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Electrophysiological patterning of the heart
Boukens, B.J.D.Publication date 2012
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Boukens, B. J. D. (2012). Electrophysiological patterning of the heart.
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Electrophysiological
patterning
of the heart
Electrophysiological patterning of
the heart
ISBN: 978-94-6190-997-8 Printed: Offpage
© 2012 Bas Boukens
No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without permission of the author
Electrophysiological patterning of
the heart
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Aula der Universiteit op woensdag 19 december 2012, te 13:00 uur
door
Bastiaan Joannes Dirk Boukens
Promotor: Prof. dr. V.M. Christoffels Co-promotor: Dr. R. Coronel
Overige leden: Prof. dr. M.J. Janse Prof. dr. A.A.M. Wilde Prof. dr. H.V.M van Rijen Prof. dr. I.R. Efimov Dr. I.P. Moskowitz
Prof. dr. N.A. Blom Faculteit der Geneeskunde
Het onderzoek dat aan dit proefschrift ten grondslag ligt is mogelijk gemaakt door een subsidie van de Nederlandse Hartstichting (2008B062).
Het verschijnen van dit proefschrift werd mede mogelijk gemaakt door de Nederlandse Hartstichting.
Additional financial support for the publication of this thesis was generously provided by the department Anatomy, Embryology and Physiology, the University of Amsterdam.
Contents
Scope of the thesis Chapter 1
Electrophysiological patterning of the heart
Pedriatic Cardiology, 2012
Chapter 2
Defective Tbx2-dependent patterning of the atrioventricular canal myocardium causes accessory pathway formation in mice
Journal of Clinical Investigation, 2011
Chapter 3
Complementary roles of Tbx3 and Nkx2-5 in the structure and function of the atrioventricular conduction system
In preparation
Chapter 4
Developmental basis for electrophysiological heterogeneity in the ventricular and outflow tract myocardium as a substrate for live-threatening ventricular arrhythmias
Circulation Research, 2009
Chapter 5
Reduced sodium channel function unmasks slow conduction in the adult right ventricular outflow tract as part of the maintained embryonic gene program
Submitted for publication
Chapter 6
Early repolarization in mice causes overestimation of ventricular activation time by the QRS duration
Cardiovascular Research, 2012
Chapter 7
Popeye proteins: muscle for the aging sinus node
Journal of Clinical Investigation, 2012
Chapter 8 Summary Chapter 9 Samenvatting Publications Dankwoord 15 31 57 75 103 121 141 151 155 159 163
Sudden cardiac death accounts for 300.000 deaths annually in the United States.1 Although the majority of sudden cardiac death is due to coronary heart disease, up to 5% is due to primary electrical or genetic ion channel abnormalities.2 To this category belong the Wolff-Parkinson-White (WPW), Brugada and Long QT syndrome as well as congenital atrioventricular (AV) block or acquired diseases of the sinus node.3 The mechanism of arrhythmia differs between these cardiac pathologies, but it may result, if untreated, in ventricular fibrillation or a-systole leading to sudden death.2 The majority of the patients with primary electrical disease or hereditary ion channel abnormalities are treated effectively by implantation of a electronic pacemaker (for sinus node dysfunction AV block or the Brugada syndrome4, 5), by radiofrequency ablation (in WPW patients6), or pharmacologically (in most Long QT syndrome patients7). In some of these patients an automatic internal cardioverter defibrillator is implanted to treat VF with electroshock. In a small percentage (<10%) of the patients sudden cardiac death is the first manifestation of the disease.8, 9 Therefore, risk stratification of patients is important. Unfortunately, we lack information on specific markers of an increased risk of death from arrhythmias in the general population and among those with nonspecific and intermediate risk profiles. The general hypothesis underlying this thesis is that understanding of the normal embryonic development of the electrical properties of the heart would provide insight into the mechanisms of abnormal development and function, and therefore can be used to define factors that are important for electrical dysfunction. Thus, the thesis seeks to bridge the gap between embryonic cardiac development and lethal function which, could lead to the identification of new specific markers for an increased risk of arrhythmias occurring in adulthood. We use Brugada syndrome, WPW syndrome, Sick sinus syndrome and AV node dysfunction as models for this approach. In chapter 1 we introduce the term ‘electrophysiological patterning’ and explain which transcription factors are involved in generating the electrophysiological properties of structures in the ventricular myocardium and the AV node. In chapter 2 we studied the role of T-box (Tbx) transcription factor 2 in patterning of the AV canal and its role in the formation of accessory pathways (related to WPW-syndrome). In chapter 3 we investigated the role of transcription factor Nkx2-5 and Tbx3 in the formation, function and maintenance of the AV conduction system. In chapter 4 we review the temporal and spatial expression of transcription factors in relation to electrophysiological heterogeneities that exist in the ventricular myocardium of the adult heart, with a main focus on the right ventricular outflow tract (RVOT, related to Brugada Syndrome). In chapter 5 we studied the differences in function and gene expression between RVOT and the right ventricle, and explored the hypothesis that the slow conduction property and gene expression program of the embryonic outflow tract are maintained in the adult RVOT. In chapter 6, an editorial, we comment on a study that introduced a new family of cAMP binding proteins called Popeye-containing-domain proteins, which is involved in age and stress
times to the surface electrocardiogram (ECG) of the mouse. In chapter 8 the thesis is summarized.
Reference List
(1) Kong MH, Fonarow GC, Peterson ED, Curtis AB, Hernandez AF, Sanders GD, Thomas KL, Hayes DL, Al-Khatib SM. Systematic review of the incidence of sudden cardiac death in the United States. J Am Coll Cardiol 2011 February 15;57(7):794-801.
