characterisation of the structural and functional
TRANSCRIPT
Characterisation of the Structural and
Functional Properties of Subsidiary Atrial
Pacemakers in a Goat Model of Sinus Node
Dysfunction
A thesis submitted to the University of Manchester for the degree of
Doctor of Philosophy in the Faculty of Medical and Human Sciences
2015
Dr Zoltan Borbas
School of Medicine
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Contents
List of Tables ..................................................................................... 6
List of Figures .................................................................................... 7
Abstract ............................................................................................ 9
Declaration ...................................................................................... 10
Copyright Statement ........................................................................ 11
Acknowledgements .......................................................................... 12
List of abbreviations ......................................................................... 13
1 Introduction .............................................................. 16
1.1 Discovery of the sinus node ................................................... 16
1.2 Confirmation of the role of the sinus node ............................. 18
1.3 Structure of the sinus node .................................................... 22
1.3.1 Basic anatomy and histology ................................................................... 22
1.3.2 Discrepancy between structure and function .......................................... 23
1.4 Cellular electrophysiology of the sinus node .......................... 26
1.4.1 “Funny current” (If) .................................................................................. 28
1.4.2 K+ currents ................................................................................................ 28
1.4.3 Na+ current ............................................................................................... 29
1.4.4 Ca++ currents ............................................................................................. 30
1.4.5 The “Calcium Clock” ................................................................................. 30
1.4.6 Electrical coupling and the connexins ...................................................... 31
1.5 Electrical mapping of the pacemaker complex ....................... 32
1.5.1 Electrode-based “traditional” mapping ................................................... 32
1.5.2 Advanced mapping techniques ................................................................ 33
1.6 Sinus node dysfunction and subsidiary atrial pacemakers ...... 34
1.6.1 Sinus node dysfunction ............................................................................ 34
1.6.2 Subsidiary atrial pacemakers ................................................................... 37
1.6.3 Paranodal area ........................................................................................ 40
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1.7 Concept of biopacemaking ..................................................... 44
1.8 Aims of thesis ........................................................................ 45
1.9 Research Hypotheses ............................................................. 46
2 Materials and Methods ............................................. 47
2.1 Overview of the research protocol ......................................... 47
2.2 Species used .......................................................................... 49
2.3 Surgical technique to expose the SN ...................................... 49
2.3.1 Anaesthesia and Surgical preparation ..................................................... 49
2.3.2 Thoracotomy, Exposing the Right Atrium ................................................ 50
2.4 Epicardial Pacemaker Implantation ........................................ 52
2.5 Mapping and ablation ............................................................ 54
2.5.1 Mapping the earliest activation within the sinus node ........................... 54
2.5.2 Ablation of the Sinus Node ...................................................................... 56
2.5.3 Follow-up Period ...................................................................................... 59
2.5.4 Mapping of the Subsidiary Atrial Pacemaker .......................................... 59
2.6 Methods of functional characterisation ................................. 60
2.6.1 Surface electrocardiogram ...................................................................... 60
2.6.2 Overdrive pacing, SN and SAP recovery time .......................................... 60
2.6.3 Monitoring the mean heart rate .............................................................. 63
2.6.4 Implantable loop recording ..................................................................... 63
2.7 Right Atrial Tissue Processing ................................................. 65
2.7.1 Harvesting and dissection ........................................................................ 65
2.7.2 Cryosectioning.......................................................................................... 66
2.8 Methods of tissue characterisation ........................................ 68
2.8.1 Histology .................................................................................................. 68
2.9 Light microscopy .................................................................... 68
2.10 Immunohistochemistry .......................................................... 70
2.10.1.1 Primary antibodies ........................................................................... 71
2.10.1.2 Secondary antibodies ....................................................................... 71
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2.10.1.3 Immunofluorescence staining protocol ........................................... 73
2.10.1.4 Immunofluorescence microscopy .................................................... 73
2.10.2 Semiquantitative immunohistochemistry ................................................ 74
2.11 3D anatomical reconstruction ................................................ 75
3 Functional characterisation of the SN and SAP .......... 76
3.1 Site of earliest activation within the SN ................................. 76
3.2 Acute Success of Sinus Node Ablation .................................... 76
3.3 Location of the Subsidiary Atrial Pacemaker .......................... 78
3.4 Electrocardiographic parameters ........................................... 81
3.4.1 Heart rate before ablation ....................................................................... 81
3.4.2 Heart rate changes following ablation of the SN .................................... 83
3.4.3 PR Interval ................................................................................................ 87
3.4.4 P wave morphology ................................................................................. 87
3.5 SN and SAP recovery time ...................................................... 89
3.6 Long term event recording data ............................................. 89
4 Structural characterisation of the SN and SAP ........... 91
4.1 Morphology of the atrial pacemaker complex ........................ 93
4.1.1 Sinus node ................................................................................................ 93
4.1.2 Paranodal area ........................................................................................ 97
4.1.3 Subsidiary atrial pacemaker .................................................................. 101
4.1.3.1 Ablated sinus node ............................................................................. 101
4.2 Extent of the atrial pacemaker complex in the goat ............. 106
4.2.1 Spatial relationship of the SN and PNA within the RA ........................... 106
4.2.2 The body of the SN ................................................................................. 106
4.2.3 The tail of the SN and the PNA .............................................................. 108
4.2.4 The head of the SN and the second PNA ............................................... 108
4.2.5 3D reconstruction of the SN and PNA .................................................... 108
4.2.6 The extent of the SAP ............................................................................. 112
4.3 Protein expression in the SN and SAP .................................. 112
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4.3.1 Connexin 43............................................................................................ 112
4.3.2 HCN 4 ..................................................................................................... 113
4.3.3 Calcium handling proteins ..................................................................... 113
4.3.3.1 Sodium-Calcium exchanger ................................................................ 113
4.3.3.2 Ryanodine receptor ............................................................................ 113
4.3.3.3 Sarcoplasmic reticulum Ca2+ ATPase .................................................. 113
5 Discussion and Summary ......................................... 115
5.1 Creation of a large animal model of SND .............................. 115
5.2 Structural characterisation of the SAP .................................. 116
5.3 Extensive distribution of the atrial pacemaker complex, the
role of the paranodal area .............................................................. 117
5.4 Limitations of the study ....................................................... 118
5.5 Future directions.................................................................. 119
6 References .............................................................. 120
7 Appendix ................................................................. 132
7.1 DVD multimedia appendix ................................................... 132
Word Count: 29361
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List of Tables
Table 1: List of primary antibodies used .............................................................. 72
Table 2: List of secondary antibodies used ........................................................... 72
Table 3 Properties of fluorochromes used ........................................................... 72
Table 4: Acute outcome in the experimental group following ablation of the sinus
node .................................................................................................................... 77
Table 5: Dependence on electronic pacemaker ................................................... 84
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List of Figures
Figure 1 A schematic diagram by M. Flack depicting the sinus node (SN). ............ 17
Figure 2a: Experiments to confirm the location of the leading pacemaker........... 20
Figure 2b: Experiments to confirm the location of the leading pacemaker .......... 21
Figure 3 Histology of the sinus node. ................................................................... 23
Figure 4: Anatomical location and size of the human sinus node ......................... 25
Figure 5: Action potentials in the right atrium ..................................................... 27
Figure 6: Different manifestations of SND in humans .......................................... 35
Figure 7a: Location of subsidiary atrial pacemakers ............................................. 38
Figure 7b: Location of subsidiary atrial pacemakers ............................................ 39
Figure 8: Human sinus node and paranodal area ................................................. 41
Figure 9: Human sinus node at different levels .................................................... 42
Figure 10: Flow chart of the research protocol. ................................................... 48
Figure 11: Right lateral thoracotomy – skin incision............................................. 51
Figure 12: The heart in the pericardial cradle....................................................... 51
Figure 13: Unipolar epicardial pacing electrode ................................................... 53
Figure 14: Bipolar epicardial pacing electrode ..................................................... 53
Figure 15: Epicardial grid to aid mapping ............................................................. 55
Figure 16: Determination of the site of earliest activation ................................... 55
Figure 17: Irrigated ablation catheter .................................................................. 57
Figure 18: Ablation in vivo ................................................................................... 57
Figure 19: Flow chart of epicardial total sinus node ablation. .............................. 58
Figure 20: Calculation of the sinus node recovery time. ....................................... 62
Figure 21: Heart rate histogram ........................................................................... 64
Figure 22: Implantable loop recorder .................................................................. 64
Figure 23: Right atrial tissue preparations ........................................................... 67
Figure 24: Masson’s trichrome histological stain ................................................. 69
Figure 25: Digital slide ......................................................................................... 69
Figure 26: Principle of immunofluorescence ........................................................ 70
Figure 27: Location of the site of earliest activation in the intact sinus node ....... 77
Figure 28: Schematic diagram of the locations of the site of earliest activation ... 79
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Figure 29: Total ablation time and success of SN ablation ................................... 79
Figure 30: Spontaneous pacemaker activity from the apparently ablated SN ...... 80
Figure 31: Heart rate variance in conscious versus anaesthetised goat ................ 82
Figure 32: Heart rate correlation in conscious and anaesthetised state prior to
ablation ............................................................................................... 82
Figure 33: HR correlation in the SN ablated state ................................................ 84
Figure 34 : Heart rate comparison pre- and post SN ablation ............................... 86
Figure 35: Long term heart rate change during the follow-up .............................. 86
Figure 36: Change in PR interval .......................................................................... 88
Figure 37: Comparison of PR intervals pre and post ablation ............................... 88
Figure 38: P wave morphology change ................................................................ 88
Figure 39: Response of SN and SAP to overdrive suppression .............................. 89
Figure 40: Asystole and bradycardia in the experimental model .......................... 90
Figure 41: Formalin vs Methanol fixation for HCN4 staining ................................ 92
Figure 42: Macroscopic appearance of the SN ..................................................... 94
Figure 43: Histology of the sinus node and the surrounding atrial myocardium. .. 95
Figure 44: The SN and right atrium - Cx43/NCX1 double labelling ........................ 96
Figure 45: Paranodal area – histological features ................................................ 98
Figure 46: Paranodal area – immunohistochemistry ............................................ 99
Figure 47: Paranodal area – moderate magnification ......................................... 100
Figure 48: Subsidiary atrial pacemaker – histological features ............................ 102
Figure 49: Subsidiary atrial pacemaker – Immunohistochemistry I. .................... 103
Figure 50: Subsidiary atrial pacemaker – Immunohistochemistry II. ................... 104
Figure 51: Histology of the ablated sinus node ................................................... 105
Figure 52: Surviving pacemaker cells following ablation ..................................... 105
Figure 53: The body of the SN ............................................................................. 107
Figure 54: Nodal cells in the interatrial groove – the second PNA ....................... 109
Figure 55: HCN4 positive nodal-like cells in the second PNA ............................... 110
Figure 56: Schematic of the SN and PNA in the goat ........................................... 111
Figure 57 : Protein expression in the goat RA and pacemaking tissue ................. 114
Figure 59: Atrial pacemaker complex in the rat .................................................. 118
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Abstract
The University of Manchester
Submission for the degree of Doctor of Philosophy, The University of Manchester
Title: Characterisation of the Structural and Functional Properties of Subsidiary Atrial Pacemakers in a Goat Model of Sinus Node Dysfunction
Dr Zoltan Borbas
March 2015
The sinus node (SN) is the natural pacemaker of the heart. In the human, the SN is surrounded by the paranodal area (PNA), the function of which is currently unknown. The PNA may act as subsidiary atrial pacemakers (SAP) and become the dominant pacemaker during sinus node dysfunction (SND). Creation of an animal model of SND allows characterisation of SAP, which can be a target for novel treatment strategies other than the currently available electronic pacemakers.
I developed a large animal model of SND by ablating the SN in the goat and validated it by mapping the location of the newly emergent SAP.
Functional characterisation of the SAP revealed reduced atrioventricular (AV) conduction time consistent with a location of the SAP close to the AV junction. SAP recovery time showed an initially significant prolongation compared to the SN recovery time, followed by a gradual decrease over 4 weeks. SAP pauses, and temporary reliance on electronic pacemaker activity have also been demonstrated then disappeared over time, suggesting possible modulation, maturation of the SAP.
Structural characterisation of the SN revealed an extensive pacemaking complex within the right atrium (RA); the SN was surrounded by the PNA, extending down to the inferior vena cava (IVC) and into the interatrial groove. The PNA had a histological appearance that is intermediate to the SN and the RA. 3D reconstruction demonstrated, for the first time in a large animal model, an extensive and almost complete circle of pacemaking tissue at the junction of the embryologically different sinus venosus and the muscular right atrium.
The SAP emerged in a location close to the IVC along the crista terminalis. Expression of key ion channel proteins in the SAP showed abundance of the pacemaker channel (HCN4) and the sodium/calcium exchanger (NCX1) compared to RA, similar to the expression pattern of the SN. The expression of the main high conductance connexin (Cx43) was not significantly different between SAP and RA, and both expressed Cx43 more abundantly than the SN.
Conclusion: Destruction of the sinus node in this experimental model resulted in the generation of chronic SAP activity in the majority of the animals. The SAP displayed maturation over time and located in the inferior part of the RA, in the same area where the PNA was found in controls, suggesting the role of PNA as the dominant pacemaker in sinus node dysfunction. The SAP in the goat constitutes a promising stable target for electrophysiological modification to construct a fully functioning biological pacemaker.
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Declaration
No 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.
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Copyright Statement
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Acknowledgements
I would like to thank several members of the Cardiovascular Research Group, who
supported and helped me during my PhD programme.
First of all, I would like to express my gratitude towards my supervisors Dr Halina
Dobrzynski, Professor Clifford Garratt and my colleague Dr Jane Caldwell for the
initial research idea, their continuous encouragement and the training provided in
immunohistochemistry and ablation techniques. I am grateful to Prof Mark Boyett,
who has always been there when guidance was needed.
I thank to my academic advisors Dr Stephen O’Neil and Professor Andrew Trafford
for their support and advice throughout my PhD.
I would like to thank Dr Joseph Yanni and Mr Andrew Atkinson for the training
provided in histology, and laboratory techniques.
I acknowledge Drs Jane Caldwell, Yawer Saeed, Brian Prendergast and Akbar Vohra
for their contribution in the creation of the animal model and Dr Sunil Logantha
who performed the sharp glass electrode intracellular recording in this work.
I would like to acknowledge the assistance provided by the Manchester X-ray
Imaging Facility, which was funded in part by the EPSRC (grants EP/F007906/1,
EP/F001452/1 and EP/I02249X/1)
Finally, I thank British Heart Foundation for financial support, Boston Scientific and
Medtronic Inc. for providing key elements of the pacing and ablation setup.
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List of abbreviations
AF Atrial fibrillation
ANOVA Analysis of variance
ANP Atrial natriuretic peptide
AV Atrioventricular
BAPTA Aminophenoxy-ethane tetraacetic acid
bpm beat per minute
BSA Bovine serum albumin
BSCL Basic cycle length
CART Corrected atrial recovery time
CSNRT Corrected sinus node recovery time
CT Crista terminalis
ECG Electrocardiogram
EP Electrophysiology
FITC Fluorescein isothiocyanate
GA General anaesthesia
HCN Hyperpolarization-activated cyclic nucleotide-gated
channels
HR Heart rate
IF Immunofluorescence
IHC Immunohistochemistry
ILR Implantable loop recorder
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IVC Inferior vena cava
LCR Local calcium release
MT Masson's trichrome
NCX Sodium-calcium exchanger
NF-M Neurofilament medium
PBS Phosphate buffered saline
PNA Paranodal area
ppm permanent pacemaker
PR atrioventricular conduction time
qPCR Polymerase chain reaction
RA Right atrium
RAA Right atrial appendage
RF Radiofrequency
RIPV Right inferior pulmonary vein
rSN recovered sinus node group
RSPV Right superior pulmonary vein
RyR2 Ryanodine receptor
SAP Subsidiary atrial pacemaker
SEA Site of earliest activation
SERCA2A Sarcoplasmic reticulum Ca2+ ATPase
SN Sinus node
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SND Sinus node dysfunction
SNRT Sinus node recovery time
SV Sinus venosus
SVC Superior vena cava
TC Terminal crest
VF Ventricular fibrillation
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1 Introduction
The cardiac sinus node (SN) or sino-atrial node is the primary pacemaker of
the mammalian heart, the source of the depolarization wave front sweeping
through the whole of the myocardium ultimately resulting in a beating heart. In
order to fulfil this role, its structure and function has to be unique; with properties
differing vastly from that of the working myocardial cells. Research, over a century,
has already elucidated the fine details of the anatomical and histological features of
the sinus node, and significant advancement has been achieved to understand the
complex interaction of its subcellular and molecular components. However, in
disease states, the sinus node may become dysfunctional, and subsidiary
pacemakers (SAP) take over, ensuring ongoing generation and propagation of the
cardiac impulse. Currently, little is known about the structural and functional
properties of these pacemakers. It is vital to understand their anatomy, histology,
electrophysiology and any possible adaptation the SAP may undergo in order to
substitute the sinus node; a knowledge that may eventually lead to better
treatment of sinus node dysfunction.
1.1 Discovery of the sinus node
The sinus node was discovered by Martin Flack and Sir Arthur Keith in 1906 and
published in 19071. Keith, an anatomy demonstrator in The London Hospital, had
been examining the functional anatomy of musculature of the heart since 1900. His
acquaintance with Dr Mackenzie, a famous contemporary physician in 1903 gave a
new impetus to his research; he was given several pathological human heart
specimens for examination. Keith, looking for a caval closure mechanism,
incidentally noticed “a small condensed area of tissue, just where the cava sank into
the auricle” 2 but failed to recognise its significance at that time. Nevertheless,
influenced by Mackenzie, his studies then concentrated on what we know today as
the cardiac conduction system.
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In 1906, he was introduced to Martin Flack, a young medical student from Oxford.
