Investigation of the role of PMCA1 in cardiac electrical function and heart rhythm stability

270 Pages·2017·6.57 MB·English

by  Claire Wilson

University of Manchester

Investigation of the role of PMCA1
in cardiac electrical function and
heart rhythm stability

A thesis submitted to the University of Manchester for the degree of Doctor of
Philosophy in the Faculty of Biology, Medicine and Health

2017

Claire Wilson

School of Medical Sciences
Division of Cardiovascular Sciences
Table of Contents
List of figures 10
List of tables 13
List of abbreviations 14
Declaration 16
Copyright statement 17
Abstract 18
Publications and Presentations 19
Acknowledgements 20
1. Introduction 21
1.1. Heart Failure 22
1.1.1. Heart failure epidemiology 22
1.1.2. Heart failure progression 23
1.1.3. Features of heart failure 23
1.1.4. Presentation of heart failure 26
1.1.5. Arrhythmias and heart failure 27
1.2. Arrhythmias 28
1.2.1. Normal cardiac conduction pathway 29
1.2.2. Monitoring heart rhythm 34
1.2.2.1. The theory of (lead II) ECG measurements 34
1.2.2.2. ECG and arrhythmia diagnosis 35
1.2.3. Mechanism of arrhythmias 37
1.2.3.1. Abnormal impulse formation 37
1.2.3.2. Abnormal impulse transduction 38
1.4. Cardiac electrical remodelling associated with arrhythmia
39
development
1.4.1. Electrical remodelling of the Na+ current 40
1.4.2. Electrical remodelling of the Ca2+ current 41
1.4.3. Electrical remodelling of the K+ current 41
1.4.4. Other related cardiac remodelling 44
2
1.4.5. Electrical remodelling associated with EAD and DAD 46
1.5. Factors influencing arrhythmia development 48
1.5.1. Impact of pathological and physiological stress on
48
arrhythmia development
1.5.2. Impact of genetics on arrhythmia development 50
1.6. Current arrhythmia genetic research 53
1.6.1. The use of animal models in arrhythmia research 53
1.6.2. The theory of MAP recordings 56
1.7 Plasma membrane calcium ATPase (PMCA) 57
1.7.1. Isoforms and diversity 58
1.7.2. Expression 58
1.7.3. Structure 59
1.7.4. Regulation 61
1.8. The involvement of PMCA in physiological processes 62
1.8.1. Role of PMCA in regulation of global and local
62
intracellular Ca2+
1.8.2. Role of PMCA in signal transduction 63
1.9. Role of PMCA in health and disease 65
1.9.1. PMCA and human disease 65
1.9.2. The role of PMCA in the cardiovascular system 65
1.9.3. The role of PMCA4 in cardiovascular health and disease 67
1.9.4. The role of PMCA1 in cardiovascular health and disease 69
1.9.5. Potential therapeutic role of PMCA1 72
1.9.6. Investigating the role of PMCA1 in arrhythmia
72
development
1.10. Hypothesis 74
1.11. Aims 74
2. Methods 75
2.1. Animal subjects 76
2.1.1. Cardiomyocyte-specific deletion of PMCA1 77
2.1.2. Heterozygous PMCA1 expression 78
2.2. Genotype analysis 79
3
2.2.1. DNA extraction 79
2.2.2. PCR genotyping 80
2.3. Animal studies 81
2.3.1. In vivo electrocardiography 81
2.3.1.1. Conscious ECG 82
2.3.1.2. Unconscious ECG 82
2.3.1.3. ECG analysis 82
2.3.2. In vivo pacing 83
2.3.3. In vivo cardiac stress models 84
2.3.3.1. Intraperitoneal injection of pharmaceutical
84
agent
2.3.3.2. Acute β-adrenergic stimulation via
85
Dobutamine administration
2.3.3.3. Brugada stress test via Flecainide
85
administration
2.3.3.4. Osmotic mini pump administration of
85
isoproterenol
2.3.4 Ex vivo monophasic action potential recordings 86
2.3.4.1. Heart isolation 86
2.3.4.2. Langendorff perfusion 86
2.3.4.3. Ex vivo monophasic action potential 86
2.3.4.4. Ex vivo MAP analysis 87
2.3.4.5 Ex vivo PES 87
2.4. Sample collection 88
2.5. Histological analysis 88
2.5.1. Sample preparation 88
2.5.2. Haematoxylin and Eosin staining 89
2.5.3. Masson’s trichrome staining 90
2.5.4. Picrosirius red staining 90
2.5.5. Connexin-43 immunohistochemistry 91
2.6. Molecular analysis 92
2.6.1. mRNA analysis by qPCR 92
2.6.1.1. RNA isolation from ventricle tissue 92
4
2.6.1.2. RNA isolation from atria tissue 92
2.6.1.3. RNA conversion to cDNA by reverse
93
transcription
2.6.1.4. Quantitative PCR 94
2.6.1.5. SYBR Green PCR 94
94
2.6.1.6. TaqMan PCR
97
2.6.1.7. Analysis of gene expression
98
2.6.1.8. Reference gene analysis using geNorm
98
2.6.2. Protein analysis by Western blot
2.6.2.1. Protein extraction and membrane 98
fractionation
99
2.6.2.2. Protein concentration
99
2.6.2.3. Western Blot
100
2.6.2.4. Analysis of protein expression
102
2.6.3. Large-scale protein analysis using mass spectrometry
102
2.6.3.1. Sample preparation for LC-MS
103
2.6.3.2. LC-MS analysis
104
2.7. Statistical Analysis
3. Establishing the role of PMCA1 in cardiac electrical properties related to
105
heart rhythm stability using an in vivo model.
3.1. Introduction 106
3.1.1. PMCA1 as a potential genetic determinant of arrhythmic
106
events
3.2. Aims 107
3.3. Method 107
3.4. Results 107
3.4.1. Genotyping of PMCA1CKO mice 107
3.4.2. In vivo analysis of cardiac electrical activity of male
108
PMCA1CKO mice under basal conditions
3.4.3. In vivo analysis of cardiac electrical activity of female
111
PMCA1CKO mice under basal conditions
3.4.4. Ex vivo analysis of cardiac electrical activity via
113
monophasic action potential recordings
3.4.5. In vivo and ex vivo ventricular pacing of PMCA1CKO mice 115
3.4.5. Analysis of cardiac structure of PMCA1CKO mice 119
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3.4.6. Genotyping of αMHC-CreTg mice 123
3.4.7. In vivo analysis of cardiac electrical activity of
125
αMHC-CreTg mice
3.4.8. In vivo ventricular pacing of αMHC-CreTg mice 125
3.4.9. Cardiac structure of αMHC-CreTg mice 126
3.5. Discussion 130