(2) Huikuri HV, Castellanos A, Myerburg RJ. Sudden death due to cardiac arrhythmias. N Engl J Med 2001 November 15;345(20):1473-82.
(3) Zipes DP, Wellens HJ. Sudden cardiac death. Circulation 1998 November 24;98(21):2334-51. (4) Antzelevitch C, Brugada P, Borggrefe M, Brugada J, Brugada R, Corrado D, Gussak I, LeMarec H,
Nademanee K, Perez Riera AR, Shimizu W, Schulze-Bahr E, Tan H, Wilde A. Brugada syndrome: Report of the second consensus conference: endorsed by the Heart Rhythm Society and the European Heart Rhythm Association. Heart Rhythm 2005 April;2(4):429-40.
(5) Mond HG, Proclemer A. The 11th world survey of cardiac pacing and implantable cardioverter-defibrillators: calendar year 2009--a World Society of Arrhythmia’s project. Pacing Clin Electrophysiol 2011 August;34(8):1013-27.
(6) Cohen MI, Triedman JK, Cannon BC, Davis AM, Drago F, Janousek J, Klein GJ, Law IH, Morady FJ, Paul T, Perry JC, Sanatani S, Tanel RE. PACES/HRS expert consensus statement on the management of the asymptomatic young patient with a Wolff-Parkinson-White (WPW, ventricular preexcitation) electrocardiographic pattern: developed in partnership between the Pediatric and Congenital
Electrophysiology Society (PACES) and the Heart Rhythm Society (HRS). Endorsed by the governing bodies of PACES, HRS, the American College of Cardiology Foundation (ACCF), the American Heart Association (AHA), the American Academy of Pediatrics (AAP), and the Canadian Heart Rhythm Society (CHRS). Heart Rhythm 2012 June;9(6):1006-24.
(7) Schwartz PJ, Crotti L, Insolia R. Long-QT Syndrome: From Genetics to Management. Circ Arrhythm Electrophysiol 2012 August 1;5(4):868-77.
(8) Berne P, Brugada J. Brugada syndrome 2012. Circ J 2012 June 25;76(7):1563-71.
(9) Bromberg BI, Lindsay BD, Cain ME, Cox JL. Impact of clinical history and electrophysiologic characterization of accessory pathways on management strategies to reduce sudden death among children with Wolff-Parkinson-White syndrome. J Am Coll Cardiol 1996 March 1;27(3):690-5.
Pediatr Cardiol. 2012 Aug;33(6):900-6.
Bas Boukens
Vincent Christoffels
Electrophysiological patterning of the heart
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Abstract
In the adult heart, electrophysiological heterogeneity is present in order to guide activation and contraction. A change in electrophysiological heterogeneity, for example during disease, can contribute to arrhythmogenesis. During development, spatial and temporal patterns of transcriptional activity regulate the localized expression of ion channels that cause electrophysiological heterogeneity throughout the heart. If we gain insight into the regulating processes that generate the electrophysiological characteristics and factors involved during development we can use this knowledge in the search for new therapeutic targets. In this review we discuss which factors guide the electrical patterning of atrioventricular conduction system and ventricles and how this patterning relates to arrhythmogenic disease in patients.
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Introduction
Rhythmic and synchronized contraction of the atria and ventricles of the heart is essential for efficient pumping of blood through the body. This process relies on the proper generation and conduction of the cardiac electrical impulse. The cardiac electrical impulse is generated in the sinus node, activates the atria, and is propagated to the atrioventricular (AV) node. Conduction of the electrical impulse through the AV node is slow, allowing the atria to pump blood into the ventricles. Subsequently, the impulse is propagated through the rapidly-conducting AV bundle, its branches and the Purkinje fibre network, from where it reaches the ventricular working myocardium. The coordinated activation pattern results in a contraction wave progressing from the cardiac apex to its base. These intrinsic electrophysiological differences between the various sections of the heart are rooted, at least in part, in the regional, compartment-specific differences in expression of genes encoding ion channels and gap junction proteins 1. This ‘electrophysiological pattern’ develops during embryogenesis and post-natal maturation of the heart (Figure 1).
In the adult heart some regions are more arrhythmogenic than others. For instance, atrial fibrillation more often originates in the pulmonary myocardium than in the atrial free wall 2, whereas in the Brugada syndrome the main origin is the right ventricular outflow tract (RVOT) and not the left ventricle 3. The underlying mechanism is multifactorial, and likely has a genetic component that predisposes for the development of these arrhythmias. Furthermore, in rare cases, even a single mutation in a gene encoding an ion channel that is important for electrical patterning has been identified to cause arrhythmia 4. A major challenge, therefore, is to understand normal and abnormal electrical patterning of the heart and to provide the mechanistic details underlying congenital and acquired arrhythmias. In this review we focus on how the AV conduction system and ventricles are electrically patterned during development and how this patterning relates to arrhythmogenic diseases in patients.