They together worked on various mammalian hearts with the intention to verify
Tawara’s discovery of the atrio-ventricular bundle. However, apart from confirming
the existence of the atrio-ventricular node, they also made an unexpected
discovery: “...Flack was the section-cutter. One evening when Celia and I returned
from a bicycle ride he showed me a wonderful structure he had discovered in the
right auricle of the mole, just where the superior vena cava enters that chamber. It
had a microscopic structure similar to that of the node in which the a-v bundle
commences-the a-v node. I immediately remembered the structure I had met with in
Mackenzie’s hearts-exactly in the same position as in the mole, but of more
restricted development. We at once set to work on the sections we had already
made, and found this structure in every one of them. We inferred that it must be in
this structure, which we named the sino-auricular node, that the heart beat is
initiated and controlled.”2(figure 1).
Figure 1 A schematic diagram by M. Flack depicting the sinus node (SN).
Superior vena cava (a), right atrial appendage (b), vestibules of the left auricle (c), arterial supply of the sinus node (f, g), inferior vena cava (i), aorta (j). 3
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In their landmark publication the authors gave a detailed anatomical and
histological description of the mammalian sinus node the details of which were
later confirmed and further refined by different researchers 1.
1.2 Confirmation of the role of the sinus node
Flack’s and Keith’s assumption regarding the origin of the heart beat was based on
the work of a British physiologist Walter Gaskell, who described the peristaltic
movement of the tortoise’s heart and identified the origin of the muscular
contraction as the sinus venosus4. Flack himself also performed animal experiments
on the in vivo heart; he found that heating the area of the sinus node produced
heart rate acceleration whereas cooling it caused bradycardia. This effect was not
seen when the thermal stimulation was applied elsewhere in the atrium, providing
further indirect evidence for the role of the sinus node 3. However, the irrefutable
confirmation did not come until 5 years later, when Sir Thomas Lewis in a series of
original and elegant experiment proved that the origin of the heartbeat indeed lies
in the sinus node.
In 1901, Willem Einthoven invented the string galvanometer and laid the
foundation of electrocardiography. The new device allowed, for the first time, the
ability to record, and to study the electric activity of the heart directly. Thomas
Lewis, a then young clinical scientist installed one of these galvanometers in his
laboratory, and encouraged by Keith’s friend Dr Mackenzie, he studied the
mechanisms of cardiac arrhythmias. In 1909, he successfully confirmed the sinus
node as the leading pacemaker of the heart. He stimulated the heart of the dog
from various sites of the atria and found that only the site near the superior vena
cava resulted in an identical surface atrial electrogram (P wave) to that generated
by a normal, native heart beat suggesting that the origin of the two electrograms
must be the same (figure 2a)5. This technique today is called pace mapping and is
commonly used in localisation of focal arrhythmias. In the second experiment he
implanted widely spaced bipolar electrodes at different sites of the right atrium and
observed that only pairs whose exploring electrode was placed over the sinus node
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showed a primary negative deflection, signifying a depolarization wave front which
never passes beneath this electrode, therefore indicating origin at that site.5 Finally,
he invented yet another principal electrophysiology technique, that is activation
mapping, by measuring the onset of the local epicardial electrogram in the dog at
various locations and finding the earliest one invariable overlying the sinus node
(figure 2b) 5.
The importance of these experiments cannot be overemphasised; they not only led
to confirmation of the physiological role of the sinus node but also founded the
principles of modern clinical electrophysiology.
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Figure 2a: Experiments to confirm the location of the leading pacemaker
P wave morphology in 13 dogs; 1st
column representing native sinus rhythm, 2nd
column pacing from superior vena cava, further columns showing various pacing sites within the atria. Each row represents a different animal. Note, that the morphology of the generated surface P wave are very
similar in all dogs in sinus rhythm (1st
column) or when pacing from the superior vena cava (2nd
column) , but differs significantly when the atria are stimulated from any other site far from the SN suggesting that the native leading pacemaker is located near the SVC. (N=sinus rhythm, SVC=superior vena cava, IVC: Inferior vena cava, PV=pulmonary veins, CS=coronary sinus, AB=base of right atrial appendage, RA and LA= tip of the right and left atrial appendage.) Adapted from 5
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Figure 2b: Experiments to confirm the location of the leading pacemaker
Demonstration of increasingly delayed activation time compared to the leading pacemaker from within the sinus node. The earliest activation marked as S.A.N line on the left and corresponds to the area of b-c on the diagram on the right . Points j to o are increasingly caudal to the sinus node, therefore display progressively later occurring activation in time, compared to the SAN line. Adapted from5.
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1.3 Structure of the sinus node
1.3.1 Basic anatomy and histology
The sinus node is a crescent shaped structure located at the junction of the superior
vena cava and the right atrium 6. Macroscopically it is paler in colour and firmer in
consistency than the surrounding myocardium due to its high collagen content. The
extent of the sinus node shows significant variation among mammalian species. In
small laboratory animals (mouse, rat rabbit) the SN occupies the full thickness of
the intercaval region adjacent to the crista terminalis(CT) extending down, almost
reaching the inferior vena cava; whereas, for example in the dog, or in the human it
is confined to a relatively short portion of the cranial intercaval region abutting the
crista terminalis and separated from the endocardium by a layer of atrial
myocardium7. In these large mammals, the sinus node comprises a wide body and a
narrower head and tail; the head and body lying closer to the epicardium, the tail to
the endocardium. Its long axis runs parallel to the terminal crest and it terminates
less than halfway toward the inferior vena cava 1,8–10. The sinus node has a unique
arterial blood supply in the form of a dedicated and disproportionately large central
artery commonly originating from the right coronary artery and entering the node
from the tail; however, wide variation in its origin, course and branching has been
described11. The artery provides a robust blood supply and acts as a core for the
structural integrity of the node 12. At a microscopic level the sinus node shows
distinctly different architecture and composition than the working atrial
myocardium (figure 3). The predominant cell type (P cell) is small, round and pale
almost “empty”, containing only limited amount of myofibrils, sarcoplasmic reticuli
and mitochondria and connected to each other but not to other cells by small and
sparse gap junctions, features in keeping with its pacemaking, non-contractile
function 13,14. Transitional or T- cells are also present within the sinus node; long
slender cells containing more myofibrils and interdigitating with the surrounding
working myocardium, but not forming specialised internodal connections (between
the sinus and atrio-ventricular node) as was thought previously 15. The cells are
embedded in connective tissue, largely collagen but also elastic fibres. Whereas the
former makes anatomical, as well as histological localisation easy, the latter has
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recently been shown to form an organised structure around and attached to the
central nodal artery and the nodal cells themselves. This extracellular matrix is
thought to be produced by a third nodal cell-type - the “spindle cell” which is a
primitive mesenchymal fibroblast, based on its histological appearance and
immunohistochemistry 13,16.
Figure 3 Histology of the sinus node. Panel A Flack’s and Keith’s original drawing from 1907 depicting the sinus node.1 musculature of superior vena cava, 2 sinus node artery, 3 position of venous valve, 4 crista terminalis, 5 adipose tissue, 6 sinus node embedded in connective tissue) Panel B Histological section of the sinus node 1,15.
1.3.2 Discrepancy between structure and function
Flack and Keith original description of the sinus node depicted it as a discrete small
nodule in all mammalian species examined 1. Several studies conducted in the 1960-
70s, based on anatomical dissection and histology only, confirmed the above
findings; and the belief of the sinus node being confined to a small, limited area of
the myocardium held for several decades. James et al. examined the sinus node in
the human and in the dog and measured its size in human as 15 mm long, 1.5 mm
thick and 5mm wide at its widest portion; the total volume of the node was very
similar in both species 8,9. Truex et al. reconstructed the sinus node in a 3
dimensional wax model and found it even smaller, only 8mm in length 10. With the
advent of electron microscopy, computerised 3D reconstruction and techniques of
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immunohistochemistry it became possible to appreciate the true extent of the sinus
node and indeed, recent human reconstruction studies described a much longer
sinus node up to 22 mm in length extending down at the posterolateral right
atrium6,17 (figure 4). However, there are various observations that suggest the
existence of an even larger sinus node, or at least, a larger area involved in
pacemaking. The rabbit sinus node extends as caudal as the inferior vena cava and
the site of earliest activation can be located anywhere within. In vivo
electrophysiological mapping of the leading pacemaker in the dog and in human
revealed a widely distributed atrial pacemaker complex. For example, Boineau et al.
placed a 156-pole custom made multi-array electrode over the right and left atrium
in man during cardiac surgery and discovered that the earliest atrial activation can
originate anywhere from a staggeringly large 7.5x1.5 cm zone in the right atrium
with the sulcus terminalis (the epicardial equivalent of the crista terminalis) in its
centre 18. Very similar observations have been made in separate experiments by
two research groups in the dog 19,20. There are also reports of total sinus node
ablation in the dog and in man requiring ablation to be carried out along the crista
terminalis from the SVC down to the ostium of the IVC in order to stop sinus rhythm
21,22. Finally, supporting evidence comes from research of the embryologic
development of the cardiac conduction system, controlled by a transcription factor
Tbx3. Tbx3 is a suppressor, promotes formation of the sinus node by repressing
gene programs responsible for creating an atrial myocardial phenotype elsewhere
in the atria 23. In the mouse, Tbx3 is exclusively expressed along a “conduction
corridor” in the heart, from the sinus node through the posterolateral right atrium
to atrio-ventricular node and ultimately to the bundle branches 24.
Why the pacemaker complex appears much more extensive than the anatomical
sinus node is far from completely understood. There have been many possible
explanations postulated based on experiments regarding the electrophysiology of
the sinus node, the potential role of hierarchical subsidiary pacemakers as well as
discovery of a unique paranodal area. These will be covered in the subsequent
chapters.
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Figure 4: Anatomical location and size of the human sinus node
The figures demonstrate the significant difference in size of the sinus node depending on the methodology used. Panel A: 3D vax model of the human sinus node by Truex et al. 10. Note the location is shown to be limited to the junction of the superior vena cava and right atrium, the length of the node is measured 7.3mm Panel B Schematic diagram of the sinus node based on computer reconstruction of histological sections by Sanchez-Quintana et al6. The long axis of the sinus node(blue area) is parallel to the terminal crest and measures a mean 13.5mm in length (SVC: superior vena cava, RAA: right atrial appendage). Panel C 3D computerised reconstruction of the human sinus node based on histological and immunohistochemical data by Chandler et al 17. The length of the node measured as 22mm.
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1.4 Cellular electrophysiology of the sinus node
Subsequent to the elegant experiments by Lewis demonstrating the initiation of the
heart beat in the sinus node, several attempts had been made to identify the
electrical phenomenon making spontaneous activity possible but it was not until
1943 that the spontaneous depolarization of the natural pacemaker was recorded
by Bozler 25. The action potentials of the sinus nodal cell and the working myocyte
are significantly different; whilst the latter has a stable negative resting potential,
the former has a less negative and gradually rising membrane potential culminating
in a propagating wave of excitation. The action potential is a result of a sum of ionic
currents; inward currents (that is positively charged ionic movement into the cell)
and outward currents (figure 5). The roles of individual ion currents in pacemaking
were elucidated by using blockers of specific ion channels in vitro, measuring
currents with voltage clamp techniques and utilising genetically modified “knock-
out” animal models. Despite extensive research, no theory of pacemaking has been
accepted universally, and the issue remains hotly debated. The next chapter
summarise the perceived contribution of various ion channels and cell coupling
proteins to impulse formation within the sinus node and its transmission to the
atrial myocardium.
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7
Figure 5: Action potentials in the right atrium
Typical action potential recordings from right atrial working myocardium (light grey) and central sinus node (dark grey). The upstroke during systole is driven by 2 different ion channel responsible for the different slope of the upstroke. Contribution of key ion channels in the development of diastolic depolarization is shown on the right side of the picture. Adapted from Boyett26
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1.4.1 “Funny current” (If)
The funny current was named after its unusual characteristic; unlike many other
currents, it activates on hyperpolarisation (i.e. at a more negative membrane
potential). It carries mostly Na+ and, to a lesser degree, also K+ and Li+ and resulting
in a net inward current 27–29. The corresponding ion channel is the hyperpolarisation
activated cyclic-nucleotide gated or HCN channel, of which 4 isoforms have been
identified (HCN1-4) 30. The mammalian heart expresses HCN 1, 2 and 4, whereas
isoform 3 is found in the brain. Working atrial and ventricular myocardium
expresses small amount of HCN2, whereas HCN 4 and 1 are found abundantly in the
sinus node suggesting an important role of the funny current in pacemaking.
Several functional experiments corroborate this possibility. Blocking the channel
specifically by Cs+ led to a significant increase in sinus cycle length in the rabbit 31.
Ivabradine, a now commercially available, If inhibitor was able to lower the heart
rate in human by an average of 9 beat/minute and gained licence for treatment of
stable angina32,33. Recently, two independent groups demonstrated, that cardio
specific, induced and temporally controlled deletion of HCN gene or destruction of
HCN positive myocytes result in profound sinus bradycardia followed by atrio-
ventricular block in the mouse, associated with high mortality 34,35. The effect of
reduction in HCN channels proved to be due to extension of the slow depolarization
phase during diastole 33. On the other hand, studies using genetically manipulated
mice for a decreased If current showed only modest bradycardia and retained
chronotropic response 36. Clearly, the funny current has a role in pacemaking, but
its exact contribution is uncertain.
1.4.2 K+ currents
As previously stated, the sinus node does not have a stable resting potential; this is
due to the lack of the “inward rectifier” K+ current (IK,1). This current, despite its
name, is an outward current under physiological conditions, which keeps the
membrane potential hyperpolarised throughout diastole in the atrial and
ventricular working myocardium preventing the cells from depolarising
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spontaneously 37. Knocking out the Kir2 channel (responsible for the IK,1 current) in
the ventricle of the guinea pig permits pacemaker activity to emerge, albeit
unreliable and slow 38. During the action potential other K+ currents are activated.
At the beginning of repolarisation, a transient outward current is measured (Ito ) ,
selective blockade of the current results an increased cycle length in isolated “cell-
balls” from inferior part of the sinus node in the rabbit, but has no effect on the
strands of tissue obtained from the more superior nodal region suggestive of
regional differences within the natural pacemaker39. The family of delayed rectifier
K+ currents (IK) activates later during the repolarisation phase, it consists of at least
3 different subtypes: ultra-rapid, rapid and slow currents; called IKur, IKr, IKs
respectively 40. Selective partial blockade of IKr reduces the slope of the action
potential, hence increases cycle length within the sinus node 41. Higher
concentration of the same blocking agent completely ceased spontaneous action
potential generation in isolated myocytes of the rabbit 42.
1.4.3 Na+ current
The inward sodium current is well known to be the responsible current for rapid
depolarization of the working myocardium throughout the heart. It is principally
carried by Nav1.5 ion channel and encoded by the SCN5A gene. According to
classical teaching it has no role in spontaneously beating pacemaker cells. However,
genetic mutation of SCN5A in human has been associated with familial sick sinus
syndrome either isolated or manifested together with long QT syndrome 43,44.
Furthermore, blocking Na+ current with tetrodotoxin reduces the upstroke velocity
of action potentials in the periphery but not in the centre of the sinus node 45. In
contrast, abolishing Ca++ current with nifedipine did not stop the sinus node firing;
only the centre of it ceased activity 46 . The implication is that sodium current is
available in the periphery, and it may be able to contribute to impulse generation as
a backup mechanism and at the same time, it helps the centre of the sinus node to
drive the heart by acting as an interface between the less negative sinus node and
the working atrial myocardium. The latter has been verified by demonstrating that
block of INa in the periphery of the sinus node causes exit block 7.
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1.4.4 Ca++ currents
Two different types of voltage dependent Ca++ channels can be found in the
mammalian heart including the sinus node: the “long-lasting” L-type Cav1.2 and 1.3
and the transient T-type Cav3.1 through 3.3 responsible for the ion current ICaL and
ICaT, respectively.47. As discussed before, upstroke of the action potential generation
in the centre of the sinus node is dependent on the L-type calcium current, whereas
the periphery can beat even if this current is blocked. Transgenic knock-out mice for
Cav3.1 have a bradycardic phenotype with sinus arrhythmia48 and blockade of the
same channel in isolated sinus nodal cells of the rabbit slows the heart rate by
14%49.
1.4.5 The “Calcium Clock”
The above described voltage dependent currents are unable by themselves to
explain the complexity and the resilience of the sinus beat; blocking currents of
apparent key importance fail to totally inhibit the pacemaker. Lakatta et al
proposed the presence of an intracellular “Ca++ clock” whose operation coupled
with, and complements the function of the “membrane clock” 50. During the late
phase of diastolic depolarization, Ca++ enters the cytosol from the sarcoplasmic
reticulum through the Ryanodine receptor (RyR2). This local calcium release (LCR) is
spontaneous, not voltage dependent, occurring even in the absence of an intact
surface membrane 51. In the sarcolemma, but in close vicinity to RyR2, there are
sodium-calcium exchangers (NCX) which are activated by LCR. The NCX is an
electrogenic pump which exchanges 1 Ca++ for 3 Na+, therefore depolarises the
membrane potential and triggers the opening of the L-type Ca++ channels which in
turn is responsible for the rapid onset of action potential and electro-mechanic
coupling by large-scale calcium induced calcium release from the sarcoplasmic
reticulum52,53. However, the theory of this coupled-clock mechanism is still
controversial and debated 26,36,54,54–56. It is speculated, that whereas the key
elements of the membrane clock are unique to the conduction system, the
intracellular Ca++ handling apparatus is not, instead it is ubiquitous throughout the
heart. Therefore, provided the calcium clock is really responsible for initiating action
potentials, its presence in non-pacing myocardium would be both arrhythmogenic
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and superfluous 56. New evidence by Himeno et al seems to support that the
“calcium clock” is not essential in pacemaking; inhibiting Ca++ transients with BAPTA
(a Ca++ a chelating buffer) did not stop electrical activity in the sinus node cell of the
guinea pig, suggesting that the spontaneous activity of the pacemaker cells solely
depends on the “membrane clock” 57. However, the experimental designs in his
work had several shortcomings, the observation made was likely secondary to a
patch seal leak current58.
