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Birth Defects Research (Part C) 69:46 –57 (2003)

Development of the Cardiac Pacemaking and Conduction System Robert G. Gourdie*, Brett S. Harris, Jaqueline Bond, Charles Justus, Kenneth W. Hewett, Terrence X. O’Brien, Robert P. Thompson, and David Sedmera The heartbeat is initiated and coordinated by a heterogeneous set of tissues, collectively referred to as the pacemaking and conduction system (PCS). While the structural and physiological properties of these specialized tissues has been studied for more than a century, distinct new insights have emerged in recent years. The tools of molecular biology and the lessons of modern embryology are beginning to uncover the mechanisms governing induction, patterning and developmental integration of the PCS. In particular, significant advances have been made in understanding the developmental biology of the fast conduction network in the ventricles – the His-Purkinje system. Although this progress has largely been made by using animal models such as the chick and mouse, the insights gained may help explain cardiac disease in humans, as well as lead to new treatment strategies. Birth Defects Research (Part C) 69: 46 –57, 2003. © 2003 Wiley-Liss, Inc.

INTRODUCTION The discovery of a speck of blood in a chicken egg is an unwelcome breakfast-time lesson in embryology with which many are familiar. As hunger usually trumps curiosity, most people do not find this a particularly instructive experience. Around 2, 350 years ago, Aristotle put down a founding marker in the natural sciences when, in a study of fertilized hen eggs, he noted that such blood specks contained a tiny, pulsating heart. The clarification of the role of the heart in blood circulation would have to await the equally keen observations of William Harvey (1628) some 2000 years later. Nonetheless, Aristotle’s comment (circa BC 345) that the barely visible heart of the 3-day-old chick embryo “moved as though

endowed with life. . .” was prescient. We now know that the heart beat is intrinsically a product of its constituent myocardial tissues. Indeed, the pacemaker driving this rhythm is but one of a set of remarkable cardiac componentry whose integrated functions are key to ensuring the pressurized and unidirectional flow of blood. This heterogeneous set of specialized cardiac tissues is collectively referred to as the cardiac pacemaking and conduction system (PCS). The organization of the PCS and its relationship to physiological characteristics, such as the electrocardiogram, were elucidated a century ago by pioneering workers including Sunao Tawara, Authur Keith and Willem Einthoven (for reviews of this history and basic PCS

anatomy see Anderson and Ho, 2002; Suma, 2001; Davies et al., 1983). While the last 100 years have also seen a wealth of information added to our understanding of the structure and function of the PCS during embryogenesis, a distinctive change has occurred in the last decade. As with the growing mechanistic emphasis in other areas of biology, the manifold and still burgeoning tools of modern biology have facilitated study of the processes regulating development and functional integration of the PCS. Thus far, the most rapid advances have been in our understanding of the development of the His-Purkinje system – the network of fast conduction tissues responsible for coordinating ventricular contraction. This work, together with a discussion of its potential clinical implications, is covered in the last half of this review. To begin with, the main structural and functional elements of the PCS — including the sinuatrial pacemaker, the atrioventricular delay generator and the His-Purkinje conduction system — will be introduced (see also Fig. 1). As is explained, the sequence of differentiation of each of these elements during cardiac development follows a distinctive pattern that

Robert G. Gourdie, Brett S. Harris, Jaqueline Bond, Charles Justus, Kenneth W. Hewett, Terrence X. O’Brien, Robert P. Thompson, and David Sedmera are from the Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina. Brett S. Harris and Terrence X. O’Brien are also from Medical Research Service, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina. Grant sponsor: NIH; Grant numbers: HD39946, HL36059, HL56728; Grant sponsor: NSF; Grant number: IBN-9734406; Grant sponsor: Office of Research and Development of the Department of Veterans Affairs. *Correspondence to: Dr. Robert G. Gourdie, Department of Cell Biology and Anatomy, MUSC, 173 Ashley Avenue, Suite 601, Charleston, SC, 29425. E-mail: [email protected] Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.10008

© 2003 Wiley-Liss, Inc.

CARDIAC CONDUCTION SYSTEM DEVELOPMENT 47

bers. This morphogenetic sequence is also conserved in mouse and humans (see Fig. 1).

The Emergence of a Cardiac Pacemaker in the Embryonic Tube Heart

Figure 1. Functional and structural components of PCS in the developing and adult chick heart. Yellow stars indicate locii of pacemaking activity. Direction and velocity of AP propagation shown by isochrones and small arrows on the different stages of the developing heart illustrated. AV, atrioventricular; Ao, Aorta; SA, sinuatrial; RA and LA, right and left atria; RV an LV, right and left ventricle.

appears to be morphogenetically conserved.

COMPONENTS OF THE PACEMAKING AND CONDUCTION SYSTEM HAVE CONSERVED FUNCTIONS While subject to neuronal modulation, the intrinsic rhythm of the heart of higher vertebrates is determined within the tissues of the cardiac pacemaker — the sinuatrial (SA) node. The SA node is situated at the inflow port of the cardiac pump, or more formally, at the boundary of the superior caval vein and the right atrium. Following initiation of a cardiac action potential (AP) within the node, activation is propagated through the muscular tissues of the atria, eventually focusing into the atrioventricular (AV) node. As its name implies, the AV node is located at the junction of the atria and ventricles, and functions as part of a mechanism for generating a momentary delay in the propagation of AP. The principal role of this AV delay generator is to separate (and to some extent insulate) the activation of the atrial chambers from that of the ventricles. Following exit from the AV node, AP rapidly propagates along the His bundle and its distal branches, finally activating the

ventricular chambers via a highly ramified network of Purkinje fibers. Together, this fast conduction system of His-Purkinje tissues forms the last of the main elements of the PCS. It should be emphasized that the above description is an oversimplification. Studies of the structure of the SA node , AV node and His-Purkinje system in the hearts of different animals reveal complex and variable organizations (reviewed Davies et al., 1983; Lamers et al., 1991; Moorman et al., 1998; Boyett et al., 2000). For example, the anatomical substrate for the AV node in the chick heart is debated (Szabo et al., 1986). The sequential contraction of the atria and ventricular chambers in this species nevertheless provides unequivocal evidence for a functional generator of AV delay. Similarly, structural and functional correlates of a pacemaker, AV delay generator and fast conducting system can be distinguished in the embryonic chick heart (reviewed Viragh and Challice 1983; Gourdie et al., 1999; Mikawa et al., 2001; Pennisi et al., 2002). The differentiation of each of these three elements of PCS function follows a repeatable sequence during cardiac morphogenesis, respectively emerging during the formation of the tubular heart, looping and septation of the cardiac cham-

