach. In human studies, the incidental occurrence of AF during an EP study has been used as a measure of atrial vulnerability203.
No standard approach exists for quantifying any AF resulting from the above measures. Experimental subjects may simply be graded as inducible or non-inducible200, 202. Alternatively, the proportion of stimulation attempts that lead to AF can be quantified199, 201. The duration of any AF induced can also be measured199, 201, 204. If extrastimuli have been employed, the range of coupling intervals that induced AF or “window of vulnerability” has been suggested as a measure of susceptibility to AF198, 205. An alternative method is the fibrillation current threshold. Using this technique, burst pacing is applied repeatedly, incrementing the applied current on each burst. The minimum current causing fibrillation is designated the fibrillation threshold. This has proven a reproducible method to assess vulnerability to ventricular fibrillation206 and has also been used to assess atrial vulnerability in rabbits207. P a g e | 85
3.1.4 Aims
The aim of this chapter is to gauge whether sheep represent a suitable model for human atrial ageing. Aspects of atrial electrophysiology that are well known to change with age in man will be assessed using techniques that are regularly employed in clinical practice, the surface ECG and non-invasive electrophysiological study. Following this, the vulnerability of the atria to fibrillation will be evaluated using burst pacing.
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3.2 Results
3.2.1 Body mass
The model of ageing used here aims to compare fully grown young adult sheep with sheep in the last quintile of life. Sheep used in this work were weighed to determine if young subjects were indeed fully grown. Young adult sheep weighed 32.9 ± 1.3kg, while aged sheep weighed 38.0 ± 2.3kg (p=NS, n = 48).
3.2.2 ECG morphology
ECGs were recorded from young and old anaesthetised sheep to look for changes in atrial conduction (Figure 3.1A). ECG morphological features are summarised in Table 3.1.
P-wave duration was 14% longer in old compared to young sheep (Figure 3.1B) (p<0.05, n = 29). The PR interval showed a similar pattern of age-dependent prolongation although this did not reach statistical significance (Figure 3.1C). P wave amplitude did not differ between groups.
ECG features pertaining to ventricular conduction and repolarisation were unchanged (Figure 3.1D and E).
Young Old Difference p-value Heart rate 89 ± 6.1bpm 90.1 ± 7.2bpm 1.2% 0.91 PR interval 101 ± 3.3 ms 109.5 ± 3.7 ms 9.0% 0.10 P duration 42.6 ± 1.4 ms 48.7 ± 1.5 ms 14.3% 0.01 * QRS duration 39.2 ± 2.4 ms 44.6 ± 3.3 ms 14.0% 0.18 QT Interval 306.1 ± 7.2 ms 290.2 ± 11.8 ms -5.2% 0.23 QTc 366.7 ± 6.4 ms 348.3 ± 13.2 ms -5.0% 0.18 P-wave amplitude 109.6 ± 15.2mV 107.2 ± 8.8mV -2.2% 0.90 R-wave amplitude 222.9 ± 31mV 195.3 ± 49.7mV -12.4% 0.62
Table 3.1 – ECG Parameters. n = 12-17 animals in each group. * p<0.05
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Figure 3.1 – ECG differences between young and old sheep. A Representative traces with P- wave duration and PR interval marked. B P-wave duration. C PR interval. D QRS duration. E Corrected QT interval. n = 12-17 animals in each group. * <0.05. NS - not significant. QTc - corrected QT interval. P a g e | 88
3.2.3 Electrophysiological parameters
Electrophysiological studies were performed in conscious sheep, the findings from which are summarised in Table 3.2.
SNRT was measured as a marker of sinus node function. Although the raw SNRT did not differ between groups, when corrected for sinus cycle length, the cSNRT was 64% longer in old compared to young sheep (Figure 3.2A)
The Wenckebach point was used to assess AVN function. No significant difference was seen in Wenckebach point with age (Figure 3.2B).
Atrial ERP was measured as a marker of atrial repolarisation. A trend was seen towards age-related ERP prolongation although this did not reach statistical significance (Figure 3.2C).
Young Old Difference p-value ERP 160 ± 7.1 ms 176.7 ± 9.1 ms 10.4% 0.16 SNRT 0.6 ± 0.1s 0.7 ± 0.1s 11.3% 0.44 cSNRT 124.3 ± 40.1 ms 203.8 ± 43.5 ms 63.9% 0.01a * Sinus cycle length 545.4 ± 34.6 ms 495 ± 25.6 ms -9.2% 0.27 Wenckebach point 245 ± 28.7 ms 280 ± 32.9 ms 14.3% 0.43
Table 3.2 – Electrophysiological parameters. n = 12-14 animals in each group. * p<0.05. a Persistently non-normally distributed data. Rank sum test applied. ERP – effective refractory period. SNRT – sinus node recovery time. cSNRT – corrected sinus node recovery time.
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Figure 3.2 – Electrophysiological parameters. A Corrected sinus node recovery time measured as time from last stimulation of 30s train to next sinus beat minus sinus cycle length. B Wenckebach point measured as longest S1S1 interval causing <1:1 atrioventricular capture and progressive PR lengthening. C Effective refractory period measured as shortest S1S2 interval that led to atrial capture following train of 8 beats. n = 12-14 animals in each group. * p<0.05. NS not significant. ECG – surface electrocardiogram. EGM – intracardiac electrogram. Representative traces smoothed using 9-point triangular filter for illustration only.
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3.2.4 Arrhythmia inducibility
Arrhythmia induction was performed at the end of the electrophysiological studies in conscious subjects. AF of at least two seconds duration was inducible on at least one attempt (Figure 3.3A) in the overwhelming majority of animals following burst pacing (13/14 young, 12/12 old, p = NS) but very rarely by extrastimulus pacing (0/14 young, 1/12 old, p = NS).
Old sheep demonstrated an increase in atrial vulnerability as shown by a greater than two-fold increase in the mean duration of AF elicited by burst pacing (Figure 3.3B). An increase in the proportion of bursts that initiated AF was also seen, although these differences did not reach statistical significance (Figure 3.3C and D). Furthermore, no difference was seen in the threshold voltage needed to induce AF (Figure 3.3E). A summary of these findings can be seen in Table 3.3.
An assessment was made of the reproducibility of the atrial vulnerability quantification methods. The coefficient of variation (COV) for the three measurements of AF duration was 101.0%, with a correlation coefficient of 0.28. The COV for the two measurements of AF threshold was 25.1% with a correlation coefficient of 0.48. These measures showed a high degree of variability suggesting that repeated testing is required to ensure a meaningful mean value.
A Bland-Altman plot was constructed of the duration of AF elicited on the first and last 5V 5s burst (Figure 3.3C). A non-significant bias of -7.2s was seen (p = NS with one-sample t-test). The plot had a funnel-shaped appearance demonstrating that the duration of AF was much less reproducible at longer durations, supporting the practice used here of curtailing the maximum duration at 180 s.
Young Old Difference p-value AF duration 4.3 ± 1.4s 15.9 ± 4.4s 267.7% 0.02a * % inductions > 2s 35.4 ± 5.3% 48.5 ± 8.4% 37.0% 0.19 % inductions > 5s 22.4 ± 3.9% 36.3 ± 7.7% 62.2% 0.10 AF threshold (mean) 3.3 ± 0.6V 3.4 ± 0.5V 3.3% 0.89 AF threshold (minimum) 3.2 ± 0.8V 3 ± 0.4V -6.7% 0.82
Table 3.3 – Arrhythmia inducibility. n =12-14 animals in each group. * p<0.05. a Data transformed by square root to achieve normal distribution. AF – atrial fibrillation.
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Figure 3.3 – Arrhythmia inducibility. A Representative ECG and EGM before, during and after 50 Hz burst pacing. B Mean duration of AF induced by 5s burst of 50 Hz stimulation at 5V. C Bland-Altman plot demonstrating variability of consecutive measurements of AF duration. D Proportion of 50 Hz bursts from 1V to 8V that led to AF of greater than 5s duration. E Proportion of 50 Hz bursts from 1V to 8V that led to AF of greater than 5s duration. n = 12-14 animals in each group. * p<0.05. NS - not significant, ECG – surface electrocardiogram, EGM – intracardiac electrogram. P a g e | 92
3.2.5 Effect of anaesthesia
In a subgroup of 6 young animals, electrophysiological studies and arrhythmia inducibility were carried out in the anaesthetised state in addition to performing conscious measurements. This data was collected to establish whether data recorded under the two conditions was similar. The results, shown in Table 3.4, demonstrate that while no significant changes were found after 6 animals were tested, a strong trend was seen towards increasing atrial vulnerability.
Conscious Anaesthetised Difference p-value ERP 165 ± 10.4 ms 183.3 ± 18.6 ms 11.1% 0.50 SNRT 650 ± 67.4s 880 ± 187.4s 35.4% 0.42 Wenckebach point 270 ± 10 ms 292.5 ± 11.1 ms 8.3% 0.34 AF duration 2.4 ± 1.6s 10.2 ± 6.4s 330.4% 0.41 % inductions >2s 28.3 ± 6% 38.2 ± 6.8% 34.8% 0.13
Table 3.4 – Effect of anaesthesia on electrophysiological parameters. n=6 animals (paired).
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3.3 Discussion The aim of this chapter was to verify that the ovine model of ageing used here is an appropriate surrogate for the changes associated with senescence in man. The principle findings are that, in comparison to fully grown young sheep, aged sheep are more susceptible to AF induced by burst pacing, have a longer P-wave duration on the surface ECG and a longer corrected sinus node recovery time.
3.3.1 The P-wave duration is prolonged in older sheep
The typical P-wave duration in man is ~90 ms 131, which is double that of the ~45 ms seen here. Despite this, the 14% increase in P-wave duration seen in this model of ageing is comparable to changes in P-wave duration seen with age in human subjects 131. The interspecies difference in p-wave duration probably represents body mass. If a similar heart weight to body weight ratio is assumed, we might expect the sheep hearts studied here to be approximately half the size of those seen in man. Smaller atria need a shorter time period to depolarise, leading to an abbreviation of P-wave duration and PR interval.
The P-wave is longer in larger atria. Atrial size increases with age in man130 and dogs208, and is a strong predictor of recurrent AF after cardioversion209. Previous work in this model has shown that left atrial diameter increases with age (unpublished data). However, visualising the atria using transthoracic echocardiography is difficult in the sheep due to the shape of the sternum, and these measurements have not been repeated in this body of work. Hypertrophy of atrial myocytes has also been demonstrated in this model (unpublished data), which may also be associated with atrial dilatation. Cell size will be examined in Chapters 4 and 6.
Slower atrial conduction also prolongs the p-wave. Atrial CV, modulated by fibrosis, INa, and gap junction expression, may decrease with age in man132, 210, although some have shown an increase in atrial CV in older humans211. Similarly, contradictory reports from animal studies have shown atrial CV to decrease212, increase213 or remain unchanged193 with age. As slower atrial conduction could explain the longer P-waves seen here, CV and its determinants will be explored in detail in Chapter 6.
Increasing P-wave duration is associated with incident AF131, 180. Mechanistically, this could be explained by slower conduction decreasing wavelength and facilitating re-entry. Additionally, increasing tissue mass may provide space for a greater number of simultaneous re-entrant circuits and thereby reduce the chance of all wavelets extinguishing at once. Furthermore, conditions that promote atrial dilatation may also cause tissue fibrosis in a patchy distribution setting the stage for micro-conduction block. P a g e | 94
Other features of the ECG did not change significantly with age. It is somewhat surprising that the PR interval was not significantly different between age groups, as P-wave contributes to the PR interval. There was, however, a trend towards increasing PR interval with a p-value of 0.10, suggesting that an inadequate sample size was responsible. The non-significant trend towards a 10% increase in PR interval is similar to that seen in man131.
3.3.2 The corrected sinus node recovery time is prolonged in older sheep
Sinus node function deteriorates with age in man, as evidenced by the prevalence of sick sinus syndrome in the elderly214. Consistent with this, the marker of sinus node function used here, the corrected sinus node recovery time, also showed evidence of sinus node impairment in older sheep. The cSNRT in young sheep was ~120 ms, similar to that seen in dogs 185 but less than the 210 ms seen in man132, but the age-related increase in cSNRT of 60% seen here is consistent across species.
From a cellular electrophysiological perspective, membrane ion channels remodel with age, as do proteins in the sarcoplasmic reticulum. Although in comparison to atrial muscle the SAN
215, 216 expresses little INa, the small amount that is present declines with age , exacerbating the tendency to conduction block and therefore impaired sinus node function. HCN4 is the channel responsible for If, the current that causes spontaneous diastolic depolarisation in pacemaker cells. SAN HCN4 mRNA decreases with age in the rat184, which would be expected to lead to bradycardia. An additional mechanism explaining SAN automaticity is the ‘calcium clock’, whereby Ca2+, spontaneously released from the SR via the RyR, is extruded via NCX causing membrane depolarisation217. RyR mRNA decreases with age in the rat SAN184, potentially impairing SAN pacemaking ability via a diminished Ca2+ clock, reflected in the increased cSNRT seen here. The compromised electrical conduction that leads to impaired SAN function could also underlie atrial vulnerability to fibrillation.
The increase in cSNRT seen with age may reflect changes in the tissue-level structure of the SAN or electrophysiological changes at a cellular level. From a structural perspective, the SAN does not appear to change in size with age in man215, although it enlarges with age in the rat216. Remodelling of the extracellular matrix occurs with age in humans and large mammals, leading to a reorganisation of collagen into a fine network surrounding SAN cells without affecting total collagen content215. This collagen network may interfere with intercellular connections, exacerbated by decreasing connexin-43 expression as shown in the ageing guinea-pig SAN218. These alterations could potentially cause a failure of impulse transmission from the SAN to the surrounding atrial myocardium and therefore increase the cSNRT. P a g e | 95
Deteriorating SAN function may represent an end result of pathology that also precipitates AF. However, some data suggests that the sinus node impairment itself, rather than the underlying pathology, can increase atrial vulnerability. It appears that regular activity from the sinus node suppresses ectopic foci in the pulmonary veins, and when the electrical connections between the two are blocked an increase in pulmonary vein ectopy results, potentially triggering AF219.
No other statistically significant differences were seen between old and young sheep. The Wenckebach point was similar between age groups, despite Wenckebach occurring at longer cycle lengths in older humans186 and rats189. This may be because even in man, the Wenckebach point is highly variable and the small sample was underpowered. However, a true difference may have been missed owing to the method of assessment used. A decision was made to avoid pacing the atria at very short cycle lengths as there was concern that this could affect assessment of AF inducibility. The shortest cycle length used was therefore 200 ms. A significant number of animals still maintained 1:1 conduction at these short cycle lengths and therefore the true Wenckebach point was not obtained. This serves to minimise any true difference between groups.
The right atrial ERP of ~160 ms is similar to that seen in humans at ~180 ms 203, but was not significantly different between age groups. However, a trend was seen towards a 10% prolongation in older subjects (p = 0.16) that is similar to the age-related increase in atrial ERP reported in some human studies88, 132. It should be noted that some well powered human studies have still failed to demonstrate a significant difference in right atrial ERP between old and young subjects220.
3.3.3 Atrial fibrillation sustains itself for longer in older sheep
Previous descriptions of AF induction in anaesthetised sheep have reported that 34% of inductions led to AF lasting >10s 221. In their small study (n=11) a significant difference in atrial vulnerability was not found between conscious and anaesthetised animals. These findings are broadly similar to those presented here. Studies in rats 194, rabbits 204 and dogs 222 have shown increased atrial vulnerability in senescent subjects compared to young adults.
Although the evidence supporting the vulnerability of elderly humans to spontaneous atrial fibrillation is incontrovertible6, 223, when AF has been induced artificially the picture has been less clear. Attempting AF induction using extrastimulus pacing, some have found no difference in atrial vulnerability between old and young88, while others have paradoxically found that younger patients were more susceptible to extrastimulus-induced AF224. A possible interpretation of these findings is that extrastimulus-induced AF is at best a weak surrogate for P a g e | 96 vulnerability to spontaneous AF. It has been suggested that this may be due to the longer ERP seen in older subjects offering a degree of protection from extrastimulus- induced AF. The data presented here, although not statistically significant, is in keeping with an age-related ERP prolongation. Despite this, atrial vulnerability to burst pacing-induced AF was greater in the old cohort. An optimistic interpretation for this could be that burst pacing represents a more realistic model of arrhythmia induction than isolated extrastimuli.
The mechanisms of atrial fibrillation, as detailed in Chapter 1.6, rest upon its initiation by abnormal automaticity or triggered activity, and its maintenance by re-entry either in the form of multiple wavelets or rotors. The ensuing chapters will explore some of the means by which AF can be perpetuated, and how this changes with age in terms of action potential duration (Chapter 4), alternans behaviour (Chapter 5) and electrical conduction (Chapter 6).
3.3.4 Limitations
One confounding variable is the effect of stress and therefore sympathetic tone. Sympathetic activation is well known to promote AF225, as well as modulate each of the parameters measured in this chapter. The consequence of subject tension was clearly seen when arrhythmia induction was attempted in the same animal in both free-roaming and gently restrained experimental conditions. During gentle restraint AF was readily provoked, while during the more relaxed state AF was non-inducible. Observing the behaviour of different subjects made it abundantly clear that some were naturally more anxious than others. Differential stress levels give the potential for a systematic bias. It is more likely, however, that fluctuating stress adds variability to the data points in a similar manner between groups, thereby masking true differences. Although the effects of stress could have been reduced by performing the studies under anaesthesia, the paired data suggests a marked difference in vulnerability to arrhythmias between the conscious and anaesthetised states. This could be due to the anaesthetic agent used, an increase in vagal tone or acute atrial inflammation due to lead implantation. Conscious measurements, it can be argues, represent a more physiological milieu.
The measurement of ECG parameters with a rounded onset and offset, such as the P-wave, is prone to observer error. This is less likely to occur with more clearly defined sharp deflections such as the QRS complex. ECG analysis was not blinded and therefore observer bias could have occurred, particularly in measuring more error-prone parameters. However, measurement of all parameters, including those marked by sharp deflections, followed a similar pattern of increasing with age, although not all reached statistical significance. P a g e | 97
As no standardised measure for AF inducibility exists, a variety of approaches were employed. Arrhythmia inducibility had not been robustly measured in our model and therefore it was not possible to prospectively specify which measure would be used as a primary outcome. The outcome of assessing these different measures is a set of discordant results. All analyses performed of atrial vulnerability have been presented here. It should be noted that the correlation between successive repetitions of measures of atrial vulnerability was poor, highlighting the need for a mean of repeated measures of the same parameter. However, this must be balanced against repeated AF induction leading to AF remodelling and a corresponding increase in vulnerability that would distort the findings. Future work will prospectively specify the endpoints to be used.
All subjects used in this work are female, for the practical considerations of supply and ease of handling. Epidemiological studies in man have shown that both males and females have an increasing incidence and prevalence of spontaneous AF with age 6. The majority of studies looking at age-related electrophysiological changes in man have not stratified their findings by sex. The limited data assessing both age and sex suggests that the cSNRT, sinus cycle length and QRS duration are shorter in females than males but gender does not influence the effects of ageing on atrial electrophysiology220.
3.4 Conclusions This set of experiments confirms that the sheep represents an appropriate model for ageing. Although the raw values for the electrophysiological parameters differ from those in humans, the age-associated changes in ECG morphology, sinus node function, AV node function and atrial ERP do not differ from those seen in humans. The atria of aged sheep, at least be some measures, are more vulnerable to fibrillation than those of young sheep.
This chapter creates a platform on which to build further experiments. The next chapter will investigate how the atrial action potential differs between young and old subjects.
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4 ACTION POTENTIAL MORPHOLOGY
4.1 Introduction
As discussed in Chapter 1, changes in the shape of the atrial AP, specifically shortening of the AP duration, are associated with atrial vulnerability. Conversely, atrial tachycardia remodelling changes the shape of the AP226. As might be expected given the strong association between ageing and AF, ageing too leads to alterations in several aspects of AP morphology. Similarly, as aberrant Ca2+ handling promotes triggered activity it is unsurprising that these properties also change with age.
4.1.1 Action potential characteristics affected by age
A less negative RMP has been found in old compared to young dogs when AP data from both atria was averaged49, and similar changes with age have been found in the rat 29 and rabbit227.
As the RMP determines the recovery from inactivation of INa, which controls the AP upstroke, it is unsurprising that these studies showed a corresponding decrease in AP amplitude and V̇max. 193 + However, others have shown no difference in RMP, AP amplitude, or V̇max with age . The K currents responsible for the RMP have not been investigated to date, and limited data concerning INa in dog atria has not shown a difference with age.
The AP duration increases with age in the left222, 227 and right49, 193 atria dogs and rabbits. However, the same changes are not seen in aged mice212, 228 and are inconsistently found in rats229, 230. The effective refractory period (ERP) is the interval following a first stimulus that a tissue will not respond to a second stimulus, and is governed in part by the AP duration. The atrial ERP is prolonged in older humans132 but not rodents194. As AP duration is determined by the balance of depolarising and repolarising currents, it might be expected that ICa(L) would increase with age. Instead, a reduction in ICa(L) with advancing age has been reported by most in dogs43, 231 and humans232, matching a reported decrease in height of the AP plateau193. However, some have reported the opposite finding of an elevated AP plateau and increase in
222 ICa with age in dogs .
An alternative explanation for a longer AP duration could be that one or more repolarising K+
+ currents may decrease. However, the transient outward K current Ito has been shown to increase with age in the right atrium of mice233 and dogs231. Correspondingly, mRNA for a protein which interacts with and upregulates Ito, KChIP2, is transcribed more with age in the 234 pig . These increases in Ito might be expected to cause AP shortening in older subjects, the reverse of what is seen. Little attention has been paid to other repolarising currents, but a P a g e | 99
233 single study showed no age-associated change in IKur in mouse atria . IK(ACh) may be more responsive to proarrhythmic vagal stimulation in aged atria28, 29 due to an increase in muscarinic receptors235, although baseline vagal activity decreases with age129, 236. Atrial pressure increases with age237, potentially affecting stretch-activated K+ channels, although this has not been specifically investigated.
4.1.2 Calcium cycling characteristics affected by age
One of the classical mechanisms of arrhythmia induction discussed in Chapter 1 is triggered activity leading to ectopic activations. Triggered activity, in the form of early and delayed afterdepolarisations, is dependent upon Ca2+ cycling, and abnormalities of Ca2+ cycling increase the likelihood of their occurrence13. Furthermore, Ca2+ cycling is affected by atrial tachycardia remodeling and is therefore markedly different in patients with a history of AF compared to those without70. It is therefore logical to assess whether the increased atrial vulnerability seen in age is related to aberrant Ca2+ handling.
Some data is available regarding how atrial Ca2+ cycling differs between the old and young, and although many aspects have been covered, the data originates from a very small number of
2+ 231 2+ published works. Ca entry is impaired, as shown by a decrease in ICa(L) , L-type Ca channel protein66 and mRNA66 in older dogs and humans232. The decrease in Ca2+ entry corresponds with a lower Ca2+ transient amplitude in older atrial myocytes232. Some have found SR Ca2+ content to decrease with age232, although the reverse of this was found in our sheep model of ageing (unpublished data). Ca2+ reuptake slows with age in rodents238 and man232, which has been associated with decreased SERCA expression by some232, but not in our model (unpublished data). Ca2+ release, as assessed by spontaneous Ca2+ waves, has not been shown to change with age232, corresponding with similar RyR expression between young and old66.
4.1.3 Aims
The changes in the shape of the AP with age are well described. However, much less is known about how ageing affects the relationship between right and left atrial APs that creates the spatial gradient of repolarisation. Furthermore, the majority of studies have recorded APs in vitro under conditions where important humoral factors are absent. The aim of this chapter is therefore to explore any changes that occur in the shape of the atrial action potential with age in female sheep, and whether similar changes occur in the left and right atria. Action potentials were recorded both in isolated atrial myocytes using perforated patch, and in vivo P a g e | 100 as monophasic action potentials. In a subset of isolated cells, basic Ca2+ handling properties were also examined. P a g e | 101
4.2 Results
4.2.1 Size of atrial myocytes
Atrial myocytes were obtained from the left and right atria of young and old sheep. The myocytes from old sheep were 12% longer and 10% wider than those from young sheep (Figure 4.1). Differences were also seen between myocytes from the left and right atria. Right atrial myocytes were 18% longer and 27% wider than those from the left atrium.