1.4.6 Electrical coupling and the connexins
Apart from transmembrane currents and intracellular Ca++ handling, appropriate
intercellular electrical coupling is a prerequisite of proper functioning of the cardiac
conduction system. Without it, synchronisation of firing within the sinus node,
capture of the atrium and propagation of the activation potential would not be
possible. The intercellular electrical coupling is achieved via gap junctions (low
resistance non-selective pores) comprising connexins (Cx). In the working atrial
myocardium the most abundant isoform is Cx43; its high conductance permits an
unrestricted route for rapid propagation of the activation wave front 59. In contrast,
within the sinus node such an arrangement would be disadvantageous; in fact some
degree of electric isolation is essential in order to protect the node from the
hyperpolarising effect of the surrounding myocytes 60. Hence it is not surprising that
Cx43 is absent from sinus node and that the predominant isoform Cx45 has both a
lower conductance per gap junctions as well as reduced density 61,62. The one order
of magnitude slower intranodal conduction velocity (compared to the atrium) is a
direct consequence of this feature. In the rabbit, toward the periphery of the sinus
node the electric coupling improves as Cx43 positive atrial myocytes interdigitate
with nodal cells. This feature may be important in enabling the small sinus node to
drive the atrium 63. Similar arrangement has been described recently in the human:
Cx43 and Cx40 connexins were detected at an intermediate level near the sinus
node in the so-called paranodal area 59. The putative role of this area will be
discussed later.
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1.5 Electrical mapping of the pacemaker complex
1.5.1 Electrode-based “traditional” mapping
So far, in this review the electrical phenomenon within a single sinus nodal cell has
been described based on ionic currents and their effect on transmembrane
potential and assumptions made how the sinus node as a whole behaves,
extrapolating the results. However, for studying the properties of intercellular
action potential propagation directly, activation mapping of the natural
pacemakers, sinus node or subsidiary pacemakers alike requires the ability of
recording in the intact tissue. Extracellular cardiac potential was utilized as early as
the beginning of the 20th century by Lewis (see section 2.2) 5. The electrode based
mapping had several shortcomings: unlike working myocardium the sinus node
produces low amplitude, low frequency signals which can be easily missed due to
erroneous filtering or obscured by the much larger electrogram produced by the
adjacent atrium 64. It is important to ensure that appropriate filtering is in place; the
low-frequency signal produced by the sinus node is easily rejected by a low cut-off
frequency filter that is set too high. Human electrophysiology studies aimed at
studying atrial and ventricular extracellular potentials usually set to filter out signals
below 30Hz. However, when one wishes to display sinus node potentials it is
necessary to use a cut-off below 0.5Hz 65. This technique is adequate for mapping
prior to ablative procedures of the sinus node as higher resolution for localization is
not necessary 21,66. The development of the microelectrode technique allowed more
precise localisation of the leading pacemaker; the closely spaced electrodes helped
excluding far-field atrial electrograms by common mode rejection 67. Sano and
Yamagishi using the microelectrodes were the first to observe highly anisotropic
conduction within the node, relatively fast activation in caudo-cranial direction and
block toward the septum in the rabbit. They proposed the presence of “preferential
conduction pathways” to explain their findings 68. Unfortunately, the use of
microelectrodes in vivo on the whole heart is not feasible due to the vigorous
movement of the beating heart.
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1.5.2 Advanced mapping techniques
Whereas the rabbit sinus node is essentially a 2 dimensional structure, researchers
experimenting with large animal models faced an additional challenge; the sinus
node is “sandwiched” between layer of myocardium more endo- then epicardially
69. This precludes the separate acquisition of the pure sinoatrial potentials,
moreover the large amplitude, high frequency atrial action potential masks the
smaller, low-frequency activation of the pacemaker cells 69. Due to these inherent
limitations of the traditional method, it was still not possible to verify whether
isolation of the sinus node with the exception of preferential conduction pathways
holds true in man. In the last two decades advanced mapping systems have been
developed allowing for high resolution, simultaneous electrophysiology mapping.
The non-contact mapping uses an inflatable balloon with several multipolar
electrodes on its surface, coupled by a computer which creates more than 3000
unipolar virtual electrograms from the chamber the array was introduced 70.The
balloon array is uniquely suited to study the activation sequence of the sinus node
and right atrium in large animals and in men in vivo. Using this technique Stiles et al
has recently confirmed that the site of earliest activation within the sinus node
fluctuates and there are preferential pathways of conduction the same way as in
smaller species 71. They showed that certain part of the atrial myocardium activates
earlier then some of the latent pacemaker cells within the sinus node due to the
fast activation of the former through the preferential pathways. Therefore, the
breakthrough atrial potentials, based purely on their early activation, may seem to
be part of the pacemaker complex; this could be an alternative explanation to why
the pacemaker complex appears larger than the sinus node 71. Fedorov used optical
mapping instead, to show virtually the same result in an isolated human sinus node
72. Optical mapping uses a voltage dependent fluorescent dye as a substitute for
direct recording of potentials. Whilst its resolution is impressive and a computerized
method effectively capable of differentiating various components within the signal
(i.e. myocardium and intramural sinus node) it is not without limitation, it cannot be
used in vivo 69. The mammalian sinus node is a surprisingly complex structure; the
combination of its anatomical, electrophysiological and cellular properties making it
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possible to play the role of the leading pacemaker. It is also apparent, that different
ionic currents are responsible for pacemaking in different region of the sinus node.
It is speculated that the above listed mechanisms are backups of each other, making
the atrial pacemaker complex a very robust structure; failure of one can be
substituted by another 7. Finally one has to remember how much larger the
functional pacemaker is than the anatomical sinus node; this may explain why the
pacemaker activity continues, even when the sinus node is diseased; albeit less
reliably and prone to intermittent failure, perhaps because the alternative,
subsidiary pacemakers do not possess the same “built-in” redundancy. Clearly,
further research is required to elucidate and clarify the exact molecular make-up in
the atria.
1.6 Sinus node dysfunction and subsidiary atrial pacemakers
1.6.1 Sinus node dysfunction
Sinus node dysfunction (SND), or sick sinus syndrome, is an abnormality of action
potential generation or propagation within the SN and or at the interface of the SN
with the working atrial myocardium. The prevalence is in about one of every 600
cardiac patients over 65 years of age 73 and in a recent world survey of pacing
accounted for 20-50% of pacemaker implants 74. The electrocardiographic features
of SND can be highly variable and manifested in several different ways (figure 6)75.
-Sinus bradycardia; a decrease in the intrinsic heart rate below the normal range.
-Ectopic atrial bradycardia; slow heart rhythm arising from a subsidiary atrial
pacemaker (SAP).
-Increase in the sino-atrial conduction time; the time taken for the action potential
to propagate from the leading pacemaker site into the neighbouring atrial muscle.
-Exit block; failure of the action potential to exit from the sinus node into the atrial
muscle. -Sinus arrest; temporary cessation of the firing of the SN
-
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Figure 6: Different manifestations of SND in humans
Panel A: Bradycardia. The heart rate is slow and falls outside the normal range.
Panel B: Ectopic bradycardia. The heart rate is not only slower but the beats originating from different pacemakers, evidenced by the different P wave morphology (red circles).
Panel C: Sinus arrest. Following a several seconds long pause, the SN still does not recover and a subsidiary pacemaker arises from the AV node preventing further asystole. Note the lack of P wave preceding the ventricular complexes (blue arrows).
Panel D: Chronotropic incompetence on a 24h ECG recording. This patient had an electronic pacemaker implanted firing at a rate of 60 beat/min. Note that the diseased SN was unable to raise the heart rate above 60 beat/min over 24h, despite physical activity.
Images are taken from the author’s own collection.
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-Increased sinus node recovery time (SNRT); a prolonged suppression of the natural
pacemaker following atrial overdrive suppression. This can be observed following
either naturally occurring paroxysmal tachycardia or temporary atrial pacing
-Tachycardia-bradycardia syndrome; propensity to develop paroxysmal atrial
arrhythmias
-Chronotropic incompetence; inability to raise the heart rate during exercise.
Sinus node dysfunction has been attributed to fibrosis since the 1950s, and backed
up in later reports 76–78. This is now disputed; no increase of collagen content was
found in the SN of the cat; whereas in humans an increase from 38% to 70%
demonstrated from childhood to adulthood, but no further increase in later life79.
Yanni et al have recently reported a decrease in collagen coding mRNA content and
a corresponding reduced protein signal density on picrosirius red staining in the
sinus node of the ageing rat. However, the same ageing rats showed an increased
sensitivity of nodal If and INa currents, to their specific blockers 80. It is now thought
that sinus node dysfunction may be related to modulation in ion channel expression
or function. Studies of transgenic mice have shown that knock-out of various ion
channels and related proteins (Nav1.5, Na+ channel β2 subunit, Cav1.3, Cav3.1,
HCN2, HCN4, ankyrin-B and Cx40) can result in sinus node dysfunction 81.
In clinical practice, sinus node dysfunction commonly affects the elderly population
and is often associated with coronary artery disease, heart failure, diabetes or atrial
fibrillation 82. Sinus node disease can also occur in the young, usually in patients
with congenital heart disease especially if corrected with surgery. Extreme
endurance sports can also induce bradycardia, which can become pathological and
symptomatic; it may be reversible on detraining or require pacemaker treatment.
Interestingly, the decades old dogma of heightened vagal tone as its cause may not
be the correct one: Katona et al examined the intrinsic heart rate of a rowing squad
and found it to be significantly lower than in the control population 83. Moreover, in
professional cyclists, even after years of detraining, higher than expected incidence
of symptomatic bradycardia occur and may warrant electronic pacemaker
therapy84. An alternative mechanism behind the training induced bradycardia has
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recently been proposed and proved; trained rats and mice displayed slowing of the
heart rate even when complete autonomic blockade was achieved. Following the
training period a significant downregulation of HCN4 was demonstrated at mRNA,
and protein level as well as a decrease of the corresponding ionic current(If) 85.
These observations in the human and research in laboratory animals support the
notion of a disease causing intrinsic remodelling of the sinus node.
The most uncommon aetiology of all is undoubtedly the inherited/familial sinus
node dysfunction. Mutations of HCN4, SCN5A, ankyrin B proteins have been
identified and linked to bradycardia, sinus dysrhythmia and exit block 81. The
importance of this group from the research point of view is high; it helps creating
better mathematical models of the sinus node, and provide useful clues regarding
the putative acquired molecular remodelling underlying the more common forms of
the disease.
1.6.2 Subsidiary atrial pacemakers
Irrespective of the pathological process, when the sinus node fails, in both clinical
and animal studies, subsidiary pacemakers take over as the leading pacemaker site
20,21,86–88. Boineau mapped a large area of the human right and left atria during
surgery and found that in a few patients the site of earliest activation lay outside
the boundaries of the sinus node and it was close to the inferior vena cava (figure 7
A)18. In the dog, sinus node dysfunction has been modelled experimentally by
surgical excision, epicardial radiofrequency catheter ablation and endocardial
radiofrequency catheter ablation of the sinus node. In all studies, after destruction
of the sinus node, subsidiary atrial pacemakers emerge 20,21,89,90. Most commonly
the leading pacemaker site is inferior to the crista terminalis and next to the inferior
vena cava (figure 7 B&C), i.e. at the same location as the leading pacemaker site in
the patient in figure 7A. Sinus node dysfunction can also appear as a result of
isolation, rather than complete destruction of the SN; despite continuing activation
of the SN, the rest of the atria can be driven by a SAP at a slower cycle length (figure
7 D) 88
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Figure 7a: Location of subsidiary atrial pacemakers
Panel A: earliest activation in a young human with a structurally normal heart in the anaesthetised state (red area near the IVC) Adapted from 18
Panel B: In vitro right atrial preparation. Dots and represent sites of earliest activation in the canine low right atrium in different animals following sinus node destruction by ligating the sinoatrial nodal artery. Adapted from 86.
Panel C: Schematic diagram of the posterolateral canine right atrium. Location of subsidiary atrial pacemakers marked by dots and stars. Shaded area represents the ablated sinus node. Adapted from 20
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Figure 7b: Location of subsidiary atrial pacemakers
Intracardiac recordings in a human with sinus node dysfunction.
Top left: The SN is isolated from the rest of the heart. Note the earliest activation marked with white colour collides with activation wavefront from the SAP and does not drive the right atrium.
Top right and bottom: A slower SAP located in the interatrial septum (white area) drives the atria and subsequently the ventricles (white arrows), whilst the SN continues to show a faster, spontaneous depolarization but fails to capture the atria (red arrow). Adapted from88.
SN:Sinus Node; SAP:Subsidiary atrial pacemaker
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These subsidiary pacemakers are functionally distinct from the sinus node in that
they give rise to a slower resting heart rate and slower exertional heart rates 91 and
at least initially are more sensitive to cholinergic agents such as acetylcholine, less
sensitive to sympathetic agents such as noradrenalin and more sensitive to
overdrive pacing. With time, these differences subside and the new pacemaker
becomes more established as the dominant pacemaker21,92,93.
Microelectrode studies of in vitro preparations of the canine inferior right atrium
have shown that the electrical activity of the subsidiary pacemaker is more akin to
that of the sinus node than to the surrounding atrial muscle; action potentials
exhibit prominent diastolic depolarization and a significantly less negative diastolic
potential, overshoot, and amplitude than typical atrial muscle89.
Kistler et al. demonstrated that in 31% of their patients the atrial tachycardia arose
from a focus in the crista terminalis – subsequently have been termed as ‘cristal
tachycardias’94,95. The cristal tachycardias are arising from the area associated
either with normal sinus rhythm or rhythm from subsidiary atrial pacemakers (see
above), i.e. an area corresponding to the sinus node and paranodal area (see
below).
1.6.3 Paranodal area
The summary above demonstrates that, although the whole of the crista terminalis,
from the superior to the inferior vena cava, is associated with physiological and
pathophysiological pacemaking, the sinus node is restricted to the upper part of this
structure only. It is possible that a newly identified paranodal area plays an
important role in pacemaker activity. Chandler et al identified in the human a
unique structure adjacent to the sinus node, which they termed the paranodal area
(figure 8 panel A)59. As seen in the figure 8A and figure4C (yellow area), the
paranodal area lies within the crista terminalis and is anatomically discrete from the
sinus node. Most importantly, the paranodal area appears to be much more
extensive than the conventional nodal tissue. In their study of healthy human heart,
the sinus node tissue had already disappeared – at the most inferior point of the
specimen (halfway between the superior and inferior vena cava), (figure. 4C)
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Figure 8: Human sinus node and paranodal area.
Panel A:Masson’s trichrome stained section through the SN and paranodal area in the human. The less densely packed paranodal area can be clearly seen.
Panel B and C: Immunolabelling of Cx43 (green signal) and ANP (red signal) in the SN, paranodal area and atrial muscle. The white arrows highlight expressing myocytes, whilst the blue arrows highlight non-expressing myocytes. From Chandler et al.59.
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Figure 9: Human sinus node at different levels
Low-power photomicrographs taken at three levels perpendicular to the crista terminalis (CT). From top to bottom: level of superior vena cava (SVC), level at midway between SVC and IVC, level of inferior vena cava (IVC). Note the pale staining and the loosely packed cells within the CT, (arrows) the appearance is identical to the paranodal area in figure 7 and it is still identifiable near the IVC. Adapted from Matsuyama96 ).
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the paranodal area was still large (figure. 4C, yellow area)17. Furthermore, in
previous studies by Sanchez-Quintana and Matsuyama the same area of loosely
packed myocytes embedded in fibro-fatty tissue was noted, but they failed to
recognize as a separate entity 6,96. Matsuyama et al showed a diagram in their
original publication on which a histologically identical structure to the paranodal
area could be identified down to the inferior vena cava (figure 9).
It is possible, therefore, that the paranodal area extends toward the inferior vena
cava. Whereas sinus node cells are small and atrial myocytes are large, the cells of
the paranodal area are intermediate in size 59. The cells of the paranodal area are
less densely packed than the surrounding atrial muscle, and appear on
immunolabelling to consist of a mixture of cells; some with the characteristics of
nodal cells and some with the characteristics of atrial myocytes. As shown (figure, 8
B,C), some of the cells of the PNA express Cx43 and atrial natriuretic peptide (ANP),
whilst others do not, like nodal cells. The authors detailed ion channel expression in
the paranodal area and it is different from that of both the sinus node and
surrounding atrial muscle. The expression pattern of many ion channels in the
paranodal area is intermediate between that of the sinus node and atrial muscle;
for example, the expression of the cardiac Na+ channel, Nav1.5, and the inward
rectifier K+ channel, Kir2.1, is intermediate between that of the SN and atrial muscle.
However, compared with both the sinus node and atrial muscle, the paranodal area
shows greater expression of Kv4.2, Kir6.1, TASK1, SK2 and MiRP2 59.
The function of this paranodal area is as yet unknown and no functional
experiments have been published. The poor expression of Kir2.1 means that the
paranodal area may be capable of pacemaker activity (see section 2.4.2 earlier).
This structure has functional characteristics intermediate to that of the adjacent
sinus node and atrial muscle and on sinus node dysfunction may well be modulated
and take on the role as the leading pacemaker site, i.e. act as a subsidiary atrial
pacemaker. In addition, with such unique electrophysiological properties, it is
possible that the cells of the paranodal area may be unstable and prone to ectopic
activity, and could be responsible for the cristal tachycardias known to originate
from this region. To date, the paranodal area has only been described in the human.