Consonant with its role initiating the heart beat, a pacemaker is the first functional element of the PCS to become recognizable in the embryo. In the chick, a pacemaker locus differentiates at around 25 to 35 hours of development in the posterior-most segment of the primitive tubular heart (Kamino et al., 1981; reviewed Kamino, 1991). In mice and humans, similar stages would correspond to embryonic ages of around 7.5 and 21 days respectively. At this early stage of morphogenesis, the tube heart has a simple organization consisting of a few concentric layers of cells — a thin outer layer of epithelioid myocytes, an intermediate stratum of acellular matrix and an inner lining of endocardial cells (reviewed Manasek et al., 1968; Fishman and Chien, 1997; Markwald and Wessels, 2001). As the rudimentary heart begins looping, rhythmic waves of excitation triggered by the pacemaker induce the propagation of slow, peristaltic contractions along the single-chambered organ (Patten and Kramer, 1933; de Jong et al., 1992). The flow of blood established by these initially modest contractions marks the appearance of the unidirectional circulatory pattern that will be maintained by the heart throughout the life of the animal. The rhythmic propagation of AP along the tube heart displays posterior-anterior polarity (Satin et al., 1988). Myocytes in the posterior, sinuatrial segment assume pacemaking dominance by virtue of having the fastest intrinsic rate. Interestingly, retinoic acid sensitive gradients of posterior-anterior gene expression have been characterized along the tube heart of zebrafish, chicks and mice (Stainier and Fishman, 1992; Yutzey, et al., 1994; Wang et al., 1998; XavierNeto et al., 1999). It remains to be determined whether the mecha-

Birth Defects Research (Part C) 69:46 –57, (2003)

48 GOURDIE ET AL.

nisms governing such gradients inform the distribution of pacemaker dominance. Nonetheless, given the potential relevance to differentiation of SA nodal function, an investigation of the relationship between retinoid signaling and dominant automaticity would seem to be an area worthy of further attention. The electrogenic mechanisms shaping the differentiation and maintenance of rhythmic AP generation are beginning to be better understood. There is now strong evidence that the hyperpolarizationactivated cyclic nucleotide-gated (HCN) family of ion channel subunits is a key determinant of pacemaker activity in the adult and embryonic heart (Baker et al., 1997; reviewed Biehl et al., 2002). Evidence for the involvement of other types of channel in pacemaking activity comes also from the observation that knockout of the NaCa exchanger gene results in lethality due to the failure of the tubular heart to establish spontaneous beating (Koushik et al., 2001).

Development of an Atrioventricular Delay in the Looping, Tube Heart Initially, the AP upstroke of all myocytes along the tubular heart has a relatively slow rate of rise — a pattern mainly accounted for by Ca2⫹ currents, including L- and T-type Ca2⫹ channels (reviewed Sperelakis, 1990; Fozzard, 2002). Consistent with this slow depolarization, the velocity of posterioanterior propagation of AP is uniformly sluggish. However, as looping proceeds, non-uniformities in velocity appear . In particular, a slowly conducting AV canal separating the activation of the faster conducting segments of the atrium and ventricle becomes distinguishable (Lieberman et al., 1965a,b; Arguello et al., 1986; de Jong et al., 1992). In the avian embryo, delay at the AV canal becomes evident from around 42 hours of development, a stage corresponding to around 8 and 25 days of embryonic age in mouse and human respectively. The AV canal is one of three regions of the looping, tube heart

(including the sinuatrial region and outflow tract), which display electro-mechanical properties similar to those of the early tube heart (de Jong et al., 1992). As the cardiovascular system grows in scale and complexity, these slow-conducting segments and associated cushion tissues function as sphincter-like valves, coordinated in their contraction and relaxation to increase the efficiency of the early blood pump. It should be emphasized that AV delay is probably not simply a function of single membrane channels. Cell-to-cell spread of cardiac activation is mediated by gap junctional connections between myocytes (reviewed Lo, 2000; Barker and Gourdie, 2002). Studies in rodents indicate that these electrotonic couplings in the tubular heart (and later in the AV canal and outflow tract) are mainly dependent on gap junctions comprised of connexin45 (Alcolea et al., 1999; Coppen et al., 2001), channels that are characterized by high voltage-sensitivity and low permeability (Veenstra et al., 1994). Such properties are likely to contribute to slow conduction in the AV canal, a point underscored by the observation that knockout of the connexin45 gene results in lethality from heart block at looped, tubular stages of heart development (Kumai et al., 2001). Connexin45 is also the most highly expressed gap junction protein within the SA node and AV node of the mouse and other mammalian species (Coppen et al., 1999; reviewed Gourdie and Lo, 2001; Severs et al., 2001). Abnormalities in AV nodal function commonly manifest as tachyarrhymias or bradyarrhythmias (particularly intra- or extranodal bypass tracts), and often require lifelong medications, electronic pacemakers or electrophysiologic procedures. Given this clinical imperative, it is surprising that the mechanism by which the cyto-architecture of the node, gap junctions and membrane channels combine to generate AV delay in the adult remains something of a mystery. The presently obscure cellular and molecular mechanisms responsible for normal and abnor-


mal AV nodal development and function would seem to hold much promise for future discovery and progress.