Data is summarised in Table 4.1.
Age Laterality Young Left Young Right Old Left Old Right difference difference Cell 114.6 ± 4.2μm 132.5 ± 6.6μm 124.7 ± 4.9μm 145.7 ± 4.8μm 11.8% * 17.8% * Length Cell 15.5 ± 0.8μm 18.9 ± 1μm 15.9 ± 0.8μm 20.8 ± 0.9μm 9.8% 27.2% *a Width 1782.3 ± 2508 ± 2006.3 ± 3054.5 ± Area 24.2% * 50.3% * 112.9μm2 201.3μm2 142μm2 188.2μm2
Table 4.1 – Atrial myocyte size. n = 20-32 cells, 6-14 animals in each group. * p<0.05, a non-normally distributed data, transformed by square root. RMP resting membrane potential.
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Figure 4.1 – Atrial myocyte size. A Left atrial myocyte from young sheep. B Right atrial myocyte from old sheep. C Mean cell length. C Mean cell width. n = 20-32 cells, 6-14 animals in each group. * p<0.05, NS - non-significant.
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4.2.2 Baseline characteristics of patch-clamped myocytes
Atrial myocytes were almost universally depolarised at rest, and under these conditions stimulation did not produce true APs (Figure 4.2A). The extent of resting depolarisation differed between age groups, and the RMP from old sheep was 17% more negative than those from young sheep (Figure 4.2C). A small holding current was therefore applied to force the
RMP to -80mV, permitting recovery from inactivation of INa and enabling APs (Figure 4.2B). Corresponding with the extent of depolarisation, the holding current required to force the RMP to -80mV was 29% smaller in older sheep (Figure 4.2D). The stimulation threshold and seal quality were similar between age groups.
Although no difference was seen in the RMP, a smaller holding current was required in right atrial myocytes (Figure 4.2 C and D). The stimulation threshold was 20% greater in right atrial myocytes. Seal quality was similar between all groups.
Data is summarised in Table 4.2.
Age Laterality Young Left Young Right Old Left Old Right difference difference RMP -39.3 ± 1.9mV -40.7 ± 2.7mV -47 ± 1.8mV -46.4 ± 2mV 17.3% * 3.3% Holding -20.7 ± 2pA -24.5 ± 2.9pA -20.4 ± 1.8pA -11.5 ± 1.9pA -28.6% φ -16.5% # current Threshold 21 ± 1.5A.U. 24.2 ± 1.8A.U. 23.1 ± 1.8A.U. 28 ± 1.3A.U. 15.2% 20.4% * 988.2 ± 929.5 ± 988.9 ± Seal 750 ± 69.8MΩ -9.4% 9.3% 61.5MΩ 98.5MΩ 77.4MΩ
Table 4.2 – Cell size and baseline parameters. n = 20-32 cells, 6-14 animals in each group. * p<0.05, # p<0.05 in right atrial myocytes only, φ p<0.05 in old myocytes only, .a non-normally distributed data, transformed by square root. RMP - resting membrane potential
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Figure 4.2 – Atrial myocyte baseline characteristics. A Voltage trace from stimulated left atrial myocyte without holding current. B Voltage trace from same myocyte following application of 20pA holding current to bring RMP to -80mV. C Mean baseline RMP for all groups. D Mean holding current applied. n = 20-32 cells, 6-14 animals in each group. * p<0.05, # p<0.05 in right atrial myocytes only, φ p<0.05 in old myocytes only, NS - non-significant, RMP - resting membrane potential.
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4.2.3 Transmembrane action potential amplitude
Atrial myocytes were stimulated at rates from 0.25 to 4 Hz. Increasing the stimulation rate led to shortening of the AP duration, depolarisation of the MDP and a decrease in AP amplitude (Figure 4.3). At low stimulation rates, each AP in the train was qualitatively similar. As stimulation rate increased, the uniform pattern disappeared. This sometimes assumed a pattern of large amplitude, small amplitude, large, small in a repeating pattern (Figure 4.3D), although occasionally patterns that repeated every third or fourth beat were seen. As the overall average of these repeating patterns did not resemble any of the individual beats, traces showing alternating patterns were not included in the analysis of AP morphology. Instead, they were analysed separately, and the results of this will be presented in Chapter 5.
Phase 0, rapid depolarisation, was examined first. The AP amplitude was calculated as the difference between the true AP peak and the MDP (Figure 4.4A), while the first differential of
the voltage trace was used to obtain V̇max (Figure 4.4B). In each case the stimulation artefact was ignored.
No significant differences between age groups were seen in AP amplitude, V̇max or time to peak when assessed using a 2-way repeated measures ANOVA (Figure 4.4C-F).
No significant differences were seen in AP amplitude, V̇max or time to peak between left and right atrial myocytes (Figure 4.4C, E and F).
Data is summarised in Table 4.3.
Age Laterality Young Left Young Right Old Left Old Right difference difference Peak 15 ± 1.9mV 15 ± 3mV 16.6 ± 2.1mV 19.8 ± 2mV 21.2% b 12.6% b Amplitude 95.8 ± 1.9mV 95.9 ± 3.3mV 97.1 ± 2.2mV 100.7 ± 2.4mV 3.1% b 2.3% -1 -1 -1 -1 a a V̇max 44.2 ± 5.4V.s 44.7 ± 7.2V.s 39.5 ± 5.8V.s 43.9 ± 5.3V.s -5.8% 5.2% Time to 14.7 ± 1.7 ms 14.4 ± 1.6 ms 17.3 ± 1.8 ms 16.4 ± 2.3 ms 15.7% b -2.2% b peak
Table 4.3 – Transmembrane action potential amplitude, time to peak and V̇max at 1 Hz stimulation in unloaded atrial myocytes. n = 20-32 cells, 6-14 animals in each group. * p<0.05. a non-normally distributed data, transformed by square root. b persistently non-
normally distributed data. V̇max - maximum rate of rise of action potential.
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Figure 4.3 – Effects of stimulation rate on action potential morphology. A Left atrial myocyte from young sheep stimulated at 0.5 Hz. B Same cell stimulated at 1 Hz. C Same cell stimulated at 2 Hz. D Same cell stimulated at 3 Hz with alternans present. E Superimposed action potentials recorded at different stimulation rates to highlight changes in action potential shape. P a g e | 107
Figure 4.4 – Transmembrane action potential amplitude, time to peak and V̇max in unloaded atrial myocytes. A Representative trace demonstrating stimulation artefact and true peak of action potential. B 1st derivative of same action potential demonstrating calculation of
V̇max. C Action potential amplitude from left and right atrial myocytes from young and old sheep at 1 Hz. D Action potential amplitude changes with stimulation frequency. E Action potential time to peak at 1 Hz. F Action potential V̇max at 1 Hz. n = 20-32 cells, 6-14 animals in each group. * p<0.05 for difference between age groups using RM ANOVA at marked frequencies only. NS - not significant, V̇max - maximum rate of rise of action potential, AP - action potential. P a g e | 108
4.2.4 Transmembrane action potential duration
The repolarisation phase of the cellular AP was examined next (Figure 4.5A). Data is
summarised in Table 4.4. Using a 2-way repeated measures ANOVA, APD50 and APD90 were
longer in myocytes from older animals (p<0.05). At 1Hz stimulation, APD90 and APD50 were 27% and 23% longer respectively in myocytes from old compared to young sheep (Figure 4.5B and C). As stimulation rate increased, AP duration declined (p<0.05, Figure 4.5D). AP duration decreased at a similar rate in myocytes from old and young sheep. The MDP was similar between age groups at low stimulation rates but depolarised at higher rates (Figure 4.5E). This depolarisation was more pronounced in myocytes from old sheep at the highest stimulation rates.
Using a 2-way repeated measures ANOVA to assess differences in APD between the left and right atria, laterality differences did not reach statistical significance overall, but post-hoc analysis of interactions showed that a significant difference was present at stimulation frequencies from 0.25 to 1Hz. Right atrial myocytes exhibited APs that were 8% and 10% longer at 50% and 90% repolarisation respectively compared to those from left atrial myocytes (Figure 4.5C). The inter-atrial differences at low stimulation rates were present in myocytes from young and old animals, although were more marked in younger animals. Maximum diastolic potential was similar between the left and right atria at low stimulation frequencies but right atrial myocytes were less able to maintain their polarity at the highest rates.
Age Laterality Young Left Young Right Old Left Old Right difference difference MDP -80.8 ± 0.2mV -81 ± 0.6mV -80.5 ± 0.6mV -80.9 ± 0.5mV -0.2% b,d 0.3% b,d b b,c APD50 88.4 ± 9.3 ms 109.3 ± 8.9 ms 126.4 ± 9.6 ms 116.5 ± 10.7ms 26.8% * 8.0% * b b,c APD90 333.1 ± 26 ms 381.8 ± 24.8ms 425.1 ± 21.4ms 436.1 ± 19.8ms 22.7% * 10.4% *
Table 4.4 – Transmembrane action potential duration and minimum diastolic potential at 1 Hz stimulation in unloaded atrial myocytes. n = 20-32 cells, 6-14 animals in each group. * p<0.05. b persistently non-normally distributed data. c significant difference using RM ANOVA including stimulation rates from 0.25 to 1 Hz. d significant at 4 Hz only. MDP - minimum diastolic potential. APD50 - action potential duration at 50% repolarisation. APD90 - action potential duration at 90% repolarisation. P a g e | 109
Figure 4.5 – Transmembrane action potential duration in unloaded atrial myocytes. A
Representative action potential demonstrating calculation of APD90. B Mean action potentials from young and old left atria at 1 Hz with standard errors. C APD90 from left and right atria of young and old sheep at 1 Hz. D Action potential duration decreases at faster stimulation rates. E MDP becomes less negative at faster stimulation rates. n = 20-32 cells, 6-14 animals in each group. * p<0.05, NS - not significant. MDP - minimum diastolic potential. APD90 - action potential duration at 90% repolarisation. P a g e | 110
4.2.5 Monophasic action potential morphology
Right atrial AP morphology was also examined in vivo. Representative MAPs can be seen in Figure 4.6A and B and data is summarised in Table 4.5.
No difference was seen in APD90, but surprisingly a trend was seen towards a shorter APD90 in the old cohort (Figure 4.6 C and D). No significant differences were seen between age groups in amplitude, V̇max or time to peak (Figure 4.6E and F).
As was seen in the cellular AP recordings, at high stimulation rates oscillations in the AP morphology developed between beats. These will be discussed in Chapter 5.
Young Old Difference p-value Amplitude 5 ± 0.4mV 4.6 ± 0.5mV -7.7% 0.60 a APD90 219.6 ± 16.5 ms 191.6 ± 9.4ms -12.7% 0.13 -1 -1 V̇max 1.6 ± 0.2 V.s 1.4 ± 0.3 V.s -13.6% 0.54 Time to peak 4.1 ± 0.7 ms 4.8 ± 0.6 ms 15.7% 0.50
Table 4.5 – Monophasic action potential morphology at 2 Hz. n = 10-11 animals in each group. APD90 - action potential duration at 90% repolarisation, V̇max - maximum rate of rise of action potential upstroke, a persistently non-normally distributed data. P a g e | 111
Figure 4.6 – Monophasic action potential morphology. A Representative trace demonstrating calculation of APD90. B Representative monophasic action potentials from the right atrium of young and old sheep. C APD90 stimulated at 2 Hz. D APD90 stimulated at 2-4 Hz. E Amplitude stimulated at 2 Hz. F Time to peak stimulated at 2 Hz. n = 10-11 animals in each group. NS - not significant, APD90 - action potential duration at 90% repolarisation.
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4.2.6 Calcium transient amplitude and rate of decay
After examining the shape of the action potential, the effect of ageing on the Ca2+ handling properties of isolated myocytes was then studied, as these too have been implicated in arrhythmogenesis.
Left atrial myocytes from six animals were loaded with the fluorescent indicator Fluo-5F to
2+ 2+ measure [Ca ]i. 20 consecutive Ca transients were averaged and a single exponential was fitted to the decay phase (Figure 4.7A). A reliable Fmax was not obtained in all cells and so data is expressed as F/F0 and summarised in Table 4.6.
No significant differences were seen in the amplitude, time to peak, or rate of decay of the Ca2+ transient between age groups (Figure 4.7A(ii), B, C, and D). However, a strong trend was seen towards a decrease in Ca2+ transient amplitude and a slower rate of decay.
Young Old Difference p-value
Amplitude (F/F0) 5.5 ± 1.5 1.9 ± 0.6 -65.7% 0.15 Time to peak 134 ± 8 ms 154.2 ± 32.9 ms 15.1% 0.39 Rate of decay 235.8 ± 27.9 ms -1 329.5 ± 24.7 ms -1 39.8% 0.08
Table 4.6 – Calcium transient amplitude and rate of decay. n = 5-14 cells, 2-4 animals in each group. P a g e | 113
Figure 4.7 – Calcium transient amplitude and rate of decay. A Representative traces. (i) Black – single calcium transient, green – average of 20 calcium transients, white – single exponential fitted to decay phase. (ii) Representative transients from young and old left atrial myocytes (iii) Normalised transients to emphasise difference in decay phase. B Calcium transient amplitude. C Calcium transient time to peak. D Rate of decay of calcium transient. n = 5-14 cells, 2-4 animals in each group. * p<0.05, NS - not significant. P a g e | 114
4.3 Discussion
The main findings presented in this chapter are that, when compared to young sheep, the atrial myocytes from old sheep are larger and have a more negative RMP at rest. The APs from old atrial myocytes have larger amplitudes at low stimulation rates but this difference disappears at higher rates. The AP duration is prolonged in older atrial myocytes. These differences were not found when right atrial monophasic APs were recorded in vivo. In both cellular and monophasic AP recordings, high stimulation rates led to an alternating pattern of long, short, long, short AP morphology, which will be explored in greater detail in Chapter 5. No significant differences were found in Ca2+ handling, but a strong trend was seen towards smaller, more slowly decaying Ca2+ transients in the small sample obtained.
4.3.1 Myocytes are larger in the right atrium than the left and hypertrophy with age
The length (~120 μm) and width (~18 μm) of atrial myocytes found here is comparable to that of previously reported sheep239, dog240 and human atrial myocytes241. Increases in atrial myocyte size in animal models of ageing have been suggested using cell capacitance as a measure of cell surface area160, 194, 233. The 17% increase in cell length between right and left atrial myocytes is consistent with previous work in pigs (sex unspecified)242, although such a difference was not found in male sheep239. Conversely, in rodents, myocytes from the left atrium are longer than those from the right242,243.
The observed increase in myocyte size with age may reflect atrial dilatation as a whole. This could occur in response to long-term changes in loading conditions, as haemodynamic overload has been shown to cause atrial myocyte hypertrophy as well as atrial dilatation241. Left atrial pressure, assessed using the echocardiographic E/e’ ratio, has been shown to increase with age in man237. These changes have been reported in subjects with normal blood pressure, but are accelerated in the presence of systemic hypertension. The prevalence of hypertension is well-known to increase with age in man244 and has also been shown in the ovine model of ageing used in this laboratory (unpublished data). While pressure in the pulmonary circulation and right ventricle increase with age245, it is uncertain if this translates into an increase in right atrial pressures246.
Cellular hypertrophy also occurs in reaction to myocyte loss. It has been shown in the human ventricle that ageing leads to a decline in the total number of cardiomyocytes with a corresponding increase in cell size247. A similar phenomenon may occur in the atria but has not yet been demonstrated in man. P a g e | 115
It is unclear why right atrial myocytes are larger than those from the left atrium. If atrial pressures were solely responsible for myocyte size then the tendency to slightly higher pressures in the LA than the RA in sheep248 and pigs249 might suggest that left atrial myocytes would be larger. No difference in myocyte size was found between atrial sides in male sheep239, raising the possibility of a sex-specific difference.
4.3.2 Isolated atrial myocytes are depolarised at rest
Atrial myocytes were almost universally depolarised under resting conditions. This is a consistent finding in our sheep model amongst atrial but not ventricular myocytes (unpublished data), and has also been reported in isolated atrial myocytes from humans250, 251 and trout252. It has been suggested that the depolarisation is due to a loss of the repolarising
+ 252 + K current IK(ACh) , most likely due to a loss of cholinergic tone but potentially due to K channel digestion during the isolation process. This is plausible as IK(ACh) is present at much greater density in the atria compared to the ventricles, explaining why this phenomenon is not seen in ventricular myocytes.
The RMP was more negative in older myocytes. This was unexpected as previous reports have
49 suggested that the RMP becomes less negative with age . The potassium current IK1 plays the greatest role in determining the RMP, but the effects of age on this specific current have not been investigated.
If we instead see these depolarised myocytes as being deficient in IK(ACh), could the more negative RMP in older myocytes reflect differences in this current? It has been suggested that aged atria are more responsive to cholinergic stimulation28, 29. This could imply a greater density of IK(ACh), which would be expected to lead to a greater depolarisation at rest if the current was lost. Alternatively, it could represent less basal IK(ACh) activation, leading to less resting depolarisation in its absence.
Alternatively, some of the resting depolarisation could arise from leak between the patch pipette and the cell membrane. A similar leak will cause more depolarisation in a smaller cell. Therefore, if leak was similar between groups but older myocytes were larger, less apparent depolarisation would occur in older cells.
The RMP plays a potent role in determining the recovery from inactivation of INa; a more negative RMP will therefore increase AP amplitude and rate of rise. Any intrinsic differences in RMP in these experiments will however be masked as RMP was consistently forced to -80 mV. P a g e | 116
4.3.3 Action potential amplitude does not change with age.
The transmembrane AP amplitude (~98mV) seen here is within 10% of that reported in previous literature using the sharp microelectrode49, 193, 222 and perforated patch techniques27. When assessed over the full range of stimulation frequencies, no differences were seen in AP amplitude between age groups. However, at low stimulation rates the AP amplitude was greater in older cells, while at faster stimulation rates AP amplitude was smaller in older cells. These opposing trends at low and high stimulation rates may reflect an inability of older myocytes to sustain an MDP. Depolarisation of the MDP occurred at lower stimulation rates in myocytes from old sheep due to the longer APs seen in older myocytes, leading to reduced recovery from inactivation of INa and therefore diminishing the AP amplitude.
Of four publications found examining the effect of age in the dog, three found no difference in AP amplitude28, 193, 222 while one showed decreased amplitude in older subjects49. It should be noted that two further reports using the same dataset as Gan et al. (2013) were ignored43, 66. Studies using rodent models of ageing have described a decrease in atrial AP amplitude with age227, 230.
The amplitude of the monophasic AP recordings was much smaller and did not show any significant age-related changes. This difference in results between recording techniques reflects the process that each parameter signifies. Unlike the transmembrane AP which directly records membrane potential, the monophasic AP records current flowing to and from a partially depolarised area of tissue directly under the electrode. The size of this current depends upon how depolarised the local tissue is, which a function of electrode contact pressure, and the thickness of the tissue253. This distinction means that while monophasic AP amplitude may be of use when examining changes within a single recording, it is far less valid when comparing traces between subjects.
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4.3.4 Action potential upstroke recorded with perforated patch does not differ with age
The measurements of V̇max do not fit well with existing literature. Using perforated patch -1 clamp, V̇max was calculated as ~40 Vs , whereas using the sharp microelectrode technique V̇max measurements are generally around three to eight-fold higher28, 193, 227. The significant underestimation seen here is likely to be due to do with the experimental technique. In the sharp microelectrode technique, tissue stimulation and recording of membrane potential are performed by separate electrodes, meaning that the upstroke of the AP is not contaminated by a stimulation artefact. In the work presented here, cell stimulation and recording of membrane potential are both performed by the same electrode, leading to superimposition of the stimulation artefact upon the AP upstroke (Figure 4.4A and B). The steepest segment of the AP upstroke occurs early, and this is masked in the recordings performed here. Contributing to the underestimation is the acquisition sampling rate. A longer sample interval underestimates V̇max compared to a shorter one. The studies mentioned above use sampling rates of up to 125 kHz227, considerably faster than the 2.5 kHz used here. Because of these flaws, it is unsurprising that wide error bars were present and no differences were seen between groups.
4.3.5 Action potential duration is longer in isolated myocytes than intact tissue
The two techniques used here to record AP duration produced quite different results. The right atrial AP duration recorded using perforated patch clamp (~300 ms at 2 Hz) was ~50% greater than that recorded using monophasic APs at a comparable rate (~220 ms at 2 Hz).
These findings are consistent with previous work in the dog, where the transmembrane AP in isolated atrial myocytes under similar experimental conditions to those used here showed an
31 APD90 of 340 ms when stimulated at 1 Hz, compared with 330 ms seen here . Comparable results have been found using the whole-cell patch clamp technique222.
Similarly, monophasic APs recorded from canine atrial tissue showed an APD90 of 170 ms at 2 Hz stimulation compared to 220 ms seen here254. Canine APs recorded using the sharp microelectrode technique in tissue strips show a comparable timecourse to monophasic APs193, suggesting that the discrepancy lies within the difference between intact tissue and isolated myocytes.
These differences could be explained by a decrease in repolarising currents in isolated myocytes. While this could be due to a loss of IK(ACh), the isolated canine myocyte studies above made no mention of RMP depolarisation. In the same vein, sympathetic tone may P a g e | 118 impact upon repolarising currents in vivo, thereby influencing MAP recordings but not measurements from isolated myocytes. This would not, however, account for the differences seen between single cells and in-vitro tissue strips. Stretch-sensitive K+ currents will likewise be activated in vivo but not in-vitro, shortening AP duration.
Alternatively, the reason could lie within the electrotonic effects between adjacent myocytes or between myocytes and fibroblasts. Electrotonic interaction between myocytes and electrically active fibroblasts has been proposed to accelerate repolarisation in silico41, although a modelling study of atrial myocyte coupling to fibroblasts predicted that coupling could lead to either shortening or prolongation of the APD depending on how many fibroblasts were involved255. However, the electrotonic effects of non-excitable cell types may slow repolarisation. The addition of mesenchymal stem cells to cultured cardiomyocytes slowed repolarisation and promoted arrhythmogenesis42. Similarly, the addition of HEK cells expressing gap junctional proteins prolonged the AP duration of cultured cardiomyocytes256. As the majority of cells in the heart (far outnumbering myocytes) are fibroblasts, the more rapidly repolarising in vivo APs are likely to reflect electrotonic coupling between myocytes and excitable myofibroblasts, an interaction that is missing in vitro.
4.3.6 Action potential duration prolongs with age in isolated myocytes
The cellular AP recordings mirror the age-related atrial AP prolongation seen in the majority of large mammalian studies28, 49, 193, 222. No significant difference between age groups was seen in the monophasic AP recordings. There are several possible causes for this. Firstly, the in vivo sample size was much smaller, raising the possibility that these experiments were underpowered to detect a true difference. If this were the sole explanation, a non-significant trend towards monophasic AP prolongation with age might be expected. However, the trend in this set of experiments was towards a shortening of AP duration with age, a reversal of the cellular findings.
A second explanation is that experiments on isolated cells do not take into account all factors that modulate AP duration, such as autonomic tone and atrial stretch. Parasympathetic tone decreases with age, potentially prolonging AP duration via IK(ACh), while atrial pressure rises, potentially shortening AP duration via stretch sensitive K+ currents. The body of published literature falls foul of this flaw, and to my knowledge there are no published studies that have investigated atrial ageing using the in vivo monophasic AP technique. As a counter to this argument, the atrial ERP, which is determined by AP duration and Na+ channel kinetics, has been shown to increase with age in vivo132, 210. Although not direct evidence, an increase in ERP is more consistent with increased AP duration. P a g e | 119
Thirdly, monophasic APs were recorded in anaesthetised subjects. The anaesthetic agent used, isofluorane, has been shown to influence the atrial and AV nodal ERP in Langendorff- perfused guinea-pig hearts, although changes in the monophasic AP duration did not reach statistical significance257. It is possible that anaesthesia could have affected atrial electrophysiology unequally between age groups.
Fourthly, monophasic APs were recorded from the posterior wall of the right atrium, while myocytes were obtained from the right and left atrial appendages. . Previous work has shown that while the electrical properties of regions of the atria are relatively similar in youth, heterogeneity develops with age49. This may be caused by a prolongation of AP duration in some regions but not others, a hypothesis that would be in keeping with the results presented here.