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For example, previous work on the rabbit and rat has shown a more extensive sinus
node rather than a discrete paranodal area 97,98. Further research is required to
clarify the role of the paranodal area.
1.7 Concept of biopacemaking
At present, the only treatment for sinus node dysfunction is the insertion of an
electronic pacemaker. The implantation of pacemakers is associated with acute
risks during implantation, such as collapsed lung (1-2%), haematoma (1%)and
cardiac perforation (≤1%) and longer-term problems of infection (1-2%),lead failure
(2-3%), lead displacement (2-4%), erosion (0.9%), lead perforation and cardiac
failure due to prolonged right ventricular pacing99–102. Thus, the development of
biological pacemakers (e.g. via genetic manipulation) is a target being actively
pursued around the world, because it potentially could circumvent many of the
limitations of electronic pacemakers 103. Miake et al attempted to generate a
biological pacemaker within the working myocardium by reducing the repolarising
current of IK,138 .41. A proof of concept experiment on transfecting murine left atrial
myocytes with HCN2 gene has been published 104. However, there are many
differences in gene expression between a pacemaker tissue and working
myocardium and arguably genetically manipulating one or two genes in a vastly
different substrate is unlikely to produce a robust pacemaker. Instead, a strategy of
either modifying the genetic program of the common myocytes or genetically
manipulating the sinus node, the subsidiary atrial pacemaker or any other part of
the cardiac conduction system is a more realistic way of generating a biological
pacemaker. An example for the former method is the expression of the
transcription factor Tbx18, that reprogram the working myocardium and convert
them into pacemaker cells105. There is published work for the latter strategy, too.
Recently, the left bundle was transfected with a combination of a pacemaker gene
(HCN2) and a sodium channel gene (SKM1), the SKM1 being responsible for
allowing spontaneous depolarization at a more negative resting membrane
potential106. In another study, an already functioning subsidiary atrial pacemaker
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was successfully accelerated by transfecting the in vitro right atrial preparation by a
chimeric HCN product 107. To pursue this concept to create a clinically useful
biopacemaker, more information about the structure and function of subsidiary
pacemakers is required.
1.8 Aims of thesis
The detailed structure and extent of the sinus node in the goat is currently
unknown. The first aim of my research was to locate and assess the extent of the
primary atrial pacemaker complex in the goat, including the sinus node and the
surrounding atrial tissue, specifically looking for an area equivalent of the paranodal
area described in human.
The structural and functional properties of subsidiary atrial pacemakers in small
laboratory animals have been investigated. However, in large animals, only limited,
mainly functional data is available. Therefore, the second aim of my work was to
create a large animal model of sinus node dysfunction by ablating the sinus node
and subsequently characterise the functional and structural properties of the
emerging subsidiary atrial pacemakers and their spatial relationship to the sinus
node.
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1.9 Research Hypotheses
1. Following ablation of the sinus node a stable subsidiary atrial pacemaker
emerges in the goat right atrium
2. The location of the SAP is anatomically different from that of the sinus node
with a predilection to be located in the caudal part of the right atrium along
the crista terminalis
3. The electrophysiological properties of the SAP, (P wave morphology, PR
interval, subsidiary atrial pacemaker recovery time) are significantly
different following ablation of the sinus node
4. There are significant differences in the abundance of various ion-channels,
connexins, between the SAP and working myocardium, allowing pacemaking
in the SAP.
5. At the protein level the characteristics of SAP is intermediate between that
of the sinus node and the atrial myocardium , with particular emphasis on
structural and ion-channel proteins known to be important in pacemaking
and action potential propagation
6. As in the human, there is a paranodal area in the goat, and it may act as SAP
Page | 47
2 Materials and Methods
2.1 Overview of the research protocol
The flow chart of the research protocol helps to understand the methods and
materials used (figure 10).
All the experimental group (n=10) underwent surgery in order to map of the site of
earliest activation within the SN and testing the SN recovery time, followed by
ablation of the SN. The animals were recovered and further tested in vivo for 4
weeks, comprising surface ECG, pacemaker telemetry and assessment of the SAP
recovery time. In addition, some of the experimental animals (n=3) have also
received an implantable ECG loop recorder to collect single lead ECG data of
suspected episodes of bradycardia (slow heart rhythm). During a second surgery the
location of the emerging SAP was mapped, then the animals were humanely killed
and their right atria harvested.
The control group (n=5) underwent a single surgery to map the site of earliest
activation within the SN and to perform intraoperative ECG and SN recovery time
assessment. Right atria have been harvested similarly to that of the experimental
group.
The right atria were dissected into preparations containing the whole postero-
lateral right atrium and frozen until used. Four control and four experimental right
atrial preparation were sectioned with cryotome for histology and
immunohistochemistry experiments.
Page | 48
Figure 10: Flow chart of the research protocol.
*Surface ECG to assess heart rate, atrioventricular conduction time and P wave morphology as well as assessment of SN/SAP recovery time following overdrive pacing
**Histology, qualitative and semi-quantitative immunohistochemistry were used, and characteristics of the sinus node, working atrial myocardium and the newly emerging atrial pacemaker were compared.
All Goats n=15
Experimental group n=10 Control group n=5
Procedure I
Epicardial ablation of the sinus node, electronic
pacemaker implant
Atrial harvesting
Tissue characterization**
Procedure II
Repeat epicardial mapping to locate the new dominant pacemaker
Tissue characterization**
Procedure I
Epicardial mapping to
confirm location of the sinus node
Atrial harvesting
4 weeks
Functional characterisation*
Page | 49
2.2 Species used
Experiments on 15 adult female goats were planned in accordance with a project
license issued by the Home office under the Animals (Scientific Procedures) Act
1986. We choose goats in this project for the following reasons:
1. It is known that the extension and structure of SN is different in small vs.
large mammals (section 1.3.1); therefore studying SN in a large animal
model may have more relevance and the results are easier to extrapolate to
human research than that of obtained from small laboratory animals.
2. To date, the PNA has only been described in human (section 1.6.3). A pilot
study performed by our research group suggested the presence of PNA in
the goat; making this animal an ideal choice.
3. The goat is also an ideal choice as we have great expertise in implanting
pacemakers, a procedure that may be necessary after ablating the SN108–110.
Although it is possible to place pacemakers in smaller animals, it is
technically very difficult and the irritation caused by the pacing leads could
cause fibrosis thus affecting the substrate we wish to study.
2.3 Surgical technique to expose the SN
2.3.1 Anaesthesia and Surgical preparation
The animals were kept free from food overnight prior to the procedure. The
operative team consisted of a cardiothoracic surgeon, an anaesthetist, an assistant
trained in animal procedures and two cardiology trainees, including myself, trained
in human cardiac ablation techniques. Following an initial training period, I became
the sole responsible operator performing the mapping and ablation procedures,
and carrying out all intraoperative electrophysiology testing. I have also performed
the tracheal intubation and jugular vein cannulation in most cases.
Anaesthesia was induced through a mask in the pen (3% isoflurane and a 2:1
mixture of O2 and N2O). The goats where then transferred to the operating theatre,
and intubated with a long intratracheal tube through which anaesthesia was
Page | 50
maintained using the same mixture of inhalative anaesthetics. The goats were
ventilated using positive pressure and placed in the left lateral position. The right
jugular vein was cannulated for administration of prophylactic antibiotic (Co-
amoxiclav), analgesia (buprenorphine) and intravenous fluid replacement. Skin
preparation was made by thorough shaving of various areas: the location of the
planned surgical incision, the place for indifferent electrode of the ablation system
and surgical diathermy, surface ECG electrodes and defibrillator patches.
Intraoperative monitoring was continuous throughout the procedure utilising pulse
oximetry, non-invasive blood pressure monitoring placed on the tail and single lead
ECG.
2.3.2 Thoracotomy, Exposing the Right Atrium
The SN is a predominantly an epicardial structure it is unlikely that we would
achieve a complete ablation of the SN via a transvenous, endocardial route.
Therefore, an epicardial approach was selected requiring thoracotomy to expose
the right atrium.
An incision was made on the right lateral thorax and soft tissue divided using
unipolar cutting diathermy (figure 11). Thoracotomy was then performed, the
intercostal space enlarged by retracting the adjacent ribs and also excising a small
portion of one of the ribs. Haemostasis was meticulously maintained throughout
the procedure with coagulating diathermy and manually ligating the subcostal
neurovascular bundle. We found that the third or fourth rib space gave the best
exposure of the posterolateral right atrium whilst utilising the smallest and
therefore least traumatic incision. Following opening the pleura and retraction of
the right lung the pericardium was opened and the edges were sutured to the
thoracic wall, creating a pericardial cradle in which the beating heart was contained.
As a result of this method, I had a clear view of the sinus venosus (postero-lateral
right atrium), right atrial appendage, superior and inferior caval veins and the
terminal sulcus (the epicardial manifestation of the terminal crest) (figure 12). At
this stage, the heart rate was recorded as preablation baseline.
Page | 51
Figure 11: Right lateral thoracotomy – skin incision
The inset diagram in the right bottom corner depicts the position of the animal on the operating table. From this exposure, the posterolateral right atrium will readily be accessible.
Figure 12: The heart in the pericardial cradle
Same orientation as figure 11 The superior and inferior vena cavae (SVC, IVC), right atrial appendage (RAA) and Sinus venosus of the right atrium (SV-RA) are clearly identified. The terminal sulcus separates the SV-RA form RAA
RAA
IVC SVC SV-RA
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2.4 Epicardial Pacemaker Implantation
In order to ensure adequate heart rate in the immediate postoperative period,
when the intrinsic heart rate provided by the SAP is expected to be unreliable and
slow, implantation of an electronic epicardial pacemaker system is necessary.
Furthermore, the pacing electrode also acted as a reference or fiducial point during
the ablation process. Finally, the pacemaker allowed me to perform programmed
extrastimuli during the follow-up period required for the assessment of the SAP
recovery time and also provided telemetry data regarding the average resting heart
rate in the conscious animals.
Initially unipolar epicardial leads were used; the electric field resulting in myocardial
stimulation occurred between the tip of the unipolar electrode placed on the
surface of the right atrial appendage/right ventricle and the pulse generator placed
in a subcutaneous pocket (figure 13). The performance of the electronic pacemaker
was tested via a pacing system analyser (Medtronic Inc.) and was considered
satisfactory if pacing threshold was less than 1.5V and sensed atrial/ventricular
amplitude>2mV. The unipolar atrial lead was attached to the mapping system to aid
the subsequent mapping procedure along with another temporary unipolar
electrode.
However, this configuration caused problems: intermittent extracardiac muscle
stimulation in one animal, intermittent loss of capture in another experiment. The
location of the pacemaker pocket was also restricted due to the unipolar pacing; as
the heart had to lie between the generator and the tip of the lead in order to be
captured. One experimental animal developed a treatment refractory ventricular
fibrillation on placement of the ventricular lead and it was lost to further
experiments. For these reasons, after the second experiment, I stopped using a
ventricular lead and replaced the atrial unipolar lead with a bipolar electrode.
sutured to the right atrial appendage placed 5 mm apart (figure 14). The bipolar
electrode also made the use of temporary electrodes redundant during mapping.
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Figure 13: Unipolar epicardial pacing electrode
The electrode was screwed into the myocardium as well as directly sutured on the epicardial surface of the tip of the right atrial appendage (RAA), dashed red circle on the diagram in the right bottom corner. The pacemaker generator was placed inferior to the incision into a subcutaneous pocket (not shown). The arrow indicates the electric field between the tip of the unipolar electrode and the pacemaker.
Figure 14: Bipolar epicardial pacing electrode
The bipolar electrodes placed onto RAA, 5mm apart. The stimulation occurs between the two bipole; therefore, the location of the generator in bipolar pacing is not crucial. The generator was placed into a superior pocket, closer to the spine aiding easier interrogation and more comfort for the animal (not shown).
RAA
IVC SVC
RAA
IVC SVC
Page | 54
2.5 Mapping and ablation
2.5.1 Mapping the earliest activation within the sinus node
Systematic epicardial mapping was performed to determine the site of earliest
activation (SEA): a quadripolar ablation catheter with 5 mm interelectrode spacing,
routinely used in human endocardial electrophysiology studies, was manually
positioned at multiple sites on the right atrial free wall such that it maps the entire
epicardially available right atrial surface. The mapping was guided by a virtual grid,
which was constructed using anatomical landmarks to aid reproducibility (figure 15,
Panel A).
The epicardial pacing electrodes sutured onto the right atrial appendage were used
as a reference point along with the onset of the P wave recorded by using a
traditional surface ECG lead (section 2.6.1). The local activation time, measured
separately at the distal and proximal bipole of the exploring catheter, was
compared to the activation time of both reference. The exploring catheter then was
moved to its next location and the measurements repeated.
The SEA is the origin of the depolarization wavefront, therefore it has to precede
any local electrograms. Furthermore, the SEA also has to be premature to the onset
of the P wave, which marks the beginning of the atrial depolarization. The most
premature local activation time was considered to be the SEA (figure 16).
Confirmation of SEA was obtained by placing the mapping electrode at the SEA ,
and demonstrating that slight movement of it to any direction resulted in a less
premature local activation time (Figure 15 panel B). SEA was marked with fine
surgical sutures in the control animals.
Electrograms were acquired using a filter and preamplifier system (Digitimer Ltd)
and a PowerLab data acquisition system (ADInstruments) at a sampling rate of 1
kHz. Band-pass filters of 0.3-300Hz, 0.1-30Hz and 30-300Hz were applied to the
surface ECG, mapping electrode and right atrial reference electrode, respectively. I
refer to section 2.5.1 as an explanation for the chosen filter settings.
Page | 55
Figure 15: Epicardial grid to aid mapping:
Right panel: The mapping catheter was placed in each area defined by the depicted virtual grid. The line of 0 (blue line) represents the level of the right superior pulmonary vein (RSPV). From this point horizontal lines are drawn cranial and caudal with 5mm spacing corresponding to the interelectrode distance of the mapping catheter. RIPV: Right inferior pulmonary vein.
Left Panel: The mapping catheter placed at the line of zero. Inserts are showing the orientation.
Figure 16: Determination of the site of earliest activation
The distal bipole of the mapping catheter records a local activation time that is clearly premature to both the intracardiac reference (red trace, RAA) as well as to the onset of P wave (purple trace) The degree of prematurity is illustrated with the horizontal black lines.
1s
SVC
RA
IVC
0 -1 -2 -3
1 2 3
A B C D
RSPV
RIPV
E
4
Page | 56
2.5.2 Ablation of the Sinus Node
Following identification of the leading pacemaker site, radiofrequency (RF) energy
was delivered, utilising a temperature controlled, power feedback ablation system
(max temperature: 45-50℃, max delivered power: 50-60W, Boston Scientific Inc.).
Tissue damage is caused via resistive heating of the immediately adjacent
myocardium and then further spread occurs via conductive heating 111. However,
unlike endocardial ablation, the epicardial approach lacks the cooling effect of
blood-flow causing premature temperature rise at the catheter-tip, which limits
power delivery precluding adequate tissue damage 112. To circumvent this problem
the same catheter used for mapping was modified; it was continuously irrigated by
room temperature 0.9% saline solution during ablation (figure 17).
The ablation at any one location was controlled under direct visualisation of the
development of an ablation burn, as well as observing a 90% reduction in the
amplitude of the local electrogram (figure 18). Earliest atrial activation during
spontaneous rhythm was remapped after each ablation, the endpoint for
radiofrequency application being a decrease in spontaneous heart rate by 50%
and/or the emergence of junctional rhythm (figure 19). This approach was similarly
used by Kalman et al in the dog, although their ablation was endocardial 21. Ablation
endpoint was also declared if, following the last ablation, all sites on the epicardial
surface of the right atrium revealed an activation time that was late compared to
the onset of the P wave, indicating shift of the leading pacemaker to the interatrial
septum or to the left atrium, where the epicardial setup did not allow access.
Following completion of the ablation procedure the pacemaker leads were attached
to pacemaker generator, which was set to dual chamber pacing in the first 2
experiments and to single chamber stimulation (atrium only) for the rest. A pacing
rate of 60- 80 beats per minute was chosen until the next morning, in order to
stabilise the haemodynamics of the animals in the immediate postoperative period.
The thoracotomy wound was closed in layers. The animals were taken back to their
pen, where they were closely monitored until they were able to stand.
Page | 57
Figure 17: Irrigated ablation catheter
The same catheter used for mapping and ablation. The mapping occurred between electrode 1-2 and 3-4. The radiofrequency ablation energy was applied between the tip electrode (1) and the large surface area patch electrode placed on the goat left flank (not shown). Continuous irrigation cooled the tip via the plastic tubing during power delivery.
Figure 18: Ablation in vivo
Single ablation burn (red circle) at the earliest activation within the sinus node; insert: schematic diagram of the posterolateral right atrium with the ablation burn (red dot). Reference electrodes (a unipolar silicone lead and a blue temporary pacing wire are also shown.
Page | 58
Figure 19: Flow chart of epicardial total sinus node ablation.
SEA: site of earliest activation
*We were unable to map the new SEA if it shifted away from the right atrial free wall to a site inaccessible to epicardial mapping, i.e. a location in the interatrial septum or left atrium.
Ablation at SEA
Visible lesion and
90% reduction of local electrogram
30s
no
50% HR reduction and/or
Junctional rhtyhm
yes
no
Remapping for new SEA
New SEA found on the right atrial epicardial surface
yes
Ablation endpoint
no
yes
Page | 59
2.5.3 Follow-up Period
On the first or second postoperative day, all pacemakers were reset to 30 beat/min
demand pacing, in order to promote intrinsic rhythm to emerge and stabilise. Once
the rhythm stabilised I programmed the pacemakers to sensing only mode, in order
to be able to observe signs of SND.
All experimental goats were followed up for 4 weeks, before the second procedure
(mapping for the subsidiary atrial pacemaker) took place. Single lead ECGs were
recorded weekly. Corrected subsidiary atrial pacemaker recovery time (CART) was
determined in the resting awake state (section 2.6.2).