Differentiation of the Fast Conduction System During Chamber Septation The fast conduction system of the ventricles is the last element of the PCS to differentiate. In chick, this is marked by an apparent reversal in the sequence of ventricular activation (Chuck et al., 1997a). Specifically, an immature base-to-apex pattern of epicardial activation switches to a mature apex-to-base pattern between the 6th to 9th day of chick embryonic development. Fundamentally, the immature pattern is a maintenance of the sequence of activation seen at earlier looped, tubular stages of development. The mature “apex-first” pattern coincides with completion of ventricular septation, and is likely to result from epicardial breakthrough near the termini of the right and left branches of the HisPurkinje system. Rentschler and co-workers (2001) have reported that apical activation in mouse is initiated around 10.5 days of embryonic development, a timing prior to completion of ventricular septation. Until recently, it was thought that lower vertebrates did not possess a fast conduction system. However, work in Xenopus and zebrafish have now confirmed that these species also demonstrate a functional equivalent of this specialized network (Sedmera et al., 2002). This observation opens up the use of models such as the zebrafish for investigations of the mechanisms underlying His-Purkinje development. Retroviral lineage-tracing studies in chick indicated that central elements of the PCS (e.g., the His bundle) differentiated independently of distally located PCS components, such as the peripheral network of Purkinje fibers (Gourdie et al., 1995). This raised the prospect that certain PCS elements may form separately and link together as development proceeds. While it remains to be established that such a


linkage process accounts for the switch in activation sequence, addressing this question may be important, as it could inform the origins of congenital AV block in humans, such as that occurring in autoimmune lupus (Askanase et al., 2002). There are other data that may have bearing on this aspect of His-Purkinje development. Work in humans has indicated that the remodeling of an initially ringlike domain, present in the looped, tubular heart, may be key to morphogenesis of the conduction system (Wessels et al., 1992). Two molecules linked to intercellular interactions, PSA-NCAM and connexin40 have been reported to show increasingly retrograde patterns of expression along the axis of atrioventricular conduction in the developing chick heart (Chuck et al., 1997; Gourdie et al., 1993). Interestingly, PSA-NCAM has been implicated in neuronal guidance (Durbec and Cremer, 2001). The gap junction protein connexin40 is highly up-regulated during development of the conduction system in birds (Gourdie et al., 1993) and mammals (Delorme et al., 1995). Coupling mediated by this high conductance channel is thought to be key to emergence of fast activation spread in the His-Purkinje system (reviewed Lo, 2000; Severs et al., 2001). Finally, lineage marking studies in chick have revealed patterns of association between extracardiac-derived populations of cells and specific parts of the developing conduction system (Gittenberger deGroot et al., 1998; Cheng et al., 1999; Poelmann and Gittenberger-de Groo, 1999). In particular, the timing of neural crest migration into the embryonic heart and its subsequent interaction with forming central conduction fascicles appears to correlate with maturation of function of the His-Purkinje system (Cheng et al., 1999; Poelmann and Gittenberger-de Groot, 1999). A caveat to note here is that such associations should not be taken as implying that PCS tissues have an extracardiac origin. Indeed, as will be discussed, lineage analyses from different groups have suggested that signif-

icant contributions by migratory cohorts, such as the neural crest, to the conduction system are unlikely, at least in higher vertebrates.

MECHANISMS OF DEVELOPMENT OF THE CONDUCTION SYSTEM The Cardiomyogenic Origin of Conduction Cells At present, the mechanisms governing ontogenesis of the pacemaker and AV delay generator are not well understood. Elegant lineage studies by Davies, Wessels and Burch using cre-lox transgenics have traced cells contributing to the AV canal (i.e., prospective AV node and ring progenitors) to the external margins of the cardiogenic fields in the mouse gastrula (Davies et al., 2000, 2001). However, with this notable exception, there is a lack of mechanistic information in areas as basic as the cellular origin of nodal tissues. In contrast to this relative paucity of data, there is a growing understanding of the molecular and cellular processes contributing to the development of the ventricular conduction system (reviewed Moorman et al., 1998; Gourdie et al., 1999; Mikawa et al., 2001; Pennisi et al., 2002). Essential to these insights have been markers of conduction tissues, in the form of both specific gene products differentially localized in specialized cells, and more recently, transgenic animals encoding reporter genes expressed in the PCS (Table 1). A further key to progress has been the advent of reliable tracers of the developmental history of cells. In particular, replication incomptetent retroviruses for lineage tracing, which have been used in the chick embryo to probe the derivation and phenotypic divergence patterns of cells contributing to the developing heart (reviewed Fischman and Mikawa, 1997; Gourdie et al., 2000). One of the first insights provided by lineage tools concerned the organization of ventricular muscle in the embryonic chick heart. Injection of low titers of retrovirus into the looped, tubular heart (with sub-

sequent development) identified segments of clonally related myocytes (Mikawa et al., 1992). In ensuing work, it was shown that some of these clonal segments contained conduction cells and working myocytes (Gourdie et al., 1995; Cheng et al., 1999); this heterotypic clonal motif could be identified at both large conduction fascicles (e.g., the His bundle), and the diffuse network of peripheral conduction cells (i.e., subendocardial and periarterial Purkinje fibers – see Fig. 2). This direct demonstration of a common origin for working and specialized myocardial cells in the embryonic ventricle went some way to settling a longstanding debate. An earlier hypothesis, based largely on the observation that PCS tissues in some species expressed neuronal markers, proposed an extracardiac neural origin for conduction cells (reviewed Gorza et al., 1988). However, the demonstration of a cardiomyogenic derivation of conduction cells, together with data from direct retroviral marking of the cardiac neural crest, indicated that any contribution by neurogenic cells to the His-Purkinje system was likely to be small (Gourdie et al., 1995; Cheng et al., 1999). Similarly, in mouse, Cre-lox marking of presumptive neural crest derivatives confirmed that there was no detectable contribution to the conduction system by neural cells in this species (Jiang et al., 2000; Epstein et al., 2000).