Development of the AV conduction system
The cardiomyocytes of the heart tube have a primary phenotype characterized by an underdeveloped sarcoplasmatic reticulum, weaker contraction and slower conduction compared to cardiomyocytes of the adult heart 5, 6. The slow conduction in the primary myocardium gives rise to a sinusoidal electrocardiogram 7. From embryonic day (E) 8.5 onwards, part of the heart tube differentiates into ventricular and atrial working myocardium. As a consequence, the atria and ventricles are separated by the myocardium of the AV canal. The conduction in the AV canal remains slow whereas the conduction velocity in the working myocardium increases 6. This results in an adult-like electrocardiogram with normal AV delay, although at this stage a fibrous annulus fibrosis has not formed yet 7. Slow conduction in the AV canal results from absence of Connexin (Cx) 40 and Cx43 that form gap junctions with high conductance, and the absence of cardiac sodium channel (sodium channel, voltage gated, type V, a [Scn5a]) 8. In addition, Cx30.2 is expressed in the AV canal and forms gap junctions
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with low conductance that lead to slow conduction 8, 9. The expression of Cx30.2 in the AV canal is regulated by transcription factors Gata4 and Tbx5 10. The expression of Cx40, Cx43 and Scn5a in the AV canal is repressed by T-box (tbx) factor 2 and Tbx3, which in turn are regulated by Wnt-, bone morphogenetic protein (Bmp) 2-, and Notch signalling 5, 11, 12.
Most of the embryonic AV canal myocardium differentiates further to ventricular working myocardium, whereas a small part forms the AV node and AV ring bundles 13-16. Nkx2-5, Tbx5, and Notch-signalling are important for normal formation of the AV node 17-19. The AV node in the adult heart can be identified based on the expression of Tbx3, hyperpolarization-activated
cyclic nucleotide-gated channel (Hcn) 4, Cx45 (and Cx30.2 in mouse), and the absence of atrial natriuretic peptide (Nppa), Cx40 and Cx43 5. It has been reported that Cx43 and Cx40 are expressed in the lower part of the AV node (lower nodal cells) 20. However, based on their origin and gene expression pattern we rather think that these myocytes are part of the AV bundle 16. The atrial myocardium is directly connected to the compact AV node, but also to part of the right AV ring bundle, which is often referred to as the inferior nodal extension, thereby creating the basis for what in the adult is commonly referred to as the fast and slow AV-nodal pathway 16, 20.
Development of the AV bundle and bundle branches
The AV bundle (bundle of His) forms at the top of the ventricular septum and forms a continuity between the AV node and the ventricular septum. The lateral subendocardial myocardium of the septum gives rise to the bundle branches. Tbx3 in the bundle and branches suppresses working myocardial gene expression (Cx43, Cx40) 21. Tbx5 and Nkx2-5 coordinate specification of the AV bundle and bundle branches through activation of Cx40 and inhibitor of differentiation protein (Id) 2 and other genes 22. However, the expression of Id2 is not dependent on Tbx3. This suggests the presence of independent pathways in specification of the AV bundle and
Figure 1. Development of the embryonic heart tube, formation of the atrial and ventricular chambers and the development of the pacemaker and conduction system. A mature type of ECG can be observed already during chamber development. l/ra = left/right atrium; l/rv = left/right ventricle; avc/n = atrioventricular canal/node; sn = sinus node.
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branches 21. Nkx2-5 interacts with Tbx5 and Tbx3, that both recognize the same DNA binding sites. Therefore, the balance between these factors may be of crucial importance for correct formation of the AV bundle and the bundle branches 21. Irx3 is also expressed in the AV bundle and bundle branches, where it represses Cx43 (mice) and (indirectly) activates the expression of Cx40 23. Furthermore, additional factors must be involved in the development of the AV node and AV bundle, because Nkx2-5, Tbx5 and Tbx3 are expressed in both structures. The observation that the AV bundle forms from the ventricular lineage whereas the AV node forms from the AV canal lineage indicates that different transcription factors have been active during the development of these structures 16. Taken together, a network of locally and more broadly expressed transcription factors control the electrical patterning of the AV conduction system.
AV conduction related arrhythmias
High-degree AV conduction block is life-threatening and is a major indication for pacemaker implantation 24. Mutations in NKX2-5 and TBX5 may cause AV block 25. Genome-wide association studies have implicated several loci in AV conduction, including TBX3/TBX5, NKX2-5, SOX5,
WNT11, SCN10A, SCN5A, CAV1-CAV2, and MEIS1 26-29. Thus, transcriptional regulators important for heart development play a major role in the function of the AV conduction system. Variations and mutations in these genes may impact on each structure involved in P-R interval (atria, AV node, AV bundle, branches, Purkinje fibres), but the underlying mechanism is unclear. Several types of arrhythmia involve AV conduction. In AV nodal re-entrant tachycardia (AVNRT), the slow and the fast pathway within the AV junction and part of the transitional myocytes in the atrium form a re-entrant circuit 30. What factors underlie the development of this type of arrhythmia is not known although in some cases familial AVNRT suggest an inheritable component 31, 32. AV re-entrant tachycardia (AVRT) results from an accessory myocardial connection between the atrial and ventricular myocardium. The myocardium of the accessory connection can have a slow conducting nodal (Mahaim) or a fast conducting working myocardial (Öhnell) phenotype 33. An accessory connection can lead to ventricular preexcitation and AVRT as seen in patients with the Wolff-Parkinson-White (WPW) syndrome 34. In the presence of atrial fibrillation it may cause ventricular fibrillation and sudden death, Not much is known about the mechanism underlying development of connections with a nodal phenotype. In the adult, the largest remnant of the embryonic AV canal is the dorsal-caudal portion of right AV ring bundle. The right-dorsal-caudal position and nodal properties of Mahaim bundles suggests that they are remnants or mis-localized AV canal tissue. Connections with a working myocardial phenotype may also be remnants of AV canal myocardium, and result from abnormal patterning of the embryonic AV canal 8, 19. Bmp2, Notch1 and Tbx2 (the latter is downstream of the BMP2) are important for correct patterning of the AV canal myocardium. Inactivation of Bmp-signalling or Tbx2, or activation of Notch-signalling in mouse causes ventricular pre-excitation 8, 19, 35. Indeed, deletions have been found in BMP2 and in JAGGED1 (Notch ligand) in patients with WPW syndrome 36, 37.