2.5.4 Mapping of the Subsidiary Atrial Pacemaker
The perioperative steps leading to exposure of the right atrial free wall were the
same as during the first procedure (section 2.5.1). Once the right atrium was
visualised and the pacing leads disconnected from the pacemaker generator 5min
ECG as recorded and CART was determined in the anaesthetised state, then
mapping of the SAP took place. In principle, this was performed by the same
method as during SN mapping. The site of earliest activation was then marked with
fine surgical sutures; 2mm cranial and caudal from it, the same way as the SEA of SN
was marked in the control group.
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2.6 Methods of functional characterisation
2.6.1 Surface electrocardiogram
Single lead ECG recording was performed in all goats (n=15) in a resting non-
anaesthetised state, within 24 hour prior to the operation as well as
intraoperatively under general anaesthesia. In the experimental group following the
ablation of the SN (Procedure I), ECGs were recorded repeatedly weekly over the 4
week follow-up using the same technique.
The leads were placed on a shaved area above the bony prominence on the right
and left shoulder and the left hip. Bipolar recordings of 30s were made
consecutively between left to right shoulder, left hip to left shoulder and, left hip to
right shoulder; analogous to the surface ECG leads I, II and III in human. Then a
continuous 5 min recording was created using the left hip- right shoulder bipole
(lead II in human). The reason for choosing this particular lead was that the electric
vector of the atrial activation is mostly parallel to this lead and therefore produced
the highest amplitude P wave (the manifestation of atrial depolarization on the
surface ECG) allowing accurate assessment of the timing and morphology of the P
wave113. The leads were connected through a bioamplifier to an analogue-digital
converter (PowerLab Data acquisition system ADInstruments) and displayed on a PC
using LabChart software (ADInstruments). Beat to beat heart rate was calculated in
a consecutive 4 min of recording and cleaned from noise/ baseline movement with
a combination of digital filtering (1-100 Hz-band pass filter) and by rejecting
distorted sections using a semi-automatic method (Beat Classifier, LabChart
ADInstruments). The mean PR interval was calculated from 30 consecutive beats. A
beat averaged ECG were created from the same 30 beats for qualitative analysis of
the morphology of the P wave.
2.6.2 Overdrive pacing, SN and SAP recovery time
The healthy SN is very resistant to overdrive suppression. The time required for the
SN to resume activity following fast atrial pacing (SNRT) is short; and its corrected
value, the CSNRT (see later), is less than 525ms in human and comparably short in
dogs93,114. This natural protective mechanism prevents prolonged SN pause, and its
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potentially catastrophic consequence, following termination of any rapid
arrhythmia. It is thought, that the almost complete lack of INa current is responsible
for this phenomenon7. In contrast, the SAP or other ectopic pacemakers arising
from the AV junction, Purkinje fibres or ventricles have been shown to be
susceptible to suppression by overdrive pacing, sometimes extremely so 92,93,115.
Two temporary epicardial unipolar electrode (control group) or a single permanent
bipolar pacing lead (experimental group) were sutured to the right atrial
appendage. The leads were placed 5 mm apart, in order to be able to record
intracardiac, local atrial electrograms as well as to perform myocardial stimulation.
Sinus node recovery time (SNRT) was assessed using Narula’s method 116. Briefly,
the right atrium was stimulated by a pacing system analyser (Medtronic Inc.)
through the epicardial electrodes for 30 seconds at different cycle lengths of drive
trains (600, 500, 450, 400, 350 ms). Upon cessation of pacing the interval between
the last paced beat and the first return of the sinus beat (SNRT) was measured 3
times at each cycle length and averaged.
The CSNRT was obtained by calculating the difference between SNRT and the base
sinus cycle length (figure 20). In other words, CSNRT measures the extra time
required for the resumption of SN activity compared to what would be expected if
there were no overdrive suppression. The longest of the averaged CSNRT value was
taken as the maximal CSNRT.
In the experimental group (n=10) corrected recovery time of the subsidiary
pacemaker (CART) was measured using the same method described above. I
assessed for CART weekly in the postablation period in the conscious animals and
during the second SAP mapping procedure under general anaesthesia.
Page | 6
2
Figure 20: Calculation of the sinus node recovery time.
The SNRT is calculated by measuring the time interval between the last atrial pacing spike (red arrow) and the onset of the atrial depolarization (P wave marked with green arrow). The P wave onset is the best surrogate for the resumption of SN activity on a surface ECG.
The corrected sinus node recovery time (CSNRT) is calculated by subtracting the pre-pacing basic sinus cycle length (BSCL) from the sinus node recovery time (SNRT). The ECG used for illustration is from one of my experiments.
Page | 63
2.6.3 Monitoring the mean heart rate
Between the ablation of the SN and mapping the SAP, the experimental group had a
4-week follow-up period. Besides the weekly single lead ECG recording and CART
assessment described in the previous sections, the animals were also monitored by
downloading data from their pacemaker producing a heart rate histogram reflecting
the distribution and variation of the heart rate for each week (figure 21)
2.6.4 Implantable loop recording
Dysfunction of the SN may not be obvious at any point in time; sinus pauses, sinus
arrest can be easily missed if the rhythm is not assessed continuously. Although the
implanted pacemaker provides information regarding the mean heart rate, and
detects even short, significant drops in heart rate and/or development of asystole
of a few seconds duration, but not designed to record this information.
To overcome this limitation, an implantable loop recorder (Reveal Dx, Medtronic
Inc.) was inserted at the end of the first procedure in three of the experimental
animals. On the opposite ends of the recorder there are two electrodes acting as a
bipolar recorder, capable of recording a 1 minute long ECG, when a predefined
criteria is met, which I chose to be either lower 30 beats/min or a pause longer than
2 sec duration (figure 22). The recorder was simply placed in a superficial
subcutaneous pocket overlying the heart.
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Figure 21: Heart rate histogram
The implanted pacemakers can be wirelessly interrogated and the automatically created heart rate histogram downloaded. Mean heart rate than can be calculated from the histogram.
Figure 22: Implantable loop recorder
A typical recording made by the loop recorder. The single lead ECG depicts a significant sinus arrest of more than 3 s duration. A picture of the loop recorder itself shown to the right.
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2.7 Right Atrial Tissue Processing
2.7.1 Harvesting and dissection
At the end of the in vivo experiment, the animals were humanely killed with a lethal
dose of pentobarbitone injection. The control group reached this stage at the end of
the first procedure following mapping and determining the SNRT. The experimental
group had undergone a 4-week follow-up period and a second surgery in order to
map the SAP before their heart was harvested.
The IVC and SVC had been ligated before all the large vessels been cut to make sure
the proximal portion of both of these vessels were included in the tissue obtained.
The heart was then quickly excised and placed in ice-cold Tyrode solution.
Dissection of the harvested heart was carried out in the protection of a
continuously refreshed ice-cold Tyrode solution bath to keep the environment
physiological in terms of electrolyte balance and pH, but to keep metabolism and
tissue degradation at the minimum.
First, the heart was separated from the remainder of lung and adjacent mediastinal
tissue then the distal 2/3 of the ventricles were cut off. The right ventricular outflow
tract and the aorta were opened with two separate longitudinal incisions which
allowed careful removal of the entire right and left ventricular musculature. The
right interatrial septum was separated from the aortic root anteriorly and from the
left atrium posteriorly, and the left atrium was removed from the right by cutting it
around the foramen ovale and dissecting the right sided pulmonary veins off the
surface of the sinus venosus leaving only the right atrial tissue within the specimen.
The tip of RA appendage was removed and the RA opened longitudinally through
the anterior aspect of the IVC-RA-SVC continuum. The final preparation contained
the crista terminalis in the middle, atrial musculature to the left, the Intercaval
region and interatrial septum to the right and remnants of SVC and IVC at the top
and bottom (figure 23). This dissection method is widely used in our research group
and the resulting tissue orientation was very similar to that of obtained in the
human, rat and rabbit17,98,107. However, I realised using histology (see later) that this
type of dissection in the goat may exclude part of the SN/SAP, hence I modified the
Page | 66
dissection method by keeping both the IVC and SVC as well as the whole sinus
venosus and its cavity intact. The modified preparation was more suitable to
characterise the spatial relationship of the SN and SAP (figure 21). The tissue was
then divided perpendicular to the terminal crest into 2 segments and to get
manageable sized pieces for later cryosectioning. Some of the remaining right
atrium, mainly a portion of the interatrial septum and the right atrial appendage
was discarded. Submerging the preparation in isopentane previously cooled to -
50℃ in liquid nitrogen froze the tissue immediately and without tissue cracking.
They were then stored at -80 ℃ in a refrigerator until further use.
2.7.2 Cryosectioning
The right atrial preparations were mounted on a metal plate, placed into the
chamber of the cryostat (Zeiss) and warmed to -17-20℃. The cutting temperature
range was set to avoid smearing of the tissue (too warm) or excessive curling and
fragmentation (too cold). The tissue was oriented in a way that sections were cut
perpendicular to the crista terminalis. For each mm of the block, 4 and 20
consecutive sections were made at 10um and 20um thickness respectively and
placed in pairs on Superfrost plus microscopy slides. The Superfrost plus slide
provides a positively charged surface for quick tissue adhesion with a reduced
chance of deformation. The slides were then stored at -80℃ until used.
Sections corresponding to the region of the earliest activation within the sinus node
in the control animals and to the subsidiary atrial pacemaker location in the
experimental animals were marked. The resolution of the mapping system is 5mm,
therefore 5 consecutive mm of sections were considered as potentially containing
the leading pacemaker within SN/SAP.
One atrial tissue from the control group was serially sectioned in its whole length in
order to be able to use it for 3D reconstruction of the SN region.
Page | 6
7
Figure 23: Right atrial tissue preparations
Panels A,B,C show SN preparations with endocardial side up in the rat ,human and goat. Note the similar configuration. CT/TC: crista terminalis, IVC:inferior vena cava SVC: superior vena cava, SN: sinus node, RAA: right atrial appendage, RA: right atrium. Panel D: Modified dissection in the goat, epicardial side The whole of the sinus venosus and caval veins remain intact..Panel A and B adapted from 17,107.
A B
1 cm
SN
RAA
SVC
IVC
C
SVC
IVC
RAA
D
Page | 68
2.8 Methods of tissue characterisation
2.8.1 Histology
I used Masson’s trichrome histology staining. The staining solutions were prepared
fresh by myself using commercially available ingredients. Sections were removed
from storage and were placed into Bouin’s fixative (Sigma-Aldrich) overnight. The
Bouin’s solution contains formaldehyde as fixative and picric acid and acetic acid to
enhance nuclear staining. The sections were rinsed in 70% ethanol three times to
precipitate the excess pycric acid before the staining process started. The slides
were first stained with Celestine blue, rinsed in distilled water and then immersed
in haematoxylin for 10 min. Haematoxylin stains nucleic acid, and therefore the
nuclei, dark blue; whereas Celestine blue increases the permanency of the nuclear
stain. Acid fuchsin was applied for 3 min as the next step that stains the cytoplasm
and collagen pink. In order to differentiate collagen, phosphomolybdic acid and
methyl blue solutions were used; the former competitively expels acid fuchsin from
collagen and the latter stains them royal blue (figure 24) 117. The stained sections
were rinsed in distilled water, 1% acetic acid and dehydrated in ethanol of
increasing concentrations (70% 1min, 90% 1 min, 100% for 2x2 min). Finally,
samples were bathed for 10 min in clearing agent (Histoclear, National Diagnostics).
Coverslips (Menzel-Glaser) were fixed to the slides using DPX (Prolabo) mounting
medium.
2.9 Light microscopy
For detailed analysis of cell morphology within the SN, SAP and RA I used traditional
light microscopy with a motorised stage (Zeiss) utilising a high power magnification
(63x) immersion objective (figure 24). However, to precisely delineate the
boundaries of the SN, SAP and PNA, visualisation of a much larger, yet relatively
high resolution field of view is necessary. A panoramic, automated light microscope
was used for this purpose creating a single digital slide at 20x magnification
covering the whole section (3D Histech). The bundled software (Panoramic Viewer)
allowed me to freely and seamlessly pan and zoom at any area of interest (figure
25).
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Figure 24: Masson’s trichrome histological stain
High power image from the right atrium of the goat (Zeiss 63x). The nuclei of the myocytes and fibroblasts are dark blue, the cytoplasm is pink and the connective tissue is royal blue (light blue).
Figure 25: Digital slide
The whole section has been digitalised creating a very large, moderate magnification image by automatically stitching individual field of views taken at 20x magnification. Despite the large area visualised, fine details are retained as it is demonstrated by the inset picture (bottom right) displaying the area corresponding to the black rectangle on the low magnification image.
100 µm
2 mm
50 µm
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2.10 Immunohistochemistry
In principle, immunohistochemistry is a technique to detect the presence of a
protein (antigen) by using antibodies (Ab). The antibodies binds to the target and
are conjugated with a marker system either directly or indirectly via a secondary Ab.
The protein-Ab-marker complex then visualised at the site of the reaction, making
possible to investigate protein localisation, their relationship to cell structures or
other proteins if double labelling is used. The marker can be an enzyme that
produce a local reaction detectable by light microscopy (immunoenzyme), or a
fluorochrome that emits light when excited detectable by a fluorescent microscope
(immunofluorescence) (figure 26). I choose the indirect immunofluorescence (IF)
method due to its ability to perform dual labelling with ease, and its superior
resolution when used in conjunction with confocal laser scanning microscope.
Semiquantification of the labelled protein is also possible using IF (see later).
Figure 26: Principle of immunofluorescence
The primary antibodies (10Ab) bind to their specific target antigens and labelled with a fluorochrome either directly (left panel) or via a secondary Ab (20Ab) (middle panel). It is possible to use two different 10Ab raised in different species on the same specimen in order to visualise two different antigens at the same time.
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2.10.1.1 Primary antibodies
Primary antibodies are immunoglubulins that bind to the antigen; in case of a
protein, the region responsible for the antigenicity called the epitope. A protein
molecule may have several epitopes that can bind different antibodies. The
antibodies can be monoclonal or polyclonal. A monoclonal antibody is produced by
hybridoma cells, that are immortalised and cloned B cells, capable of producing only
one type of antibody, therefore can bind to only one epitope giving a very specific
staining. Polyclonal antibodies are produced in vivo, by injecting the antigen into a
host species, and obtaining and purifying antibodies developed by the immune
system of the host. Polyclonal antibodies will target multiple epitopes of the same
protein; they may be able to label a protein even if some of the epitopes are
unavailable (confirmation change during fixation), hence polyclonal antibodies have
higher sensitivity to detect the target and give more intense signal, at the expense
of a potentially lower specificity.
I used both monoclonal and polyclonal commercially available primary antibodies in
this study (table 1).
2.10.1.2 Secondary antibodies
The secondary antibodies were all raised in a host species different from that of the
primary antibodies were obtained from (table 2). For example, a secondary Ab
raised in donkey against a primary Ab raised in mouse targeting connexin 43 . Their
target is the non-variable region of the primary antibody; the same secondary
antibody can be used in conjunctions with different primary antibodies raised in the
same species. All secondary antibodies were conjugated with either fluorescein
isothiocyanate (FITC) or indocarbocyanine (Cy3) fluorochrome (table 3)
Page | 72
Primary antibodies used
target source isotype class manufacturer product No
HCN4 rabbit IgG polyclonal Alomone Labs APC-052
NCX1 mouse IgM monoclonal Thermo Scientific MA3-926
SERCA2 mouse IgG monoclonal Thermo Scientific MA3-910
RYR2 mouse IgG monoclonal Thermo Scientific MA3-916
Cx43 rabbit IgG polyclonal Sigma C6219
Cx43 mouse IgG monoclonal Millipore MAB 3068
HCN4 rabbit IgG polyclonal Millipore MAB 5808
Table 1: List of primary antibodies used
Secondary antibodies used
target source fluorochrome conjugate manufacturer product
No
Mouse IgG donkey Cy3 polyclonal Millipore AP192C
Mouse IgM donkey FITC polyclonal Sigma F9259
Rabbit IgG donkey FITC polyclonal Millipore AP182F
Table 2: List of secondary antibodies used
Fluorochrome absorption Peak emission peak
FITC 492 nm 520 nm
CY3 550 nm 570 nm
Table 3 Properties of fluorochromes used
FITC= fluorescein isothiocyanate, Cy3= indocarbocyanine
Page | 73
2.10.1.3 Immunofluorescence staining protocol
Most IF experiments were carried out according to an established protocol widely
used in our laboratory59,80,107. Slides were removed from the freezer, allowed to dry
for 1 min and tissue sections demarcated with a hydrophobic pen (Sigma-Aldrich).
The sections were then immersed in 10% buffered formalin for 30 min to fix the
tissue and rinsed three times in 0.01M phosphate buffered saline (PBS).
Permeabilisation of the membrane was achieved with detergent (0.1% Triton-X100,
Sigma-Aldrich) followed by repeated rinses in PBS. In order to reduce non-specific
binding the preparations were treated with 1% bovine serum albumin (BSA).
Primary antibodies (diluted in a range of 1:50-1:800) were carefully applied within
the boundaries of the circles created by the PAP pen and incubated overnight at 4
°C degree. Following the incubation period antibodies were removed and recycled
for further use. The slides were washed in PBS three times before and after the
secondary antibodies applied and incubated for 90 min at room temperature. FITC
conjugated antibodies were diluted to 1:100 and Cy3 conjugated ones to 1:400.
Finally, the stained slides were mounted in Vectashield ant-fade medium
(Vectorlabs) covered with coverslips and sealed with nail varnish.
Methanol fixation was used as an alternative approach, when the usual protocol did
not result in specific staining. The slides were air-dried for 5 min and fixed in
absolute methanol for 5 min at -20 °C. Methanol also permeabilise the cell
membrane, so the detergent step was omitted and the slides from that point on
were prepared the same way as the formalin fixed sections.