Different Models for Developmental Elaboration of the Conduction System While lineage tracing has demonstrated that conduction cells are derived from progenitors in the early embryonic heart, there are different ways of conceiving how such cells could give rise to a structure as complex as the mature conduction system (Cheng et al., 1999; Rentchler et al., 2000). On the one hand, the His-Purkinje system may form by proliferative outgrowth from a pool of differentiated conduction cells. On the other hand, it could elaborate by localized patterns of cellular recruitment to


50 GOURDIE ET AL.

TABLE 1. Gene Expression and Transgenic Markers of the Conduction System In Mouse and Chick Gene

Species

Localization

Onset

Mouse Mouse/Chick

Entire PCS Mainly H-P

Tubular Looped Tubular

Chick Chick Chick *

AVN-R H-P H-P P

Looped Tubular “ “ “

Mouse/Chick Mouse Mouse Chick Chick Mouse/Chick Mouse Chick Chick Chick

AVN, H-P Entire PCS H-P H-P P H-P H-P(decreased) H-P H-P

Tubular “ “ “ “ — Looped Tube Late Gest. “ “

Chick Mouse/Chick Chick Chick

AVN-R Entire PCS P P

Looped Tubular “ — —

Transgenic

Construct/Gene

Localization

Onset

cGATA-6-LacZ

GATA-6 Enhancer Desmin Enhancer Troponin I Enhancer Engrailed-2 Enhancer K⫹ channel modulator Transcription factor

AVN-R

Pre-Tubular

H-P

Tubular

AVN-R

“

Entire PCS

Looped Tubular

H-P

“

H-P

“

Gap Junctional Connexin 45 Connexin 40 Neuronal/Signaling Related HNK-1/Leu-7 Acetylcholinesterase Polysialated-NCAM ANP Structural ␣ MHC B MHC Desmin Transitin (EAP-300) Neonatal skeletal MHC ␣ Smooth muscle actin Cardiac troponin I Cardiac myosin binding protein-C Slow tonic MHC (sMHC) Myosin binding protein H Transcription Factors Msx2 (Hox8) Nkx2.5 Gata6 MyoD

DES1 TroponinI-LacZ Engrailed 2 - LacZ fusion minK knockout LacZ knock in HF-1b knockout LacZ knock in

A few pointers to note. “Onset” refers to onset of expression within the developing heart and not necessarily the timing of specific localization to the PCS. Some of the gene markers listed are initially expressed in larger domains and converge into the conduction system with development (e.g., transitin), while others are not necessarily exclusively expressed in conduction tissues (e.g. ␣- and ␤-MHC). Cardiac myosin binding protein-C expression is marked by down-regulation, rather than upregulation. *ANP has not been reported to be specifically expressed in chick and mouse, but is an important PCS marker in various other species including rat, pig and human. It should also be noted that some of the transgenics demonstrate insertion-dependent transgene expression patterns (e.g., engrailed-2-LacZ and troponin I-LacZ), while others display insertion-independent expression profiles (e.g. cGATA6-LacZ). PCS, pacemaking and conduction system; H-P, His-Purkinje system; P, peripheral Purkinje fibers; AVN-R, atrioventricular node and ring. V. Sept, timing around ventricular septation; Post V Sept, following ventricular septation; Late Gest, just prior to hatching. References see text and Moorman et al., 1998; Gourdie et al., ’99; Schiafinno et al., ’97; Di Lisi et al., ’00; Rentchler et al., ’00; Machida et al., ’00; Davies et al., ’01; Franco and Icardo, ’01; Pennisi et al., ’02.

the growing network from progenitors that have not yet terminally differentiated. While it may turn out that the developmental elaboration of the conduction system is a multigenic process, there are a number of considerations suggesting that inductive recruitment is at least one

contributing mechanism. First, it is relatively straightforward to conceive how localized recruitment could lead to myocardial clones (i.e., as defined by retroviral marking) containing working myocytes and conduction cells (e.g., Fig. 2). Second, factors identified as


prompting differentiation of conduction cells in vitro (e.g, endothelin in chick and neuregulin in mouse), appear to directly recruit embryonic heart cells to specialized phenotypes without changing rates of division of the converted cells (Gourdie et al., 1998; Rentschler et


1978; Thompson et al., 1990, 2000; Cheng et al., 1999). This appears to be a consistent feature of both avian and mammalian species.

The Avian Periarterial Purkinje Fiber as a Paradigm for Mechanistic Studies of Conduction System Development

Figure 2. Localized and progressive recruitment of periarterial Purkinje fiber cells within a myocyte lineage. A: Periarterial Purkinje fibers are the terminal cells of the His-Purkinje system in the developing chick heart. B: Segment of a retrovirally defined myocardial clone containing myocytes (M) and periarterial Purkinje fibers (PPF). Cells infected with defective retrovirus have a nuclear LacZ tag (blue nuclei). C: Model for generation of the heterocellular clone seen in B. Box A-Retrovirus infected myocyte divides. Box B Daughter myocyte generated by division is proximal to a newly generated branch of a coronary artery and inductively recruited to undergo differentiation. Box C – Purkinje fiber withdraws from proliferation and continues differentiation while sister myocytes continue proliferation.

Figure 3. Model of the hemodynamic and molecular factors involved in the induction of subendocardial and periarterial Purkinje fiber cells in chick. ECE-1; endothelin converting enzyme-1, ET-1; endothelin-1, ET-R; endothelin receptor (Kanzawa et al., 2002, indicates that the ET-1 signal is likely to be transduced via the ET-A receptor isotype).

al., 2001). Finally, and perhaps most significantly, the potential for outgrowth from a differentiated pool of cells is limited by the fact

that commitment to non- or slowed proliferation is one of the earliest differentiating characteristics of conduction cells (Rumyantsev,

The conduction system of the avian heart displays a number of distinctive features. In particular, its terminal elements include Purkinje fibers which penetrate deep into muscle in intimate association with coronary arteries– i.e., periarterial Purkinje fibers (see Fig. 2). An elegant study by Francis Davies published in 1933 provided the first description of these cells in various species, including birds that are common to London parks, such as the pigeon and black swan. Periarterial Purkinje fibers in the chick have been singularly influential in shaping our ideas about the factors involved in development of the fast conduction system. Among other insights, these tissues provided the first evidence that recruitment of cells to the elaborating conduction system occurred progressively (reviewed Gourdie et al., 1999). In the initial lineage studies, it was found that the proportion of clones containing periarterial conduction cells increased during the last half of the 21-day period of incubation in ovo (Gourdie et al., 1995). This result suggested that as the vascular tree continued to grow and ramify in the developing chick heart, there was ongoing recruitment of Purkinje fibers to sites around newly generated arterial branches (e.g., Fig. 2). A series of experiments in which coronary arterial branching was either inhibited or activated in vivo subsequently confirmed this hypothesis (Hyer et al., 1999). Evidence suggesting that assembly by progressive recruitment was not simply confined to terminal parts of the PCS came from birth-dating studies using radioactive pulse labeling (Cheng et al., 1999). Birth dating indicated that conscription


52 GOURDIE ET AL.

of cells to structures such as the His bundle, initiated at looping and continued until shortly after completion of ventricular septation. In general agreement with the pattern of recruitment resolved by retroviral tagging, the birthdates of peripheral conduction cells consistently fell after completion of septation.