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Patterning of the ventricular wall
In the adult heart, the ventricular wall is composed (from the lumen to the outside) of endocardium, Purkinje fibres and trabeculae, compact myocardium and epicardium. In addition, many other cell types are found, including fibroblasts and cells of the coronary vasculature. The formation of the ventricular wall starts after E8.5 with development of the trabeculae that clonally expand from myocytes of the early chamber myocardium. This process is regulated by signalling molecules and receptors in the myocardium and the underlying endocardium, including Notch and Neuregulin-1 38, 39. The myocardial gene program, including that of the chamber myocardium, requires key cardiac transcription factors
Tbx5, Nkx2-5 and Gata4 40-42. Other transcription factors and signalling molecules are also involved in chamber development and expansion. For example, Tbx20 is required for chamber development, and in addition represses the expression of Tbx2, thereby allowing differentiation to working myocardium 43. Furthermore, Hey2, encoding a helix–loop–helix transcription factor, represses the expression of Tbx5, Nppa and Cx40 in the compact myocardium 44. The compact myocardium starts to form after E11.5 under influence of signalling molecules from the epicardium 45. The ventricular wall expresses Cx43 and Scn5a, which are required for the fast conduction in this compartment. Both genes show transmural differential expression patterns (e.g. Scn5a expression is higher in the trabecular component than in the subepicardium). The factor(s) that regulate the patterned expression of Scn5a and Cx43 are not known, although Tbx5 is a likely candidate regulator of the transmural differential expression pattern of Scn5a (unpublished). Genes encoding K+ channels are important for repolarization and are heterogeneously expressed throughout the ventricular wall 46. However, the only factor that is currently known to generate this patterned expression is transcription factor Irx5, itself expressed in a transmural pattern (high in the subendocardium, low in the subepicardium). Irx5 represses the expression of kcdn2 that encodes Kv4.2, a subunit of the ion channel that carries the transient outwards current, ITO. This leads to a characteristic notch of the subepicardial action potential shape and to a longer action potentials in the subendocardial than in the subepicardial myocardium. Therefore, Irx5 is an important factor in realizing a repolarization gradient in the ventricular wall of mice 47. Genes for both transcription factors and ion channels have been implicated in arrhythmogenesis. Common variants located in or near genes for cardiac transcription factors TBX5, TBX3 and TBX20 have been associated with prolonged QRS duration, a predictor for sudden death 26-29. The working myocardial gene program includes genes that encode ion channels that are important for conduction, such as Na+ channels, and repolarization, such as K+ channels 1, 48. Mutation in genes encoding K+ channels including KVLQT1, KCNJ2 and HERG cause prolongation of the action potential and are directly associated with life threatening arrhythmias as in the long QT syndrome 49-51. Mutations in genes encoding Na+ channels are found in patients with prolonged repolarization 52, 53 but also in patients with conduction disturbances 52, 54. The clinical outcome of mutations in genes encoding for a single ion channel supports the importance of transcription factors that guide the expression of these ion channels.
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The ventricular conduction system
The Purkinje fibre network is the most distal part of the conduction system and functionally connects the conduction system to the ventricular myocardium 55. Cx40 expression is considered the best specific marker for this network in the ventricles 56. The Purkinje fibres develop from the trabeculae. Tbx5 and Irx3 are involved in the regulation of expression of Cx40 and other genes in the trabeculae and Purkinje fibres 23, 47. In chicken, endothelin-1 expressed in the underlying endocardium induces Cx40 and specification of the Purkinje fibres 57. However, in mice, neuregulin influences the ventricular conduction pattern during development, probably through its role in trabecular development 39. An alternative model for Purkinje fibre development involves formation of the compact myocardium. The reasoning is as follows. The entire ventricular wall expresses Cx40 early during development (approx. until mouse E10.5-11.5). However, from E11.5 onwards, the compact myocardium is formed and increases in size. At this point in time Cx40 expression decreases, whereas the Cx40-positive trabeculae do not increase in size 58. This enlargement of the compact myocardium will leave a relatively small Cx40-positive trabecular cell population at the endocardial side of the right and left ventricular walls. Clonal analysis data is in line with this model 47, 59. After birth, under influence of Nkx2-5, the trabecular layer transforms into the 1-2 cell layer thin, Cx40-positive Purkinje fibre network in the adult heart 56, 60.
Because the Purkinje fibre network is electrically insulated from the ventricular myocardium, it may serve as substrate for re-entry 61. Furthermore, Purkinje fibre myocytes can induce polymorphic ventricular tachycardia due to abnormal calcium handling 62. To our knowledge, no mutations in genes involved in formation of the Purkinje fibres have been reported in arrhythmia patients.
The ventricular outflow tract
The myocardium of the RVOT of the adult heart is derived from the embryonic outflow tract (OFT) 63, 64. The embryonic OFT expresses Tbx2 but does not express Tbx5. This probably underlies its primary myocardial phenotype, expression of Cx30.2 and the absence of expression of Cx40 and Cx43 46 (our unpublished observations). This primary phenotype results in slow conduction, which is maintained until late in development. In the RVOT of the adult heart, the conduction velocity is not slow compared to in the ventricular myocardium (our unpublished data in mice and pigs). Whether gene expression in the myocardium of the RVOT is different from that of the right or left ventricular myocardium is not known. However, reduced expression of Cx43 in the RVOT myocardium compared to the right and left ventricular myocardium has been reported 65.