The mechanisms underlying the increase in AP duration at the cellular level could involve a decrease in depolarising or an increase in repolarising currents. The major depolarising
222, 231, 232 currents in atrial myocytes are ICa(L) which decreases with age , ICa(T) which has not been studied in age, and INa, a single study of which did not find a significant difference between age groups160. None of these results explain a prolongation of APD. The current state of knowledge regarding age-related changes in repolarising currents is equally unenlightening.
231, 233 The major repolarising currents in atrial myocytes are Ito which increases with age and 233 would therefore be expected to shorten AP duration; IKur which does not change with age ; and IKr, IKs, IK1,and IK(ACh) which have not been investigated for age-related changes. INa as a possible contributor to AP duration prolongation will be further investigated in Chapter 6.
AP prolongation could contribute to atrial vulnerability through triggered activity. EADs are most commonly seen in cells with longer APs that permit the recovery from inactivation of ICa(L) and subsequent re-activation. Although afterdepolarisations were not looked for in this work, limited data from rodent studies suggests that older atrial myocytes are more susceptible to EADs when stressed by glycolytic inhibition238. Sympathetic activation may also promote EADs, and studies of heart rate variability suggest that sympathetic tone increases while parasympathetic tone decreases in older persons127, 129.
However, triggered activity generating ectopic beats could be seen more as a mechanism for initiating AF rather than sustaining it. In Chapter 3 we showed that AF was more sustained in older sheep. AP prolongation increases the wavelength of re-entrant arrhythmias, which if the multiple wavelet theory is to be believed decreases the number of potential simultaneous re- entrant circuits, decreasing vulnerability to AF. AP prolongation does not, therefore, explain why AF lasts longer in older sheep. P a g e | 120
4.3.7 Action potential duration is longer in myocytes from the right atrium than the left at low stimulation rates
The AP duration in myocytes from the right atrium was 10% longer than that in left atrial myocytes at low stimulation rates, but that difference disappeared when myocytes were stimulated at more than 1 Hz. As distance from the SAN increases, atrial AP duration decreases in many species258-260, but this has not been previously demonstrated in the sheep.
Inter-atrial differences in IKr distribution, but not ICa(L), IK1 or Ito have been shown to be responsible for the longer right atrial AP duration in dogs18.
This spatial gradient means that myocytes upstream of the depolarising wave are refractory for longer than those downstream, which some suggest smoothes the wave of depolarisation and reduces the likelihood of re-entry258. Accordingly, in silico modelling predicts that a greater spatial gradient protects against re-entry and fibrillation, and that this benefit is magnified when the baseline AP duration is longer261. Interestingly, the difference in AP duration between the left and right atria decreased with age. In younger animals right atrial AP durations were 15% longer than those in the left, but in older animals there was only a 3% difference. A gradient was however still present in myocytes from older animals. The diminution of spatial gradient with age could therefore contribute to atrial vulnerability. This contrasts with previous work performed in dogs which suggested that the dispersion of repolarisation both between and within the atria increased with age49. This data should therefore only be viewed as hypothesis-generating, as the gradient within each atrium may be of greater importance.
4.3.8 Differences in calcium transient amplitude and rate of decay did not reach statistical significance
After examining how the shape of the AP changes with age, we sent on to assess the differences in Ca2+ cycling. Ca2+ cycling is closely intertwined with AP shape and is strongly implicated in the genesis of many arrhythmias.
The observed trend towards a slowing of the Ca2+ transient rate of decay and decreasing Ca2+ transient amplitude with age are in keeping with the limited published work concerning age-related changes in Ca2+ cycling232. These observations also concord with previous work performed on this ovine model of ageing that is as of yet unpublished. Although the differences in Ca2+ transient amplitude and rate of decay were large, the small sample size (14 young myocytes but only five old) precluded statistical significance. P a g e | 121
A slowing of Ca2+ transient decay could reflect decelerated Ca2+ extrusion via NCX or reuptake via SERCA. Both of these factors may be affected by the total myocyte Ca2+ buffering power262 as an increase in buffering will reduce the apparent concentration gradient driving the removal of Ca2+ from the cytosol. A decrease in SERCA activity might also lead to a fall in SR Ca2+ content, in turn producing the observed trend towards declining Ca2+ transient amplitude. An increase in leak from the RyR will also cause an apparent slowing of the net removal of Ca2+ from the cytosol. A second explanation for the diminished Ca2+ transient amplitude lies with the well-described decrease in atrial ICa(L) seen with age, in turn leading to impairment of CICR.
As age-related changes in atrial Ca2+ cycling have already been extensively explored in this laboratory, these parameters will not be investigated further in this project. However, Ca2+ cycling will be revisited in the context of alternans behavior in Chapter 5.
4.3.9 Limitations
As all myocytes were depolarised at rest, a small holding current averaging -20pA was needed to bring membrane potentials to -80 mV. This repolarising current will serve to abbreviate AP duration. Furthermore, more holding current was required to polarise young myocytes than was needed for old myocytes. This additional current could contribute to the shorter action potentials seen in the young. However, a repolarising current of some form must be present to sustain the RMP and therefore the holding current represents only a replacement of something lost, in essence recreating a physiological state.
Quality control criteria were used to exclude unhealthy cells. Recordings were rejected if the holding potential needed was >0.06nA, the seal was <500MΩ or if the peak potential was more negative than 0 mV. It is possible that some healthy myocytes failed to meet these criteria, potentially biasing the results.
As stimulation frequency was increased, myocytes developed a beat-to-beat variation in AP duration with a regular 2:1 oscillation referred to as alternans. Recordings from a myocyte at frequencies showing alternans were excluded from analysis. Although this did not affect low stimulation frequencies, a large proportion of recording were rejected higher rates. The net effect of this is that at higher stimulation frequencies a comparison was made only of non-alternating young vs. non-alternating old myocytes. Myocytes were more likely to alternate if they had longer baseline AP duration. Rejecting alternating cells therefore rejects cells with longer action potentials, thereby excluding more cells in one group than another and masking any true difference. Alternans will be examined further in Chapter 5. P a g e | 122
All experiments were performed unblinded. It is possible that the results could be biased by selecting cells to patch that fit the existing pattern such as longer myocytes from old sheep. This form of bias is less likely to have occurred in the monophasic AP recordings.
The atrial extracellular matrix composition changes with age263, although total atrial collagen content was not found to differ between old and young sheep in our model (unpublished data). A difference in matrix may have required alterations to the cell isolation protocol such as a longer digestion, something that was not systematically assessed. A longer digestion may lead to exacerbation of off-target proteolytic effects such as ion channel damage.
4.4 Conclusions
In this chapter we have seen that, as in other large mammalian models of ageing, the cellular AP morphology changes with age, leading to a prolongation of AP duration. Also consistent with the existing body of literature is the observation of atrial myocyte hypertrophy with age. Although right atrial myocytes are larger and exhibit longer AP durations, ageing affects myocytes from both atria to a similar extent. In contrast to previous work, an increase in AP amplitude at low stimulation rates was observed. The differences in cellular AP morphology were not replicated when the in vivo monophasic AP technique was used.
The following chapter will examine the transition from the uniform trains of APs seen at low stimulation rates to the alternating pattern seen at faster rates.
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5 ALTERNANS
5.1 Introduction
In Chapter 4 we saw that at higher stimulation rates, a series of action potentials lost its uniform shape from beat to beat, but instead developed a pattern of alternating long and short action potentials. In this chapter we will explore alternans in greater depth in terms of its cellular origin, its relationship to arrhythmias, and how it changes with age.
5.1.1 What is alternans?
The term ‘alternans’ refers to consecutive measurements that flip between two contrasting states. This phenomenon can be found in many aspects of cardiac physiology. Function of the entire heart may alternate, which can be observed as a physical sign such as pulse pressure264, measured as cardiac output265, or inferred from alternation of electrical activity on the surface ECG266. Alternans can also be seen in the APs and Ca2+ cycling of individual cells267 and is even seen at a sub-cellular scale268. Far from being a mere curiosity, alternans can be used to predict which patients are more likely to suffer a fatal cardiac arrest269, and in some cases even underlies the development of arrhythmias270.
5.1.2 Historical perspective
An alternation of cardiac output was first noted by Traube in 1872264, who while palpating the pulse of a patient with heart failure observed a strong beat, weak beat, strong, weak pattern, and recognised that this physical sign conveyed a poor prognosis. The link between an alternation of pump function and the underlying electrical disturbance was noticed nearly 40 years later. In 1910, the association was made between alternans of arterial pressure with alternans of ECG features, specifically the R-wave (corresponding to ventricular depolarisation) and the T-wave (corresponding to ventricular repolarisation) in both dogs271 and man272. These early reports identified that electrical alternans was rate-dependent, occurring at higher heart rates. In addition, pathology could reduce the stimulation rate that generated alternans (the alternans threshold) so that, for example, ischaemic myocardium manifested alternans at resting heart rates. Other factors modifying alternans behaviour include intoxication with drugs, acquired cardiomyopathies, and genetic conditions such as long QT syndrome273.
The majority of subsequent work focussed on T-wave alternans. Computational advances permitted the detection of subtle, or microvolt, T-wave alternans (mTWA) that was invisible to the naked eye. It was demonstrated that experimental conditions that made it easier to P a g e | 124 induce ventricular fibrillation in dogs, such as ischaemia or hypothermia, also produced mTWA on the surface ECG274. Similarly, the presence of mTWA or alternans of the QRS complex during ventricular pacing was shown to predict vulnerability to ventricular arrhythmias in man275, 276. Although large-scale clinical trials have produced mixed results, several have shown that patients displaying mTWA are more likely to suffer from ventricular arrhythmias or die during follow-up269, 276-278. Many trials have used mTWA as a categorical variable, being either normal, abnormal or indeterminate. Despite this, the magnitude of mTWA has been shown to be a continuous variable, whereby increasing alternans magnitude predicts a poorer prognosis279.
As electrical activity seen on the surface ECG represents the summation of APs from a multitude of myocytes, alternans on the surface ECG represents the summation of alternating APs. Cellular AP alternans was shown resembles alternans affecting unipolar electrograms recorded from intact papillary muscles280, and this link was conclusively demonstrated by simultaneously measuring T-wave alternans and AP repolarisation alternans using optical mapping270. The AP rate of rise, amplitude and duration may alternate independently281, and if seen together, alternans of depolarisation and repolarisation may exist in or out of phase with each other282. Some have suggested that depolarisation alternans may be better predictive of fibrillation than repolarisation alternans283.
5.1.3 Concordant and discordant alternans Why should alternation of membrane repolarisation lead to arrhythmias? Let us imagine a wall of myocardial tissue comprised of cellular bricks, each of which is following a similar pattern of long and short APs. If every cell is alternating, the mental picture that first springs to mind is that each cell manifests a similar AP at the same time, so all cells have short APs together and all have long APs together. This is referred to as spatially concordant alternans (SCA) and is illustrated in Figure 5.1A, left. An alternative scenario is that myocytes in one region of the heart may follow a long, short, long, short pattern while another region follows a short, long, short long pattern (Figure 5.1A , right). All of the tissue may be alternating, but the phase of the oscillation may differ between regions. This is referred to as spatially discordant alternans (SDA).
Both of these situations have been observed experimentally in the same tissue at different times270. As pacing rate increases, a characteristic pattern is seen from no alternans, to concordant alternans, to discordant alternans, to fibrillation. When discordant alternans occurs, marked variation is seen in the repolarisation state of regions of the heart. The distance between these regions may be very small. A region that repolarises quickly is P a g e | 125 therefore able to conduct the next impulse even if it occurs prematurely. A neighbouring region which repolarises slowly will still be refractory to a premature impulse. It is easy to imagine that areas of blocked conduction can lead to a break in the wavefront of excitation (Figure 1.5), and that this sets the stage for re-entrant circuit formation as discussed in Chapter 1.5.4.
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Figure 5.1 – Spatially concordant and discordant alternans. A An illustration of alternans that can be spatially concordant (left) or spatially discordant between sites 1 and 2 (right). B A potential mechanism explaining the transition of concordant to discordant alternans. Long APs at site 1 lead to slower conduction between sites. After several cycles, a change in phase at site 2 relative to site 1 occurs.
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The transition from SCA to SDA may occur in response to an increase in pacing rate or following a single premature impulse. Most explanations as to what brings about this transition rely on the changes in AP duration or CV that occur when the interval between impulses changes, referred to as restitution. As the interval between two impulses (the
+ coupling interval) shortens, there is less time available for the Na current INa to recover from inactivation. This decreases INa and therefore slows CV. This is known as decremental conduction or CV restitution. The related AP restitution (sometimes referred to as electrical restitution) affects AP duration. In this case, a shorter coupling interval impairs the recovery from inactivation of Na+, Ca2+ and K+ currents, decreasing the AP duration of the second beat (Figure 5.2A and B), and will be discussed further later.
The development of SDA can be explained by CV restitution284, illustrated in Figure 5.1B. A longer AP on the first beat slows conduction on the second beat. The slowly conducted second beat prolongs the diastolic interval, which obeying AP restitution prolongs the AP duration of the third beat. This pattern of fast and slow conduction can bring downstream regions of the propagation wave out of phase with upstream regions. An alternative explanation for the transition has been proposed that relies upon Ca2+ cycling285.
There are elegant theoretical reasons to view the discordance of alternans as the key driver of arrhythmogenesis, and in some models SDA has been shown to precede every recorded episode of ventricular fibrillation270, 286, 287. Despite this, others have associated a stronger association between SCA than SDA with vulnerability to ventricular fibrillation288.
5.1.4 Alternans in the atria Although much of our understanding of cardiac alternans has been gleaned from study of the ventricles, alternans is also seen in atrial tissue. A growing body of works suggests that atrial alternans is linked to the development of AF. In animal models, conditions that increase atrial vulnerability simultaneously promote alternans. This association has been shown in rabbits post-myocardial infarction207, and in sheep subjected to atrial tachycardia remodelling98. The latter study has close parallels with human studies comparing patients with and without a history of AF. Atrial alternans develops at rapid pacing rates in all patients. However it occurs at lower rates in patients with a history of paroxysmal AF, and lower rates still in those with a history of persistent AF97, 289. In the latter case, alternans can even occur at the resting heart rate. Furthermore, in those patients in whom AF can be induced by rapid pacing, alternans of the atrial monophasic AP duration is always seen prior to AF onset, whether looked for in the right290, 291 or left97 atria. In those patients whose atria resist AF induction, alternans may be seen, but is exclusively present at high pacing rates and is of low amplitude97, 291. P a g e | 128
Figure 5.2 – The action potential restitution curve. A Cartoon of action potentials illustrating that a premature impulse shortens the diastolic interval and the duration of the subsequent action potential. B Superimposed final action potentials from (A). C Plotting APD90 against diastolic interval produces the restitution curve. The slope is flat at long but steep at short diastolic intervals. D Simulated constant restitution slope <1 causes oscillations to decrease over time. E Simulated constant restitution slope >1 causes oscillations to increase over time. APD90 – action potential duration at 90% repolarisation, DI – diastolic interval. P a g e | 129
5.1.5 Action potential restitution as a cause of alternans The AP alternans of single myocytes that underpins alternans at a tissue scale originates from how membrane currents recover from inactivation, and also from how intracellular Ca2+ cycling interacts with membrane potential. Early explorations of alternans mechanisms focused on the former, suggesting that alternans could be fully explained by the AP restitution described above. Nolasco and Dahlen292 investigated the dependence of AP duration upon the preceding diastolic interval. When a long diastolic interval was abbreviated, AP duration only shortened a little. However, further reducing an already short diastolic interval shortened AP duration a lot. Plotting the relationship between these parameters generated a curve, nearly flat at long diastolic intervals and steepening at shorter intervals (Figure 5.2C). As diastolic interval is measured as the time from the end of repolarisation to the start of the next AP, the relationship is two-way, as at a constant pacing rate a short AP duration creates a longer diastolic interval.
Creating analogies to electronic circuits that can be described using simple mathematics, the authors postulated that if the AP duration shortened by less than the change in diastolic interval, any sudden change in rate would cause a brief oscillation that converged to a stable equilibrium. This can be expressed graphically on a restitution curve as a gradient of <1 (Figure 5.2D). Conversely, if AP duration shortened by more than the change in diastolic interval, the resulting oscillation would increase in size over subsequent beats and diverge to alternans, shown graphically as a gradient >1 (Figure 5.2E). In silico models of membrane current dynamics also predicted that alternans would occur at restitution curve gradients >1293, and that this would lead to wavebreak of reentrant spiral waves294.
The restitution theory was supported by experimental data showing that pro-arrhythmic maneuvers such as applying quinidine simultaneously increased the steepness of the AP restitution curve and promoted alternans295. In contrast, drugs that flattened the restitution
296 2+ 297 slope such as the IKr inhibitor E-4031 , the Ca channel inhibitor verapamil , and the excitation-contraction uncoupler diacetyl monoxime297 (also a nonspecific blocker of Na+, K+, and Ca2+ channels) were shown to suppress alternans and ventricular fibrillation. The putative relationship between restitution, alternans, and wavebreak was strengthened when the K+ channel inhibitor bretylium, which also flattens the restitution slope, was directly shown to inhibit wavebreak298.
A potential flaw common to all of these studies is that association does not equate with causality. While measures were taken to reduce the effect of compounding such as the effects of drugs on AP duration, a direct effect was very difficult to prove. Evidence suggesting that AP P a g e | 130 restitution could not account for all alternans behavior came from experimental work showing that in ventricular muscle, alternans was seen at stimulation rates at which the slope of the restitution curve was <1295, 299. Furthermore, the changes in AP duration on successive beats were not found to correlate well with the preceding diastolic interval300. While isolated myocytes frequently manifest alternans, investigators found that restitution slopes in the same myocytes were never >1301. Others showed that more alternans was seen in ischaemic tissue as time passed, but this was accompanied by a flattening of the restitution slope302, and that the regions of the heart exhibiting the steepest restitution slopes were separate from those most prone to alternans303. Although AP restitution plays a part, the alternans engine needed another component.
5.1.6 The interplay between calcium cycling and action potential alternans The missing component may be intracellular Ca2+ cycling. If we look inside a myocyte whose
2+ APs are alternating, we see that alternans of [Ca ]i develops at approximately the same time303. How do these two facets of alternans interact?
As described in Chapter 1.3.1, the Ca2+ transient is generated through the release of Ca2+ from the SR via ryanodine receptors (RyRs). This, in turn, is triggered by the sarcolemmal Ca2+
2+ 2+ current ICa(L), in a process known as Ca -induced Ca release (CICR). Along with other factors such as SR Ca2+ content and the properties of the RyR, the amplitude of the Ca2+ transient is
2+ 13 determined by the size of the trigger – increasing ICa(L) will create a bigger Ca transient . ICa(L) is affected by the size and shape of the AP. An abnormally low amplitude AP will initiate less
2+ ICa(L), thereby producing a smaller Ca transient (Figure 5.3A). Furthermore, increasing AP plateau duration will increase the size of the Ca2+ transient54. It can therefore be imagined that an alternating train of APs, in particular if depolarisation alternans is present, could produce
2+ 2+ [Ca ]i alternans, with small and short APs corresponding with small Ca transients.
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Figure 5.3 – Interactions between calcium cycling and the action potential. A Action potentials with greater amplitudes and longer durations produce more calcium influx via
ICa(L), triggering more calcium release from RyRs. B Higher cytosolic calcium concentrations generate more current through NCX but lead to greater inactivation of ICa(L), limiting calcium influx. LTCC – L-type calcium channel, NCX – Na+ / Ca2+ exchanger, RyR – ryanodine receptor, SERCA – sarco-endoplasmic reticulum Ca2+ ATPase.
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In return, Ca2+ transient amplitude can affect AP duration, as several membrane currents are
2+ + 2+ 2+ sensitive to [Ca ]i (Figure 5.3B). The Na / Ca exchange current INCX, which removes Ca from 2+ the cytosol during diastole, generates a net inward current. In the presence of elevated [Ca ]i 304 a greater INCX will result, prolonging the AP . INCX is enhanced at more negative membrane potentials, so the region of the AP most affected by changes in NCX is late phase 3, affecting
2+ 2+ APD90. In contrast, the L-type Ca current ICa(L) is subject to Ca -dependent inactivation, 2+ meaning that in the presence of elevated [Ca ]i a smaller ICa(L) will result, shortening the AP.
ICa(L) is seen during phase 2 and 3 of the AP, and therefore most affects the plateau and APD50. The balance between these opposing influences which serve to prolong or shorten the AP
2+ 305 duration in response to changes in [Ca ]i varies between tissues . During alternans, in 2+ myocytes expressing relatively little ICa(L), beats with a large Ca transient manifest longer APs, referred to as electromechanically concordant alternans. Conversely, if copious ICa(L) is present, beats with large Ca2+ transients manifest shorter APs, known as electromechanically discordant
306 2+ alternans . While this explains how [Ca ]i alternans can produce AP repolarisation alternans, another step is needed to understand how depolarisation alternans arises. If the oscillations in repolarisation are large enough and the diastolic interval is very short, repolarisation may not complete before the next stimulus arrives (Figure 5.4). In this case, the less negative membrane potential prevents INa from recovering adequately from inactivation, impairing AP 2+ amplitude. We can therefore see that at lower stimulation rates, primary [Ca ]i alternans can produce AP repolarisation alternans, and at faster rates depolarisation alternans.
Figure 5.4 – Repolarisation alternans precipitates depolarisation alternans
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The two-way relationship between membrane currents and Ca2+ cycling makes it unsurprising that alternans of the two occurs simultaneously, but raises the question – does AP alternans drive Ca2+ alternans, does Ca2+ alternans drive AP alternans, or are the two aspects synergistic?
2+ Several lines of evidence showed that [Ca ]i alternans can exist without AP alternans. Chudin 2+ et al. rapidly paced isolated rabbit ventricular myocytes, showing that AP alternans and [Ca ]i occurred simultaneously307. They went on to demonstrate that when the AP morphology was
2+ artificially kept the same from beat to beat (an AP clamp), [Ca ]i alternans was still present. This concept was extended by the observation that an AP clamp that kept depolarisation similar between beats, but created alternans of the terminal portion of repolarisation, did not
2+ 308 generate [Ca ]i alternans at low stimulation rates .
2+ 2+ [Ca ]i alternans may indeed be essential for AP alternans to occur. When Ca cycling was disrupted in rabbit ventricular myocytes using the SERCA inhibitor thapsigargin, the Ca2+ chelator BAPTA or the RyR inhibitor ryanodine, AP alternans disappeared299. Similarly, in silico modeling using parameters derived from human ventricular myocytes predicted that disruption of Ca2+ cycling would abolish AP alternans309. Ca2+ cycling is also responsible for the hysteresis effect, whereby the rate threshold for alternans generation decreases with successive attempts at alternans induction310, due to Ca2+ accumulation.
More recent work has harmonised these two schools of thought. Using a sophisticated in silico
2+ approach that quantified the relative contribution of [Ca ]i and AP restitution to alternans, it 2+ was found that at low stimulation rates [Ca ]i alternans was the primary driver, agreeing with data showing alternans at rates where the restitution slope was <1311. As pacing rate increased, the relative contribution of the surface membrane currents increased until AP restitution became the dominant force at very rates.
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5.1.7 Mechanisms underlying alternans of intracellular calcium 2+ If alternans of [Ca ]i can drive AP alternans at low stimulation rates, we need to explore how 2+ [Ca ]i arises, independently of changes in sarcolemmal currents. As alluded to above, a major determinant of Ca2+ transient amplitude is the SR Ca2+ content whereby if all else is equal, elevating SR Ca2+ content will increase the size of the Ca2+ transient312.
A feedback loop exists between SR content and Ca2+ transient amplitude313:
(i) High SR content produces a large Ca2+ transient. 2+ (ii) An elevation in [Ca ]i increases INCX and decreases ICa(L). (iii) On this beat, more Ca2+ is removed from the cell than flows in. 2+ 2+ (iv) Reduced [Ca ]i means that less Ca can be returned to the SR. (v) The next beat starts from a position of a lower SR Ca2+ content, reversing the process. In a manner very similar to the restitution curve hypothesis of AP alternans generation, the extent to which an increase in SR content increases Ca2+ transient magnitude is crucial. This is referred to as the ‘gain’ of the system. If an increase in SR content produces a small change in Ca2+ transient amplitude (low gain), the changes in these parameters will decrease over subsequent beats until equilibrium is reached. If the change in Ca2+ transient amplitude is large (high gain), oscillations will increase and produce alternans. Such a steep dependence of Ca2+ release on SR content has been demonstrated during periods of alternans312.