Endothelin-Induced Recruitment of Conduction Cells in Chick Perhaps the main insight into mechanism arising from studies of the Periarterial Purkinje fiber has been the potential linkage inferred between conduction tissue differentiation and hemodynamic factors. Periarterial Purkinje fibers are only found next to coronary arteries, and are never seen adjacent to capilliaries or cardiac veins — i.e., lower tension vessels distal from the aortic root (Davies, 1933; Gourdie et al., 1993; TakebayashiSuzuki et al., 2000). The timing of differentiation of Purkinje fibers around arteries shows a conspicuous relationship with the timing of initiation of blood flow within the embryonic arterial tree following its connection to the aortic root (Gourdie et al.,1995; reviewed Gourdie et al., 1999; Mikawa et al., 2001). Such observations led to the hypothesis that paracrine signaling by arterial tissues, particularly those signals elicited by hemodynamic changes, might have a role in the induction and patterning of the conduction system. Support for this idea came from experiments in which embryonic myocytes were exposed to endothelin-1 (ET-1) in vitro, a shear-stress responsive cytokine prominently expressed in the coronary arterial bed (Yanagisawa et al., 1988; Yoshizumi et al., 1989; Yoshisue et al., 2002). Treatment with ET-1 caused changes in gene expression (e.g., increases in sMHC and connexin40) consistent with the differentiation of Purkinje fibers (Gourdie et al., 1998). Further support for the ability of ET-1 to promote differentiation of Purkinje fibers has been provided by elegant work undertaken mainly

by Kimiko Takebayashi-Suzuki in the laboratory of Takashi Mikawa (Takebayashi-Suzuki et al., 2000; 2001). Modification of PreproET-1 to active ET-1 is a two-step process (Xu et al., 1994). The first step involves a relatively non-specific cleavage of PreproET-1 to bigET-1. This is then followed by a highly specific step, whereby Big-ET-1 is cleaved to active ET-1 via the Endothelin Converting Enzyme ECE-1. Direct expression of an active ET-1, missing the N-terminal Prepro sequence, was found not to cause changes in myocyte phenotype in chick embryonic hearts in vivo, possibly due to defects in secretion of the truncated gene product (Takebayashi-Suzuki et al., 2000) . However, when retroviral gene vectors were used to generate overlapping domains of exogenous ECE-1 and PreproET-1, ectopic and precocious differentiation of Purkinje fibers was found to occur. This experimental paradigm suggested a mechanism by which the timing and location of Purkinje fiber differentiation might be regulated during normal cardiac morphogenesis. Namely, the ET-1 function may not be constrained by the expression pattern of the ligand per se, but by the distribution of its activating enzyme, ECE-1. Consistent with this, in situ hybridization revealed that ECE-1 is exclusively expressed in endothelial cells of the endocardium and coronary arteries, but not veins or capilliaries (TakebayashiSuzuki et al., 2000, 2001). Moreover, the timing of ECE-1 expression at these two loci appears to correlate well with the differentiation of sub-endocardial and Periarterial Purkinje fibers respectively. Preliminary evidence indicates that ECE-1 expression itself may be subject to modulation by hemodynamics. Increasing load on the chick embryonic ventricle (by conotruncal banding) leads to upregulated ECE-1 expression (Mikawa et al., 2003) and precocious differentiation of His-Purkinje function (Sedmera et al., in press). Thus, evidence is growing that the response of endothelial tissues to physical force during cardiac morphogenesis may be central to selec-


tion of specialized myocardial fate and generation of a conduction system pattern (see Fig. 3). This interplay between biophysical factors and paracrine signaling may provide new avenues for exploring the origins of plasticity leading to congenital abnormalities in cardiac structure and function.

Neuregulin-Induced Recruitment of Conduction Cells in Mouse The work of others in mouse has suggested a role for another factor secreted by endothelial cells in promoting the differentiation of PCS tissues (Hertig et al., 1998; Rentschler et al., 2002). Neuregulin signaling has been shown to have important functions during cardiogenesis, in processes such as trabeculation of the ventricles and cardiac valve formation (Meyer and Birchmeier, 1995; Camenisch et al., 2002). Using cultured mouse embryos, Hertig and co-workers (1998) found that neuregulin-1 (NG-1) treatment induced increases in ventricular trabeculation and upregulation of acetylcholinesterase (a transient marker of PCS differentiation). Interestingly, these changes occurred without alterations in proliferation rate, suggesting that NG-1 had recruited cells to the inner trabecular layer of the ventricular wall, otherwise destined to join the outer compact layer. These observations were extended in a recent study by Rentchler and co-workers (2002) involving a transgenic line of mice that expresses LacZ in the developing PCS (see Table 1). In addition to inducing increases in trabeculation, this study reported that NG-1 treatment of heart explants and cultured embryos resulted in striking increases in LacZ expression in the ventricle, and electrophysiological changes consistent with precocious maturation of conduction system function. There are parallels and relationships between the endothelin-andneuregulin signaling pathways, other than common expression by endothelial tissues, that perhaps merit discussion. Like ET-1, NG-1


can upregulate atrial natiuretic peptide (ANP) in neonatal ventricular myocytes cultured from rodents (Zhao et al., 1998). Pertinently, ANP is transiently expressed during normal development of the conduction system in some species (reviewed Moorman et al., 1998; Gourdie et al., 1999). The ability of NG-1 and ET-1 to recruit cells to conduction lineages in vitro is restricted to the same post-looping window of cardiac morphogenesis in chick and mouse (Gourdie et al., 1998; Rentschler et al., 2002). In this respect, it is relevant that ET-1 has been shown to directly upregulate NG-1 expression in endothelial cells (Zhao et al., 1998), suggesting potential for cross-talk between these two pathways. Indeed, mechano-induced endothelin signaling as an intermediary to NG-1 would seem an interesting prospect for future study. Finally, there is growing evidence that interplay between endothelin and neuregulin is key to regulation of other developmental processes, including during differentiation of peripheral nervous system the major glial cell of the Schwann cells (Jessen and Mirsky, 2002).