The adult RVOT is the origin of arrhythmias in the Brugada syndrome 3. Several mutations in genes encoding ion channels, like in SCN5A, KCNE3, CACNA1C and CACNB2b, have been found in a minority of patients with the Brugada syndrome 54, 66, 67. A plausible mechanism underlying arrhythmias in the Brugada syndrome involves conduction delay or block within the RVOT due to subtle structural abnormalities 68. This process most likely is facilitated by variations or mutations in ion channel and other genes. The embryonic origin of
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the RVOT may explain why it is sensitive for arrhythmogenesis 46. For instance, the working myocardial gene program is fully activated in the OFT just before birth (unpublished data). Furthermore, in Brugada Syndrome patients, the expression of several genes active during the development of the RVOT is downregulated 69. Therefore, we speculate that the maintenance of the working myocardial gene program is affected in Brugada Syndrome patients.
Conclusion
Heterogeneous transcriptional activity mediates the electrophysiological patterning of the AV conduction system and ventricular myocardium, which is crucial for correct function of these structures in the adult heart (Figure 2). Furthermore, mutations in target genes encoding ion channels that determine the electrophysiological properties of the myocardial structures can lead to life-threatening arrhythmias. Experimental studies, such as optical analyses of heart function in (mutant) animals and genetic studies such as genome-wide association studies will reveal novel genes and genomic loci important for cardiac electrophysiology and its patterning. Tools to assess heterogeneous transcriptional activity, function of the (non-coding) genomic loci and target gene expression have emerged, including chromatin immunoprecipitation-sequencing to define the genome-wide binding of transcription factors and epigenetic modifications and RNA-sequencing to define transcriptomes in specific cardiac lineages and domains. Combined, these data will provide insight into the mechanisms underlying the establishment and maintenance of the electrophysiological pattern of the heart, and may provide leads for the development of new therapeutic targets for arrhythmias in patients.
Figure 2. Electrical patterning of the chamber myocardium. During development, transcription factors regulate the expression of ion channels in the ventricular wall and each component of the atrioventricular conduction sys-tem. This leads to slow conduction in the AV node and fast conduction in the AV bundle and bundle branches and Purkinje fibres and, to a lesser extent, also in the ventricular wall. Cv = conduction velocity; ► = expression higher in subendocardium than subepicardium, ◄ = expression higher in subepicardium than in subendocardium; l/ra = left/right atrium; l/rv = left/right ventricle; avn = atrioventricular node.
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Acknowledgments
We thank Dr. Ruben Coronel for critical reading the manuscript.
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Journal of Clinical Investigation. 2011 Feb 1;121(2):534-44
Wim TJ Aanhaanen*
Bastiaan JD Boukens*
Aleksander Sizarov
Vincent Wakker
Corrie de Gier-de Vries
Antoni C van Ginneken
Antoon FM Moorman
Ruben Coronel
Vincent M Christoffels
* These authors contributed equallyDefective Tbx2-dependent patterning of the
atrioventricular canal myocardium causes
accessory pathway formation in mice
2
Abstract
Ventricular preexcitation, a feature of Wolff-Parkinson-White syndrome, is caused by accessory myocardial pathways that bypass the annulus fibrosus. This condition increases the risk of atrioventricular tachycardia and, in the presence of atrial fibrillation, sudden death. The developmental mechanisms underlying accessory pathway formation are poorly understood, but are thought to involve primarily malformation of the annulus fibrosus. Before birth, however, slowly conducting atrioventricular myocardium causes a functional atrioventricular activation delay in the absence of the annulus fibrosis. This myocardium remains present after birth, suggesting that disturbed development of atrioventricular canal myocardium may be primarily involved in the formation of rapidly conducting accessory pathways. We show that myocardium-specific inactivation of Tbx2, a transcription factor essential for atrioventricular canal patterning, leads to the formation of fast-conducting accessory pathways, malformation of the annulus fibrosus and ventricular preexcitation in mice. The accessory pathways ectopically express proteins required for fast conduction (Cx40, Cx43 and Scn5a/Nav1.5). Additional inactivation of Cx30.2, a subunit for gap-junctions with low conductance expressed in the atrioventricular canal and unaffected by loss of Tbx2, did not affect the functionality of the accessory pathways. Our results suggest that malformation of the annulus fibrosus and preexcitation arise from disturbed development of the atrioventricular myocardium.
2
Introduction
In the heart, the atrial and ventricular muscle masses are electrically insulated from each other by the annulus fibrosus. Normally, the only muscular connection that crosses this insulation is formed by the atrioventricular (AV) node and AV bundle. In the general population, 1-3 in 1000 individuals have accessory myocardial pathways that bypass the insulation, known as bundles of Kent.1,2 These accessory connections may lead to preexcitation of the ventricle, circus movement tachycardia and even life-threatening ventricular fibrillation in the presence of atrial fibrillation, as seen in Wolff-Parkinson-White (WPW) syndrome patients.3-5 The mechanism by which these accessory myocardial connections develop, and the molecular properties of these connections are largely unknown.
Accessory myocardial connections that can lead to preexcitation are commonly thought to result from malformation of the annulus fibrosus.6-9 However, during development, the AV canal myocardium causes an adequate AV delay to allow synchronized alternating contraction of the atria and ventricles10 in the absence of an annulus fibrosus. Remnant strands of this AV myocardium have still been observed in normal hearts around and after birth, which disappear when the annulus fibrosus is fully formed.6,11 Usually, these strands do not lead to preexcitation, indicating they maintain slow conduction properties of the AV myocardium. Furthermore, in the adult heart, after formation of the annulus fibrosus has been completed, slow-conducting AV canal-type myocardium remains present around the orifices of the mitral and tricuspid valve.12-14 Together, these data suggest that AV canal myocardium plays a central role in the AV delay, and that defects in AV canal myocardium could underlie formation of functional accessory pathways.