Gain can also be a function of Ca2+ extrusion. If all Ca2+ is returned to the SR following a Ca2+ transient, then no matter what size the transient SR content will always remain stable. On the other hand, if most of the Ca2+ is extruded via NCX, larger transients will decrease SR Ca2+ content more than smaller transients. The balance between SR reuptake and Ca2+ extrusion via NCX might therefore be predictive of alternans behavior.
5.1.8 Impaired calcium reuptake as a mechanism of alternans Supporting this view, beat to beat changes in SR Ca2+ content have been clearly demonstrated in some models312. As SR Ca2+ content is determined in part by the rate of refilling, some have suggested that if the filling mechanism is overwhelmed by too short a diastolic interval or too slow a pump, instability may result. Could impaired Ca2+ reuptake contribute to alternans?
To investigate this, the inherent variability between regions of the heart in their Ca2+ handling properties has been exploited. In both guinea pigs314 and dogs315, ventricular subendocardial myocytes are more susceptible to alternans than those from the subepicardium, and also return Ca2+ more slowly to the SR. These differences are associated with decreased subendocardial SERCA314, 315 expression and may relate to an increase in NCX314. Similar differences are seen between ventricular myocytes from dogs with and without heart failure, P a g e | 135 again showing an association between slower Ca2+ reuptake and increased alternans activity286. Strengthening this viewpoint, when SERCA is partially inhibited AP alternans is potentiated in silico316 and experimentally317, whereas upregulating SERCA using viral transfection reduces the
2+ 318 incidence of [Ca ]i alternans, AP alternans, and ventricular fibrillation .
Although the evidence for a role of Ca2+ reuptake in the genesis of alternans has mainly been derived from work in the ventricles, there is complementary data from atrial myocytes319 and cultured atrial monolayers320. Despite this, AF can either enhance46, leave unchanged73, or slow Ca2+ reuptake71 depending on the duration of remodeling, raising the possibility that any increase in alternans seen in AF might be due to another mechanism.
5.1.9 Altered calcium release as a mechanism of alternans 2+ Although fluctuations in SR content are often seen during [Ca ]i alternans, they are not essential. A similar SR content between large and small alternating beats has been shown in atrial321, 322 and ventricular myocytes323, and inferred from work in intact tissue324. SR Ca2+ content does not seem to fluctuate at stimulation rates only just high enough to produce alternans, but fluctuations appear at higher rates.
If SR Ca2+ content fluctuations are not necessary to produce alternans, the proportion of Ca2+ released with each beat must change. Confirming the suspicion that impaired Ca2+ release may cause alternans, it was shown that the Ca2+ transients from myocytes that are more prone
314 to alternans are smaller and rise more slowly . In theory alternating ICa(L) magnitude could bring about alternans. If this were the case, beats with a large ICa(L) would correspond with large Ca2+ transients. This has does not appear to be the case in practice, as larger Ca2+
2+ 321, 323, 325 transients correspond with smaller ICa(L) due to enhanced Ca dependent inactivation . Instead, the proportion of RyRs that open with each beat may change, which could occur due to a delay in RyR recovery from inactivation.
In silico modeling has implicated RyR refractoriness as a culprit for alternans in single cells326
327 2+ and atrial tissue and that this parameter alone can generate [Ca ]i alternans without fluctuations of SR Ca2+ content where few T-tubules are present. Experimental evidence for RyR refractoriness comes from studies of isolated myocytes subjected to rapid pacing. Refractory RyRs would be expected to show less spontaneous release in the form of Ca2+ sparks or Ca2+ waves. When myocytes are stimulated rapidly, producing alternating large and small Ca2+ transients, and then stimulation is paused: less spontaneous Ca2+ release is seen immediately after a large transient (suggesting refractory RyRs) than a small transient321. Further evidence comes from pharmacological manipulation of RyR opening. Low dose caffeine, which increases open probability thereby reducing RyR refractoriness, inhibits P a g e | 136 alternans, decreasing alternans magnitude and increasing the alternans rate threshold321, 324. Conversely, measures which reduce RyR open probability and increase refractoriness promote alternans, such as tetracaine and acidosis328.
However, alternans caused by RyR refractoriness, making RyRs abnormally unlikely to open, only represents half of the picture. Alternans has also been described under conditions where RyRs are abnormally likely to open, or leaky. In dogs recovering from myocardial infarction, a proportion was found to be prone to ventricular fibrillation (VF). When tissue and cells from these animals were compared to control, VF-prone tissue was more susceptible to alternans and VF-prone cells had RyRs that were more likely to spontaneously open despite a lower SR Ca2+ content329. Having excluded differences in Ca2+ reuptake, the authors attributed these differences in part to oxidation of RyRs leading to leakiness. Further evidence for a role of RyR leak potentiating alternans came from studies manipulating calstabin-2, a calcium stabilising protein that binds to RyRs and prevents spontaneous opening. Calstabin-2 knockdown mice were more susceptible to alternans and showed more spontaneous Ca2+ release, suggesting RyR leak330. Furthermore, treatment with a drug that increases binding of calstabin-2 to RyRs (JV519) prevented alternans.
5.1.10 Subcellular alternans
So far we have envisaged the SR and membrane as behaving like single homogenous structures through which ions flow uniformly. To do so is simplistic. The RyRs studding the SR form Ca2+ release units (CRUs) that can all act independently. Each brief release of Ca2+ from a CRU is
2+ known as a Ca ‘spark’, which may be spontaneous, triggered by ICa(L) (a primary spark), or even triggered by a Ca2+ spark from a neighbouring CRU (a secondary spark). Ca2+ spark occurrence has a strong element of randomness to it, although this randomness can be influenced. For example, a single CRU that is not currently exposed to Ca2+ influx has a small, random, probability of opening spontaneously. If a local L-type Ca2+ channel opens and exposes the CRU to Ca2+, it may still remain closed but the probability of it opening is greatly increased. This randomness is referred to as stochasticity. Only when many random openings are summed does the illusion of order become apparent.
When we look at this subcellular scale, we see that during alternans the SR function is far from uniform. Even within a single cell, alternating Ca2+ release can be spatially discordant, with some regions of the cell releasing a large amount of Ca2+ while others release little, only for this pattern to reverse on the next beat328. This spatial discordance can mask alternans of the overall Ca2+ transient, and ‘microscopic’ alternans of regions of a single cell has been shown to precede ‘macroscopic’ alternans detectable in the Ca2+ transient as a whole268. P a g e | 137
Experimental328 and in silico331 studies have suggested that the large Ca2+ transients seen during alternans represent a combination of primary Ca2+ sparks followed by a slowly propagating wave of secondary sparks, whereas the small transients reflect a failure of propagation of the secondary spark wave. Some have, however, disputed the notion that slowly propagating Ca2+ waves are a significant component of alternans323, arguing that they are only observed under non-physiological conditions. Weiss et al. have presented a unified theory of Ca2+ release to explain alternans at a subcellular and cellular scale, pruning the complexities back to the probability of primary sparks occurring, the recovery of CRUs following a spark, and the coupling between adjacent CRUs90.
5.1.11 Modulation of alternans As described by the earliest observers, alternans does not occur solely in response to tachycardia, but also when ‘the heart shows evidence of embarrassment’272. If a myocyte is starved of energy, alternans frequently results. The energy supply can be reduced experimentally by inhibiting glycolysis, which reduces with Ca2+ reuptake by decreasing SERCA activity319, and impairs Ca2+ release322. Ischaemia, a classical cause of alternans, reduces energy availability and acidifies the cytoplasm, which by itself decreases RyR opening and promotes alternans328.
The autonomic nervous system can modulate alternans. Somewhat counterintuitively as sympathetic activation is often seen as pro-arrhythmic, activation of β-adrenergic receptors using isoprenaline decreases the susceptibility to alternans in the atria322 and ventricles317 in most studies. Conversely, blocking β-adrenergic receptors using propranolol increases alternans in vitro317. The anti alternans effect of β-adrenergic stimulation can be explained by the acceleration of Ca2+ reuptake and increased RyR sensitivity to Ca2+ in response to β-adrenergic stimulation13.
However, β-adrenergic stimulation also increases the AP restitution slope, potentially explaining why some have found that β-adrenergic stimulation promotes alternans in embryonic ventricles332 and β-adrenergic blockade reduces T-wave alternans in patients with LV dysfunction333.
The parasympathetic nervous system, although less well studied, has also been shown to influence alternans. Stimulation of the vagus nerve reduces the slope of the atrial AP restitution curve and inhibits alternans334, despite increasing atrial vulnerability to fibrillation. Blocking parasympathetic influence using atropine does not significantly affect T-wave alternans333. P a g e | 138
5.1.12 Higher order periodicities 2+ All discussion so far has focussed upon alternans, an oscillation of AP duration or [Ca ]i that repeats after two beats. We can call this a 2:1 oscillation, or say that the period of this oscillation is two. Regular oscillations with a period of three of four, or more complex rhythms have also been described in vivo335 and in cultured cells336, but have been studied to a lesser extent than classical 2:1 alternans. Some have suggested that these higher order periodicities represent a further step along the road from organisation to chaotic fibrillation, and limited data suggests that their occurrence predicts arrhythmias better than alternans does335.
5.1.13 Alternans and ageing There is limited data exploring how the susceptibility to alternans changes with age. In the
2+ 337 intact ventricle, older rats develops more [Ca ]i alternans in response to rapid pacing , while 2+ ventricular myocytes isolated from older rats are more susceptible to ischaemia-induced [Ca ]i alternans338, 339.
In the atria, no detailed studies have been conducted upon the effects of age on alternans
2+ behaviour. However, two studies of atrial ageing have touched upon alternans. AP and [Ca ]i alternans were seen in pulmonary vein sleeve myocytes from older rabbits but not in young, associated with decreased SERCA but increased RyR expression340. Similarly, the alternans observed in older rat atria was more pronounced that that in the atria of young rats238. No studies have been performed upon the effects of age on alternans behaviour in large mammals in the ventricles or atria, and none have looked at higher order periodicities.
5.1.14 Aims
This chapter will investigate how alternans changes with age in the atria of sheep, as the very limited previous work has only involved rodents. Alternans will be explored both in isolated myocytes and in vivo. A detailed assessment of alternans behaviour will be carried out, including the rate threshold for initiation of alternans, the magnitude of alternans and the presence of higher order periodicities. Following this, the mechanisms underlying action potential alternans in the atria will be investigated, with particular reference to the interplay between alternans of intracellular calcium concentrations and the shape of the action potential.
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5.2 Method of alternans quantification
Gross alternans of the AP shape is easily identified in clean traces. However, subtle alternans, particularly in traces contaminated by noise, is harder to reliably distinguish from random fluctuations. Several methods have been used to aid in this task. One of the most commonly used is the spectral method. This technique was originally used to identify microvolt T-wave alternans due to its ability to discriminate small oscillations from noise274, but has been adapted for the study of AP alternans316.
The method relies on the regularity of the oscillations present in alternans. Oscillations due to alternans occur exclusively with a period of two. These 2:1 oscillations can be separated from oscillations occurring with any other period, can be quantified, and if they reach a predetermined size are classed as alternans. How can this separation of frequencies be accomplished?
Most of us have seen the technique used to separate out a frequency band from many in the form of the graphic equaliser on a music system, whereby a series of columns representing high, mid or low tones bob along to what we are listening to. The mathematical operation used to achieve this is called the discrete Fourier transform.
In the same way that a colourful scene can be painted using a small range of hues depending on how the colours are mixed, any complex waveform can be built from a series of sine waves depending on how they are scaled. A mathematical operation called a discrete Fourier transform can be used to deconstruct a waveform into its component sine waves. The Fourier transform tells us the frequencies of sine waves that are needed, and how much of each is required to reconstruct any waveform we give it, and is calculated using
Equation 5.1
where Fn is the nth item in the output array fn is the nth item in the input array N is the total number of samples m is the frequency where m/N is the samples per cycle i is the imaginary number corresponding to √-1
To apply this to the study of action potentials, in its simplest form we first reduce each AP in a series (Figure 5.5A) to a number, for example the membrane potential at a particular P a g e | 140 timepoint within each AP (Figure 5.5B, dashed line). We can then plot this list of values against beat number (Figure 5.5C). The Fourier transform, when applied to this list, tells us which sine waves at which frequencies are needed to reconstruct the list. The sine wave periods are measured as the number of beats needed to return to the initial value or complete a cycle, quantified as beats / cycle. The frequency is the reciprocal of the period, so is measured in cycles / beat. The amplitude of each sine wave needed is plotted against frequency to give a magnitude spectrum (Figure 5.5D). The oscillations we are interested in repeat after two beats, therefore the sine wave has a period of two and a frequency of 0.5 cycles/ beat. The peak in the magnitude spectrum at 0.5 cycles / beat is therefore referred to as the alternans voltage (VAlt), which corresponds to half the difference between the average odd and even sweeps (Figure 5.5C).
Beyond applying the Fourier transform to a single timepoint, additional information can be obtained by looking at the voltage of every timepoint within the AP. A Fourier transform is performed on each timepoint within every AP in the series, and the results summated for a specific AP phase or the whole AP. In this work, the depolarisation phase (phase 0-1) was defined as timepoints from the AP onset to 10ms after the average AP peak. The repolarisation phase (phase 2-3) was defined as timepoints from 10ms after the average AP peak to APD90. MDP was defined as timepoints from 10ms before AP onset to AP onset.
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Figure 5.5 – Quantification of alternans. A A series of action potentials is recorded. B The action potentials are superimposed and the membrane potential at a particular timepoint (dashed line) is recorded. C The membrane potential at the specified timepoint is plotted for each action potential against beat number, creating a beat series. D A discrete Fourier transform is used to calculate the frequencies at which oscillations occur within the beat series, plotted as a magnitude spectrum. VAlt alternans magnitude. Adapted from Pearman (2014)341.
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The likelihood that any oscillations we see are caused by alternans rather than random fluctuations can be calculated by comparing different frequencies within the magnitude spectrum. The alternans frequency is 0.5 cycles per beat, while random oscillations are evenly distributed amongst the remaining frequencies. By convention, the magnitude at 0.5 cycles / beat is compared against the magnitude from 0.34 – 0.49 cycles / beat, referred to as the ‘noise band’ (Figure 5.1D). If the 2:1 oscillations exceed the noise band by a predetermined number of standard deviations (k>3 is usually deemed significant266), alternans is said to be present. This is calculated as the k-score (Equation 5.2)
Equation 5.2 where ΣT is the spectral magnitude at 0.5 cycles / beat
µnoise is the mean spectral magnitude from 0.33 to 0.49 cycles / beat σnoise is the standard deviation of the spectral magnitude from 0.33 to 0.49 cycles / beat
The spectral method can also be used to identify higher order periodicities such as 3:1 (a peak at 0.33 cycles / beat) or 4:1 (a peak at 0.25 cycles / beat). Corresponding k3 and k4 scores can be calculated, by shifting the noise band outside the peak of interest.
To automate this process, software was custom written using Visual Basic for Applications to run within Excel. This work has been published341, and can be found in Appendix 1.
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5.3 Results
5.3.1 Alternans threshold in isolated myocytes
We firstly explored whether the minimum rate at which alternans developed differed with age or between the left or right atria (with laterality) in isolated myocytes. Myocytes were stimulated from 0.25 – 4 Hz. At low stimulation rates, trains of APs were generated that were similar from beat to beat (Figure 5.6A). At higher rates, AP morphology alternated between two contrasting states (Figure 5.6B). The alternans threshold was defined as the minimum stimulation rate that generated a k-score of ≥3 with a VAlt of ≥0.5 mV. The threshold was calculated for alternans affecting the whole trace, phase 0-1 alone, phase 2-3 alone, or MDP alone.
Alternans occurred at lower simulation rates in old compared to young myocytes (Figure 5.6C and D). No significant difference was seen in alternans threshold between left and right atrial myocytes (Figure 5.6C).
Data is summarised in Table 5.1
Age Laterality Young left Old left Young right Old right difference difference Whole trace 1.9 ± 0.1 Hz 1.6 ± 0.1 Hz 2 ± 0.2 Hz 1.5 ± 0.1 Hz -21% * a -2% a Phase 0-1 1.9 ± 0.1 Hz 1.7 ± 0.1 Hz 2.1 ± 0.2 Hz 1.5 ± 0.1 Hz -20% * a -3% a Phase 2-3 1.8 ± 0.1 Hz 1.6 ± 0.1 Hz 1.8 ± 0.2 Hz 1.4 ± 0.1 Hz -16% * -7% MDP 2.1 ± 0.1 Hz 1.9 ± 0.2 Hz 2 ± 0.2 Hz 1.7 ± 0.1 Hz -12% -10%
Table 5.1 – Alternans threshold in isolated myocytes. n = 14-18 cells, 5-8 animals per group. * p<0.05 for differences between age groups using 2-way ANOVA. a non-normally distributed data, transformed using square root.
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Figure 5.6 – Alternans threshold in isolated myocytes. A (from left to right) Train of action potentials stimulated at 2 Hz, superimposed means of odd and even sweeps, magnitude spectrum demonstrating no alternans. B (From left to right) Train of action potentials stimulated at 2.25 Hz, superimposed means of odd and even sweeps, magnitude spectrum demonstrating clear alternans. C Mean alternans thresholds from isolated myocytes. D Proportion of cells having alternated by a given frequency. n = 14-18 cells, 5-8 animals per group. * p<0.05 for differences between age groups using 2-way ANOVA.
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5.3.2 Alternans magnitude in isolated myocytes
When alternans occurs, the differences between odd and even beats may be subtle or very pronounced. This difference can be quantified as the alternans magnitude (VAlt), defined as the spectral magnitude at 0.5 cycles / beat (Figure 5.5). We next explored whether the magnitude of alternans differed with age or laterality.
In the first analysis, VAlt was calculated for all cells at all stimulation rates (Figure 5.7C). Alternans magnitude was greater in atrial myocytes from old sheep (Figure 5.7A and B), but did not differ between the left and right atria. VAlt at a single stimulation frequency is summarised in Table 5.2.
These results could be obtained by virtue of the fact that more cells were alternating at a given frequency in one group than another. To explore whether a true difference in alternans magnitude was present, a second analysis was performed including only cells that were alternating (whole cell k-score >3) at a given rate. The age-related increase in VAlt persisted in this second analysis (Figure 5.7D).
Age Laterality Young left Old left Young right Old right difference difference Whole trace 2.4 ± 0.7 mV 4.4 ± 1.2 mV 2 ± 0.6 mV 5.2 ± 1.1 mV 115% * a 11% a Phase 0-1 4 ± 1.4 mV 7.1 ± 1.8 mV 1.9 ± 0.5 mV 7.9 ± 2.2 mV 145% a -7% a Phase 2-3 2.8 ± 0.8 mV 5.4 ± 1.4 mV 2.6 ± 0.8 mV 6.1 ± 1.3 mV 114% * a 10% a MDP 0.9 ± 0.3 mV 1.3 ± 0.4 mV 0.6 ± 0.2 mV 2 ± 0.5 mV 119% * a 20% a
Table 5.2 – Alternans magnitude in isolated myocytes stimulated at 2 Hz. Statistical significance determined by 2-way repeated measures ANOVA from 0.5 – 4 Hz. n = 14-18 cells, 5-8 animals per group. a persistently non-normally distributed data. * p<0.05 P a g e | 146
Figure 5.7 – Magnitude of alternans in isolated cells. A Representative trace showing superimposed mean odd and even sweeps from young left atrial myocyte (above) and corresponding magnitude spectrum (below). B Representative trace showing superimposed mean odd and even sweeps from old left atrial myocyte (above) and corresponding magnitude spectrum (below). C VAlt for all cells at all frequencies. D VAlt for cells with a k- score of >3 at each frequency. n = 14-18 cells, 5-8 animals per group. * p<0.05,
VAlt - alternans magnitude taken as spectral magnitude at 0.5 cycles/beat. P a g e | 147
5.3.3 Presence of higher order oscillations in action potentials from isolated myocytes
Alternans, a beat-to-beat variability that repeated after every 2nd beat (a 2:1 oscillation) was the most commonly seen disturbance of AP trains. However, beat-to-beat variability that repeated after a greater number of beats (showing a higher periodicity) such as 3:1 or 4:1 oscillations were also seen. 3:1 oscillations were marked by a peak in the magnitude spectrum at 0.33 cycles / beat (Figure 5.8A), while 4:1 oscillations were marked by a peak at 0.25 cycles / beat.
3:1 oscillations were defined as a k3-score of ≥3 with a spectral magnitude at 0.33 cycles / beat ≥0.5 mV. These were rarely seen and occurred exclusively at high stimulation rates (Figure
5.8B). 4:1 oscillations were defined as a k4-score of ≥3 with a spectral magnitude at 0.25 cycles / beat ≥0.5 mV. These commonly occurred at the same time as 2:1 oscillations, and were uniformly distributed across the range of stimulation rates studied.
The number of cells showing higher order oscillations at any stimulation rate did not differ significantly with age (Figure 5.8C).
Data is summarised in Table 5.3
Young Old Difference p-value
3 to 1 1 / 29 cells 2 / 35 cells 66% 1.00 4 to 1 9 / 29 cells 18 / 35 cells 66% 0.13
Table 5.3 – Prevalence of higher order oscillations in isolated myocytes. n = 14-18 cells, 5-8 animals in each group. Significance determined using Chi-squared or Fisher’s exact test.
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Figure 5.8 – Higher order oscillations in isolated myocytes. A (From left to right) Example of 3:1 oscillations in an atrial myocyte, superimposed traces of mean of every 1st, 2nd and 3rd sweep, and magnitude spectrum showing peak at 0.33 cycles/beat. B Proportion of cells at each stimulation frequency manifesting 2:1, 3:1 and 4:1 regular oscillations. C Proportion of cells manifesting 3:1 and 4:1 oscillations at any frequency stratified by age group. n = 14-18 cells, 5-8 animals in each group. NS - not significant.
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5.3.4 Alternans threshold in vivo
We next explored whether the differences in alternans behaviour seen in isolated myocytes would be reflected in vivo. Monophasic APs were recorded from the posterior wall of the right atrium in anaesthetised sheep, while the atria were stimulated from the right atrial appendage.
Examples of monophasic AP alternans can be seen in Figure 5.9A and B. It can be clearly seen that alternans was much more subtle in vivo than in isolated myocytes. Additionally, the monophasic AP signals obtained were of much lower amplitude than APs from isolated myocytes. The definition of alternans was therefore adjusted to a k-score >3 and a VAlt of 0.03 mV.
As was seen in isolated myocytes, the in vivo atrial alternans threshold was 13% lower in old sheep compared to young (Figure 5.9C and D). Data is summarised in Table 5.3.
Young Old Difference p-value Whole Trace 3.5 ± 0.1 Hz 3 ± 0.1 Hz -12.6% 0.002 * Phase 0-1 3.4 ± 0.1 Hz 3 ± 0.1 Hz -10.0% 0.02 * Phase 2-3 3.5 ± 0.1 Hz 3 ± 0.1 Hz -12.2% 0.003 *
Table 5.4 – In vivo alternans thresholds. n = 10-11 animals in each group. * p<0.05 for differences between age groups using unpaired t-test.
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Figure 5.9 – In vivo alternans thresholds. A (From left to right) Train of action potentials stimulated at 2.5 Hz, superimposed means of odd and even sweeps, magnitude spectrum demonstrating no alternans. B (From left to right) Train of action potential stimulated at 3.3 Hz, superimposed means of odd and even sweeps, magnitude spectrum demonstrating clear alternans. C Mean in vivo right atrial alternans thresholds. D Proportion of atria having alternated by a given frequency. n = 10-11 animals in each group. * p<0.05 for differences between age groups using unpaired t-test.
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5.3.5 Alternans magnitude in vivo
Next, the magnitude of in vivo right atrial alternans was examined. Representative MAP traces recorded from young and old sheep can be seen in Figure 5.10A and B.
VAlt from all atria was recorded at all stimulation frequencies (Figure 5.10C). VAlt increased as stimulation frequency increased (p<0.05 using repeated measures ANOVA). At the lowest stimulation rates, no difference in VAlt was seen between age groups. In the mid-range of stimulation frequencies studied, VAlt was greater in old compared to young atria. At the highest stimulation frequencies this effect disappeared and showed a trend to reversal. When a RM ANOVA was applied across all stimulation frequencies, the differences between curves did not attain statistical significance. However, when only mid-range frequencies were included in the analysis (2.9 to 3.7 Hz), VAlt was found to be greater in old compared to young atria, although the difference was present at only some stimulation rates. This difference was present when the whole trace or phase 2-3 alone was analysed, but not when phase 0-1 alone was assessed. Data is summarised in Table 5.4.