MECHANISMS OF PCS DEVELOPMENT AND CARDIAC DISEASE Genetic Disorders Can understanding the mechanisms that contribute to development of the PCS teach us anything about the origins of cardiac disease in humans? Many types of congenital heart disease manifest conduction abnormalities. In the adult, cardiac arrhythmias are the most common cause of cardiovascular death. To give a recent example, Purkinje fibers were mapped as the predominant origin of arrhythmias in human patients with recurrent idiopathic ventricular fibrillation (Haissaguerre et al., 2002). Around 0.2% of the population has WolffeParkinson-White syndrome (WPW), and a greater number have AV nodal and accessory bypass tracts are commonly found in supraventricular tachycardias (reviewed

Cain et al., 1992). Exploration of whether abnormalities in PCS development contribute to such syndromes is a hypothesis worthy of further exploration. While comprehensive understanding is probably some way off, mechanistic studies of relatively rare genetic diseases often provide insights into more prevalent diseases. The recent discovery of a gamma-2 regulatory subunit of AMP-activated protein kinase (PPKAG2) mutation associating with a familial form of WPW and progressive conduction system disease is one such example (Gollob et al., 2002). Other important opportunities for ongoing focus include the so-called “channelopathies” where genetic abnormalities of a variety of ion channels predispose individuals to arrhythmias and sudden death (reviewed Marban, 2002). The long QT syndrome with its characteristic ECG abnormality and propensity for fatal ventricular tachyarrhythmias at a young age has been linked to four IK current subunit genes and the Na⫹ channel gene, SCN5A. Also, mutations in the T-box gene TBX5 are associated with Holt-Oram syndrome, a rare genetic disease characterized by limb and cardiac defects, including conduction abnormalities (Basson et al., 1997). TBX5 has been reported to be expressed in the human AV node (Hatcher et al., 2000), and haploinsufficiency of this gene in mice leads to downregulation of the atrial and conduction system associated genes ANF and connexin40 (Bruneau et al., 2001). Similar to the phenotype observed in the connexin40 knockout (reviewed Lo, 2000), mice with a single Tbx5 allele display abnormalities in ventricular activation including AV block. As is known in other genetic diseases, several unique mutations of a critical gene may lead to similar phenotypes. Over 10 different mutations in the cardiac transcription factor NKX2.5 have been shown to be associated with several phenotypes of atrial septal defects and AV block (Schott et al., 1998). Interestingly, NKX2.5 also illustrates the poorly understood interplay of multiple genetic factors. A sequencing

survey of children with several different congenital heart diseases involving septal abnormalities (e.g., tetralogy of Fallot, ventricular and atrial septal defects) showed that about 4% had NKX2.5 mutations (Goldmuntz et al., 2001). NKX2.5 is expressed at elevated levels in the embryonic human PCS (Thomas et al., 2001), and studies in animal models indicate that Nkx-2.5 may have significant functions during PCS development (Thomas et al., 2001; Takebayashi-Suzuki et al., 2001; O’Brien et al., 2001). Progressive AV conduction defects and heart failure have been reported in transgenic mice expressing one of the mutant forms of NKX2.5 identified in humans (Kasahara et al., 2001). Increased Nkx2.5 expression has also been reported in animal models of adult cardiac hypertrophy (Thompson et al., 1998), and thus in addition to its roles in early cardiogenesis and PCS differentiation, Nkx2.5 may function in adaptive responses of working myocardial tissues to certain disease states.

Is There a Link between the Differentiation of Conduction Cells and Pathological Electrophysiological Heterogeneity? The HF-1B knockout mouse is a model that may provide one link between conduction cell differentiation and heart disease (NguyenTran et al., 2000). Mice deficient in HF-1b survive to term, but develop defects to cardiac activation, often dying suddenly and unexpectedly from ventricular arrhythmias. Analyses of single cells have revealed that markers distinguishing ventricular and conduction lineages are disrupted in these animals, with increased heterogeneity in myocyte AP characteristics. A hypothesis suggested to account for these observations proposed that knockout of the HF-1B gene effected mechanisms determining transition between myocyte and conduction cell fate in the embryonic ventricle. While there has been no direct con-


54 GOURDIE ET AL.

Figure 4. Model for differentiation of arrhythmogenic myocyte heterogeneity in diseased hearts. Ventricular myocytes (M) in certain diseases show upregulation of Purkinje fiber differentiation markers (e.g., Cx40, ANF, Nkx-2.5), suggestive of the generation of a transitional (T) phenotypes . Does this suggest the possibility of recapitulation of signaling mechanisms involved in the embryonic development of Purkinje fibers during the generation of arrhythmogenic heterogeneity within the ventricular wall of the diseased heart?

firmation of this hypothesis, there is much evidence indicating that variation in myocardial phenotype within the normal and diseased heart defies simple classification. In the failing human heart, connexin40 is disturbed from its normal sub-endocardial localization in Purkinje fibers and becomes ectopically expressed in the ventricular wall (Dupont et al., 2001). In the normal pig ventricle, Toshimori and co-workers (1988) have described cells transitional between myocytes and conduction cells. Similarly, in the rat, transitional Purkinje fibers have been reported around arteries in distributions that are strikingly reminiscent of periarterial Purkinje fibers in chick (Cantin et al., 1989). Electrophysiological studies in various mammalian species have turned up populations of myocytes termed M cells, with AP characteristics that (in some respects) appear to be intermediate between working myocytes and Purkinje fibers (Antzelevitch et al., 1999; Anyukhovsky et al., 1999). That AP heterogeneity is an important predisposing factor in cardiac arrhythmia is widely accepted. It is thus surprising that relationships between transitional cells in the normal ventricle (such as those around coronary arteries) and pathological AP heterogeneity remain largely unexplored. Could it be that signaling processes primarily active during embryonic differentiation of conduction tissues are recapitulated in certain disease states in the

adult (see Fig. 4)? As outlined above, the abnormal expression of markers of conduction cell differentiation (e.g., Cx40, Nkx-2.5, ANF) does seem to be a feature common to different diseases of the heart. Further work is necessary to determine whether this hypothesis is an avenue that could provide fresh insights into the origins and eventual treatment of conduction disorders of the human heart.