2.10.1.4 Immunofluorescence microscopy
The slides were kept in a refrigerator at 4 °C in the dark after staining to avoid
fading of the fluorochrome. I used a confocal laser scanning microscope (Zeiss
LSM5) to visualise the immunostained sections at a magnification range between
10-63x. The confocal microscope has the advantage over epifluorescent
microscope, that is the ability to focus only 1-3 µm thick part instead of the full
thickness of 20 µm. The thinner plane of focus results in higher quality image due to
Page | 74
the lack of superimposition of structures in the z plane. The scanning laser excites
all fluorochromes in the line of the light, but only those emitted from the desired
depth are allowed to reach the detector through a pinhole placed in front of the
detector. The microscope was fitted with 2 different laser to match the excitation
peak of the two fluorochrome (FITC,CY3) used for labelling.
For selected slides I also used the same panoramic microscope as for histology, but
equipped with an epifluorescent objective (3D Histech). The technique has the
advantage of visualising the whole immunofluorescent section at moderate
magnification. Comparison between sister slides prepared for IF and light
microscopy allowed studying the extent of the SN , PNA and SAP more precisely.
2.10.2 Semiquantitative immunohistochemistry
Slides obtained from the leading pacemaker sites in the experimental group and the
control group underwent IF staining protocol as described above. Even
commercially available antibodies show significant difference in intensity and
specificity of the staining when the antibodies used are from different batches of
production, especially if the antibodies are polyclonal. Furthermore, specimens
fixed and permeaibilised in different experiments may react with the antibodies to
more or less extent making direct comparison unreliable.
The sections in the semiquantitative experiments, therefore, were of equal
thickness and I stained them simultaneously using the same batch of primary and
secondary antibodies. The slides were imaged within one day using confocal laser
microscope. The size of pinhole, the energy output of the lasers were all kept
constant, in this way the resulting amount of immunofluorescence is directly
proportional to the amount of protein expressed in the given tissue, thus allowing
semiquantitative analysis of the target protein59. High power images were
digitalised at 10-bit colour depth and loaded into the analysing software (Volocity),
which measured the sum of pixel intensities expressed on an arbitrary scale. The
values were compared by one-way ANOVA statistical analysis.
Page | 75
2.11 3D anatomical reconstruction
One right atrial specimen was used to create a 3D reconstruction of the goat SN,
PNA and the surrounding right atrial myocardium. The right atrium was dissected
and frozen as previously described (section 2.7). However, before cryosectioning,
the whole block of tissue was placed into a very high-resolution computer
tomography scanner (Manchester X-ray imaging facility). The block was mounted on
a heat sink, which was placed in a bowl of dry ice and surface temperature was
monitored to maintain frozen state during the scan (at least -20 C ,to prevent
protein degradation). The raw data was uploaded to a 3D reconstruction software
(Avizo) where the first step of reconstruction was made by rendering a 3D volume
and creating 2000 grayscale virtual slides. The virtual slides had the same
orientation as the later cryosectioned true sections.
The whole specimen were then cryosectioned at 0.5mm intervals at 10 µm
thickness and stained with Masson’s trichrome histological stain. A total of 112
sections were processed. Digital slides were created of all sections (3D Histech).
Where the morphology of the preparation changed significantly an additional slide
was stained using IF double labelled with HCN4 (marker of sinus nodal cells) and
Connexin43 (marker of atrial myocardium).
During cryosectioning, a small degree of deformation is unavoidable, therefore all
section were aligned to its computer topographically created virtual counterpart
using elastic alignment (Image J). The SN and PNA was manually delineated in each
sections (Panoramic Viewer) and the sections are placed into a virtual stack, re-
aligned and the volume of SN, PNA and the outline of each section rendered (Image
J, TrackEM2 plugin). Finally, a movie was created visualising the spatial relationship
of the SN and PNA within the RA.
Page | 76
3 Functional characterisation of the SN and SAP
3.1 Site of earliest activation within the SN
In each animal the SEA was determined, either before harvesting the atria (control
group n=4) or before ablating the SN (experimental group n=11). Previous studies in
the dog, rabbit and human have consistently demonstrated that SEA of SN is
commonly originating from a confined area at the junction of the SVC and the crest
of the RA7,18,19,118. In the goat, the SEA was identified from within a 1.5 cm2 area at
the SVC-RA junction and its location did not differ in the control and experimental
group (figure 27).
3.2 Acute Success of Sinus Node Ablation
Ablating the sinus node is a time consuming procedure for multiple reasons. The SN,
as previously described (section 1.3), is an extensive structure requiring a large area
to be destroyed; in a human study, using non-contact mapping and endocardial
ablation, an average of 25 lesions had to be applied to achieve subtotal SN
abaltion118. The SN is not only large but also shielded from radiofrequency energy
by a layer of atrial myocardium as well as by the heat sink effect of high blood flow
within the central SN artery22,119. Finally, mapping the SN potential requires a low
“high-pass” filter setting, when catheter stability is paramount to avoid baseline
shift, making the mapping process laborious (section 2.5.1).
The acute endpoint of 50% reduction in heart rate or a pacemaker shift away from
the epicardially available right atrium has been achieved in all experiments (table 4).
The mean procedure time was 286±78 min (range: 165-455min), there were 40±27
(range: 18-77) lesions created and the mean time of power delivery was 26.8±20
min (range: 5.1-67.7min). There was one major complication: one animal developed
treatment refractory ventricular fibrillation at the end of the procedure and had to
be humanely killed. Paroxysmal atrial fibrillation developed in two animals, one of
which required direct current cardioversion and one goat had its right atrium
perforated and had to be treated with a surgical suture. All, but one animal (VF
arrest) recovered from the surgery (n=10).
Page | 77
Figure 27: Location of the site of earliest activation in the intact sinus node
Each SEA in the experimental group (left panel) and the control group (right panel) are marked with X on the diagrams. The area containing all SEA (middle panel, shaded area) was only 1.5 cm2.
No (n=11)
Reduction in HR (%)
post ablation rhythm endpoint
1 67 junctional >50% HR
2 59 junctional >50% HR
3 50 atrial >50% HR
4 52 atrial >50% HR
5 36 atrial pacemaker shift
6 100 none >50% HR
7 32 atrial pacemaker shift
8 53 atrial >50% HR
9 69 atrial >50% HR
10 50 atrial >50% HR
11 53 atrial >50% HR
Table 4: Acute outcome in the experimental group following ablation of the sinus node
Predefined endpoint was achieved in all cases. In six cases, ablation was ended when 50% rate drop was observed with atrial escape rhythm; in three cases no atrial escape rhythm was observed. On two occasions only modest heart rate reduction was achieved due to a pacemaker shift away from the right atrial free wall where further mapping and ablation was not possible (animals no 5 and 6).
SVC
RA
IVC
0 -1 -2 -3
1 2 3
A B C D
RSPV
RIPV
E
4
X
X X X
SVC
RA
IVC
0 -1 -2 -3
1 2 3
A B C D
RSPV
RIPV
E
4
X X X X X
X
X
X
X X 1.5 cm2
Page | 78
3.3 Location of the Subsidiary Atrial Pacemaker
After 4-week follow-up, during the second surgery, the location of the leading
pacemaker was mapped. I was able to map SEA in 9 out of 10 animals. In one
experiment, there was no signal found satisfying the criteria of being premature to
both the intracardiac reference electrode and to the onset of the P wave on the
surface ECG, a finding that can only be explained by a new SEA emerging from the
interatrial septum or the left atrium. The schematic diagram in figure 28 shows the
areas where the sites of earliest activation were distributed to. In a total of five
experiments the location of the emerging SAP was very different from that of the
sinus node and it was clustered along the caudal part of the crista terminalis
adjacent to the IVC. Similar location has been shown as the site of the leading
pacemaker in previous experiments in the dog (in vivo) and the rat (in vitro) and
also in mapping a human patient with sinus node disease92,107,120. The remaining
four goats had their SEA in an area identical to where all the pre-ablation leading
pacemakers had been situated, suggestive of recovery of the sinus node itself.
Failure to totally ablate the SN in 40% of the experiments was not unexpected. In
the dog, Kalman et al. found a cranial shift of the SEA several weeks after ablation
compared to the acute result21. In human studies, carried out in patients with
inappropriate sinus tachycardia, the late clinical recurrence rate was between 23-
70% following sinus node modification22,121–123. Besides the explained difficulties of
SN ablation (section 3.2) there may be another issue when making a very extensive
ablation set; the more ablation one performs on the same area of tissue, the more
tissue oedema develops, thus preventing subsequent burns to reach their intended
target resulting in incomplete tissue necrosis124,125. The total area ablated was not
different within the experiments, and were confined to the cranial half to cranial
2/3 of the posterolateral RA adjacent to the CT by visual inspection. However, I
observed a trend (p=0.09) of inverse correlation between the total time of ablation
power applied and the success of SN ablation (figure 29). In one experiment, where
SEA remained within the SN, spontaneous action potentials were recorded in vitro
from the excised SN (figure 30 , Dr Sunil Logantha). This can demonstrate that, even
after ablation, some SN tissue can remain viable.
Page | 79
Figure 28: Schematic diagram of the locations of the site of earliest activation
Left panel: The shaded squares correspond to regions of the right atrium where the individual sites of earliest activation emerged from. There are two well separated areas, one is close to the junction of the inferior vena cava and right atrium (black) and the second situating in the RA/SVC junction (dark grey) SEA of 2 more goats were not obtained (1 lost during the ablation and 1 had SEA outside the accessible epicardial RA surface, see text)
Right panel: Location of SEA prior to ablation in all experimental goats (n=11). The shaded area (light grey) is in the same anatomical region as that of the group of animals with recurrence of SN function in the left panel.
Figure 29: Total ablation time and success of SN ablation
Goats with an emerging new SEA showed a trend of shorter ablation time (14.16 ± 3.64 min, n=5) compared to those with SEA within SN (35.60 ± 11.83 min, n=4). (unpaired Student’s T-test, p=0.09). Excessive ablation may have caused tissue oedema preventing effective power delivery.
Page | 8
0
Figure 30: Spontaneous pacemaker activity from the apparently ablated SN
The photos on the right side show a right atrial preparation of a goat with postablation SEA within the SN (rSN). The tissue was perfused with 37 °C Tyrode solution and sharp, glass microelectrodes inserted into individual cells. On the left hand side there are intracellular electrograms obtained from two slightly different locations, both within the ablated area (dashed white line) and identical to where the SEA was mapped in vivo (white circle). Spontaneous diastolic depolarization was obtained consistent with pacemaker activity. The experiment was performed by Dr. Sunil Logantha.
Page | 81
3.4 Electrocardiographic parameters
3.4.1 Heart rate before ablation
ECGs were taken prior to ablating the SN, both in the conscious goats as well as
under general anaesthesia (GA) and 4 min recordings were analysed (see section
2.6.1).
The mean HR in the conscious and anaesthetised animals were 86.7±19.9 beat/min
(bpm) and 83.9±14.6 bpm respectively, similar to that reported in the
literature126,127
Although the mean HR did not differ significantly between the conscious and
anaesthetised goat, the heart rate variation revealed marked differences. Under the
influence of the inhalative anaesthetic isoflurane, the beat to beat HR was uniform
with a narrow deviation around the mean HR. In contrast, HR recorded in the
conscious state showed a more dispersed distribution (figure 31). The observed
significant difference can be explained by two mechanisms. Under GA with
Isoflurane, both the cardiac parasympathethic and sympathetic activity is directly
depressed, resulting a decreased heart rate variability, as shown in the rat, dog and
human studies128–130. The interaction of these opposing forces is complex, for
example, anaesthetic also had an effect on the HR, that was dose and time
dependent and in HR can change in both direction in the cat131. On the other hand,
the conscious ECG was recorded in the presence of a human operator, who has a
profound and variable effect (observer effect) on the autonomic balance of the
individual goats 132,133.
Despite the difference in the HR distribution, the mean values in conscious and
anaesthethised animals correlated well comparing the two methods; linear
regression analysis revealed a significant correlation (figure 32).
Page | 82
Figure 31: Heart rate variance in conscious versus anaesthetised goat
Heart rate histogram computed from continuous 4 min ECG in the same goat, same day prior to ablation. The conscious recording (left) has a much wider beat to beat variation compared to the measurements taken under general anaesthetic. The dispersion of data points in the conscious goat is visually obvious, and confirmed by a markedly higher standard deviation.
0 50 1000
50
100
R2=0.77
HR in concious goat
HR
un
der
GA
Figure 32: Heart rate correlation in conscious and anaesthetised state prior to ablation
Linear regression analysis shows strong correlation of the mean HR in anaesthetised and conscious state (Pearson’s linear regression, R2=0.77, P=0.008).
Anaesthetised
60 80 100 120 1400
10
20
30
40
Heart Rate (bpm)
Fre
qu
en
cy (
%)
Concious
60 80 100 120 1400
1
2
3
4
Heart Rate (bpm)
Fre
qu
en
cy (
%)
Page | 83
3.4.2 Heart rate changes following ablation of the SN
In the immediate postoperative period (48 hours), ECG recordings were unsuitable
for analysis, as the goats required pacing to provide a stable rhythm aiding
recovery. The electronic pacemakers were checked daily in order to reprogram it to
a demand pacing mode at 30 bpm and as soon as it was safe to do so, i.e. when the
animals showed a stable intrinsic rhythm (section 2.5.3). All goats became
independent from their electronic pacemaker within 2 days (table 5).
In addition to the 4 min ECGs in the conscious/anaesthetised animals, the HR was
also evaluated by the telemetry facility of the implanted pacemaker(ppm) (section
2.6.3). The obvious advantage of this approach is the ability to assess the HR long
term in the resting animal and without being observed. The data provided by the
pacemaker are likely the most physiological representation of the HR as opposed to
the 4 min ECG methods. However, the pacemakers were inserted during the first
procedure, hence direct comparison of the pre- and postablation HR was not
possible. The telemetry data was, therefore, only used to observe HR changes over
the course of 4 weeks and to assess for atrial arrhythmias.
To test the suspected disagreement between the methods of HR assessment, linear
regression analysis was performed. Unlike in the goats with an intact SN, in the
ablated animals there was a very weak correlation between the HR assessed under
GA compared to both methods of conscious recordings (figure 33). The 4 min
conscious HR and the pacemaker telemetry data, however, showed an almost
perfect correlation (figure 33). It has been shown in the dog that the SAP is much
more reliant on intact sympathethic/parasympathethic innervation than the SN;
anaesthesia can cause both enhanced automaticity of the SAP as well as depression
of its pacemaker activity130,134. In my own experiments I observed sudden onset of
junctional rhythm in one goat at the beginning of anaesthesia, whilst in another
case an already detected paroxysmal atrial tachycardia became more prevalent
during the procedure under GA.
Page | 84
goat No Days after ablating the SN
day 0 day 1 day2
1 100 % paced 100 % paced free
2 100 % paced 100 % paced free
3 AF 50% paced free
4 100 % paced free free
5 AF 70% paced + 30% AF free
6 100 % paced 10% paced free
7 100 % paced 15% paced free
8 100 % paced 100 % paced free
9 100 % paced free free
10 100 % paced 80% paced free
Table 5: Dependence on electronic pacemaker
Following ablation of the SN the pacemakers were set to 80 bpm atrial pacing. Apart from the two cases of fast (>80 bpm) postoperative atrial fibrillation (AF) the goats were paced continuously on the day of the surgery. On the first postoperative day the dependence on pacing decreased and eventually all animals developed an intrinsic rhythm sufficiently stable to allow turning the device to 30 bpm on-demand pacing. (Black cells: pacemaker turned off; grey cells: intermittent pacing; white cells: no pacing).
0 50 100 1500
50
100
150
R2=0.18
HR under GA
Co
ncio
us 4
min
HR
0 50 100 1500
50
100
150
R2=0.32
HR under GA
HR
fro
m p
pm
tele
metr
y
0 50 100 1500
50
100
150
R2=0.79
concious 4 min HR
HR
fro
m p
pm
tele
metr
y
Figure 33: HR correlation in the SN ablated state
The heart rate was assessed in post ablation state via three different methods (see text). Strong correlation was found only between the short term vs. long term HR measured in the conscious animal (R2=0.79 , P=0.0002)
Page | 85
As previously discussed, immediately following the ablation of the SN, the mean HR
was reduced by at least 50% in the majority of the experiments (section 3.2).
However, the atrial rhythm quickly recovered and no statistically significant
difference was observed between the preablation and postablation state, both
when measured during the first and second procedure or in the resting awake
animals (figure 34). When assessing the HR separately in the animals where the
leading pacemaker was located outside the SN area (SAP group n=6) and for those
where SN recovered (rSN group; n=4) a significant difference was still not observed.
The result is surprising as it is generally accepted that the SN has the most rapid
firing rate in the hierarchy of atrial pacemakers. Jones et al. excised progressively
bigger portions of proximal posterolateral RA in the dog demonstrating a steady
decline in the HR generated by the remainder of the pacemaking tissue135. In vitro
studies in the rat, where a SAP was created by separating the SN from the RA, also
showed a significant HR reduction97,107. However, these studies were designed to
assess short term HR changes (immediate to 2 days), the exact same period when I
also observed significant bradycardia in the goats. Two long term experiments in
the dog, using similar protocol to this current work, revealed a rapid restitution (2-4
days) of the normal HR which remained comparable to the SN rate in the first 3-4
weeks and only showed a significant reduction 4 to 8 weeks postablation. Both of
these studies also reported a gradual increase in HR after 8 weeks and Littman et al.
were even able to demonstrate normalisation of the average HR at 6 months20,90.