3.5.1. Cardiomyocyte-specific deletion of PMCA1 results in
131
abnormal heart rhythms
3.5.2. Cardiomyocyte-specific deletion of PMCA1 results in
132
increase in arrhythmia susceptibility
3.5.3. Changes in heart rhythm related to
cardiomyocyte-specific PMCA1 deletion occur in the 133
absence of any structural cardiomyopathy
3.5.4. Expression of αMHC-Cre transgene does not influence
134
heart rhythm stability
3.6. Conclusion 134
4. Assessing the extent of molecular remodelling relating to heart rhythm
136
control as a result of disrupted PMCA1 expression.
4.1. Introduction 137
4.1.1. Molecular changes in arrhythmia development 137
4.1.2. PMCA1 as a possible mediator of cardiac signalling
137
related to heart rhythm stability
4.2. Aims 138
4.3. Method 138
4.4. Results 139
4.4.1. Cardiac reference gene analysis of PMCA1CKO animals 139
4.4.2. Ventricle gene expression of cardiac electrical system
141
components in PMCA1CKO mice
4.4.3. Ventricle protein expression of cardiac electrical system
143
components in PMCA1CKO mice
4.4.4. Ventricle gene expression of cardiac structural proteins
147
in PMCA1CKO mice
4.4.5. Ventricle protein expression of cardiac structural
147
proteins in PMCA1CKO mice
4.4.6. Large-scale proteomic analysis of ventricles following
149
cardiomyocyte-specific PMCA1 deletion
4.4.7. Ventricle gene expression of cardiac electrical system
152
components in αMHC-CreTg mice
4.5. Discussion 154
4.5.1. Cardiomyocyte-specific deletion of PMCA1 influences
154
cardiac ion channel expression
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4.5.2. Cardiomyocyte-specific deletion of PMCA1 results in
157
differential gene and protein expression
4.5.3. Cardiomyocyte-specific deletion of PMCA1 does not
influence cardiac structure associated with heart 158
rhythm control
4.5.4. Large scale proteomic analysis identified altered
expression of multiple proteins following 158
cardiomyocyte-specific PMCA1 deletion
4.5.5. Expression of αMHC-Cre does not influence the
162
expression of cardiac ion channels
4.6. Conclusion 162
5. Determining the requirement for PMCA1 in maintaining ventricular
164
electrical function during physiological and pathological stress conditions.
5.1 Introduction 165
5.1.1. Physiological stress associated with arrhythmia
165
development
5.1.2. Pathological stress associated with arrhythmia
165
development
5.1.3. Modelling cardiovascular stress in an animal model 167
5.1.4. The role of PMCA1 in development of cardiovascular
168
stress conditions
5.2. Aims 169
5.3. Methods 169
5.4. Results 170
5.4.1. Impact of abnormal PMCA1 expression on heart rhythm
170
stability in an aged model
5.4.1.1. Genotyping of PMCA1Ht mice 170
5.4.1.2. Analysis of cardiac parameters of young adult
171
and aged PMCA1HT mice
5.4.1.3. In vivo analysis of cardiac electrical activity of
172
PMCA1HT mice under basal and aged conditions
5.4.1.4. In vivo ventricular pacing of PMCA1HT mice at
175
basal and aged condition
5.4.2. Determining if heart rhythm stability in relation to
cardiomyocyte-specific deletion of PMCA1 is associated with 177
underlying Brugada syndrome.
5.4.2.1. Response of PMCA1CKO mice to pro-arrhythmic
177
agent flecainide
5.4.3. Investigating the effect of acute sympathetic stimulation
on heart rhythm stability following cardiomyocyte-specific 181
PMCA1 deletion.
5.4.3.1. Expression of sympathetic nervous system
181
signalling components in PMCA1CKO mice
5.4.3.2. Response of PMCA1CKO mice to acute β-
182
adrenergic stimulation
7
5.4.4. Investigating the effect of chronic sympathetic
stimulation on heart rhythm stability following cardiomyocyte- 187
specific PMCA1 deletion.
5.4.4.1. Response of PMCA1CKO mice to chronic β-
187
adrenergic stimulation
5.4.5. Investigating the involvement of PMCA1 in heart failure
190
development
5.4.5.1. Expression of PMCA1 in heart failure models 190
5.4.5.2. ECG parameters of PMCA1CKO animals
192
following TAC
5.5. Discussion 193
5.5.1. Abnormal PMCA1 expression influences arrhythmia
193
development under aged conditions
5.5.2. Prolonged QT intervals resulting from cardiomyocyte-
specific deletion of PMCA1 is not an indicator of 195
underlying Brugada syndrome
5.5.3. Chronic sympathetic stimulation influences abnormal
heart rhythms following cardiomyocyte-specific 197
PMCA1 deletion
5.5.4. PMCA1 may play a role in the development of heart
200
failure
5.6. Conclusion 202
6. Investigating the role of PMCA1 in atrial cardiac electrical properties 203
related to heart rhythm stability using an in vivo model.
6.1 Introduction 204
6.1.1. Molecular relationship between atria and ventricular
204
arrhythmias
6.1.2. Role for PMCA1 in the atria 206
6.2. Aims 206
6.3. Methods 207
6.4. Results 207
6.4.1. in vivo atrial pacing of PMCA1CKO mice 207
6.4.2. Atrial gene expression of cardiac electrical system
211
components in PMCA1CKO mice
6.4.3. Atrial gene expression of cardiac structural proteins in
213
PMCA1CKO mice
6.4.4. Atrial gene expression of cardiac electrical system
214
components in αMHC-CreTg mice
6.5. Discussion 216
6.5.1. Cardiomyocyte-specific deletion of PMCA1 results in
217
increased susceptibility to AV node
6.5.2. Cardiomyocyte-specific deletion of PMCA1 influences
218
cardiac ion channel expression in the atria
8
6.5.3. Cardiomyocyte-specific deletion of PMCA1 influences
219
caveolae expression in the atria
6.5.4. Expression of αMHC-Cre does not influence the
221
expression of atria cardiac ion channels
6.6. Conclusion 221
7. General discussion, future work and limitations 222
7.1. General Discussion 223