LITERATURE CITED Alcolea S, Theveniau-Ruissy M, JarryGuichard T, Marics I, Tzouanacou E, Chauvin JP, Briand JP, Moorman AF, Lamers WH, Gros DB. 1999. Downregulation of connexin 45 gene products during mouse heart development. Circ Res 841:365–379. Anderson RH, Ho SY. 2002. The morphology of the specialized atrioventricular junctional area: the evolution of understanding. Pacing Clin Electrophysiol 25:957–966. Antzelevitch C, Shimizu W, Yan GX Sicouri S, Weissenburger J, Nesterenko VV, Burashnikov A, Di Diego J, Saffitz J, Thomas GP. 1999. The M cell: its contribution to the ECG and to normal and abnormal electrical function of the heart. J Cardiovasc Electrophysiol 10:1297–1299. Anyukhovsky EP, Sosunov EA, Gainullin RZ, Rosen MR. 1999. The controversial M cell. J Cardiovasc Electrophysiol 10:244 –260. Arguello C, Alanis J, Pantoja O, Valenzuela B. 1986. Electrophysiological and ultrastructural study of the atrioventricular canal during the development of the chick embryo. J Mol Cell Cardiol 18:499 –510. Aristotle. Historia Animalium. circa BC 350. transl. D.W. Thompson, Oxford, 1910.


Askanase AD, Friedman DM, Copel J, Dische MR, Dubin A, Starc TJ, Katholi MC, Buyon JP. 2002. Spectrum and progression of conduction abnormalities in infants born to mothers with anti-SSA/Ro-SSB/La antibodies. Lupus 11:145–151. Baker K, Warren KS, Yellen G, Fishman MC. 1997. Defective “pacemaker” current (Ih) in a zebrafish mutant with a slow heart rate. Proc Natl Acad Sci USA 94:4554 – 4559. Barker RJ, Gourdie RG. 2002. JNK bond regulation Why does the mammalian heart invest in Cx43? Invited editorial. Circ Res 91:556 –558. Basson CT, Bachinsky DR, Lin RC, Levi T, Elkins JA, Soults J, Grayzel D, Kroumpouzou E, Traill TA, LeblancStraceski J, Renault B, Kucherlapati R, Seidman JG, Seidman CE. 1997. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nat Genet 15:30 –35. Biel M, Schneider A, Wahl C. 2002. Cardiac HCN channels: structure, function, and modulation. Trends Cardiovasc Med 12:206 –212. Boyett MR, Honjo H, Kodama I. 2000. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res 47:658 – 687. Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE, Seidman JG. 2001. A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106:709 –721. Cain ME, Luke RA, Lindsay BD. 1992. Diagnosis and localization of accessory pathways. Pacing Clin Electrophysiol 15:801– 824. Camenisch TD, Schroeder JA, Bradley J, Klewer SE, McDonald JA. 2002. Heartvalve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors. Nat Med 8:850 – 855. Cantin M, Thibault G, Haile-Meskel H, Ding J, Milne RW, Ballak M, Charbonneau C, Nemer M, Drouin J, Garcia R. 1989. Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles. Cell Tissue Res 256: 309 –325. Cheng G, Litchenberg WH, Cole GJ, Mikawa T, Thompson RP, Gourdie RG. 1999. Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development 126: 5041–5049. Chuck ET, Freeman DM, Watanabe M, Rosenbaum DS. 1997. Changing activation sequence in the embryonic chick heart: Implications for development of the cardiac conduction system of the chick. Circ Res 81:470 – 476. Chuck ET, Watanabe M. 1997. Differential expression of PSA-NCAM and HNK-1 Epitopes in the developing car-

CARDIAC CONDUCTION SYSTEM DEVELOPMENT 55 diac conduction system of the chick. Dev Dyn 209:182–195. Coppen SR, Gourdie RG, Severs NJ. 2001. Cx45 is the first connexin expressed in the central conduct. system. Exp Clin Cardiol 6:7–23. Coppen SR, Severs NJ, Gourdie RG. 1999. Cx45 expression delineates and extended conduction system in the embryonic and adult rodent heart. Develop Genetics 9:82–91. Davies F. 1933. The conducting system of the bird’s heart. J Anat 64:129 – 146. Davies MJ, Anderson RH, Becker AE. 1983. The Conduction System of the Heart. London, UK: Butterworths. Davis DL, Edwards AV, Juraszek AL, Phelps A, Wessels A, Burch JB. 2001. A GATA-6 gene heart-region-specific enhancer provides a novel means to mark and probe a discrete component of the mouse cardiac conduction system. Mech Dev 108:105–119. Davis DL, Wessels A, Burch JB. 2000. Nkx-dependent enhancer regulates cGATA-6 gene expression during early stages of heart development. Dev Biol 217:310 –322. de Jong F, Opthof T, Wilde AAM, Janse MJ, Charles R, Lamers WH, Moorman AF. 1992. Persisting zones of slow impulse conduction in developing chicken hearts. Circ Res 71: 240 –250. Di Lisi R, Sandri C, Franco D, Ausoni S, Moorman AF, Schiaffino S. 2000. An atrioventricular canal domain defined by cardiac troponin I transgene expression in the embryonic myocardium. Anat Embryol 202:95–101. Delorme B, Dahl E, Jarry-Guichard T, Marics I, Briand JP, Willecke K, Gros D, Theveniau-Ruissy M. 1995. Developmental regulation of connexin 40 gene expression in mouse heart correlates with the differentiation of the conduction system. Dev Dyn 204: 358 –371. Dupont E, Matsushita T, Kaba RA, Vozzi C, Coppen SR, Khan N, Kaprielian R, Yacoub MH, Severs NJ. 2001. Altered connexin expression in human congestive heart failure. J Mol Cell Cardiol 33:359 –371. Durbec P, Cremer H. 2001. Revisiting the function of PSA-NCAM in the nervous system. Mol Neurobiol 24:53– 64. Epstein JA, Li J, Lang D, Chen F, Brown CB, Jin F, Lu MM, Thomas M, Liu E, Wessels A, Lo CW. 2000. Migration of cardiac neural crest cells in Splotch embryos. Development 127:1869 – 1878. Fischman DA, Mikawa T. 1997. The use of replication-defective retroviruses for cell lineage studies of myogenic cells. Methods Cell Biol 52:215–227. Fishman MC, Chien KR. 1997. Fashioning the vertebrate heart: earliest embryonic decisions. Development 124: 2099 –2117.