As with the data obtained from isolated myocytes, a second analysis was performed, this time including only data points in which the k-score was >3 (Figure 5.10D). Although qualitatively similar to the previous analysis, the difference between groups no longer reached statistical significance (p=NS).
Young Old Difference p-value Whole Trace 0.02 ± 0.01 mV 0.08 ± 0.02 mV 252% 0.04 * Phase 0-1 0.05 ± 0.02 mV 0.17 ± 0.04 mV 266% 0.13 Phase 2-3 0.02 ± 0.01 mV 0.09 ± 0.02 mV 265% 0.02 *
Table 5.5 – In vivo right atrial alternans magnitude at 3.3 Hz. n = 10-11 animals per group. * p<0.05 using 2-way repeated measures ANOVA including frequencies between 2.9 and 3.7 Hz.
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Figure 5.10 – Magnitude of in vivo right atrial alternans. A Representative trace showing superimposed mean odd and even sweeps from right atrium of young sheep (above) and corresponding magnitude spectrum (below). B Representative trace showing superimposed mean odd and even sweeps from right atrium of old sheep (above) and corresponding magnitude spectrum (below). C VAlt for all cells at all frequencies. D VAlt for cells with a k- score of >3 at each frequency. n = 10-11 animals per group. * p<0.05, VAlt – alternans magnitude taken as spectral magnitude at 0.5 cycles / beat. P a g e | 153
5.3.6 Higher order oscillation of action potentials in vivo
As was seen in the recordings from isolated myocytes, in addition to the typically described 2:1 alternans, regular oscillations were also observed at higher periodicities. Figure 5.11A shows a monophasic AP train that clearly switches in shape between three contrasting forms. The corresponding magnitude spectrum shows a peak at 0.33 cycles/beat, representing a 3:1 oscillation.
3:1 and 4:1 oscillations were observed in a substantial minority of animals, occurring across the range of stimulation rates studied (Figure 5.11B), and were more frequently observed in vivo than in isolated myocytes (p<0.05).
No significant differences were seen between age groups in the proportion of animals exhibiting higher order oscillations, although a trend was seen towards a greater incidence in the old cohort (Figure 5.11C). Data is summarised in Table 5.6.
Young Old Difference p-value
3 to 1 2 / 11 animals 4 / 10 animals 120% 0.36 4 to 1 1 / 11 animals 4 / 10 animals 340% 0.15
Table 5.6 – Prevalence of higher order oscillations in vivo. n = 10-11 animals per group. Significance determined using Fisher’s exact test.
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Figure 5.11 – Higher order oscillations in vivo. A (From left to right) Example of 3:1 oscillations in right atrial monophasic action potential, superimposed traces of mean of every 1st, 2nd and 3rd sweep, and magnitude spectrum showing peak at 0.33 cycles/beat. B Proportion of animals at each stimulation frequency manifesting 2:1, 3:1 and 4:1 regular oscillations of right atrial monophasic action potentials. C Proportion of animals manifesting 3:1 and 4:1 oscillations of right atrial monophasic action potentials at any frequency, stratified by age group. n = 10-11 animals per group. NS - not significant.
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5.3.7 Relationship between in vivo alternans and arrhythmia inducibility
As previous work has suggested a link between alternans and arrhythmogenesis, we investigated whether a relationship between the two was present in this model. Eight of the animals that had undergone electroanatomical mapping were also part of the cohort investigated for arrhythmia inducibility. Associations between parameters were investigated using Pearson’s correlation. The small sample meant that the statistical power was only sufficient to detect the strongest of correlations.
A scatterplot of VAlt at 300ms stimulation against AF duration is shown in Figure 5.12A. While this does not show a statistically significant association (p=NS), a trend can be seen whereby as
VAlt increases, the duration of AF increases. A similar result is seen when the proportion of bursts generating AF is plotted against VAlt (Figure 5.12B).
No significant relationship was seen between alternans threshold and atrial vulnerability (p=NS), nor were associations seen between the presence of higher order oscillations and atrial vulnerability (p=NS) (data not shown).
Data is summarised in Table 5.7
Parameter 1 Parameter 2 Correlation coefficient (R) p-value Alternans threshold AF duration -0.47 0.24 Alternans threshold Proportion AF >5s -0.55 0.16
Alternans magnitude AF duration 0.23 0.58 Alternans magnitude Proportion AF >5s 0.15 0.72
Table 5.7 – Associations between arrhythmia inducibility and alternans. n = 8 animals.
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Figure 5.12 – Relationship between alternans behaviour and arrhythmia inducibility. A Scatter plot showing duration of induced AF against right atrial alternans magnitude at 300ms stimulation. Dashed grey line represents linear regression. B Scatterplot showing proportion of bursts that led to AF of greater than 5s duration against alternans magnitude at 300ms stimulation. n = 1-7 animals in each group. NS - not significant, AF - atrial fibrillation, VAlt - spectral magnitude at 0.5 cycles / beat.
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5.3.8 Regions of the action potential most affected by alternans.
Some phases of the AP were more affected by alternans at low stimulation rates, while some were more affected at higher rates. Figure 5.13A shows a progression from no alternans at 2 Hz, to alternans almost exclusively affecting repolarisation at 2.5 Hz, to alternans predominantly affecting depolarisation at 3 Hz. This pattern was seen in some but not all cells, as alternans predominantly affecting depolarisation occurred in some cells as the first manifestation of alternans.
This behaviour was quantified by comparing VAlt within phase 0-1 (from 10ms prior to the AP peak to 10ms after) to VAlt within phase 2-3 (from 10ms after the AP peak to APD90). In recordings with a k-score >3, if VAlt in phase 0-1 was greater than VAlt in phase 2-3, the alternans was described as “depolarisation dominant”. Conversely, if VAlt in phase 2-3 was greater, alternans was described as “repolarisation dominant”.
At the minimum (initial) stimulation rate leading to alternans, 33/67 cells manifested repolarisation dominant alternans (Figure 5.13B). As stimulation rate increased, cells were more likely to manifest depolarisation alternans. At the maximum (final) stimulation rate that still generated alternans, only 10/67 cells were repolarisation dominant (p<0.05). No significant differences were seen between age groups.
The timepoint within the AP that manifested the greatest VAlt timepoint was tracked for all cells with a k-score >3 at each stimulation frequency. This timepoint shifted to earlier in the AP as stimulation rate increased (p<0.05, Figure 5.13C). Once again, no significant differences were seen between age groups.
Data is summarised in Table 5.7
Young Old Difference p-value Repolarisation dominance Initial 18 / 31 cells 15 / 36 cells -28.2% 0.27 Final 5 / 31 cells 5 / 36 cells -13.90% 1.00
Timepoint of maximum V Alt 1 Hz 159.8 ± 61.3ms 166.7 ± 34.6ms 4% 0.85 2 Hz 47.2 ± 7.3ms 61.9 ± 9.5ms 31% 0.36 3 Hz 37.3 ± 9.2ms 42 ± 9.3ms 12% 0.84
Table 5.8 – Region of action potential most affected by alternans. n = 14-18 cells, 5-8 animals in each group. Significance determined using Chi-squared test. P a g e | 158
Figure 5.13 – Region of action potential most affected by alternans. A Example of cellular transmembrane action potentials stimulated from 2 Hz to 3.5 Hz. At low stimulation rates, alternans predominantly affects repolarisation (repolarisation dominant). At higher rates, alternans predominantly affects depolarisation (depolarisation dominant). B Proportion of cells initially manifesting repolarisation dominant alternans and at highest frequency before alternans degenerated. C Timepoint in action potential at which maximal alternans was seen. n = 14-18 cells, 5-8 animals in each group. * p<0.05, VAlt - alternans magnitude.
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5.3.9 Simultaneous alternans of membrane potential and [Ca]i
We wished to explore the relationship between AP alternans and alternans of [Ca]i. Atrial myocytes were loaded with the fluorescent indicator Fluo-5F to enable simultaneous measurements of [Ca]i and membrane potential and stimulated from 1 Hz to 3 Hz. Examples of these recordings can be seen in Figure 5.14.
As seen previously, low stimulation rates generated trains of APs and Ca2+ transients that were similar from beat to beat (Figure 5.14A). In 3/15 cells studied, a slight increase in the pacing rate produced subtle alternans of [Ca]i without detectable alternans (k-score <3) of the AP (Figure 5.14B). In the remaining 12/15 cells, and at higher stimulation rates, simultaneous alternans of [Ca]i and membrane potential occurred (Figure 5.14C).
As seen before in cells that had not been loaded with fluorescent indicators, AP alternans could be classified as depolarisation or repolarisation dominant. When depolarisation dominant alternans was present, larger Ca2+ transients corresponded with APs of higher amplitudes and longer durations. When repolarisation dominant alternans was present, larger
2+ Ca transients corresponded with APs that had a shorter plateau phase but a longer APD90. In 2+ this set of experiments, AP alternans was never seen without concomitant alternans of [Ca ]i.
2+ 2+ In some recordings, the Ca transient was affected by alternans but the diastolic [Ca ]i returned to the same stable value between beats. This occurred exclusively during repolarisation dominant alternans at low stimulation rates (Figure 5.14B). At higher rates or
2+ during depolarisation dominant alternans, diastolic [Ca ]i was also seen to alternate (Figure 5.14C).
Higher order oscillations were also infrequently observed. At these times, the periodicity of
2+ [Ca ]i oscillations matched that of the membrane potential.
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Figure 5.14 – Simultaneous membrane potential and intracellular calcium alternans. All traces in this figure obtained from the same atrial myocyte. Original traces (left) and superimposed means of odd and even beats (right). A Calcium transients and action potentials showing a uniform response when stimulated at 1 Hz. B When stimulated at 1.25 Hz, subtle alternans of calcium transients occurs while variation in action potentials falls below the detection threshold for alternans. C At 1.5 Hz stimulation, pronounced calcium alternans occurs, marked by repolarisation dominant alternans of the action potential.
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2+ 5.3.10 Disruption of [Ca ]i alternans
2+ As was demonstrated in Figure 5.11B, [Ca ]i alternans can exist in the absence of AP alternans. 2+ We questioned whether the inverse was true – could AP alternans exist without [Ca ]i alternans? To investigate this, the SERCA inhibitor thapsigargin was used to disrupt Ca2+ cycling. Following completion of the protocol used in section 5.3.9, 10 µM thapsigargin was added to the superfusate and myocytes were stimulated at 1 Hz for 10 minutes.
Figure 5.15A and B show an atrial myocyte stimulated at 1.5 and 2 Hz demonstrating repolarisation and depolarisation alternans respectively. Following the application of
2+ 2+ thapsigargin (Figure 5.15C), Ca cycling was severely impaired with minimal residual [Ca ]i fluctuations seen caused by ICa(L) and NCX. APD50 increased, which is likely to be due to a 2+ decrease in Ca dependent inactivation of ICa(L). Alternans which had been present at low stimulation rates was no longer seen.
However, AP alternans was still present at higher stimulation rates in four out of five cells despite abolition of Ca2+ cycling with thapsigargin (Figure 5.15D). The alternans threshold increased by 14% (1.75 ± 0.1 Hz to 2.0 ± 0.1 Hz, p=0.08). Only depolarisation dominant alternans was seen under these conditions. Alternans was completely abolished at all stimulation frequencies in one out of five cells studied. In this cell, extensive irregular beat to beat variability of AP duration was seen but this did not coalesce into a regular oscillation.
2+ These experiments demonstrate that, while closely linked, [Ca ]i alternans can exist independently of AP alternans at low stimulation rates, while AP alternans can exist
2+ independently of [Ca ]i alternans at high stimulation rates. P a g e | 162
Figure 5.15 – The effect of SERCA blockade on alternans. Original traces (left) and superimposed means of odd and even beats (right). A 1.5 Hz stimulation generates alternans of intracellular calcium and action potentials under control conditions. B Alternans is also present at 2 Hz stimulation. C Application of the SERCA inhibitor thapsigargin abolishes calcium transients, disrupting alternans at 1.5 Hz stimulation. At 2 Hz stimulation, action potential alternans is seen despite the abolition of calcium transients by thapsigargin. P a g e | 163
5.3.11 Correlations between cellular alternans and Ca handling, APD, cell size
It was shown that AP alternans could occur when Ca2+ cycling was disrupted. We therefore investigated a potential association between AP duration and alternans threshold. In vivo, longer AP durations were associated with a lower alternans threshold when all data was grouped (p<0.05). To examine whether this relationship reflected a true association between the parameters rather than representing the effects of age on two unlinked variables, the data from each cohort was analysed separately (Figure 5.16A). The relationship persisted within the old cohort but was no longer significant within the young.
In isolated myocytes, a weak association between AP duration and alternans threshold was found when the data from all myocytes was grouped (p<0.05). However, when the age cohorts were assessed separately, no significant relationship was seen (Figure 5.16B).
Having also demonstrated that Ca2+ cycling can produce alternans at stimulation rates where AP alternans is absent, an association was looked for between measures of Ca2+ reuptake and alternans threshold. Figure 5.16C shows a scatterplot of the rate of decay of the Ca2+ transient
2+ against alternans threshold. Adjusting the dataset by using the [Ca ]i alternans threshold rather than the Em alternans threshold and excluding cells that initially manifested depolarisation alternans resulted in a stronger trend that still did not reach statistical significance. The sample size was too small to enable comparisons within age groups.
Data is summarised in Table 5.9.
Parameter 1 Parameter 2 Group Correlation coefficient (R) p-value In vivo
APD90 Alternans threshold All 0.7 <0.01 * Young 0.47 0.14
Old 0.64 0.048 *
Cellular
APD90 Alternans threshold All -0.32 <0.01 * Young -0.28 0.12
Old -0.18 0.31
τdecay Alternans threshold All 0.21 0.47 Adjusted 0.46 0.14
Table 5.9 – Correlations between alternans threshold and potential determinants. n = 10-11 animals in each group (in vivo); 33-34 cells, 12-13 animals in each group (cellular APD90); 4- 10 cells, 2-6 animals in each group (cellular τdecay). * p<0.05, NS - not significant, τdecay - time constant of decay of calcium transient, APD90 - action potential duration at 90% repolarisation. P a g e | 164
Figure 5.16 – Correlations between alternans and potential mechanisms. A Scatter plot of alternans threshold against in vivo monophasic action potential APD90 stimulated at 2.5 Hz (n = 10-11 animals in each group). B Scatter plot of alternans threshold against cellular
APD90 stimulated at 0.5 Hz (n = 33-34 cells, 12-13 animals in each group). C Scatter plot of alternans threshold against time constant of decay of calcium transient (n = 4-10 cells, 2-6 animals in each group). * p<0.05 within old cohort only, NS - not significant, APD90 - action 2+ potential duration at 90% repolarisation, [Ca ]i - intracellular calcium concentration, τdecay - time constant of decay of calcium transient. Dashed lines represent linear regressions of the datapoints from the corresponding age groups. P a g e | 165
5.4 Discussion
In this chapter it has been demonstrated that older myocytes from the left and right atria are more susceptible to alternans than their younger counterparts. Alternans is induced at lower stimulation rates and is of greater magnitude. A similar propensity to alternans has been shown in the right atrium of older sheep in vivo. Higher order periodicities were also shown in isolated myocytes and in vivo, and although the incidence of these was higher in old sheep, the difference did not reach statistical significance due to the low event rate.
The mechanisms responsible for alternans in the atria were explored next. Repolarisation alternans was found in many cases to precede depolarisation alternans. In myocytes loaded
2+ with a fluorescent indicator, alternans of [Ca ]i was shown to exist alongside AP alternans and in some cases occurred at lower stimulation rates than those that elicited action potential alternans. Disruption of Ca2+ cycling using the SERCA inhibitor thapsigargin greatly reduced Ca2+ transient amplitude and abolished alternans at low stimulation rates. However, at higher rates, AP alternans still occurred in some cells, and was exclusively of the depolarisation type.
5.4.1 Alternans occurs at lower stimulation rates in isolated myocytes than in vivo
The overall cellular AP alternans threshold was ~1.7 Hz and ranged from 1.25 Hz to 3.5 Hz, similar to previous work performed in rabbit atrial myocytes in which alternans developed at rates from 1 Hz to 2.5 Hz 321. In comparison, the overall in vivo alternans threshold was nearly double this at ~3.2 Hz, similar to that reported in conscious sheep at ~3.3 Hz 98, and of a similar order to that reported in humans of ~4.5 Hz 97. Why should there be a twofold difference in the alternans threshold of isolated myocytes compared to intact tissue?
Firstly, the in vivo experiments were performed under anaesthesia, and isofluorane, the anaesthetic agent used, could affect alternans dynamics. While this is possible, it seems unlikely as a similar alternans threshold has been reported in vivo in conscious sheep98. Secondly, isolated myocytes are deprived of the electronic effects that shorten the AP duration and potentially influence the restitution curve. Again, it seems unlikely that factors modulating membrane currents will play a major role in the alternans threshold as the alternans seen at lower stimulation rates is more often Ca2+ driven. This may, however, have contributed to the cells manifesting depolarisation dominant alternans at threshold. Thirdly, isolated myocytes are not exposed to important humoral factors. This seems much more likely, as both sympathetic322 and parasympathetic334 activation have been shown to increase atrial alternans threshold. P a g e | 166
5.4.2 Alternans threshold decreases with age
A decrease in alternans threshold was seen with age in isolated myocytes and in vivo, which is a novel finding. Given that the three principal mechanisms of alternans relate to AP restitution, Ca2+ reuptake and Ca2+ release, the effects of age on each of these could contribute to the observed decrease in alternans threshold.
AP restitution was not specifically studied in the work presented here, making it difficult to firmly accept or reject it as a cause for the difference in alternans threshold. A hint that membrane properties play a role in alternans threshold can be inferred from the correlation in vivo whereby atria manifesting shorter APs alternated at lower stimulation rates. This correlation is consistent with previous observations showing that regions of the intact guinea pig ventricle manifesting shorter AP durations are also more prone to alternans303, and that the K+ channel blockers tertiapin342 and bretylium298 simultaneously prolong the AP and suppress alternans. However, in vivo AP durations did not differ significantly with age, and AP duration in isolated myocytes increased with age, which if this correlation were to be followed would be expected to increase the alternans threshold with age, the reverse of what was seen. A potent role for restitution changes driving the decrease in alternans threshold also seems less likely as at threshold, alternans is often Ca2+ driven.
Could slowed Ca2+ reuptake be responsible for the decreased alternans threshold? In Chapter 4.2.6, a trend towards slower Ca2+ reuptake was seen in older myocytes. This parameter has been studied extensively during previous work in this our laboratory (as yet unpublished), confirming that age leads to a slowing of Ca2+ reuptake in atrial myocytes from older sheep. When investigated in more detail, no difference was found in SERCA activity, SERCA protein
2+ expression, or INCX, but that the slowing was due to an increase in Ca buffering with age. The effects of Ca2+ buffering have been investigated in silico, with results suggesting that far from promoting alternans, an increase in buffering might prevent alternans, as this will impair the coupling between CRUs343. These findings are far from definitive, as an effect of increasing buffering was only simulated during clamped SR Ca2+ content thereby limiting the effects on Ca2+ reuptake, and simulations with unclamped SR content only investigated a decrease in buffering. Limited experimental evidence can be found in a study of mouse ventricle, describing how when buffering was increased using low dose EGTA, the rate threshold for alternans induction increased344. The mouse ventricle may be quite different from large mammalian atria, not least because alternans was exclusively seen at pacing rates of 10 Hz and above. P a g e | 167
Contributing to the slower Ca2+ reuptake in isolated myocytes, further slowing may occur in vivo due to haemodynamic loading. Atrial stretch has been shown to slow Ca2+ reuptake in cultured atrial monolayers, and also to reduce the alternans threshold320. The dilated atria seen in the old will, following Laplace’s law, experience more stretch even under similar loading conditions, and this will be exacerbated by any increase in atrial pressure with age. By this mechanism, any underlying cellular tendency towards slow Ca2+ reuptake will be multiplied, promoting alternans.
Undermining the suggestion that slower Ca2+ reuptake in age is responsible for the decrease in alternans threshold, no association was found between Ca2+ transient rate of decay and alternans threshold. This could, however, be due to the small sample size, but qualitative inspection of the scatterplots in Figure 5.16 does not immediately suggest a trend. It should be noted that these plots use Ca2+ transient decay stimulated at only 1 Hz, whereas an inability to accelerate Ca2+ reuptake at high rates may be the key problem. Studies in rat atria have suggested that the differences in Ca2+ reuptake with age are more prominent at faster rates and under conditions of glycolytic inhibition238, potentially suggesting that energy supply may be inadequate in older myocytes and thereby slowing Ca2+ reuptake.
Instead, impaired Ca2+ release could underlie the lower alternans threshold seen in age. If RyR opening was diminished then Ca2+ transient amplitude would be expected to fall. In keeping with this, Ca2+ transient amplitude tended to decreased with age (Figure 4.7B), although this did not reach statistical significance. This explanation is not fully consistent with work performed in myocytes isolated from human atria, in which the observed decrease in Ca2+ transient amplitude with age could be attributed to a combination of lower ICa(L) and lower SR Ca2+ content232. However, previous work in this sheep model of ageing suggests that spontaneous Ca2+ spark frequency decreases with age, an observation that could correlate with increased RyR refractoriness.
5.4.3 Alternans magnitude increases with age
In addition to the decrease in alternans threshold with age, alternans magnitude increased with age. This is similar to previous findings in rat atria, in which older atria exhibited alternans of greater magnitude both before and after glycolytic inhibition238.
There is evidently a link between alternans threshold and magnitude, so do they represent two ways of measuring the same thing? Could an apparent increase in magnitude just represent more alternating compared to non-alternating myocytes at a given rate, spuriously reflected in the mean as a difference in magnitude? When looking at the alternans of individual cells, the P a g e | 168 difference in magnitude persists even when non alternating cells are excluded as shown in Figure 5.7D, rejecting this claim. However, when viewing alternans at a tissue scale this effect is likely to be important as the alternans magnitude represents a sum of alternating and non- alternating cells.
As alternans magnitude climbs as stimulation rate increases, does an increase in magnitude simply reflect a leftward shift in the curve? This is possible, but comparison of alternans threshold suggests a 0.4 Hz shift with age, whereas an equivalent alternans magnitude to that seen in old myocytes at 1.75 Hz stimulation is only seen at 3.5 Hz in young myocytes.
Apparent alternans magnitude assessed over the whole AP is greater when alternans of depolarisation and repolarisation occur together rather than alternans of repolarisation alone. Does the apparent difference in magnitude then represent a greater proportion of older cells showing depolarisation rather than repolarisation alternans? As shown in Figure 5.13C, the timepoint within the AP showing the greatest VAlt was similar between age groups, countering the suggestion that alternans of depolarisation predominated in one group but not another.
The AP alternans seen in isolated myocytes was easily identifiable with the naked eye, with a typical VAlt of 3 mV for a 100 mV amplitude AP (3%). By comparison, in vivo atrial alternans was subtle, with a typical VAlt of 0.02 mV for a 5 mV amplitude AP (0.4%), and required computation to reliably distinguish alternans from noise. One explanation for this discrepancy comes from the electrotonic effects present in tissue. At any given stimulation rate, the variability between myocytes in a tissue block mean that some, if acting independently, would alternate while others would not. The electrical coupling between myocytes serves to smooth out differences between cells, meaning that alternans becomes more subtle.
Differences in Ca2+ cycling between old and young myocytes may be responsible for the increased alternans magnitude. The slowing of Ca2+ reuptake with age suggested here and confirmed by previous work performed in this model may contribute, as slower Ca2+ reuptake and increased alternans magnitude have been associated in other models315.
Alternans magnitude may also results from changes in SR Ca2+ content. A 5% fluctuation in Ca2+ content of a well-filled SR will result in a greater difference between the amplitude of large and small Ca2+ transients than a 5% fluctuation in an almost empty SR. Previous work in our laboratory has shown that atrial SR Ca2+ content increases with age (unpublished data).