7.1.1. Cardiomyocyte-specific PMCA1 deletion results in
224
abnormal heart rhythms related to ventricular
Repolarisation dysfunction
7.1.2. Cardiomyocyte-specific PMCA1 deletion results in
225
increased susceptibility to arrhythmic events
7.1.3. Heart rhythm abnormalities associated with
cardiomyocyte- specific PMCA1 deletion occur in the 226
absence of structural cardiomyopathy
7.1.4. PMCA1 influences expression of key cardiac proteins
226
involved in heart rhythm control
7.1.5. Physiological and pathological stress can influence the
heart rhythm stability related to altered PMCA1 228
expression
7.1.6. Expression of the Cre promotor does not influence heart
230
rhythm under basal conditions
7.2. Future work 230
7.2.1. Determine if PMCA1 influences cardiac ionic currents
231
underlying heart rhythm
7.2.2. Examine Ca2+ dynamics related to heart rhythm
232
following cardiomyocyte-specific PMCA1 deletion
7.2.3. Further assessment of the conduction properties
233
following cardiomyocyte-specific PMCA1 deletion
7.2.4. Assess the role of PMCA1 in maintaining heart rhythm
234
stability during heart failure development
7.3. Limitations 235
7.4. Significance of the study 238
7.5. Final conclusion 241
8. Bibliography 242