The AV canal myocardium is specified early in cardiac development. Bone morphogenetic protein 2 (Bmp2) is expressed in the AV canal myocardium progenitors in the early heart tube where it stimulates the expression of Tbx2,15 a T-box factor required for the development of the AV canal.16-18 Repressors Tbx3 and Msx2, Notch signaling, and Hey transcription factors further establish the AV canal phenotype.19-22 Other factors involved in formation and regulation of gene expression of the AV canal and AV node include the more broadly expressed transcription factors Nkx2-5, Gata4 and Tbx5, which interact or compete with the localized repressors.16,23-27 To explore the possible role of the AV canal myocardium in the formation of accessory connections, we studied morphology and function of mice in which Tbx2 was specifically inactivated in selected tissues. Our results indicate that defective patterning and gene regulation within the AV canal myocardium may lead to malformation of the annulus fibrosus, to formation of accessory AV connections and ventricular preexcitation.
2
Methods
Transgenic mice
The Tbx2tm1.1(cre)Vmc (synonyms: Tbx2Cre, Tbx2-), Tbx2fl2, Myh6-Cre, Tie2-Cre, Wnt1-Cre,
and Cx30.2lacZ alleles have been described previously.18,28-32 All mice were held on FVB/N background, except for Cx30.2LacZ, which was held on C57BL/6 background. Experiments
with Tbx2Cre/+;Cx30.2LacZ/+ double transgenic mice were performed in mixed FVB/N;C57BL/6
background. Animal care was in accordance with national and institutional guidelines.
Human embryos
Human embryos were collected from medically induced abortions performed for social reasons at the Gynaecology Department of the Tartu University Hospital, Estonia. Collection and use of the human embryonic material for research presented here were approved by the Medical Ethics Committees of the Universities of Tartu, Estonia, and Amsterdam, the Netherlands. Subsequent processing has been previously described.33
BrdU assay
Pregnant females were injected intraperitoneally with 50 mg of 5’-bromo-2’deoxyuridine (BrdU) / kg bodyweight (Sigma B5002) dissolved in 0.9% NaCl. After 1 hour of BrdU exposure the mice were killed by cervical dislocation. The embryos were isolated on ice-cold PBS and further processed for immunohistochemistry.
Immunohistochemistry and in situ hybridization
Embryos were fixed in 4% formaldehyde, embedded in paraplast and sectioned at 7-8 μm for immunohistochemistry and at 10-14 μm for in situ hybridization. In situ hybridization was performed according to a previously described method.34 Probes have been described previously.18,35-37 Rehydration, unmasking, blocking and washing steps were performed according to the protocol of the tetramethylrhodamide based amplification kit (Perkin Elmer). Primary antibodies used for mouse sections were: cTnI rabbit polyclonal (1:250; Hytest Ltd); Tbx3 goat polyclonal (1:500; Santa Cruz Biotechnology); Cx40 mouse monoclonal (1:250; US Biological); Cx43 mouse monoclonal (1:250; BD Transduction); Scn5a rabbit polyclonal (1:250; Alemone labs); Nkx2.5 goat polyclonal (1:250; Santa Cruz Biotechnology); Hcn4 Goat polyclonal (1:250 Santa Cruz Biotechnology); BrdU rat polyclonal (1:600; AbD serotec), Cx30.2 rabbit polyclonal (1:200; gift from K. Willecke). For apoptosis detection we used the Cleaved Caspase 3 antibody (rabbit polyclonal, 1:250; Cell Signaling Technology). Primary antibodies used for human sections were: Tbx2 mouse monoclonal (1:100; gift from Colin Godin), TBX3 goat polyclonal (1:250 Santa Cruz), CX40 rabbit polyclonal (1:250; Santa Cruz). Secondary antibodies when using amplification were: Biotinylated donkey-anti-goat (1:250; Jackson Immunology); biotinylated rabbit (1:250; DAKO); biotinylated
goat-anti-2
mouse (1:250; DAKO). For visualization without the amplification step, secondary antibodies coupled to an Alexa fluorescent (1:250; Invitrogen) were used.
3D reconstruction
Image acquisition, processing and subsequent 3D reconstruction was performed according to a previously described method.38 Serial sections were stained for cTnI (labeling all cardiomyocytes) and Cx40 and reconstructed.
Preparation of the hearts and recording of electrograms and optical action potentials
Adult hearts
Electrocardiograms were recorded for a period of 5 minutes during 1.5 % isoflurane anesthesia. Signals were averaged after which RR, PQ, QRS, QT and QTc were calculated.
For the local recordings mice were anesthetized by an intraperitoneal injection of pentobarbital, after which the heart was excised, cannulated, mounted on a Langendorff perfusion set-up, and perfused at 37°C with Tyrode’s solution ((in mmol/L) 128 NaCl, 4.7 KCl, 1.45 CaCl2, 0.6 MgCl2, 27 NaHCO3, 0.4 NaH2PO4, and 11 glucose (pH maintained at 7.4 by equilibration with a mixture of 95% O2 and 5% CO2)). After that, the hearts were incubated in 10 ml Tyrode’s solution containing 15 μM Di-4 ANEPPS and subsequently placed in a optical mapping setup. Excitation light was provided by a 5 Watt power LED (filtered 510 +/- 20 nm). Fluorescence (filtered > 610nm) was transmitted through a tandem lens system on CMOS sensor (100 x 100 elements, MICAM Ultima). Activation patterns were measured during sinus rhythm, ventricular and atrial pacing at a basic cycle length of 120 ms, (twice the diastolic stimulation threshold). The effective refractory period of the atrioventricular node was determined by atrial pacing and reducing the coupling interval of a premature stimulus (after trains of 10 stimuli at basic cycle length 120 ms) in steps of 5 ms until activation of the ventricle failed. Optical action potentials were analyzed with custom software.