2+ Additionally, the interplay between [Ca ]i and AP shape could play a role. For a given 2+ magnitude of oscillation in [Ca ]i, the effects on AP shape will depend on the properties of 2+ 2+ Ca sensitive currents. If ICa(L) is increased, or if it is more sensitive to Ca -induced P a g e | 169 inactivation, then a greater difference in plateau duration will be seen between beats.
Similarly, if INCX is increased then a greater difference in the duration of the terminal region of the AP will be seen. However, previous work performed in our laboratory on this model of ageing suggests that atrial ICa(L) decreases with age, and INCX does not differ between age groups (unpublished data).
5.4.4 Depolarisation and repolarisation alternans occur separately
In isolated myocytes, alternans was shown to affect repolarisation alone, or depolarisation and repolarisation together, similar to previous findings281. Both of these were seen alongside
2+ 2+ with alternans of [Ca ]i. During repolarisation alternans, large Ca transients coincided with APs showing a shorter plateau but slower terminal repolarisation and minimal change in AP amplitude, similar to that reported in atrial myocytes from rabbits308. The plateau duration,
2+ determined by ICa(L), is likely to be shorter during large Ca transients as ICa(L) inactivates faster 2+ in the presence of higher [Ca ]i. At the same time, the terminal phase of repolarisation is longer during large transients as the inward current produced by INCX is enhanced by higher 2+ 305 2+ [Ca ]i . These observations are in keeping with [Ca ]i alternans being the primary driver of AP alternans when repolarisation alternans is seen, supported by the findings in some cells
2+ that repolarisation alternans was preceded by [Ca ]i alternans without any notable alternans of the AP shown here, and by others345.
Conversely, during depolarisation alternans, a significant alternation of AP amplitude occurs. Low amplitude APs exhibit both a shorter plateau and shorter terminal repolarisation. During high amplitude APs, incomplete repolarisation occurs so that the MDP immediately preceding small APs is less negative than that preceding large APs. This suggests that the smaller APs are caused by incomplete recovery of INa from inactivation, in turn caused by slowed repolarisation. In turn, the decrease in AP amplitude produces less ICa(L) on small beats. Low amplitude, short duration APs are associated with smaller Ca2+ transients, potentially due to less CICR from decrease ICa(L).
An interpretation of these observations is that depolarisation dominant alternans is primarily
2+ AP driven, with [Ca ]i occurring as a secondary phenomenon. This is supported by the continued presence of depolarisation dominant AP alternans at high stimulation rates despite disruption of Ca2+ cycling by thapsigargin. This finding contrasts with the work of Goldhaber et al., who showed that thapsigargin completely abolished AP alternans in rabbit ventricular myocytes299, but is complementary to the work of de Diego et al. who found that alternans was still present in embryonic mouse ventricles despite thapsigargin application332. P a g e | 170
The procession from depolarisation alternans to repolarisation alternans has been shown in APs from intact ventricular tissue and alternans on the surface ECG270. This pattern was, in general, found here as shown by the earlier timepoint within the AP showing the greatest VAlt at higher stimulation rates. However, in a significant minority of the isolated myocytes studied here, the first appearance of alternans was depolarisation dominant. Depolarisation alternans occurred in cases where long APs prevented full repolarisation before the next stimulus. As shown in Chapter 4, APs recorded from isolated myocytes are markedly longer than those found in intact tissue. Depolarisation dominant alternans may therefore not occur at low stimulation rates in intact tissue due to the shorter APs.
5.4.5 The relationship between alternans and atrial fibrillation
As shown in Chapter 3, older sheep were more susceptible to atrial fibrillation than younger sheep. We tried to establish a link between arrhythmia inducibility and alternans behaviour, but have only collected data on a limited number of animals so far. No statistically significant relationship was seen between the vulnerability to AF and alternans threshold, magnitude or the presence of higher order periodicities. However, inspection of the scatterplots in Figure 5.9 shows a trend whereby alternans of greater magnitude is associated with AF that is more frequently induced and is of longer duration.
The lack of statistical significance may simply relate to the small sample size. Another explanation may be that when recorded from a single site, no information can be obtained regarding the spatial concordance of alternans. A better predictor of alternans may come from multisite recordings, enabling the rate threshold of discordant alternans to be measured.
5.4.6 Limitations
The alternans studied may not be directly relevant to alternans seen during arrhythmia initiation. Compared to the typical dominant frequencies seen in AF of 7-10 Hz, the alternans threshold seen here was ~1.7 Hz in vitro and 3.3 Hz in vivo. Additionally, while overall the alternans magnitude was greater in old atria than young, at the highest stimulation rates tested (4 Hz) a trend was seen towards greater alternans magnitude in the old. Despite the discrepancies between the experimental conditions used here and what is likely to be seen in AF, alternans behaviour at low stimulation rates has been shown to reflect how far atrial tachycardia remodelling has progressed289.
The spectral technique of alternans analysis suffers from some drawbacks. It assumes that oscillations that occur will persist throughout the recording period, rather than coming and going or changing phase (referred to as non-stationarity). If alternans changes phase during a P a g e | 171 recording, caused by for example an extrasystole, the quantification of alternans magnitude and the discrimination from noise may be impaired346. To mitigate this, recordings of short duration have been used (32 beats), limiting the chance of these non-stationarities occurring. It is unlikely that the accuracy of this technique differed between age groups.
Data was collected and analysed without blinding. However, the threshold and magnitude of alternans was assessed computationally, reducing potential for user bias. Furthermore, in vivo recordings could not be assessed in real time during acquisition, limiting any potential for selection bias.
5.5 Conclusions
In this chapter we have seen that older atria are more prone to alternans, both in vivo and in isolated myocytes. The rate threshold to generate alternans decreases with age, and when alternans is seen it is of greater magnitude. 3:1 and 4:1 oscillations are also seen, but the incidence of these did not differ with age. Alternans affecting depolarisation and repolarisation were shown to exist independently, with repolarisation dominant alternans occurring at lower rates and being primarily Ca2+ driven, while depolarisation dominant alternans occurred more at higher rates and had a greater contribution from AP restitution.
The next chapter will explore how atrial conduction changes with age.
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6 ATRIAL CONDUCTION
6.1 Introduction In Chapter 3 we saw that the P-wave on the surface ECG was longer in older sheep, as is seen in older humans. The myocyte hypertrophy seen with age in Chapter 4, coupled with previous work in this model suggests that the atria dilate with age in the sheep. We also questioned whether a change in atrial conduction might be responsible for the P-wave prolongation of senescence.
6.1.1 Conduction velocity
Conduction velocity (CV) refers to the speed at which a wave of electrical excitation spreads through the myocardial tissue, typically 0.8-1ms-1 in man347. The CV parallel to fibre orientation is not equal to CV perpendicular to fibre orientation, a phenomenon known as anisotropy74. Increasing anisotropy has been linked to re-entrant circuit formation75. There is a two-way relationship between CV and arrhythmias, as CV slowing promotes AF348 while atrial tachycardia remodelling leads to a slowing of CV51. Atrial CV has in general been found to decrease with age in animal349 and human studies132, 210. However, this finding is not universal as some groups have shown no change193 or an increase in longitudinal CV213 with age.
6.1.2 Cable theory
As a starting point to explore the determinants of conduction velocity, we can begin with a simple biophysical model of electrical impulse propagation. Perhaps the most basic is one-dimensional cable theory. In this model, structurally complex myocytes are reduced to cylinders that can be described in terms of the resistance and capacitance of the cell membrane and cytosol. For simplicity, it is assumed that the resistance between cells (inter-cellular) is similar to that found within cells (intra-cellular). As with all simple models, important factors have been omitted. Inter-and intracellular resistance are not in reality the same, and myocardial tissue is clearly a three-dimensional rather than a one-dimensional structure. Models of increasing complexity incorporate these and other biophysical phenomena350. However, this simple model provides a foundation from which the modulators of CV can be discussed.
The cell membrane is described as having a membrane resistance Rm and capacitance Cm. The intracellular resistance Ri is assumed to be uniformly distributed along the cell. Some of these parameters are interlinked. Increasing cell radius, an important parameter in its own right, will also enlarge the surface area of the cell membrane. Capacitance is directly proportional to P a g e | 173
surface area, and Cm therefore rises in line with radius. The opposite is true of intracellular resistance, which is inversely proportional to cross sectional area, and Ri therefore falls as radius increases.
In the same way that a the central pole of a teepee tent lifts not only the middle of the fabric but also that which surrounds it, depolarisation of the membrane at one point will influence neighbouring regions passively, i.e. without the need for any ionic currents. Nearby regions will be depolarised to a large extent while more distant regions are less affected. The distance encompassed by this spread of depolarisation can be expressed as the space constant λ, representing the distance at which depolarisation has decayed to 37% of its peak value. Further depolarisation is achieved actively by the opening of Na+ channels once they have been brought to a sufficiently depolarised threshold potential. An increase in λ means that regions further from the initial depolarisation are brought to threshold earlier, thereby increasing CV. λ is calculated by equation 6.1.
푅푎푑푖푢푠 . 푅푚 휆 = √ 2. 푅푖
Equation 6.1 – Space constant where λ is the space constant
Rm is the membrane resistance per unit surface area Ri is the uniform intracellular resistivity per unit myocyte cable length Radius is the radius of the myocyte
Using this mental image, it is easy to imagine that a longer tentpole will lift fabric at a greater distance from the centre. Similarly, a larger initial depolarisation will bring membrane at a greater distance from the origin to threshold. This depolarisation is governed by INa, reflected in the amplitude and rate of rise of the AP. Increasing INa therefore accelerates conduction in theory, and has been shown to do so in practice351.
If we instead turn our attention to equation 6.1 we see that enlargement of the cell radius increases the numerator, and therefore increases λ. According to this simple model, therefore, tissue comprised of myocytes with a greater radius will exhibit a faster CV, an observation that has been borne out by experimental studies352.
Equally, factors that modulate Ri can be predicted to modulate CV. Intracellular connections are formed by gap junctions. Gap junctions that are more permeable and present in greater P a g e | 174
353 abundance decrease Ri and have experimentally been shown to increase CV . Likewise, 122 connective tissue acts as an electrical insulator, and fibrosis increases Ri, slowing conduction .
For completeness, it should be noted that the passive change in membrane potential is not instantaneous, and can be described using the charging time. A shorter charging time equates to a faster CV. The charging time is calculated using equation 6.2
−푡⁄퐶푚.푅푚 푉푡 = 푉0(1 − 푒 )
Equation 6.2 – Charging time
where Vt is the membrane potential at time t V0 is the initial membrane potential t is the timepoint
Cm is the membrane capacitance Rm is the membrane resistance
+ The key modulators of CV, being the Na current INa, gap junctions, and fibrosis will be explored in more depth in the following sections.
6.1.3 INa
To approach INa from the perspective of a mere determinant of conduction velocity risks severely underestimating its importance. The majority of excitable tissues require a tightly regulated influx of Na+ to fulfill their primary function – to be excitable. When control is lost and there is insufficient or excessive Na+ influx, a current that requires too large a trigger for activation or is activated too easily; in these cases coordinated activity breaks down. This is
354 355 reflected in the mutations affecting INa that lead to pathology in the heart , brain and 356 skeletal muscle , and has been made use of through pharmacological manipulation of INa that has been extensively employed for antiarrhythmic, antiepileptic and anaesthetic purposes355, 357.
6.1.3.1 Role of INa in the action potential
INa causes the rapid upstroke of the action potential (Phase 0) in atrial and ventricular myocytes, but not in the sinus or atrioventricular nodes which instead rely on Ca2+ influx.
Increasing INa increases the rate of rise of the AP. However, the relationship between the two 358 is non-linear, so AP rate of rise is not a suitable surrogate for INa . As discussed above, a faster depolarisation of greater magnitude will depolarise regions further from the point of origin, bringing these portions of the membrane to threshold potential earlier. Increasing INa therefore leads to faster conduction of the depolarising wave. P a g e | 175
13 INa activates and inactivates very rapidly, leading to a very transitory but large inward current as was seen in Figure 4.1. Incomplete inactivation of Na+ channels can lead to a sustained ‘late Na+ current’, causing prolongation of the AP plateau that is significant for arrhythmias and angina359.
6.1.3.2 Sodium channel structure
The plethora of excitable tissues that make use of INa fulfil very different functions, and there + are therefore understandable differences in how these tissues control their flow of Na . INa is carried by a family of Na+ channels, and specific tissues express a complement of channels tailored to their needs.
Each complete Na+ channel contains three different subunits, making it a heterotrimer. These subunits fall into two classes: α-subunits provide the central pore for ionic passage360, while β-subunits fulfil a dual role of modulating the gating functions of the complete Na+ channel, and simultaneously assist with cell-to-cell adhesion361.
Figure 6.1 – Sodium channel structure. Adapted from Catterall (2005)362
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The protein that forms Na+ channel α-subunits loops back and forth across the membrane (Figure 6.1). These are organised such that six of these loops (membrane-spanning helices) form a domain, while four domains form a complete subunit362. The arrangement of amino acids within the protein confers specific properties upon the subunit, enabling categorisation of Na+ channel α-subunits into the nine varieties have been discovered so far. The α-subunits are referred to as NaV1.1 to NaV1.9 and are encoded by the genes SCN1A to SCN11A. Neural tissue preferentially expresses NaV1.1, NaV1.2, NaV1.3, and NaV1.6 channels in the central nervous system, while NaV1.7, NaV1.8 and NaV1.9 are the dominant forms expressed in the peripheral nervous system. In skeletal muscle, the principal α-subunit expressed is NaV1.4362.
In cardiac muscle, the primary and historically believed sole Na+ channel α-subunit is NaV1.5, encoded for by the SCN5A gene. However, NaV1.1 to NaV1.6 have also been shown to be present in human atrium363 and mouse ventricle364. Current carried by NaV1.5 can be isolated from current carried by the remaining Na+ channel α-subunits seen in cardiac muscle by its relative resistance to tetrodotoxin (TTX). Making use of this property, it has been estimated
363 that up to 25% of INa seen in human atrium is carried by non-NaV1.5-containing channels .
There are four Na+ channel β-subunits, referred to as β1 to β4 and encoded for by the genes SCN1B to SCN4B. The complete Na+ channel heterotrimer is formed from one α-subunit associated with one β1/β3-subunit and one β2/β4-subunit365.
β-subunits regulate Na+ channel expression and modify the activation and inactivation kinetics of NaV1.5 366 and TTX sensitive Na+ channels367. In addition to their channel-modulating functions, β-subunits play a role in cell adhesion. β1- and β2-subunits have been shown to aggregate rat neurons361 although other reports have suggested a role in cellular repulsion368. All four β-subunits have been detected using immunofluorescence in human atrial tissue363. However, in the sheep atrium, β1-subunits have been found along with NaV1.5, but β3 were absent369.
It has been suggested that the variety of Na+ channel subunits present within cardiomyocytes may each have a specific role to play. It might be assumed that this would be associated with differences in the localisation of specific subunits, but reports concerning this are contradictory, possibly due to inter-species variation. In mouse and rat ventricular myocytes, NaV1.5 is found more at the intercalated disks, while the TTX sensitive channels are thought to localise to the T-tubule network364, 370. Conversely, in human and dog atria, NaV1.5 is found at the cell surface while NaV1.2 is localised to the intercalated disks160, 363. P a g e | 177
Some have suggested that Na+ channels located at the intercalated disk provide a means by which conduction can pass from cell to cell without the involvement of gap junctions, referred to as ephaptic coupling371. According to this theory, the narrow cleft between adjacent myocytes becomes depleted of Na+ ions during phase 0 of the AP. This loss of positive charge effectively reduces the voltage difference between the inside and outside of the adjacent cell, activating INa.
6.1.3.3 Modulation of INa
Once Na+ channel mRNA has been transcribed, expression of the functional channel is determined by interactions with a host of other proteins. Ankyrin-G is thought to play a key role in targeting of NaV1.5 to specific cellular locations372, while NaV1.5 targeting for degradation occurs via ubiquitination, controlled by Nedd4-2373. Other interacting proteins affecting NaV1.5 expression include syntrophin, plakophilin-2 and α-actinin-225.
Once the channel has been expressed, modulation of current can occur via phosphorylation. Phosphorylation by protein kinase A (PKA) influences how Na+ channels are trafficked from the endoplasmic reticulum to the cell surface, and also how, once expressed, INa responds to changes in membrane potential. PKA phosphorylation encourages the gating mechanism that permits the flow of Na+ to open at more negative potentials but then also shut at more negative potentials. In other words, the half-maximal voltages of activation and inactivation of
374 INa are shifted towards more negative potentials in response to PKA . Sympathetic activation, 25 mediated by PKA, increases INa under physiological conditions . Conversely, phosphorylation + by protein kinase C (PKC) leads to a decrease in INa due to reduced Na channel trafficking, while simultaneously shifting inactivation towards more negative potentials375. Phosphorylation can also occur via tyrosine kinases and CaM kinase, but study of the effects of these has so far shown inconsistent results25.
Na+ channel function can also be modulated by glycosylation376. Glycosylation with sialic acid
(sialylation) has been reported to cause a shift of INa towards more negative potentials of activation and inactivation in atrial myocytes without affecting peak current density377.
6.1.3.4 Sodium channels and arrhythmias
The consequences of Na+ channel malfunction can be lethal. Mutations in SCN5A may cause long QT syndrome type III (LQT3), an inherited arrhythmia syndrome leading to sudden cardiac
354 death . Mutations typically cause incomplete inactivation of INa, leading to a persistent late Na+ current. This causes prolongation of the ventricular AP, predisposing to P a g e | 178 afterdepolarisations and the lethal arrhythmia Torsades de Pointes. Patients suffering from LQT3 are also more likely to experience AF378.
In contrast to the increase in INa seen in LQT3, a pathological decrease in INa due to mutations affecting NaV1.5 or NaV1.8 underlies more than 30% of cases of Brugada syndrome, a genetic disorder causing ventricular tachyarrhythmias that arise from the right ventricle (RV)379, 380. Two complementary mechanisms suggested to underlie this are (i) a dispersion of repolarisation in the RV or (ii) impaired right ventricular CV. AF has been reported as affecting up to 50% of these patients381. As well as the AF that is seen in conjunction with these lethal ventricular arrhythmia syndromes, mutations in SCN5A have also been shown to be a rare cause of familial lone AF382.
While mutations that pathologically increase or decrease INa predispose to AF, atrial 52 tachycardia remodelling has repeatedly been shown to decrease INa , leading to a reduction in atrial CV51.
+ The relationship between INa, CV and arrhythmias is complicated by the role of Na currents in determining cellular excitability. Blockade of INa prolongs the ERP with only minimal change to AP duration, referred to as ‘post-repolarisation refractoriness’. As discussed in Chapter 1, increasing ERP prolongs the wavelength of a re-entrant circuit, potentially rendering it unsustainable. However, blockade of INa could also slow CV to some extent, shortening wavelength which might stabilise the re-entrant circuit. The net effect of partial INa blockade by the commonly used antiarrhythmic flecainide is to increase ERP more than CV is slowed,
383 therefore increasing re-entrant circuit wavelength in canine atria . INa blocking agents such as lidocaine inhibit INa to a greater extent at faster stimulation rates, a phenomenon known as “use dependence” that has been exploited in the treatment of tachyarrhythmias384. The consequence of use dependence is that a drug can be given at a concentration that has minimal effect during normal slow sinus rhythm, but that exerts a profound effect during the pathologically rapid rhythms of AF.
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6.1.3.5 INa changes with age
Few studies have looked at the changes in Na+ channel expression or Na+ current density with age. In rodents, an increase in NaV1.5 expression was seen in left atrial tissue from old wildtype mice compared to young228, while no difference in NaV1.5 expression was seen between left atrial specimens from young and old rabbits349. In large mammals, no significant difference was found in INa between atrial myocytes from young and old dogs, with the exception of enhanced use-dependence leading to a reduction of INa at high stimulation rates in old right atria160. Despite these headline figures, close inspection shows that a trend towards increasing INa with age was seen that did not reach statistical significance. No human studies were found comparing INa between senescent and young adult populations. However,
INa was shown to increase during development between paediatric (mean age 3.4) and adult (mean age 43) patients385.
6.1.4 Connexins
Conduction velocity is also related to intercellular connectivity, facilitated by gap junctions. These are comprised of two hemichannels called connexons, themselves formed from proteins known as connexins386. Of the 21 connexins identified, four are present in the mammalian heart: Cx37, Cx40, Cx43 and Cx45387. In man, Cx43 is the most abundant and is uniformly expressed throughout all four chambers. Cx40 is found preferentially in the atria and more so in the right atrium than the left. Cx37 is found predominantly in the vascular endothelium but is also present at low levels in the chamber endothelium of both atria and ventricles. Cx45 is found at low levels in all chambers but is primarily expressed in specialised conduction tissue387.
6.1.4.1 Connexin 43
The relationship between connexin expression and conduction velocity is complex. Complete absence of Cx43 proves fatal in mice while partial (heterozygous) Cx43 knockdown was shown to slow conduction in the ventricles388. While this presents a clear story within the ventricles, the atrial picture is murkier. The same group found that heterozygous Cx43 knockdown did not affect atrial conduction, similar to findings in man that Cx43 expression was not correlated with in vivo conduction velocity in sinus rhythm389. Contradicting this, Beauchamp et al. showed that Cx43 knockdown leads to CV slowing in synthetic atrial tissue strands353.
Although its position in healthy sinus rhythm is uncertain, Cx43 appears to play a significant part in AF remodelling. Chronic left atrial pressure overload has been shown to decrease atrial Cx43 expression in the rat, with an associated increase in vulnerability to AF390. The reduction P a g e | 180 in Cx43 is non-uniform and areas of tissue become devoid of this protein while others are unaffected391. When Cx43 expression was enhanced in swine models through the use of gene transfer, atrial CV increased and pigs were less vulnerable to AF392, 393.
6.1.4.2 Connexin 40
The role of Cx40 in atrial conduction has been questioned. A progressive increase in CV through synthetic atrial tissue strands was seen between wildtype, +/- knockout, and -/- knockout mice353, suggesting that the presence of Cx40 slowed conduction. Additional in vitro evidence comes from human atrial tissue in which increased Cx40 is associated with increased intracellular resistivity and slower conduction394. This view is supported by human in vivo evidence whereby greater Cx40 expression is associated with a slower CV389. It appears that some Cx40 is still necessary, as complete Cx40 knockout leads to an increase in P-wave duration suggesting a slowing of atrial conduction395, although atrial dilatation cannot be excluded as a cause for this.
Why should augmented expression of a gap junction increase resistivity and reduce CV? Gap junctions may be formed from a single variety of connexin (homotypic gap junctions), but may consist of more than one variety (heterotypic gap junctions). It has been proposed that heterotypic junctions may have different conductance properties to homotypic junctions and that the raw quantification of a single protein does not take into account how it may be combined with other proteins. Some investigators have therefore suggested that the ratio of Cx40/Cx43 is a more useful parameter396, although others have failed to replicate these findings, demonstrating no correlation between Cx40/Cx43 ratio or their individual levels with overall atrial CV76, 397.
Complete absence of Cx40 may be detrimental to conduction, while in the presence of some Cx40 then ‘less is more’. This is noteworthy, as Cx40 may be very unevenly distributed within tissue. AF leads to heterogeneity of Cx40 expression398, 399, creating islands of Cx40 absence within goat atrial tissue. It is easily envisaged how these slowly conducting islands might promote wavebreak and re-entrant circuit formation. In light of this, total Cx40 expression may miss the point. This paradigm us rationalise why studies into how Cx40 changes in AF patients have shown increased76, unaffected396, or decreased400 levels.
Despite the apparent decrease in CV associated with Cx40, enhancing Cx40 expression using gene transfer has been shown to diminish atrial vulnerability 392. Although not proven, it can be imagined that this intervention prevented the formation of Cx40-absent islands.
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6.1.4.3 Modulation of connexin permeability
Connexin permeability is modulated in part by phosphorylation. Cx43 and Cx40 can be phosphorylated by protein kinase A401 and protein kinase C402 leading to increased conductance. These properties have been exploited pharmacologically. Rotigaptide, whose mechanism of action involves connexin phosphorylation403, has been shown to increase right atrial conduction velocity in the rat404 and reduce atrial vulnerability in Langendorff-perfused heart models405. Clinical trials of rotigaptide for prevention of atrial and ventricular arrhythmias are on-going.