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List of figures
Figure 1.1: Heart failure progression can result in pump failure or sudden cardiac
25
death.
Figure 1.2: The cardiac conduction system is governed by the propagation of
31
electrical impulses called cardiac action potentials.
Figure 1.3: The cardiac action potential is governed by several key ionic currents. 32
Figure 1.4: Cardiomyocyte contraction is governed by excitation contraction
33
coupling.
Figure 1.5: Electrocardiography is used to clinically detect cardiac arrhythmias. 36
Figure 1.6: Arrhythmias can arise due to abnormal impulse transduction. 39
Figure 1.7: Comparison of cardiac ionic currents, cardiomyocyte action potentials
55
and electrocardiograms are between human and mouse.
Figure 1.8: PMCA structure allows for Ca2+ extrusion and has areas of possible
61
variance.
Figure 1.9: PMCA is involved in global and local Ca2+ handling. 63
Figure 1.10: PMCA structure allows for numerous functional protein interactions
64
related to signalling.
Figure 2.1: Breeding diagram of the generation of PMCA1CKO animals and controls. 78
Figure 2.2: Breeding diagram of the generation of PMCA1HT and controls. 79
Figure 2.3: A typical mouse ECG traces depicting the parameters measured in the
83
study.
Figure 2.4: A typical mouse MAP trace depicting the parameters measured in the
87
study.
Figure 3.1: Confirmation of cardiomyocyte-specific Cre-mediated recombination in
108
PMCA1CKO mice using PCR.
Figure 3.2: Basal general cardiac parameters of 3 month old PMCA1CKO male mice
110
and corresponding controls.
Figure 3.3: Basal general cardiac parameters of 3 month old PMCA1CKO female
112
mice and corresponding controls.
Figure 3.4: Ex vivo electrophysiological parameters of PMCA1CKO mice. 114
Figure 3.5: Ventricular in vivo electrophysiological characterisation of PMCA1CKO
116
mice.
Figure 3.6: Ex vivo electrophysiological characterisation of PMCA1CKO mice. 118
Figure 3.7: Cardiomyocyte cell size in 3 month old PMCA1CKO mice. 120
Figure 3.8: Level of interstitial fibrosis in 3 month old PMCA1CKO mice measured
121
using Masson’s trichrome and Picrosirius red.
Figure 3.9: mRNA expression of hypertrophy and fibrosis markers in 3 month old
122
PMCA1CKO mice
Figure 3.10: Expression of connexin-43 in 3 month old PMCA1CKO mice. 123
Figure 3.11: Confirmation of αMHC-Cre expression in αMHC-CreTg mice using PCR. 124
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