The only study where the HR fell within 2 weeks employed intracardiac ablation of
the SN21. The postoperative stress is higher and the recovery longer after open
chest SN destruction (either by ablation or excision) resulting in a higher
sympathethic tone that can explain why the HR is not reduced for a short period.
Alternatively or additionally, the cardiac autonomic innervation may be disturbed
by an epicardial approach to ablation and until subsequent re-innervation takes
place the heart remains sensitive to circulating catecholamines. Consistent with the
above studies I also found a significant reduction in mean HR from week 1 towards
week 4 in the goats where the leading pacemaker was located outside the area of
the SN, ie. representing a true SAP (figure 35).
Page | 86
Figure 34 : Heart rate comparison pre- and post SN ablation
There were no significant change observed in HR comparing the baseline HR to the follow-up measurements (Repeated measures ANOVA and paired Student’s T test).
The first row shows HR in the conscious goats prior to ablation and then weekly. The second row displays the HR under GA prior to ablation and at the end of the 4 week Subgroups of animals with new SAP and recovered SN (rSN) are also analysed separately (2nd and 3rd graphs).
Figure 35: Long term heart rate change during the follow-up
Continous HR monitoring provided by the electronic pacemaker telemetry facility indicates a significant HR drop in the third and fourth week of the postablation in the goats where emergence of SAP was found (repeated measures ANOVA, **P=0.0003, ***P<0.0001). No such change was observed in the goats with recovery of SN function.
Conscious state
SAP n=6
preop
wee
k1
wee
k2
wee
k3
wee
k4
0
50
100
150
ALL n=10
preop
wee
k1
wee
k2
wee
k3
wee
k4
0
50
100
150H
R b
eat/
min
rSN n=4
preop
wee
k1
wee
k2
wee
k3
wee
k4
0
50
100
150
rSN n=4
Pre
abla
tion
Post
abla
tion
0
20
40
60
80
100
120
ALL n=10
Pre
abla
tion
Post
abla
tion
0
20
40
60
80
100
120
HR
beat/
min
SAP n=6
Pre
abla
tion
Post
abla
tion
0
20
40
60
80
100
120
Under GA
rSN n=4
Pre
op
Wee
k 1
Wee
k 2
Wee
k 3
Wee
k 4
0
50
100
150
N/A
SAP n=6
Pre
op
Wee
k 1
Wee
k 2
Wee
k 3
Wee
k 4
0
50
100
150
N/A
ALL n=10
Pre
op
Wee
k 1
Wee
k 2
Wee
k 3
Wee
k 4
0
50
100
150
N/A
HR
beat/
min
Long term HR over 4 weeks
** ***
Page | 87
3.4.3 PR Interval
The PR interval measures the time required for the depolarization wave front to
reach the ventricular tissue from the onset of the atrial depolarization. It is a sum of
the intraatrial and atrioventricular nodal conduction times. It was assumed that the
AV conduction would not change following ablation, as the ablation was carried out
very far from the compact AV node, and hence the PR interval was considered a
surrogate for the intraatrial conduction time. The closer the leading pacemaker is to
the ventricle, the shorter the PR interval will become (figure 36). Previous studies
demonstrated that the PR interval when a SAP, is active rather than SN is indeed
shortened136.
The PR intervals were measured in each experiment pre and post ablation,
averaging 30 consecutive beats, and the significance of the change was assessed by
paired Student’s t-test (figure 37). The PR interval showed a significant reduction
postablation in those goats where the SAP was mapped close to the IVC (135.5±
23.91 ms vs. 117.2± 20.7 ms, p=0.04), consistent with the anatomically different
location of the subsidiary atrial pacemaker compared to the sinus node.
3.4.4 P wave morphology
The P wave is the ECG representation of the atrial depolarization, and within it, the
first half predominantly corresponds to the activation of the right atrium. In normal
sinus rhythm, the depolarization wavefront travels from cranio-caudal direction
inscribing a positive deflection on the surface ECG in lead II (section 2.6.1). If the
activation of the right atrium is reversed, ie.caudo-cranial, as is the case when the
leading pacemaker is close to the IVC, then the P wave or at least the first right
atrial component of it is expected to be inverted. I observed such a change in 3 out
of the 6 succesfully ablated animals; the 3 cases where the SAP was mapped the
most caudally (figure 38)
Page | 88
Figure 36: Change in PR interval
The interatrial conduction time is the first component of the PR interval (white arrow) and it shortens when the leading pacemaker is closer to the atrioventricular node (AV). Left: PR interval in the intact SN. Right: PR interval postablation. Middle: corresponding ECGs taken from the same goat pre-and postablation. The PR interval is between the vertical dashed lines.
Figure 37: Comparison of PR intervals pre and post ablation
The PR intervals, as expected, shortened significantly in the SAP group only. (paired t-test p=0.04)
Figure 38: P wave morphology change
There was a significant inversion of the first portion of the P wave (black arrow) postablation in the first goat (top row). No such shift observed in another goat with a recovered SN (bottom row).
All n=10
prepost
0
50
100
150
PR
in
terv
al
(ms)
rSN n=4
prepost
0
50
100
150
SAP n=6
prepost
0
50
100
150*
Page | 89
3.5 SN and SAP recovery time
Overdrive pacing performed from the RA temporarily suppresses the function of the
native pacemaker (2.6.2). Corrected recovery time of both the sinus node (CSNRT)
and the subsidiary atrial pacemaker (CART) were calculated, intraoperatively as well
as weekly in the 4 week follow-up period. The degree of suppression has been
shown to be greater in sinus node dysfunction than in healthy state in human and
an abnormal CSNRT is considered diagnostic for SND 116.
The CSNRT was very short (84.3±16.6 ms) at baseline. In the first week the CART
became more prolonged in all experimental models whose leading pacemaker had
been mapped to the SAP area (n=6). The observed trend of extreme CART
prolongation initially followed by normalisation over 2 to 4 weeks was similar to
that had been published in the literature (figure 39).
Figure 39: Response of SN and SAP to overdrive suppression
The successfully ablated group (n=6, SAP group) showed an extreme prolongation of the CART in the first week of follow-up (p=0.04), which was not observed in the recovered SN group (n=4). From the second week on, the CART values diminished and became no longer significantly longer than SNRT at baseline.
3.6 Long term event recording data
Three animals received an implantable loop recorder (ILR) to detect significant
intermittent bradycardia and/or asystole. In one animal the ILR did not show any
significant abnormalities. In that goat the SEA was located within the apparently
ablated SN (recovered SN). In the other 2 experiment the ILR registered multiple
episodes of significant bradycardia (<30 bpm) and periods of asystole up to 6 s
(figure 40). However, the abnormalities completely disappeared in the third week of
follow-up, suggestive of stabilisation of the SAP.
ALL n=10
pre 1 2 3 4post
0
1000
2000
3000
Reco
very
tim
e (
ms)
rSN n=4
pre 1 2 3 4post
0
1000
2000
3000
SAP n=6
pre 1 2 3 4post
0
1000
2000
3000
4000
5000
Page | 9
0
Figure 40: Asystole and bradycardia in the experimental model
Examples of significant asystole in one goat (top row) and bradycardia in another (bottom row). On the right there are printouts from the ILR, demonstrating multiple episodes of asystole/bradycardia. All of these abnormalities ceased by the third week of follow-up (not shown)
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4 Structural characterisation of the SN and SAP
The morphological analysis were carried out using a combination of histology and
immunohistochemistry (IHC). The addition of IHC has been shown to improve one’s
ability to delineate the boundaries of the pacemaking tissue, when appropriate
negative and positive markers of the SN are used. In addition to histology,
Dobrzynski et al utilised antibodies against Cx43 and ANP as a negative marker of
SN and anti-neurofilament-M Ab (NF-M) as a positive marker to create an accurate
reconstruction of the whole SN in the rabbit98. In a study of the human SN Chandler
et al applied anti-ANP and Cx43 antibodies for the same purpose. The reason for
not using a positive marker was that NF-M does not appear to stain the SN tissue
specifically in other species than the rabbit. An obvious second choice for positively
identifying pacemaker cells could have been targeting the HCN4 protein that is
responsible for the pacemaker-current, however, HCN4 antibodies are polyclonal
and resulted in a high background staining59.
In the goat, as in human, NF-M did not give specific staining pattern. However,
HCN4 antibody was successfully used by modifying the IHC protocol. With our
standard formalin fixation method I observed a very high background staining that
did not yield reliable results. In contrast, when the tissue was fixed with methanol
the staining pattern and specificity improved (figure 41).The reason for the
increased specificity may be that methanol fixes the tissue without crosslinking,
therefore without potential conformational change that can mask the epitope137.
The permeabilisation of the membrane is also different; this way the intracellular
targets may be more readily available to be bound with the antibody.
Complimentary to histology I used antibodies against-HCN4 as positive SN marker,
Cx43 as a negative marker and NCX1 that stains all myocytes, but does not other
cell types. The IHC staining was applied only selectively, when the morphology
would have been otherwise difficult to assess. IHC was particularly useful in
assessing the PNA and SAP (discussed later).
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Figure 41: Formalin vs Methanol fixation for HCN4 staining
Top row: When the sections were immunostained with anti-HCN4 Ab with methanol fixation the resulting staining pattern was much more selective and the background staining remained acceptable. The expected specific membrane labelling is demonstrated within the SN (top left)
Bottom row: Unspecific background staining of both tissue type with formalin fixation.
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Atrial tissue have been cryosectioned and stained serially at 1 mm intervals with
Masson’s trichrome histological stain according to a protocol described earlier
(section 2.8.1). I processed the RA of nine goats:
SN and PNA preparations: in 4 control goats
SAP preparation: in 4 ablated goats, where the SAP was found
outside the SN + 1 goat where SN function
returned.
4.1 Morphology of the atrial pacemaker complex
4.1.1 Sinus node
Analysis of serial sections confirmed that the sinus node in the goat is similar to
other mammalian species described in the literature (section 1.3.1). The body of the
SN occupies the full thickness of the intercaval region abutting the crista terminalis
(CT) like in the rabbit but does not extend across the sinus venosus toward the
septum more akin to the sinus node in the man or the dog. The large, sinus node
artery runs along the CT, antero-medial to the body of the SN rather than in the
centre of the node as observed in the dog or human. The gross area of the SN can
be visually distinguished from the working myocardium even without magnification
and sometimes even without staining, as it is paler compared to the adjacent
working myocardium (figure 42). The pale appearance caused by a combination of
the surrounding abundant extracellular matrix and the lack of dense myofibrils
within the SN cells. The SN cells are loosely packed, have paler cytoplasmic staining
at high power magnification and not organised in tight bundles like the atrial
myocardium (figure 43). The cell diameter within the SN is significantly smaller than
in the surrounding atrial myocardium (10.1± 0.66µm vs. 13.83±0.56 µm, P=0.008).
It is widely accepted fact, that the SN in all species investigated lacks expression of
the high conductance connexin, that is Cx43 (section 1.4.6). IHC double labelling
with Cx43 and NCX1 confirms the accuracy of the histological stain to delineate the
centre of the SN (figure 44).
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Figure 42: Macroscopic appearance of the SN
Digital histology slide (top) and photograph (bottom) of the SN at the same level in the same goat. The section is perpendicular to the crista terminalis. The area of the SN (within the dotted line) is clearly identifiable on the histology slide by its pale appearance. On the corresponding photograph the SN is recognisable.
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Figure 43: Histology of the sinus node and the surrounding atrial myocardium.
The connective tissue stains royal blue, the cytoplasm pink and the nuclei dark blue.
Left: cross-section perpendicular to the CT at the level of the dashed line on the diagram. The SN occupies the full thickness of the intercaval region (outlined with red); Between the SN and the working atrial myocardium is the paranodal area (PNA) which is thin an arbitrary at this level.
Right Panels from top to bottom: High magnification images of the atrium, PNA and SN. The SN cells are smaller, paler and embedded in rich extracellular matrix compared to the atrial myocardial cells. The appearance of the cells within the PNA is a mixture of the SN and atrium.
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Figure 44: The SN and right atrium - Cx43/NCX1 double labelling
The SN is clearly identified by the lack of Cx43 staining, but retained labelling of NCX1. Note the accurate match with histology sections on figure 42 and 43.
Crista Terminalis
Epicardium
Endocardium
1000 µm
20 µm
20 µm
Sinus node
Atrium
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4.1.2 Paranodal area
At the interface of the SN with the right atrium there is a zone where the cells of
both types are mixed together (figure 43). It is similar in appearance to the
periphery of the SN described in small mammals, such as the rabbit and rat (section
1.3.1). This zone is thin at the level of the SN body, close to the RA-SVC junction and
the surface at which the nodal cells interacting with atrial myocytes is limited.
However, as the SN starts to taper down toward the IVC, the intermediate zone
becomes more widespread replacing the SN; the caudal portion of the intercaval
region along the CT appears to have very different composition than the more
cranial part. One cannot observe a confluent area of nodal cells that could be
termed as SN anymore. Instead, they form small “islands” of nodal like cells; they
are paler, smaller than the atrial myocytes that occupy the space between the
groups of nodal-like cells (figure 45). Based on the morphological resemblance to
the recently discovered paranodal area in the human I termed this zone the
paranodal area in the goat. The PNA in the goat is an extensive structure extending
the length of the pacemaker complex caudally as well as dorsally. In chapter 5 the
spatial relationship and extent of the SN and PNA will be discussed in detail.
As previously described, the SN area is relatively easy to distinguish from the RA
using histological techniques alone. However, in the PNA it can be very difficult to
identify the subtle clues that Masson’s trichrome stain can provide. IHC proved to
be very useful in this setting: the nodal-like cell groups are Cx43 negative and
HCN4/NCX1 positive, similar to the myocytes within the SN. Once an area was
identified as PNA with the help of IHC, it served as a learning tool and I was
retrospectively able to recognise PNA structure on many histological slides that had
been missed before (figure 46, 47). It is possible that the main reason why the PNA
has not been shown in previous histology studies in large animals and in humans is
the lack of employing IHC and relying only on histology, therefore potentially grossly
underestimating the true extent of the pacemaker complex.
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Figure 45: Paranodal area – histological features
This section was cut perpendicular to the CT close to the IVC (see insert diagram). At this level, nodal like cells are grouped in small aggregates and embedded in atrial myocardium. Note, how difficult it is to differentiate these areas even on the magnified image in top right corner.
1000 µm
Level of section
Cri
sta
Term
inal
is
Inte
rcav
al r
egio
n
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Figure 46: Paranodal area – immunohistochemistry
Sister slide to the histology section shown in figure 45. On this IHC slide double labelled with Cx43 (red) and NCX (green) the nodal like cell-islands are readily recognised
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Figure 47: Paranodal area – moderate magnification
These sister slides are taken from a control animal, at and stained with MT (right) and double labelled with anti-HCN4/Cx43 antibodies (left). The nodal like cells can be recognised on histology only in retrospect, after assessing the same area
Histology HCN4 / Cx43
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4.1.3 Subsidiary atrial pacemaker
In the successful experimental models the caudal half of the RA preparation was
sectioned serially with the leading pacemaker (SEA) in the centre of the series. In
addition to the SAP area, representative sections were cut through the ablated SN
to check for transmurality of the lesions in 4 goats, one of which had the leading
pacemaker mapped to within the ablated SN during the second procedure.
The location of the SAP in these goats were found in the caudal half of the
intercaval region adjacent to the CT; broadly the same anatomical area as that of
the PNA in the control goats (section 3.3 and 4.1.2). Morphological features of the
SAP were also similar to the PNA. Embedded in a large mass of atrial myocardium
there were thin strands of nodal-like cells recognisable at the level of the mapped
SEA . These cells consisted of small islands and had an intimate large-surface
connection with the surrounding atrial myocytes. They were smaller and paler then
on the MT histological stain and had strong HCN4 and NCX1 labelling on IHC (figure
48, 49, 50). Recognition of the nodal-like myocytes on histology preparation was
just as difficult as in the case of the PNA; the accompanying immunological stains of
adjacent slides were necessary to correctly identify the nodal cells within the SAP
region in many cases.
4.1.3.1 Ablated sinus node
Histological sections of the ablated SN revealed a complete destruction of the
cellular structure. The pacemaking tissue has been replaced by granulation tissue
and fibrosis. There were no surviving nodal cells identified in the models where the
SEA was mapped to SAP and the RF lesions appeared transmural (figure 51). In
contrast, in one experiment with recovered SN (postablation SEA from SN area),
there was an endocardial strip of surviving nodal cells identified (figure 52). The
sections shown in figure 52, were harvested from the same animal and from the
same area where spontaneous diastolic depolarization were recorded ex vivo
(section 3.3, figure 30). This finding confirms, that incomplete ablation of the SN
was responsible for a SEA that did not shift away from the anatomical area of the
SN.
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Figure 48: Subsidiary atrial pacemaker – histological features
The section was cut perpendicular to the CT at the level of the leading pacemaker identified in one of the experimental model (see diagram in the left bottom corner) The nodal like cells within the SAP area are marked (yellow dashed line). The SAP cells stain subtly less intense then the atrial myocytes. Note, the section was 3 mm caudal to the border of the ablated area (not shown).
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Figure 49: Subsidiary atrial pacemaker – Immunohistochemistry I.
The section is 0.4 mm caudal to the histology slide on figure 48. The tissue is double labelled with HCN4 and Cx43. The pacemaker cells are HCN4 positive (bright green) and Cx43 negative (lack of red signal). The boundaries of the nodal-like cells are very distinct, unlike those observed on the histology slide. Further observation also reveals a very tight contact with the atrial myocytes that is much more pronounced than at the level of the SN (yellow arrows).
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Figure 50: Subsidiary atrial pacemaker – Immunohistochemistry II.
Sister section to that shown on figure 48. The tissue is double labelled with NCX1 and Cx43. The pacemaker cells Cx43 negative (lack of red signal) and all myocytes are NCX1-positive (green). The Cx43 signal is much stronger compared to figure 48, due to the use of rabbit polyclonal antibody instead of mouse monoclonal antibody. .