6.1.4.4 Connexin changes with age
Mean atrial Cx43 expression decreases with age in the rabbit204, 349 and in adult compared to juvenile guinea pigs391. When the distribution of Cx43 was examined, a redistribution of Cx43 from the cell sides to the poles was seen with age213 but regions devoid of Cx43 were not391. Conflicting reports have been published regarded Cx40 changes in aging with some groups reporting a decrease in Cx40 expression in older rabbits204 whereas others have not shown a change349. No change in the distribution of Cx40 has been reported with age349.
6.1.5 Fibrosis
Atrial fibrosis, representing an abnormal increase in volume of the extracellular matrix, is closely associated with conduction and AF. The matrix acts as an electrical insulator between myocytes, impairing conduction. In addition, myofibroblasts, in addition to depositing excess matrix, can modulate CV via their electronic effects.
Connective tissue increases the resistance between adjacent myocytes, slowing conduction as predicted by the cable theory above. If sufficient fibrosis is present, or if propagation is
406 compromised due to decreased INa, conduction is blocked . If block occurs in a very small area, the wave of depolarisation can continue through the tissue, but needs to adopt a slower ‘zigzag’ route407. The distribution of fibrosis within tissue may be more important than total collagen content. Atrial tissue containing thick fibrous bands, while representing an overall increase in tissue collagen content, showed faster conduction than tissue with thin fibrous bands123.
Myofibroblasts also affect conduction directly. Compared to the cardiomyocyte RMP of -80mV, myofibroblasts are markedly depolarised with a typical RMP of -30 mV. When electrically coupled to myocytes, these electrotonic effects slightly depolarise myocytes and
255 limit the recovery from inactivation of INa . Increasing the number of myofibroblasts coupled P a g e | 182 to myocytes therefore slows conduction40. Factors that promote fibrosis can therefore slow conduction by stimulating proliferation of myofibroblasts.
Reports on the effect of ageing on atrial fibrosis in animal models have been contradictory. Some studies have shown an increase in fibrosis193, 233 whereas others have not204, 349. This discordance may reflect differences in the techniques used to assess the extent of fibrotic changes, or may relate to inter-species variation. An increased atrial susceptibility to fibrillation with age has been shown to occur in rabbits despite similar levels of atrial fibrosis between age-groups185, demonstrating that fibrosis is far from the sole determinant of senescent atrial vulnerability.
Atrial fibrosis has been examined previously in the ovine model of ageing in our laboratory and found not to differ between age groups (unpublished data) and will not be explored further in this work.
6.1.6 Aims
The aim of this chapter was to build on the ECG changes that were seen with age in Chapter 3, specifically the age-related prolongation of the P-wave, seeking to determine if this prolongation was due to a slowing of atrial conduction. Direct measurements of in vivo atrial conduction velocity were made. We then examined the determinants of conduction velocity,
+ specifically the Na current INa and incorporated the differences in AP duration seen in Chapter
4 into our analysis of INa behaviour. Finally, the expression of proteins relevant to INa and inter-cellular coupling was examined.
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6.2 Results
6.2.1 Conduction velocity
In view of the increase in P-wave duration found on the surface ECG described in Chapter 3.2.2, in vivo measurements of right atrial CV were made in anaesthetised sheep. Initially, a 40mm Constellation catheter was used. Recordings were, however, of insufficient quality to allow analysis. It was suspected that recording quality would be enhanced with the use of a larger catheter, enabling better apposition of the electrodes against the atrial myocardium. Using a 48mm Constellation catheter, a better signal to noise ratio was achieved. Usable recordings were obtained from 11 of the 20 animals studied with a 48mm catheter. Results are summarised in Table 6.1.
CV was first measured along the axis of the atrium, by stimulating from an electrode pair at the distal end of the catheter. EGMs were analysed from the three remaining electrode pairs along the stimulating spline. Representative traces from young and old sheep are shown in Figure 6.2A. Activation times were calculated and CV was calculated using linear regression (Figure 6.2B) and plotted as a contour chart (Figure 6.2C). Axial CV was 36% greater in old sheep compared to young (p<0.05) (Figure 6.3B).
Stimulation was then performed from an electrode pair at the middle of the catheter to measure circumferential CV. Endocardial electrograms (EGM) were analysed from the corresponding pair on each spline. CV was, as before, calculated using linear regression, and contour maps were plotted (Figure 6.3A), using fluoroscopic images of the recording catheter to calculate basket expansion and therefore inter-electrode spacing. Although a similar change in CV was seen in the circumferential direction, the difference did not reach statistical significance (Figure 6.3C). Catheter expansion, as a surrogate for atrial size, showed a weak trend towards atrial dilatation with age.
Anisotropic ratio was calculated by dividing the circumferential CV by the axial CV for each animal, and did not differ between groups (Figure 6.3D).
Young Old Difference p-value Axial conduction velocity 0.7 ± 0.1ms-1 1.0 ± 0.04ms-1 36.2% 0.004 * Circumferential conduction 1.1 ± 0.2ms-1 1.3 ± 0.1ms-1 21.6% 0.25 velocity Anisotropic ratio 1.5 ± 0.3 1.3 ± 0.1 -13.6% 0.48 Circumferential inter- 9.4 ± 1.1mm 10.4 ± 1mm 10.8% 0.52 electrode spacing
Table 6.1 – Conduction velocity measurements. n = 5-6 animals in each group. *p<0.05 P a g e | 184
Figure 6.2 – In vivo conduction velocity recordings. A Representative traces when stimulated from 1st electrode pair and recorded from (i) 2nd (ii) 3rd and (iii) 4th electrode pairs. B Plots of activation time against distance from stimulation site demonstrating the linear regression used to calculate conduction velocity. C Contour plots following stimulation from proximal electrode pair. P a g e | 185
Figure 6.3 – In vivo conduction velocity results. A Contour plots following stimulation from middle electrode pair. B Axial conduction velocity. C Circumferential conduction velocity. D Anisotropic ratio. n = 5-6 animals in each group. * p<0.05, NS - not significant.
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6.2.2 Sodium current recordings – baseline whole cell patch clamp parameters
In order to explore whether INa was responsible for the changes seen in atrial conduction, currents were recorded from isolated left atrial myocytes using the whole cell perforated patch technique. Initially, recordings were made in the presence of 5mM extracellular Na+. It was found that some cells, particularly those that were large and from old sheep, exhibited such large INa that adequate voltage clamp was unachievable. All recordings used in this analysis were therefore made in the presence of 3mM extracellular Na+.
In keeping with the changes in cell size described in Chapter 4.2.1, cell capacitance was 29% greater in left atrial myocytes from old sheep compared to young (p<0.05). Pipette resistance, access resistance, series resistance compensation and error and peak current were all similar between groups and are summarised in Table 6.2.
Young Old Difference p-value Capacitance 68.1 ± 2.8pF 87.9 ± 6.1pF 29.1% 0.001 * Pipette resistance 2.4 ± 0.1MΩ 2.2 ± 0.1MΩ -5.8% 0.27 Access resistance 6.1 ± 0.4MΩ 6.3 ± 0.4MΩ 3.1% 0.75 Series resistance compensation 74.5 ± 1.4% 77.8 ± 1.1% 4.4% 0.12 Error at peak current 2.4 ± 0.3pA 3.6 ± 0.6pA 52.3% 0.053
Table 6.2 – Cell and pipette parameters for whole-cell recordings. n = 21-38 cells, 8-14 animals in each group. * p<0.05
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6.2.3 INa amplitude and kinetics
The peak current of INa was recorded using a 200ms square wave pulse from -100 to -30 mV and corrected for cell capacitance (Figure 6.4A). Representative currents from young and old left atrial myocytes are shown in Figure 6.4B.
Peak INa was 29% greater in myocytes from old sheep compared to young (p<0.05). The time constant of decay, calculated by fitting a single exponential to the decay phase of INa, did not differ between age groups, nor did the time to peak (Figure 6.4D and E).
Data is summarised in Table 6.3.
Young Old Difference p-value Peak current -20.6 ± 1.4 pA.pF-1 -26.5 ± 2.5 pA.pF-1 29.1% 0.03 * Time to peak 2.4 ± 0.1ms 2.5 ± 0.2ms 4.5% 0.59 τ decay 1.9 ± 0.1ms-1 1.7 ± 0.1ms-1 -12.5% 0.10
Table 6.3 – INa amplitude and kinetics. n = 21-38 cells, 8-14 animals in each group. * p<0.05 P a g e | 188
Figure 6.4 – Change in peak INa with age. A Voltage protocol used to elicit INa. B Representative traces of INa. C Amplitude of INa. D Time to peak of INa. E Time constant of decay of INa. n = 21-38 cells, 8-14 animals in each group. * p<0.05, NS - not significant.
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6.2.4 Current / voltage relationship of INa
The current / voltage (I/V) relationship of INa was investigated next to see if the difference in current was preserved across a range of voltage steps. In order to reduce capacitance artefacts and 50 Hz noise, a double pulse digital subtraction protocol was used (Figure 6.5Ai). From a holding potential of -80 mV, cells were depolarised using a 195ms square wave voltage pulse to potentials of -70 to +5 mV in 5 mV increments. The potential was returned to -80 mV for 5ms before an identical square wave pulse was applied. While the first pulse generated both INa and a capacitance artefact (Figure 6.5Aii), during the second pulse INa was inactivated and therefore only the artefact remained (Figure 6.5Aiii). Digital subtraction of the second pulse from the first led to a clearer current recording (Figure 6.5iv). The 200ms time difference between the first and second pulses meant that any 50 Hz electrical noise was in phase between pulses and therefore digital subtraction led to its cancellation.
Representative traces of left atrial I/V plots from young and old sheep are shown in Figure 6.5B and mean data is presented in Figure 6.5C.
When the I/V plots were compared using a 2-way repeated measures ANOVA, the curves were found to be different (p<0.05). However, analysis of interactions showed that the difference depended on the voltage step applied and was only present at steps to -35 mV, -30 mV, -25 mV, -20 mV, -15 mV and -5 mV (Figure 6.5C). P a g e | 190
Figure 6.5 – Current / voltage (I/V) plots of peak INa. A Voltage protocol used to generate IV plots. (i) Two 200ms pulse separated by a gap of 5ms. (ii) Recording from 1st pulse. (iii) nd Recording from 2 pulse shows capacitance artefact only with no INa seen. (iv) Digital subtraction reveals INa without contamination from artefact. B Representative traces of INa from old and young atrial myocytes. C Current / voltage plots. n = 21-38 cells, 8-14 animals in each group. * p<0.05. P a g e | 191
6.2.5 Activation and inactivation curves for INa
Activation and inactivation curves were constructed for INa. Maximal conductance of INa was determined by finding the steepest region of the I/V curve. This represented the point where
+ all Na channels were open and INa was solely dependent on the driving force of the applied potential. Maximal current at a given potential could then be calculated. The I/V curves were then converted into activation curves by dividing observed current by maximal current at that potential. The data was then fitted with a Boltzmann curve (Figure 6.6B).
Inactivation curves were constructed using the voltage protocol shown in Figure 6.6Ai. From a holding potential of -80 mV, a 1000ms square wave pulse was applied at potentials from -140 mV to -40 mV in 5 mV increments. This was immediately followed by a depolarising pulse to -30 mV. Inactivation curves were formed by plotting INa at each step normalised to the maximal INa seen, and fitting these results to a Boltzmann curve (Figure 6.6B).
No differences were seen in the half-maximal voltages (V0.5) of activation (Figure 6.6C) or inactivation (Figure 6.6D) between young and old sheep nor in the slope factor (k) of activation or inactivation. Data is summarised in Table 6.4.
Young Old Difference p-value
Activation V0.5 -29.3 ± 0.7 mV -29.9 ± 0.7 mV 2.2% 0.57 Activation k 7.2 ± 0.2 7.3 ± 0.4 1.8% 0.75
Inactivation V0.5 -85.2 ± 0.6 mV -84.5 ± 0.9 mV -0.8% 0.51 Inactivation k 5.1 ± 0.1 5.1 ± 0.1 -0.1% 0.98
Table 6.4 – Activation and inactivation of INa. n = 21-38 cells, 8-14 animals in each group.
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Figure 6.6 – Activation and inactivation curves for INa. A Voltage protocols for (i) activation and (ii) inactivation. B Activation and inactivation curves. C Half maximal voltage of activation. D Half maximal voltage of inactivation. n = 21-38 cells, 8-14 animals in each group. * p<0.05, NS - not significant.
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6.2.6 Time-dependent recovery from inactivation and use dependence of INa
The time-dependent recovery from inactivation of INa was investigated next. From a holding potential of either -80 mV or -100 mV, two 200ms square wave pulses to -30 mV were applied. These pulses were separated by a return to -80 mV of 20-1000ms in 20ms increments (Figure 6.7Ai). The data was plotted as the amplitude of the second pulse normalised to the first pulse, against interpulse interval. It was found that single exponentials fitted these curves well and were therefore employed in all cases. Data is summarised in Table 6.5.
INa recovered faster from inactivation at a holding potential of -100 mV as shown by the shorter time constant of recovery (p<0.05). However, no significant difference was found with a holding potential of -80 mV (Figure 6.7B and C). The absolute difference in current magnitude was relatively small, with only a 5% difference in current amplitude at an interpulse interval of 60ms diminishing to no difference at interpulse intervals greater than 120ms (Table 6.7).
In light of these findings, in a subset of cells the effect of stimulation at rates typical for those seen during AF was observed. From a holding potential of -80 mV, 50ms square wave pulses to -30 mV were applied at 7.5 Hz (Figure 6.7Aii). The mean current at each beat is shown in
Figure 6.6D, showing a similar pattern of increased INa with age. Due to the smaller number of cells studied (n=19), the observed difference did not reach statistical significance, but was qualitatively similar to the changes in peak current already described. The degree of use dependence was quantified by normalising the amplitude at beat 50 to the amplitude at beat 1. This analysis showed no difference in use dependence with age (Figure 6.7E).
Young Old Difference p-value Time constant of recovery 148.2 ± 6.3ms-1 135 ± 6.9ms-1 -8.9% 0.19 from inactivation at -80 mV Time constant of recovery 33.1 ± 1.8ms-1 24.9 ± 2.3ms-1 -24.6% 0.01 * from inactivation at -100 mV
Normalised INa at 60ms 0.85 ± 0.01 0.9 ± 0.02 5.5% 0.04 *
Normalised INa at 100ms 0.93 ± 0.01 0.96 ± 0.01 3.1% 0.02 *
Normalised INa at 140ms 0.96 ± 0.01 0.97 ± 0.01 0.6% 0.51
7.5 Hz INa beat 50 -14.2 ± 1.7 -17.3 ± 2.2 22.0% 0.27 pA.pF-1 pA.pF-1
7.5 Hz normalised INa beat 50 0.64 ± 0.05 0.65 ± 0.04 1.2% 0.91
Table 6.5 – Time dependant recovery and use dependence of INa. n = 12-19 cells, 7-10 animals in each group for time dependent recovery from inactivation. n = 9-10 cells, 3-4 animals in each group for use dependence. * p<0.05. P a g e | 194
Figure 6.7 – Time-dependent recovery from inactivation and use dependence of INa. A Voltage protocols for (i) time dependent recovery from inactivation (ii) use dependence. B Time-dependent recovery from inactivation. C Time constants of time-dependent recovery from inactivation. D Use dependence of INa at 7.5 Hz stimulation. E Ratio of amplitude of 50th beat to 1st beat during stimulation at 7.5 Hz. n = 12-19 cells, 7-10 animals in each group for time dependent recovery from inactivation. n = 9-10 cells, 3-4 animals in each group for use dependence. * p<0.05, NS - not significant. P a g e | 195
6.2.7 Effects of action potential duration on INa
As described in Chapter 4.2.3, the atrial APD is longer in old myocytes compared to young. A prolongation of the APD leads to a shorter time for Na+ channels to recover from inactivation between stimuli. The effects of an alteration in APD were therefore explored using an action potential clamp method.
Averaged APs from young and old left atrial myocytes recorded at 0.5 Hz, 1 Hz and 2 Hz stimulation were used to create AP trains each lasting two seconds. The trains were adjusted so that the maximum diastolic potential was exactly -80 mV and stimulation artefacts were removed. The two second train was followed by a pair of depolarising voltage steps similar to those used to record the I/V plots in Chapter 6.3.2.2 and are demonstrated in Figure 6.8A. As described in Chapter 4.2.3, APD90 increased by 30%, 28% and 42% at 0.5 Hz, 1 Hz and 2 Hz respectively in left atrial myocytes from old compared to young sheep.
To mitigate against the effects of cell run-down causing a reduction in INa, protocols were applied so that young received the young AP protocol first and the old AP protocol second, while old cells received the old AP protocol first and the young second.
Comparing cells which received both a 1 Hz train of old APs and a 1 Hz train of young APs, the old AP train led to a decrease in the maximum INa of 9% compared to the young AP train (p<0.05 with paired t-test) (Figure 6.8B).
When action potential trains that corresponded to their cell group were used (i.e. young cell, young AP train compared to old cell, old AP train), no difference in peak INa was seen between groups (Figure 6.8C).
When 0.5 Hz, 1 Hz and 2 Hz AP trains were compared, increasing stimulation rate led to a decrease in INa (p<0.05 with repeated measure ANOVA) with no significant differences between age groups (Figure 6.8D).
Young Old Difference p-value All cells, AP trains -6.5 ± 0.8 pA.pF-1 -6 ± 0.8 pA.pF-1 -9.0% 0.003 *
Corresponding AP 0.5 Hz -5.9 ± 1.1 pA.pF-1 -7.0 ± 1.8 pA.pF-1 18.9% 0.59 Corresponding AP 1 Hz -6.3 ± 1 pA.pF-1 -6.5 ± 1.2 pA.pF-1 2.8% 0.91 Corresponding AP 2 Hz -4.0 ± 0.6 pA.pF-1 -5.3 ± 1.3 pA.pF-1 33.0% 0.31
Table 6.6 – The effects of an action potential clamp on INa. n = 13-18 cells, 4-6 animals in each group. * p<0.05 P a g e | 196
Figure 6.8 – Effects of action potential duration on INa. A Voltage protocol showing 2 s train of young (black) or old (orange) APs followed by 195 ms pulse and post-pulse. B The effect of application of young APs and old APs to young myocytes. C The effect of applying young APs to young myocytes vs. old APs to old myocytes. D The effects of AP train stimulation rate. n = 13-18 cells, 4-6 animals in each group. * p<0.05, NS - not significant, AP – action potential. P a g e | 197
6.2.8 Protein expression
To determine whether the observed changes in INa were due to changes in expression of the proteins that comprise the Na+ channels, western blotting was performed to detect Na+ channel α-subunits. An antibody directed against the SP19 epitope was used (#ASC-003, Alomone laboratories, Jerusalem, Israel) which is a highly conserved region of the voltage sensor present in all mammalian Na+ channel α-subunits408. A goat anti-rabbit secondary antibody was used (SC-2004, Santa Cruz Biotechnology, Texas, USA).
Blots showed two bands at ~250kD, potentially representing glycosylated and non-glycosylated α-subunits (Figure 6.9Ai). Incubation of the antibody with an SP19 blocking peptide removed the 250kD bands (Figure 6.9Aii). No non-specific binding was seen in the region of interest when the secondary antibody was applied alone.
No difference was seen in the relative abundance of Na+ channel α-subunits between young and old samples of left atrial appendage (2.3 ± 0.6 vs. 2.0 ± 0.3, compared against sheep left ventricle, p = NS, n = 8 animals in each group) (Figure 6.9B).
To further explore the mechanisms of faster atrial conduction with age, we next explored the expression of gap junction proteins. There was insufficient time to repeat these blots in triplicate and therefore Cx43 blots were performed twice while Cx40 was only performed once.
A rabbit anti-Cx43 antibody was used (C6219, Sigma Aldrich, Saint Louis, Missouri, USA), with the same goat anti-rabbit secondary antibody used for assessing NaV (SC-2004, Santa Cruz Biotechnology, Texas, USA). Cx43 expression decreased by 50% in older atrial tissue (p<0.05)(Figure 6.9B).
A rabbit anti Cx40 antibody was used (AB38580, Cambridge, UK), with the same goat anti- rabbit secondary antibody used for assessing NaV (SC-2004, Santa Cruz Biotechnology, Texas, USA). Cx40 expression did not differ between age groups (Figure 6.9C). P a g e | 198
Figure 6.9 – Expression of proteins relevant to cardiac conduction. A (Left) Representative Western blot of pan-specific NaV1.x expression with (i) primary antibody alone (ii) antibody plus inhibitory peptide. (Right) Na+ channel α-subunit relative abundance. B Cx43 relative abundance. C Cx40 relative abundance. n = 8 animals in each group. * p<0.05, NS - not significant.
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6.3 Discussion The main findings from this chapter are that despite the increase in P-wave duration seen in
Chapter 3, in vivo conduction right atrial CV increases with age. At a cellular level, INa current density increases without any change to its activation or inactivation kinetics. INa also recovers faster from inactivation, but this does not change use-dependence. Application of an AP clamp showed that under these experimental conditions the prolongation of AP duration seen with age impairs INa and that, when changes in AP duration are taken into account, no apparent difference is seen in INa between age groups. Finally, protein quantification using Western blotting did not detect a difference in NaV α-subunit expression. The relevance of each these findings will be explored and compared to previous work.
6.3.1 Right atrial conduction velocity increases with age
Right atrial CV was faster in the circumferential (medio-lateral) than in the axial (cranio-caudal) direction, suggesting that myofibrils in the sheep atria are oriented in a direction perpendicular to the long axis of the heart rather than parallel to it. This is consistent with previous work showing that the predominant fibre orientation in the body of the right atrium is circumferential in both sheep and human atria409, although a complex web was seen at the inter-atrial septum and junctions between atrial myocardium and the vena cavae and pulmonary veins. The significant increase in axial CV with age seen here therefore represents a difference in conduction between fibres, rather than along them. Although the differences in circumferential CV did not reach statistical significance, this may represent a lack of statistical power. The circumferential recordings relied upon estimates of inter-electrode spacing obtained using fluoroscopy, representing an additional source of error and reducing the likelihood of detecting a true difference.
When comparing the raw CV seen here to those previously reported, distinctions should be made between those recorded in vivo as performed here, and in-vitro using strips of myocardium. In right atrial muscle strips from dogs, CV during steady state pacing was ~1.1ms-1 193. In vivo right atrial CV has been recorded in man, with typical values of 0.8-1.2 ms-1 reported in both conscious and anaesthetised subjects410-412. These values are similar to those presented in this work.
Several studies have investigated the effects of age on CV. There was no difference in the CV of atrial muscle strips from young and old dogs during steady stimulation, although a greater reduction in CV was found in response to closely-coupled extrastimuli in old dogs193. Other have found that in-vitro atrial CV increased in the direction of fibre orientation with age in the P a g e | 200 atrial epicardium of dogs, although it decreased perpendicular to fibre orientation213, contrasting with the findings presented here. A potential explanation for these differences could be that we recorded CV from the epicardial surface, whereas other animal work has recorded from the epicardium or used muscle strip. Atrial CV has been shown to differ between epi- and endocardial recordings413, although the effects of age upon these differences have not been studied.
Only one group has looked at the effect of age on atrial CV in man. Kistler et al.132, found right atrial CV to be ~2.0ms-1 in human subjects aged <30, while in those aged 30-60 and >60 CV was ~1ms-1, similar to the values presented here. However, in this study data no mention was made of which direction CV had been measured in. All subjects recruited to this study were undergoing electrophysiological studies for supraventricular tachycardia (SVT), and it is therefore possible that a young cohort suffering from SVT may have unusually rapid atrial conduction. The same group reported nearly identical values in a later paper210, but it is unclear if this represented the same study population or a separate set.
The age-associated increase in atrial CV may have been influenced by the increase in myocyte width described in Chapter 4.2.1, complemented by the increase in capacitance presented in this chapter. As predicted by the cable theory described above, increasing cell radius will tend to increase the space constant, thereby increasing CV. A greater cell length will also increase the proportion of the myocardial fibre taken up by low resistance sarcolemma compared to high resistance inter-cellular connection. However, increasing cell size may also manifest as an increase in t-tubule density414. More t-tubules will serve to increase cell surface area, leading to a decrease in Rm and potentially offsetting any increase in CV with cell size. Drivers of hypertrophy may also modulate gap junction resistivity in unexpected ways, influencing Ri. However, when these theoretical concerns have been addressed in more sophisticated models with experimental verification, the overall impact of cell size appears to be that CV increases in line with myocyte length and width352.
