Electrophysiological patterning of the heart - Chapter 1: Electrophysiological patterning of the heart

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Electrophysiological patterning of the heart

Boukens, B.J.D.

Publication date

2012

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Citation for published version (APA):

Boukens, B. J. D. (2012). Electrophysiological patterning of the heart.

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Pediatr Cardiol. 2012 Aug;33(6):900-6.

Bas Boukens

Vincent Christoffels

Electrophysiological patterning of the heart

1

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.

1

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

_{. 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

_{, whereas in the Brugada syndrome the main origin is the right ventricular outflow}

tract (RVOT) and not the left ventricle

_{. 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

_{. 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

_{. The slow conduction in the primary myocardium gives}

rise to a sinusoidal electrocardiogram

_{. 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

_{. This results in an adult-like electrocardiogram with normal AV delay, although}

at this stage a fibrous annulus fibrosis has not formed yet

_{. 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])

_{. In addition, Cx30.2 is expressed in the AV canal and forms gap junctions}

1

with low conductance that lead to slow conduction

_{. The expression of Cx30.2 in the AV}

canal is regulated by transcription factors Gata4 and Tbx5

_{. 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

_.

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

_{. Nkx2-5, Tbx5,}

and Notch-signalling are important for normal formation of the AV node

_{. 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

_{. It has been reported that Cx43 and Cx40 are}

expressed in the lower part of the AV node (lower nodal cells)

_{. However, based on their origin}

and gene expression pattern we rather think that these myocytes are part of the AV bundle

_.

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

_.

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)

_{. 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

_{. 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.

1

branches

_{. 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

_{. Irx3 is also expressed in the AV bundle}

and bundle branches, where it represses Cx43 (mice) and (indirectly) activates the expression

of Cx40

_{. 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

_{. 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

_{. Mutations in NKX2-5 and TBX5 may cause AV block}

_{. Genome-wide association}

studies have implicated several loci in AV conduction, including TBX3/TBX5, NKX2-5, SOX5,

WNT11, SCN10A, SCN5A, CAV1-CAV2, and MEIS1

_{. 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

_{. What factors underlie the development}

of this type of arrhythmia is not known although in some cases familial AVNRT suggest an

inheritable component

_{. 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

_{. 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

_{. 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

_{. Indeed, deletions have been}

1

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

_{. The myocardial gene}

program, including that of the chamber myocardium, requires key cardiac transcription factors

Tbx5, Nkx2-5 and Gata4

_{. 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

_{. Furthermore, Hey2, encoding a helix–loop–helix transcription}

factor, represses the expression of Tbx5, Nppa and Cx40 in the compact myocardium

_.

The compact myocardium starts to form after E11.5 under influence of signalling molecules from

the epicardium

_{. 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

_{. 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, I

.

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

_.

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

_{. 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

_{. 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

_{. Mutations in genes encoding Na}

_channels

are found in patients with prolonged repolarization

_{but also in patients with conduction}

disturbances

_{. The clinical outcome of mutations in genes encoding for a single ion channel}

1

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

_{. Cx40 expression is considered}

the best specific marker for this network in the ventricles

_{. 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

_{. In chicken, endothelin-1 expressed in the underlying}

endocardium induces Cx40 and specification of the Purkinje fibres

_{. However, in mice,}

neuregulin influences the ventricular conduction pattern during development, probably through

its role in trabecular development

_{. 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

_{. 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

_{. 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

_.

Because the Purkinje fibre network is electrically insulated from the ventricular

myocardium, it may serve as substrate for re-entry

_{. Furthermore, Purkinje}

fibre myocytes can induce polymorphic ventricular tachycardia due to abnormal

calcium handling

_{. 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

_{(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

_.

The adult RVOT is the origin of arrhythmias in the Brugada syndrome

_{. 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

_{. A plausible}

mechanism underlying arrhythmias in the Brugada syndrome involves conduction delay or

block within the RVOT due to subtle structural abnormalities

_{. This process most likely is}

1

the RVOT may explain why it is sensitive for arrhythmogenesis

_{. 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

_{. 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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