Fetal hearts
The hearts where removed from the embryo and incubated for 5 minutes with Tyrode’s solution containing 5 μM Di-4 ANEPPS at 37 oC. After incubation fetal hearts were superfused with Tyrode’s solution and placed on the stage of an inverted microscope set-up for recording optical signals.
Statistics
Group comparisons were performed using ANOVA. Values are given as mean +/- SEM. Genotype and phenotype frequencies were tested with a Chi-square test. A P-value of 0.05 was considered statistically significant.
2
Figure 1. In the left side of the atrioventricular (AV) canal of Tbx2-/- fetuses working myocardial genes are
ectopi-cally expressed and connect the left atrium with the left ventricle while the AV node and AV bundle are unaffected. Panel A, C and D show in situ hybridization analyses of serial sections wild type (left) and Tbx2-/- fetuses (right)
at E17.5. (A) In wild type the left AV canal myocardium does not express Cx40 and Cx43. Tbx2-/- ectopically
expressed Cx40 and Cx43 in the left AV canal (arrowheads). Furthermore, the AV canal in Tbx2-/- fetuses was
broader. (B) 3-Dimensional reconstructions of the heart of wild type (left) and Tbx2-/- (right) fetuses at E17.5.
Green represents all Cx40-positive myocardium and grey represents Cx40-negative myocardium. In Tbx2-/- fetuses
a Cx40-positive myocardial connection formed through the left AV canal. (C) In the upper panels, cTnI reveals the myocardium. The lower panels show the AV node based on Hcn4 expression and location. The AV node is not affected in Tbx2-/- fetuses. (D) In the upper panel, cTnI reveals all myocardium. The lower panels show the AV
bundle based on Cx40 expression and location. The AV bundle is not affected in Tbx2-/- fetuses. la, left atrium;
lv, left ventricle; ra, right atrium; rv, right ventricle; avb, atrioventricular bundle; avn, atrioventricular node; cs, coronary sinus; lsh, left sinus horn.
2
Results
Tbx2-deficiency does not affect the AV conduction axis, but causes formation of a
myocardial connection in the left atrioventricular junction
Heterozygotes containing the Tbx2Cre allele on an FVB/N background are viable, fertile, and
display no obvious phenotypic abnormalities. Tbx2Cre/Tbx2Cre mutants (Tbx2-/-) develop cleft palate and were not recovered after birth.14 Tbx2-/- fetuses collected at E14.5 and E17.5 from
Figure 2. In the left side of atrioventricular (AV) canal of Tbx2-/- fetuses working myocardial proteins are
ectopi-cally expressed. The proliferation rate in the epicardium is not different between wild type and Tbx2-/- fetuses. (A)
Immunohistochemical analyses of serial section of E17.5 wild type and two Tbx2-/- fetuses (f1,f2). In wild type
Cx30.2, is expressed in the AV canal complementary to Cx40. In Tbx2-/- fetuses Cx40 is expressed ectopically in
the AV canal, and Cx30.2 is still expressed in the AV canal myocardium. Notice the variable size and morphology of the aberrant myocardial connection. (B) Schematical representation of the expression profiles of connexins in the left AV canal myocardium in wild type and Tbx2-/- fetuses. Note that Cx40 expression withdraws from the
compact myocardium, however Cx43 remains present. (C) Immunohistochemical analyses of BrdU incorporation in epicardial cells at the left side of the AV canal in a wild type (upper) and Tbx2-/- fetus (lower). The right panel is
a magnification of the area within the white squares in the left panel. (D) A bar graph representing the proliferation rate based on BrdU incorporation in the myocardium of the left ventricle and in the epicardium at the right and left side of the AV canal. la, left atrium; lv, left ventricle; avc, atrioventricular canal; mv, mitral valve; sm, sulcus mesenchyme; lavc, left atrioventricular canal; ravc, right atriovnentricular canal.
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heterozygous intercross matings were present at less than expected on Mendelian inheritance (Supplemental Figure 1C). Fetal death of Tbx2-/- fetuses was less severe than found previously
for another Tbx2-null allele on the mixed C57BL/6/129/ICR background.17 Surviving E14.5 and E17.5 fetuses were of normal size and were used for further analysis. We assessed whether chamber-specific genes involved in conduction of the electrical impulse are ectopically expressed in the AV canal of Tbx2-/- fetuses. In wild type fetuses, Cx40 and Cx43 were not expressed in
the AV canal myocardium at E14.5 and E17.5. In contrast, Cx40 and Cx43 were ectopically expressed in the left side of the AV canal myocardium in all Tbx2-/- littermates (n=18; Figure
1A and Supplemental Figure 1A). The right AV canal myocardium was not affected in mutants (Supplemental Figure 1B). The right side of the AV canal is presumably to a larger extent under control of Tbx3 compared to the left side.18 The redundant function of Tbx3 is illustrated by the ectopic expression of Cx40 in the epicardial side of the left AV canal, complementary to that of Tbx3 at the endocardial side (Supplemental Figure 1A). Scn5a, encoding the major cardiac sodium channel (Nav1.5) that is required for fast conduction, was expressed in the AV junction
Figure 3. Typical example of an activation pattern in a wild type (left) and Tbx2-/- (middle and right) heart at E14.5.