The observed acceleration of CV could be caused by an increase in INa. At first glance this seems a very plausible explanation. However, the AP clamp data suggests that when the effect of AP prolongation is taken into account, the true INa seen under physiological conditions may not differ. These findings must be interpreted with caution before being extrapolated to what may be seen in vivo. Firstly, the recordings of INa were performed at room temperature in order to decrease the amplitude and slow the kinetics of the current. This artificial impairment of time-dependent recovery from inactivation of INa therefore exaggerates any changes wrought by AP morphological differences. P a g e | 201
Secondly, the APD recorded by monophasic APs suggests that the true APD is significantly shorter than the recordings from isolated myocytes suggest. This again lessens the impact on
INa as with even a 20% increase in APD, a long diastolic interval allowing full recovery from inactivation will still be present in the old. In vivo, the effects of an increased APD may well be of little consequence, meaning that the increase in INa seen here could still be the cause of the accelerated CV.
Furthermore, INa is steeply dependant on the resting membrane potential. The body of literature suggests that RMP is unchanged193 or may depolarise with age49. It would wrong to extrapolate the more negative RMP seen in isolated myocytes from aged atria described in
Chapter 4.2.1 to that in life. However, this finding shows the uncertainty at how INa may manifest in vivo when all factors are taken into account.
The discrepancy between the age-related increase in CV found here and the decrease with age seen in human studies may reflect alteration in the development of senescent fibrosis. In the ovine model used in our laboratory, pathology such as heart failure has been shown to lead to atrial fibrosis but age alone has not (unpublished data). The human ageing condition could be seen as more than an extended chronology, but instead an accumulation of pathologies that may lead to a fibrotic endpoint. In an animal model less likely to spontaneously develop cardiovascular morbidity, the underlying changes of age alone are revealed. In this healthy ageing phenotype, the acceleration of atrial conduction may be a compensatory mechanism for atrial dilatation.
6.3.2 Left atrial myocyte capacitance increases with age
Atrial myocyte capacitance measurements increased with age by 29% (88 vs. 68pF). Capacitance can be taken as a surrogate for cell surface area, and this therefore complements the age-related increase in myocyte dimensions that were presented in Chapter 4.2.1. Human young adult atrial myocyte capacitance has been reported as ~70pF385 , similar to the values seen here. This suggests that ovine atrial myocytes are of a similar size to those found in man.
Hypertrophy of atrial myocytes with age has been described in the mouse233, rat 194, and dog160 although atrial myocyte atrophy has been reported in man415.
As discussed in Chapter 4, atrial myocyte hypertrophy could occur due the increasing LA pressure seen in age237, or due to reactive hypertrophy following myocyte loss, as seen in the ventricle247. As capacitance reflects cell surface area, an increase in capacitance may reflect greater T-tubule density as well as myocyte size. Preliminary work in this model suggests that this may be the case (unpublished data). P a g e | 202
6.3.3 INa density increases and recovers faster from inactivation with age
In this sheep model of ageing, the density of INa increased by 29% with age from 21 to 27 pA.pF-1. The half-maximal voltage of activation did not change with age at -29 mV, nor did the half-maximal voltage of inactivation at -85 mV. INa recovered faster from inactivation, reflected in the 25% decrease in the time constant of inactivation from 33 to 25ms.
We can compare the results presented here to those obtained by other groups, with the caveat that care must be taken if the setting of different experimental conditions. The solutions used here were adapted from Baba et al.160, making their work a good place to start.
This group recorded INa from the left and right atrial myocytes of dogs using 5mM extracellular + [Na ]. They reported similar values to those shown here for activation V0.5 of -32 mV, inactivation V0.5 of -82 mV and peak INa of 25 pA/pF in left atrial myocytes. These values are 385 similar to those seen in human atrial myocytes , with the exception of a more negative V0.5 of inactivation at -60 mV.
Results can also be compared to previous work performed in the sheep. Martins et al.
239 recorded INa from the left and right atria of 6-8 month old sheep . Using 5mM intra- and + extra-cellular [Na ], they found peak INa to be significantly higher than the values shown here at ~100pA/pF. Activation and inactivation kinetics were not reported but I/V plots showed peak current occurring at -40 mV, representing a 20 mV leftward shift in activation compared to that shown here. These differences are potentially explained by the differences in
416 experimental conditions used: (i) 4-AP, which has previously been shown to affect INa was absent from their extracellular solutions. (ii) The extracellular and pipette solutions used were more acidic and alkaline respectively than those used here, potentially affecting the activation and inactivation kinetics which are known to be dependent on pH417.
Little published work has examined the effects of age on atrial INa. The work by Baba et al. described above found no statistically significant changes in INa with age at physiological pacing rates, although a non-significant increase in peak INa of 22% was seen in old compared to young left atrial myocytes. This paper did describe an age-related increase in use-dependence in the right atrium but not the left.
At a protein level, no difference was found between NaV1.5 expression in 54-months vs. 6-month old rabbits349 but NaV1.5 expression has been shown to be significantly greater in 12-month wildtype mice compared to 3 month228.
No studies have been performed on human tissue comparing young adult with senescent atrial
+ 385 INa or Na channel protein expression. However, Cai et al. looked at the effects of P a g e | 203
development on INa by comparing paediatric (mean age 3) with adult (mean age 47) samples of right atrial appendage, finding that INa increased from childhood to adulthood.
+ One might expect increased Na channel α-subunit expression to parallel enhanced INa, but this was not seen. This could be due to limitations in the technique used. As can be seen from the representative blots, protein bands detected were of low intensity and therefore prone to quantification error, adding to noise. The nature of Western blotting means that small changes in protein expression may be missed. Additionally, only eight samples were used in each group and the inherent variability of unpaired young and old cohorts means that the analysis may be underpowered.
An alternative explanation is that the observed difference in INa is caused by something other than α-subunit expression disparity. β-subunit expression influences INa amplitude and therefore could be a potential culprit. However, most studies looking at sodium channel β-subunit function have found an effect on activation and inactivation kinetics which was notably absent in this work.
The total cellular Na+ channel protein content may be similar between old and young but channel function could be impaired, leading to lower single channel conductance. It is possible that, as up to 25% of INa is carried by non-NaV1.5 channels, an alteration in the balance of α-subunit isoforms occurs with age. Such a difference would not be picked up using a pan-specific antibody as used here.
A difference in channel function could also be due to post-translational modification by glycosylation. Total glycosylation of plasma proteins has been shown to change with age418 but the specific effects of age on sodium channel glycosylation have not been explored. However, addition of sialic acid residues to sodium channels has been shown to modify the
377 activation/inactivation kinetics of INa without affecting peak current density which would not be consistent with the results presented here.
Additionally, differential phosphorylation by PKA, PKC or CaMKII may occur between age groups, as all of these have been shown to modulate INa. Isolated myocytes bathed in similar solutions are less likely to show differences in kinase activity due to the lack of neurohumoral activators. Both groups have also undergone intracellular dialysis with similar pipette solutions, which could wash out these modulators. Once again, phosphorylation has previously been reported to shift activation/inactivation kinetics, differences which were not found here. P a g e | 204
+ In summary, an increase in INa density without a change in total Na channel α-subunit expression could be caused by a change in proportional expression of Na+ channel α-subunit isoforms, differences in channel glycosylation or phosphorylation.
6.3.4 P-wave prolongation despite faster atrial conduction may reflect atrial dilatation
Chapter 3.3.2 showed that P-wave duration increased with age. This seems to contradict the increase in CV and INa described in this chapter. However, age has been associated simultaneously with both an increase in NaV1.5 expression and P-wave duration in mice228.
P-wave duration is a function of both CV and atrial size. An increase in atrial size that exceeded the increase in CV could reconcile these observations. Deployed basket catheter dimensions showed a non-significant trend towards increasing by 11%. However, this underestimates true changes in atrial size by ignoring any change in axial dimensions. Secondly, the sample is distorted by including only cases where good contact was achieved, thereby excluding larger atria. Myocyte dimensions also increased by 11%. This can at best be taken as supportive evidence for a change in atrial size as a whole and cannot be extrapolated quantitatively.
D’Andrea et al. showed that left atrial diameter increased by 20% between patients aged 20-30 and patients aged >50130. A similar pattern of increase was seen up the 9th decade but this was not specifically quantified.
Measurements of atrial size have proven difficult in the sheep. The most commonly used measure of atrial size, echocardiography, is hampered in the sheep by the shape of the ovine sternum. The makes it very difficult to obtain the four-chambered view necessary to accurately quantify atrial size. Ex-vivo measurements following extraction of the heart are difficult to interpret as a physiological filling pressure is required to accurately determine the size of the distensible atria.
An alternative explanation may be due to the non- uniform conduction between the atria. It is possible that while CV may increase within the RA with age, the same changes may not be found within the LA. Equally, the specialised tissue linking the two atria or Bachmann’s bundle that modulates inter-atrial conduction may age differently to the RA. If either of these factors were to be the case, an acceleration of RA conduction might only make a small contribution to P-wave duration.
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6.3.5 Increased action potential amplitude is due to augmented INa
In Chapter 4.2.3 it was shown that, while AP amplitude did not differ between age groups overall, at the lowest stimulation rates APs were of larger amplitude in myocytes from old sheep compared to young. This difference was not seen at stimulation rates >1 Hz. An increase in INa might be expected to increase AP amplitude if fully recovered from inactivation during very slow pacing. However, as suggested by the AP clamp data in section 6.3.2.5, at faster stimulation rates the shorter diastolic intervals seen in older myocytes outweigh a tendency to an increase in INa.
Corresponding changes might have been expected in the V̇max data, but were not found. An explanation for this could lie within the contamination of the AP upstroke by the stimulation artefact imposed by use of the perforated patch technique. This makes measurement of V̇max more open to error, masking any true differences.
INa may contribute to the differences in AP duration seen in Chapter 4.2.4. As APD reflects the balance between depolarising and repolarising currents, enhancement of an inward current may be expected to prolong APD. The precedent for this comes in the form of LQT3 whereby mutations in SCN5A lead to ventricular APD prolongation and arrhythmias. However, the key aspect of the LQT3 phenotype is the presence of persistent Na+ influx that extends through to the latter stages of the AP, and late INa has not been explored in this work.
6.3.6 Cx43 expression decreases with age in the left atrium while Cx40 does not change
The gap junctional protein Cx43 was less expressed in older left atrial tissue. The 50% decrease seen here is similar to the 60% decrease seen with age in rabbits204, 349. Decreasing Cx43 would be expected to slow atrial conduction, the reverse of what was found here. However, Cx43 expression was measured in left atrial tissue, whereas CV was recorded from the right atrium. Previous work has, though, shown that changes in connexin expression are similar in the left and right atria204.
Conversely, no significant difference was found in Cx40 expression. This experiment does however require a greater sample size, as due to time constraints blots were not repeated in triplicate. In rabbits, some have shown Cx40 to decrease with age204, which would be expected to increase CV, although some have not found a difference349. P a g e | 206
6.3.7 Limitations
6.3.7.1 In vivo conduction velocity
In vivo measurements of atrial CV were difficult to obtain. Approximately 50-60% of recordings had a poor signal to noise ratio and were therefore unsuitable for analysis. Good quality recordings require adequate contact between the electrodes and the atrial wall. A possible explanation for the large proportion of poor quality recordings was that the right atrial size in some subjects was too large to allow good contact. The samples used, therefore, are biased towards subjects with smaller atria. This will minimise any observed difference in atrial size between groups, potentially explaining why no significant difference was seen. This bias may also result in a comparison between young sheep with small atria vs. large sheep with small atria and therefore may not be representative of the true ageing process.
A difference was seen in axial CV but not circumferential conduction. This may be because the sample was underpowered, but could also be due to the confounding errors associated with measuring electrode expansion. These measurements have assumed a symmetrical, circular expansion whereas the true cross section may well be elliptical. This is unlikely to have led to a systematic bias between groups but does represent another source of error.
As can be seen from the representative traces, defining the onset of each electrogram was not clear cut. Although steps were taken to increase reliability such as using a semi-automated EGM detection system with decision rules for EGM onset, the potential for observer bias when measuring unblinded data remains.
All in vivo conduction was generated from the right atrium alone due to practical difficulties in accessing the left atrium with such a large catheter. Changes in CV could therefore be limited to the RA. CV measurements were all performed in the anaesthetised state and changes could therefore represent differences in sensitivity to the anaesthetic agent used. This is, however, a limitation common to all animal in vivo work and it may be argued compares favourably against in vitro studies that lose important physiological differences such as adrenergic tone.
6.3.7.2 Sodium current recording
+ The concordance between INa measured in isolated cells with Na channel function in vivo is imperfect. Humoral factors which may well change with age such as adrenergic tone are not present. The myocyte isolation process uses agents that may interfere with channel function such as BDM which acts as phosphatase inhibitor, while proteolytic enzymes also degrade ion channel proteins, potentially degrading channels that contribute to INa. Variation in the P a g e | 207 composition of the extracellular matrix with age could necessitate longer digestion times in older subjects, exposing channels to greater risk of degradation. However, the same enzymes were used for both age groups, reducing the chances of a differential effect on channel degradation, and more extensive channel degradation due to longer cell isolations would be expected to decrease any current observed, which is not consistent with the results seen here.
The whole cell technique suffers from the disadvantage of cytoplasmic dialysis. Second messengers which may play crucial roles in INa modulation are washed out, potentially hindering the extrapolation of cellular to in vivo findings.
Only myocytes from the left atrial appendage were used to study INa. While this hampers comparison between left atrial cellular findings and right atrial in vivo findings, the LA is arguably more important to the genesis of AF. In canine atrial myocytes, left atrial INa current density was greater than that found in the right atrium160. In that canine model, the only age-dependant changes were only found within the RA. However, previous work in young
239 sheep atria has found no between right and left atrial INa current density .
Whilst we have endeavoured to use animals in the first and last quintiles of life, establishing the age of sheep from their dentition is imprecise. It is possible that we have instead compared adolescent animals with those that are fully grown, thereby inadvertently studying the effects development rather than senescence. Human studies have shown that INa increases between the very young and adult groups385. However, as shown in Chapter 3.2.1, the body mass of sheep from the young and old cohorts did not differ significantly, suggesting that young sheep were truly adult.
6.4 Conclusions In this ovine model of ageing, right atrial axial CV increased with age, corresponding with an increase in Na+ current density. However, no change was seen in Na+ channel α-subunit protein expression. These unexpected findings do not agree with the conventional association of an inverse relationship between CV and atrial vulnerability, suggesting that other factors may be of greater import.
The final chapter will aim to synthesise the individual strands of the work presented here and point towards further research goals.
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7 GENERAL DISCUSSION
In this final chapter, the strands of work presented in this thesis will be woven together, and future research opportunities brought to light.
7.1 Sheep are a valid model for human atrial ageing
The aim of Chapter 3 was to assess whether sheep represented a suitable model for the effects of ageing in human atria. This is a valid question, as many of changes seen in association with human ageing may be due to cumulative effect of diseases that are less likely to be encountered in sheep such as diabetes mellitus, systemic inflammation and a western diet high in fat and processed sugars419.
It was demonstrated that older sheep are more vulnerable to induced AF than younger sheep, through the proportion of bursts that generated AF and its resulting duration. Further evidence for the similarities between human and ovine ageing came from the increase in P-wave duration that is well-established in man131. In humans, the increasing P-wave duration in older subjects is associated with an increase in atrial size130, 132. Although atrial size was not assessed in this work, previous work in our laboratory has confirmed that, as in humans, the atria dilate with age in sheep (unpublished data). Atrial dilatation may promote AF as a greater tissue mass can harbour more simultaneous re-entrant circuits.
The validity of the sheep model of ageing was strengthened by the prolongation of the cSNRT in older subjects, suggesting declining SAN function with age similar to that seen in humans132, 214. Although other aspects of atrial electrophysiology that are accepted to change with age in man such as deteriorating AVN function186, 190 and increasing ERP88, 132 did not show a statistically significant difference in this model, this probably represents inadequate sample size as trends in the expected direction were observed.
These findings support the use of sheep as a model for human ageing, and also highlight the difference in experimental approaches to assessing atrial vulnerability. Even in human work, where the vulnerability to spontaneous AF incontrovertibly increases with age, assessing atrial vulnerability by inducing AF artificially using an extrastimulus approach has failed to show an age-related increase88, 224. Inducing AF using burst pacing may potentially provide more meaningful results, although to my knowledge no head-to-head comparisons of these methods have been undertaken. An area of future study could be to clarify the best method for assessing atrial vulnerability against the gold standard of spontaneous AF. This could be P a g e | 209 achieved by performing electrophysiological studies in patients during pacemaker implantation. Long-term assessment of arrhythmia burden could be accomplished using the recording capabilities of the implanted devices.
7.2 The atrial action potential increases with age in vitro but not in vivo
In Chapter 4, age-related changes in the shape of the AP were assessed in myocytes from both atria. These results showed that, as in dogs49, AP duration prolongs with age in both sheep atria. However, in contrast to dogs, the spatial gradient between the two diminished with age. These findings are new and useful as the only large mammalian work assessing the effects of age on isolated myocytes has used dogs. Complementary data obtained using a second large mammalian species is beneficial as there are no studies assessing the effects of senescence on spatial AP gradients in isolated human myocytes. The cellular AP prolongation could facilitate AF by promoting early afterdepolarisations, but could also inhibit AF by increasing arrhythmia wavelength and impeding re-entry.
In comparison, in vivo right atrial monophasic action potential duration did not differ with age. This is an interesting finding as there is little data comparing cellular and in vivo action potentials from the same model. While it could represent an underpowered sample size, it may reflect the importance of humoral factors missing from isolated cell studies.
The small age-related differences in AP amplitude that were only seen at low stimulation rates are not, by themselves, of particular importance. Despite this, they complement the data presented in Chapter 6 showing that INa increases with age.
Measurements of Ca2+ handling characteristics were performed in a small subset of myocytes, but no differences were seen that attained statistical significance. This is likely to reflect the small sample size, as the general pattern was similar to that shown in human atrial myocytes of slower Ca2+ reuptake and smaller Ca2+ transients in older subjects232.
These findings open the door to further research as to why AP changes were seen in isolated cells but not in vivo. This could be accomplished by assessing the effects of stretch in vitro in old and young myocytes. In vivo, right atrial pressures could be compared between young and old sheep and then manipulated by infusing saline. The contribution of parasympathetic tone to in vivo atrial AP duration could be assessed by using an anti-cholinergic drug such as atropine. Furthermore, the mechanisms underlying the differences in AP duration in cells could be explored by assessing the contributions of IK(ACh), IKr, IKs, and Ito to repolarisation in old and young myocytes. P a g e | 210
7.3 Ageing promotes action potential alternans
An enhancement to existing techniques for quantifying alternans was presented in Chapter 5. Using this technique it was demonstrated that alternans of the atrial AP occurs at lower stimulation rates and is of greater magnitude in older sheep both in vitro and in vivo. These findings are novel as the very limited previous work on the effects of ageing upon alternans behaviour had been performed in rats238 and rabbits340, species which exhibit significant electrophysiological differences to humans. For the first time, 3:1 and 4:1 oscillations were systematically searched for and quantified in atrial tissue. These occurred rarely, and therefore did not differ significantly between age groups. The mechanism of alternans was explored and the dual role of Ca2+ cycling and membrane currents was demonstrated in the genesis of atrial alternans. The tendency of older cells and tissue to alternate promotes AF by creating a spatial dispersion of repolarisation which sets the stage for wavebreak and the formation of re-entrant circuits.
This work can be expanded by exploring what makes older myocytes more susceptible to alternans. The Ca2+ release properties of atrial myocytes could be investigated to see whether excessively leaky or excessively refractory RyRs were responsible for the increase in alternans. This could be accomplished by modulating the RyRs with tetracaine, to impair RyR opening, and low dose caffeine, to increase RyR opening. These drugs could be combined at a selection of ratios and the ratio that minimised alternans determined. The ratios could be compared between old and young myocytes. If the ratios were similar this might suggest that RyR opening was not responsible for the differences in alternans. However, if older myocytes needed a ratio that favoured RyR closure to minimise alternans, this would implicate RyR leak as responsible for the difference in alternans behaviour.
One strand of evidence implicating dysfunctional Ca2+ release as a source of alternans is the mini-waves seen during alternans induced by small depolarising pulses in rat myocytes267. These have not been universally accepted as a mechanism for alternans induced by rapid pacing, and could be studied in atrial myocytes using a combination of patch clamping and confocal microscopy. Furthermore, the expression of the RyR stabilising protein calstabin could also be explored.
Another avenue of research could explore whether impaired Ca2+ reuptake is responsible for the increased alternans seen in older subjects. Low dose thapsigargin could be applied to young myocytes at a concentration that slowed Ca2+ reuptake to the same extent as seen in older myocytes. Alternatively, a compound that slowed Ca2+ reuptake by enhancing buffering P a g e | 211 power such as EMD-57033 could be employed. If, by equalizing the rate of Ca2+ reuptake between old and young cells this eliminated any difference in alternans, Ca2+ reuptake could be seen as the main determinant of alternans susceptibility seen in older cells. The expression of proteins that modulate Ca2+ reuptake such as sarcolipin63 and regucalcin420 could also be explored.
The relevance of alternans to AF should also be explored further. In the work presented here, no significant correlation was shown between alternans magnitude and vulnerability to AF, which may be due to the small sample size. This work could be extended, and monophasic APs could be recorded at two sites thereby determining if the threshold for discordant alternans predicted atrial vulnerability better than the threshold for concordant alternans. This could be extended to human studies during pulmonary vein isolation procedures to see if alternans susceptibility predicted recurrence of arrhythmias.
7.4 Right atrial conduction velocity and INa increase with age
Chapter 6 showed that right atrial CV increases with age. This finding, although not unprecedented213, contrasted with previous work showing similar193 or slower conduction in
132, 210, 349 older subjects . In association with this, a novel finding was that INa density increased and recovered faster from inactivation in older left atrial myocytes. It is unclear why enhanced atrial conduction itself should promote AF, as this should increase wavelength and discourage re-entry. Acceleration of conduction may instead occur as an adaptive response to atrial dilatation which is well known to be pro-arrhythmic. Na+ channel α-subunit expression did not change, suggesting that altered function rather than quantity of the pore-forming subunits could be causing the increased current density. Gap junctional protein expression was assessed, showing a decrease in Cx43 with age, a change that would be expected to slow atrial conduction. Cx40 expression was not significantly different between age groups.
The claim of an increase in atrial CV with age could be strengthened by measuring CV in the left atrium. CV was recorded at a single stimulation rate and the data may be more robust if explored at a range of stimulation rates or in response to an extrastimulus protocol. The suggestion that the faster CV may be an adaptation to atrial size could be evaluated by assessing atrial dimensions and CV in the same animals. It has proven difficult to do this using echocardiography and so an alternative technique such as fluoroscopy combined with injection of a radiopaque contrast medium could be used.
The increase in atrial INa could be further explored by using cells from the right atrium. As INa is carried by several channels, it may be instructive to discover whether a different balance of P a g e | 212
channel isoforms is responsible for INa in older myocytes. This could be performed by assessing the response to tetrodotoxin (TTX) of INa in myocytes from each age group. As TTX blocks all Na+ channels expressed in the atria bar Nav1.5, application of this drug would separate out which channels were carrying the enhanced current in older cells. The relevance of the enhanced recovery from inactivation in older myocytes could be established by repeating experiments at higher temperatures. Na+ channel β-subunit expression could be quantified. The sample size for connexin expression needs to be increased before firm conclusions are drawn, and this data would be complemented by assessing connexin distribution using immunofluorescence.
7.5 Conclusions
Older sheep, like older humans, are more susceptible to AF. While some age-associated alterations of atrial physiology promote arrhythmias such as heightened alternans activity, other might be expected to protect against it such as enhanced atrial conduction. Others, such as the action potential prolongation in older myocytes, might at the same time favour the initiation of arrhythmias but hinder their maintenance.
We are heading towards an era of individualised medicine. Evidence based medicine has historically over-represented younger patients in clinical trials, who may not be representative of those we need to treat. Instead of a one-size-fits-all approach, these fresh understandings of atrial ageing highlight how the optimal management of arrhythmias may differ in the old.
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9 APPENDIX
The following is a reprint of an original paper entitled “An Excel-based implementation of the spectral method of action potential alternans analysis” that was published in Physiological reports, 2014. 2(12).