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Figure 51: Histology of the ablated sinus node
Histology section at the level of the destructed SN, Masson’s trichrome stain. The completely structure-less ablated area encircled (red dashed line) incorporates the whole of the approximate SN location (yellow dashed line). The RF lesion is transmural; there is no surviving myocardium from the epi- to endocardium.
Figure 52: Surviving pacemaker cells following ablation
In this experiment, the leading pacemaker remained within the anatomical SN area. The corresponding histological section confirms a thin endocardial strip of surviving nodal cells. (same experimental animal as depicted in figure 30.)
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4.2 Extent of the atrial pacemaker complex in the goat
All control right atrial preparation (n=4) were sectioned serially from the SVC to the
IVC, in order to investigate the extent of the SN and PNA in the goat for the first
time. The sections were cut at 1 mm intervals and with 10 µm thickness. In our lab
we usually use 20 µm thick slides, but I found the thinner sections resulted in a
higher quality histological stain. Initially, the right atrial preparation (n=2) was
created by dividing the great veins (IVC and SVC) according to standard practice in
our laboratory and also widely used in works published worldwide. The remaining
tissues (n=2) were prepared with a modified approach keeping the great veins of
IVC and SVC intact (section 2.7, figure 23 panel C and D). The histology slides then
were digitized and the dimension of the SN measured as well as its spatial
relationship to the surrounding RA analysed.
4.2.1 Spatial relationship of the SN and PNA within the RA
The SN in the goat consists of a thick body and a tapering head and tail, similar to all
other species previously studied in the literature (section 1.3).
4.2.2 The body of the SN
The body of the SN is thickest 2-3 mm caudal to the junction of the anterior portion
of the SVC with the RA (crest). From this point the SN extends down toward the IVC
and does not start tapering for another 22±5 mm. The mean width of the SN body is
3.9±0.5mm. The SN body occupies the full thickness of the intercaval region. In the
postero-lateral direction, it ends by intermingling with the atrial myocardium, but
only over a minimal distance of 0.5 mm (figure 53). Antero-medially, on the
opposite side, the SN abuts the thick muscle of the CT and propagates on its
endocardial surface as a thin tongue of nodal tissue for 3-4 mm. The surface area
meeting the atrial myocardium is more extensive, although the nodal cells more or
less remain separated from the atrial myocytes by connective tissue (figure 53).
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Figure 53: The body of the SN
The body of the SN occupies the full thickness of the intercaval region. The SN extends into the adjacent atrial myocardium on both sides and creates interdigitating interface with the atrial myocytes through the paranodal area. The mixture of two different cell types in the PNA is demonstrated on inserts taken at 40x magnification.
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4.2.3 The tail of the SN and the PNA
The SN starts tapering at halfway between the IVC and the SVC, gains an
endocardial layer of atrial myocardium and within 3-4 mm loses its compactness.
The last portion of the nodal tissue is dispersed into thin “fingers” of nodal tissue
that is the paranodal area. The pacemaker cells in this region are more extensively
intermingled with atrial myocytes compared to more proximal sites (figures 45-47).
However, at the resolution of the light microscope it cannot be established whether
the different type of cells have a tight desmosomal connection, i.e. whether an
electronic coupling exists between them.
4.2.4 The head of the SN and the second PNA
In the traditionally dissected two preparations (SVC divided), the SN started to taper
as it was directing dorsally (postero-medially) toward the interatrial groove and
across the crest. However, instead of gradually disappearing it abruptly ended at
the margin of the section, suggesting the possibility that the SN may continue
further (figure 54). Indeed, this was the case; the two remaining control specimens
confirmed that the SN continues downward into the interatrial groove for 6-7 mm
before it is converted into a second paranodal area for another 12-13 mm (figures
54,55). The second PNA resembles its counterpart on the opposite wall; the nodal-
like cells are paler and smaller than the atrial cells, they do not express Cx43 but
expresses HCN4 (figure 55). In other words, there are tracts of nodal-like cells in the
interatrial groove reaching almost as caudally as on the opposite side along the
crista terminalis creating an inverted U shaped pacemaker complex. In a sense, it
seems more appropriate to refer to the SN in the goat as a structure with a central
body and two tails, instead of a head and a tail.
4.2.5 3D reconstruction of the SN and PNA
One RA preparation was selected for 3D reconstruction of the whole pacemaker
complex including the SN and the PNA. For this purpose I chose one of the modified
preparations in order to retain the physiological integrity of the RA (with the
exception of a collapsed cavity). The 3D model was created according to the
method described in section 2.11. The model confirmed what has been observed on
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Figure 54: Nodal cells in the interatrial groove – the second PNA
The orientation is different from previous images; the RA cavity is kept intact, with the exception the removal of the RA appendage toward the right side of the image. The SN is on top right side of the section (yellow dashed line). There is a second, large area on the other side of the RA cavity (red dashed line)
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Figure 55: HCN4 positive nodal-like cells in the second PNA
Similar section and orientation as in figure 54, but taken from another control animal. The insert of an IHC slide (adjacent to the histology slide) reveals HCN4 positive/Cx43 negative cells from the area of the second PNA.
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the two dimensional slides and described earlier in this chapter. The SN in the goat
is a very extensive structure extending caudally toward the IVC anteriorly and
toward the atrioventricular node in the interatrial groove. The total volume of the
SN and PNA was measured as 314.5 and 100.7mm3 respectively. At the tail ends of
the SN there are the paranodal areas. The two tails converges toward each other as
they descend inferiorly creating an almost complete circle of pacemaking tissue in
the right atrium. Figure 56 shows the extent of the SN and PNA in the goat on a
schematic cartoon. A movie created from the 3D model can be found on the DVD
disc attached to this thesis at the back.
Figure 56: Schematic of the SN and PNA in the goat
The SN (yellow) and the PNA (brown) together create an extensive atrial pacemaker descending from the crest both anteriorly and posteriorly.
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4.2.6 The extent of the SAP
The SAP, by definition, was localised electrophysiologically rather than histologically
with determining the site of earliest activation (section 3.1). However, once the SEA
is located, a correlation between the functional SAP and the structure of the tissue
the SEA was found can be drawn. In the successfully ablated group (n=6) five had a
leading pacemaker that I could map from an epicardial approach. Out of these five
experiments, structural analysis was performed in four and confirmed in all cases
that the anatomical site of the SAP closely approximates the area of the PNA in the
control goats. As described earlier (section 4.1.3), not only the location but also the
histological features are very similar between the PNA and the SAP. Perhaps, in the
single experimental model where the SEA could not be mapped to the epicardial
surface; the leading pacemaker may have been originated from the second PNA in
the interatrial groove.
4.3 Protein expression in the SN and SAP
The final step in structural characterisation is to determine whether differences in
expression of proteins exist between the leading pacemakers in the control and the
experimental group. I selected proteins that have a key role in the generation and
propagation of the spontaneous diastolic depolarization. Control and experimental
animals (n=8) were compared using an immunofluorescence semiquantitative
method (section 2.10.1). The specificity of the antibodies used has been previously
tested in our laboratory and the same number of samples has also been successfully
used in previous works published by our group59,138,139. Statistical analysis was
performed comparing RA to SAP in the experimental group, and RA, SN and PNA to
each other in the control group, using paired Student’s t test and repeated measure
ANOVA, respectively.
4.3.1 Connexin 43
In the goat Cx43 was expressed abundantly in the working atrial myocardium (RA),
but not in the SN. The PNA in the control group showed reduced expression of Cx43
compared to the atrium (RA>PNA>SN), whereas the SAP area in the experimental
group showed no significant difference compared to the atrium (RA=SAP).
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4.3.2 HCN 4
The protein responsible for the pacemaking funny current is the HCN, and its most
abundant isoform in the atrial pacemakers is HCN4. The expression of HCN4 in the
control group showed a significant difference between the regions (RA<PNA<SN). In
the ablated group HCN4 expression was significantly higher in the SAP than in the
working atrial myocardium (RA<SAP).
4.3.3 Calcium handling proteins
Besides the funny current as the principal ionic current of the “membrane-clock”
the other important mechanism behind spontaneous pacemaking activity is the
Ca2+-clock, therefore I investigated three main components of it.
4.3.3.1 Sodium-Calcium exchanger
The NCX1 was more abundant in the control SN and PNA than it was in the atrium
(SN=PNA>RA). Similarly, in the experimental group expression was higher in the SAP
(SAP>RA).
4.3.3.2 Ryanodine receptor
The cardiac isoform of the ryanodine receptor (RYR2) was tested. There was no
significant difference found, RYR2 expressed equally in all tissue types (RA=SN=PNA
and RA=SAP).
4.3.3.3 Sarcoplasmic reticulum Ca2+ ATPase
The other important calcium-handling protein in the sarcoplasmic reticulum is the
SERCA2A. No significant difference was found in either group (RA=SN=PNA and
RA=SAP)
The results of the expressions of proteins are displayed in figure 57.
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Figure 57 : Protein expression in the goat RA and pacemaking tissue
The SN is more abundantly expresses HCN4 than any other region, and the SAP and PNA has intermediate level of expression.
NCX1 is expressed less in the RA than in the pacemaking tissue.
Cx43 is highly expressed in the RA and also in the SAP tissue, whereas the SN and PNA has low level of expression.
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5 Discussion and Summary
In this current work, I successfully created a large animal model of sinus node
dysfunction and characterised the emerging subsidiary atrial pacemaker. I was also
able to establish a link between SAP function and structure by identifying cells from
the SAP area with molecular and morphological characteristics of pacemaker cells.
To my knowledge, the combined investigation of structure and function of an in
vivo artificially created SAP has not been undertaken previously. I also described the
morphology and extent of the goat sinus node for the first time. Finally, I discovered
a much larger than suspected native atrial pacemaker complex that contains the SN
and the paranodal area extending not only toward the IVC but also dorsally in the
interatrial groove. This type of arrangement has not been reported in any large
animal before.
5.1 Creation of a large animal model of SND
In the goat, SAP takes over the role of the leading pacemaker of the heart,
subsequent to destruction of the SN. The SAP is predominantly found in the caudal
half of the posterolateral RA along the crista terminalis. After an initial phase of
instability, marked by bradycardia, atrial arrhythmias, increased suppression by
overdrive pacing and intermittent asystoles , the SAP eventually becomes stable
and capable of generating a HR similar to that of the SN. The perhaps surprising
finding of no significant change in resting heart rate following SN ablation is in
keeping with previous published studies conducted in the dog, where authors found
that the resting HR does not change in the first 3 to 4 weeks and even after this
period the reduction is minor89,92,135,140. In contrast, Tse et al. demonstrated a
significant drop in heart rate when the SN was ablated endocardially in the pig141.
Their result may reflect the different extent of the porcine SN compared to that of
the dog or goat. Although the SAP is able to generate a sufficient HR at rest, it is
dysfunctional when there is a demand; chronotropic incompetence to exercise and
blunted response to chemical sympathethic stimulation both been described in the
dog87,91,136. I have not repeated these experiments nor did I extended the follow-up
to several weeks, as the primary goal of the study was different. I investigated the
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structure of the SAP and SN with the aim of potentially finding a link between
structural/morphological data and function rather than replicating the results of
previous studies.
5.2 Structural characterisation of the SAP
Whilst functional studies are plentiful, the structure of the created SAP has been
studied only by a few authors. Morris et al recently created an in vivo model of SND
in the rat by excising the cranial part of the RA and with it the SN107. The main
research objective was to create a robust biopacemaker by enhancing the existing
SAP function with a chimeric HCN product. The work was successful in this regard,
but he could show HCN4 activity in the SAP region only in 1 out of 6 experiments
using immunofluorescence method. This finding maybe genuine or HCN4
expression was simply below detection level in thin rat tissue. Another work in the
rabbit mapped the HCN4 positive regions in the RA and concluded that it is found
only in the centre of the SN and not elsewhere in the RA, although the SAP region
has not been specifically sampled142. Yet, the SAP in the goat clearly expresses HCN4
at a level that is intermediate between the SN and the atrium consistent with the
spontaneous pacemaker activity of the SAP.
Beyond the membrane-clock, the other important component of the coupled-clock
theory of pacemaking activity is the Calcium-clock comprising the voltage
dependent Ca channels, Sodium-calcium exchanger (NCX1) in the membrane, and
the Ca handling proteins of SERCA and RyR2 in the sarcoplasmic reticulum143.
Lyashkov et al showed in the rabbit that NCX1 indeed has an increased expression
level in the SN compared to the atrial cells and although RyR2 and SERCA expression
remained the same across all regions RyR2 showed strong submembrane co-
localisation with NCX1 in the SN, in keeping with its’ suggested role in the Ca++-
clock144. My results are in concert with the above; NCX1 is highly abundant in all
pacemaking tissue (SN, PNA, SAP) and less abundant in the RA. SERCA2A and RyR2
protein expressions are not significantly different across the regions.
High conductance connexins, such as Cx43 are ubiquitous in the working atrial
myocardium, but have been shown to be absent in the sinus node of the human,
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rat, mouse (section 1.4.6). Cx43 expression in the goat is no exception, Cx43 is more
prevalent in the RA than it is in the SN or PNA. However, the SAP shows a
considerable amount of Cx43 staining, in fact it is not significantly different from
what has been detected in the RA. It has been suggested by modelling work
recently that a gradually increased gradient of Cx43 toward the atrium from the SN
may actually be a requisite for the SN to be able to drive the heart145.
5.3 Extensive distribution of the atrial pacemaker complex, the role of the paranodal area
The location, size and extent of the SN has been the target of intensive research
ever since Keith and Flack discovered its existence. Depending on the methodology
and/or species used the length of the SN varied considerably. In large mammals, like
the dog, pig and human, the SN has been shown to end at one third to half of the
length of the crista terminals (CT), whereas in smaller laboratory animals, like the
rabbit and mouse, the SN can be followed much more caudally reaching the point
where the IVC enters the RA6,17,96,98,146–148. What almost all studies agreed on is that
the SN has a central body that tapers down in both cranial and caudal direction
creating the head and the tail of the SN respectively. The head of the SN extends
over the junction of the SVC with RA (the crest), but does not spread more dorsally.
There have been two published works, both in the rat, that suggested otherwise.
Yamamoto et al showed an extensive area in continuation of the SN head toward
the dorsal interatrial groove that was both Cx45 and HCN4 positive suggesting that
the cells in this region may have pacemaker activity. Together with the SN it
composed an inverted U shaped atrial pacemaker complex (figure 58). When the SN
was separated from the interatrial groove, the region kept beating and the SEA was
originating from within directly proving its capability as a SAP97. Yanni in the old and
young rat found an identical structure. In his experiments, the interatrial groove
preparation kept beating, but some of the central SN preparation did not show
spontaneous depolarization without being attached to its peripheral/paranodal
region80. In the goat I confirmed the presence of a very similar inverted U shaped
atrial pacemaker complex consisting of the compact SN and the paranodal area at
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both of its ends (figure 56 in section4.2.4). I also demonstrated that majority of the
successfully ablated experimental model had an emerging SAP detected with the
Figure 59: Atrial pacemaker complex in the rat
Left atrial preparation of the rat shown from anterior (left) and posterior (right) aspect. The black area on both diagram delineate the extent of the pacemaking tissue. The long dorsal extension in the interatrial groove has not been described in any other species until now. Adapted from Yamamoto et al.97
leading pacemaker located to the same area where the PNA was found in the
control goat. The physiological role of the PNA perhaps is multiple; it may help the
SN to drive the more hyperpolarised atria by offering an interface to it, at the same
time protecting the node from being hyperpolarised and therefore rendered
dysfunctional and finally it is likely the main locus of the atrial subsidiary
pacemakers. Apart from a physiological role, PNA may also be important in
arrhythmia genesis; many of the focal atrial tachycardias are originating from the
crista terminalis and the interatrial septum, sites where HCN4 positive pacemaking
tissue exists in the goat.
5.4 Limitations of the study
Ablation and mapping was carried out in an epicardial fashion, therefore, it was not
possible to characterise the functional aspect of the newly discovered
dorsal/interatrial extension to the SN. One of the experimental models may have
developed SAP function in this area, but in the current study setup, I could not
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prove it. The mapping system itself had an inherent limitation defined by the 5 mm
interelectrode distance. It is possible, that the pacemaker cells identified in the area
of the SAP are not the origin of the spontaneous depolarization. However, the
consistency of finding these cells at every successfully located SAP region suggests
that the accuracy of the mapping catheter was sufficient to identify SAP.
Protein expression was measured by a semi-quantitative method using IF
technique. The number of proteins investigated was limited by the available
antibodies that worked in this species. At the time of the study was conducted, the
goat genome was unknown, therefore, no molecular characterisation was carried
out at the mRNA level. The goat genome was published in 2013 and new
technologies such as next generation sequencing are now a reality; future
experiments in the goat will have a more robust array of molecular biological
methods149.
5.5 Future directions
The goat, too, has a paranodal area adjacent to the SN, like the human. The
composition of the goat PNA is somewhat different, seems to be more pronounced
toward the tails, unlike in the human where it extends into the crista terminalis. The
extent of the goat SN also appears much more extensive. However, the paranodal
area in the human may extend more caudally then discovered. Chandler et al in
their paper describing the PNA emphasised the fact that the SN preparation was
incomplete; neither the caudal part of the CT, nor the extreme cranial part of the
crest or the interatrial septum were included. Therefore, it is possible that the
human atrial pacemaker complex has a similar extent. Work is underway in our
group to investigate this possibility. The goat model of SND may well be used in the
future to develop and test strategies of biopacemaking.
Page | 120
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7 Appendix
7.1 DVD multimedia appendix
The attached DVD contains a movie of the 3D reconstruction of the right atrium in
the goat embedded in a Microsoft PowerPoint presentation. The individual sections
the model had been built from are also included as a single, large stacked image file
(tiff).