In the wild type (left), activation starts in the atria, and after a delay of 50 ms the ventricles are activated within 3 ms after the first moment of activation of the apex of the left ventricle. In the middle, an activation pattern of a Tbx2-/- heart is shown. The activation starts in the atria and after a normal atrioventricular delay the ventricles
are activated from apex to base, after which the atria are activated for the second time via the left side. The right panels show an example of ventricular preexcitation in a Tbx2-/- heart. The activation starts in the atria after which
the base of the left ventricle is activated with an atrioventricular delay of 8 ms. Complete activation of both the left and right ventricle is within 15 ms. ra, right atrium; la, left atrium; lv, left ventricle; rv, right ventricle; t, time; ms, millisecond.
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of Tbx2-/- but not in wild type animals. Heterozygous Tbx2 mutants were not different from wild type (data not shown).
In the Tbx2-/- fetus, the compact AV node (Hcn4 positive) and AV bundle (Cx40-positive) were not affected (Figure 1C and 1D) and were not in contact with the aberrantly formed Cx40-positive (and Cx43-Cx40-positive) myocardial connection. Three-dimensional reconstructions of a E17.5 wild type and a Tbx2-/- fetus (Figure 1B) show the Cx40-positive pathway in Tbx2-/- that is not connected to the AV conduction axis (Figure 1B).
To assess whether the patterns of Tbx2 and Cx40 are conserved in human, we analyzed the expression of these proteins in a human fetus of Carnegie stage 14 (comparable to mouse E11.5). As in mouse, TBX2 and TBX3 are expressed in the AV canal myocardium, whereas CX40 is strictly absent from the AV canal myocardium but expressed in the atrial and ventricular myocardium (Supplemental Figure 2).
Next, we investigated the formation of the annulus fibrosus in Tbx2-/- embryos. In wild type embryos of E14.5, when annulus fibrosus formation is in progress, a narrow myocardial AV
Figure 4. Genes typical for the AV canal and genes typical for the working myocardium are simultaneously ex-pressed in the left side of the atrioventricular (AV) canal of Tbx2-/- fetuses. Images of in situ hybridization analyses
in sections of wild type (A,B) and Myh6-Cre;Tbx2fl/fl (C,D). B and D are magnifications of the black squares in,
respectively, panel A and C. (A,C) cTnI labels all myocardium. (B) In wild type fetuses, the AV canal myocardium did not express Cx40 and Scn5a, genes associated with fast conduction. The AV canal myocardium did express typical AV canal genes associated with slow conduction (Cacna1g, Cacna2d2), automaticity (Hcn4) and AV con-duction system maturation (Id2). (D) In Tbx2-/- fetuses Cx40 and Scn5a are ectopically expressed in the left AV
canal myocardium. The AV canal specific genes are still expressed and even found in the left ventricular wall in some cases (red arrowheads). la, left atrium; lv, left ventricle.
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junction connected the atria with the ventricles. At E17.5, the atria and ventricles where almost completely separated by the annulus fibrosus, although many small myocardial connections were still observed. In Tbx2-/- animals at E17.5, however, a myocardial connection in the left AV junction was formed. The size and morphology of the connection varied between animals (Figure 2A,B). Non-myocardial cells intermingled with the persisting myocardial connection, but did not form a complete separation. The right AV junction developed normally.
In Tbx2-/- animals, the sulcus mesenchyme is normally formed at the right side of the AV canal, while it is not formed at the left side. The proliferation rate of the epicardial sulcus was found to be similar between wild type (0.36 +/- 0.019) and Tbx2-/- animals (0.38 +/- 0.036) (Figure 2C,D). Previously we established an increased proliferation rate in the left AV canal of
Tbx2-/- at E9.5.18 At E11.5 the epicardial side of the left atrioventricular canal myocardium of the Tbx2-/- animals had an increased proliferation profile compared to the wild type (Figure 2C). Neither wild type (n=3) nor Tbx2-/- embryos (n=3) showed apoptosis in the right AV junction myocardium and epicardium at E11.5 (data not shown). These data suggest that the annulus fibrosus is malformed due to altered migration or dysmorphogenesis of the sulcus mesenchyme.
Preexcitation of the ventricles and retrograde activation of the atria in Tbx2-/- mice
In all E14.5 wild type (n=5) and Tbx2+/- fetuses (n=6), the left ventricle was activated from
apex to base within 2 ms, after an AV delay of 67 +/- 18 ms during sinus rhythm. However, we observed a functional accessory pathway in 6 of 14 Tbx2-/- fetuses. 5 of 14 Tbx2-/- fetuses showed
retrograde activation of the atria after a normal AV delay. In 1 of 14 Tbx2-/- fetuses we observed
that the ventricular myocardium was completely activated via the accessory pathway during sinus rhythm, after an AV delay of only 8 ms (Figure 3, Supplemental Figure 3 for movies). The location of the earliest ventricular activation coincided with the location of the accessory
{
{
{
FVB/N mixed FVB/N; C57BL/6 FVB/N -- Myh6-Cre;Tbx2 fl/fl Myh6-Cre;Tbx2 +/fl 0 (n=5) 7 (n=7) 5 (n=8) 6 (n=9) 7 (n=11) -- - -Tbx2 ;Cx30.2 -/- +/LacZ Tbx2 ;Cx30.2 -/- LacZ/LacZ Tbx2 ;Cx30.2 -/- +/+ - - Tbx2 wild type 0 (n=6) Tbx2 0 (n=8) 1 (n=6) 0 (n=5) 0 (n=6) 6 (n=13) 0 (n=5) -/-+/- - -Table1 1. Presence of a functional accessory pathway