Fozzard HA. 2002. Cardiac sodium and calcium channels: a history of excitatory currents. Cardiovasc Res 55:1– 8. Franco D, Icardo JM. 2001. Molecular characterization of the ventricular conduction system in the developing mouse heart: topographical correlation in normal and congenitally malformed hearts. Cardiovasc Res 49:417– 429. Gittenberger-de Groot AC, Vrancken Peeters MP, Mentink MM, Gourdie RG, Poelmann RE. 1998. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res 82: 1043–1052. Goldmuntz E, Geiger E, Benson DW. 2001. NKX2.5 mutations in patients with tetralogy of Fallot. Circulation 104:2565–2568. Gollob MH, Green MS, Tang AS, Roberts R. 2002. PRKAG2 cardiac syndrome: familial ventricular preexcitation, conduction system disease, and cardiac hypertrophy. Curr Opin Cardiol 17:229 –234. Gorza L, Schiaffino S, Vitadello M. 1988. Heart conduction system: a neural crest derivative? Brain Research 457: 360 –366. Gourdie RG, Cheng G, Thompson RP, Mikawa T. 2000. Retroviral cell lineage analysis in the developing chick heart. Methods Mol Biol 135:297–304. Gourdie RG, Green CR, Severs NJ, Anderson RH, Thompson RP. 1993. Evidence for a distinct gap-junctional phenotype in ventricular conduction tissues of the developing and mature avian heart. Circ Res 72:278 –289. Gourdie RG, Kubalak S, Mikawa T. 1999. Conducting the embryonic heart: orchestrating development of specialized cardiac tissues. Trends Cardiovasc Med 9:18 –26. Gourdie RG, Lo CW. 2000. Cx43 gap junctions in cardiac development and disease. In: C Perrachia, editor. Current Topics in Membranes, vol 49, Gap Junctions Academic Press, pp 581– 602. Gourdie RG, Mima T, Thompson RP, Mikawa T. 1995. Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system. Development 121:1423–1431. Gourdie RG, Wei Y, Kim D, Klatt SC, Mikawa T 1998. Endothelin-induced conversion of embryonic heart muscle cells into impulse-conducting Purkinje fibers. Proc Natl Acad Sci USA 95:6815– 6818. Gourdie RG, Wei Y, Kim D, Klatt SC, Mikawa T. 1998. Endothelin-induced conversion of embryonic heart muscle cells into impulse-conducting Purkinje fibers. Proc Natl Acad Sci USA 95:6815– 6818. Haissaguerre M, Shah DC, Jais P, et al. 2002. Role of Purkinje conducting system in triggering of idiopathic ventricular fibrillation. Lancet 359:677– 678.

Harvey William. Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus. Published in Latin at Frankfort in 1628. Hatcher,CJ, Goldstein MM, Mah CS, Delia CS, Basson CT. 2000. Identification and localization of TBX5 transcription factor during human cardiac morphogenesis. Dev Dyn 219:90 –95. Hertig CM, Kubalak SW, Wang Y, Chien KR. 1999. Synergistic roles of neuregulin-1 and insulin-like growth factor-I in activation of the phosphatidylinositol 3-kinase pathway and cardiac chamber morphogenesis. J Biol Chem 274:37362–37369. Houweling AC, Somi S, Van Den Hoff MJ, Moorman AF, Christoffels VM. 2002. Developmental pattern of ANF gene expression reveals a strict localization of cardiac chamber formation in chicken. Anat Rec 266:93–102. Hyer J, Johansen M, Prasad A, Gourdie RG, Mikawa T. 1999. Induction of Purkinje fiber differentiation by coronary arterialization. Proc Natl Acad Sci USA 96:13214 –13218 Jessen KR, Mirsky R. 2002. Signals that determine Schwann cell identity. J Anat 200:367–376. Jiang X, Rowitch DH, Soriano P, McMahon AP, Sucov HM. 2000. Fate of the mammalian cardiac neural crest. Development 127:1607–1616. Kamino K, Hirota A, Fujii S. 1981. Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye. Nature 290: 595–597. Kamino K. 1991. Optical approaches to ontogeny of electrical activity and related functional organization during early heart development. Physiol Rev 71:53–91. Kanzawa N, Poma CP, Takebayashi-Suzuki K, Diaz KG, LaylievJ, Mikawa T. 2002. Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression. Development 129:3185–3194. Kasahara H, Wakimoto H, Liu M, Maguire CT, Converso KL, Shioi T, Huang WY, Manning WJ, Paul D, Lawitts J, Berul CI, Izumo S. 2001. Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein. J Clin Invest 108:189 –201. Koushik SV, Wang J, Rogers R, Moskophidis D, Lambert NA, Creazzo TL, Conway SJ. 2001. Targeted inactivation of the sodium-calcium exchanger (Ncx1) results in the lack of a heartbeat and abnormal myofibrillar organization. FASEB J 7:1209 –1211. Kumai M, Nishii K, Nakamura K, Takeda N, Suzuki M, Shibata Y. 2000. Loss of connexin45 causes a cushion defect in early cardiogenesis. Development 127:3501–3512. Lamers WH, De Jong F, De Groot IJ, Moorman AF. 1991. The development


56 GOURDIE ET AL. of the avian conduction system, a review. Eur J Morphol 29:233–253. Lieberman M, Paes de Carvalho A. 1965a. The electrophysiological organization of the embryonic chick heart. J Gen Physiol 49:351–363. Lieberman M, Paes de Carvalho A. 1965b. The spread of excitation in the embryonic chick heart. J Gen Physiol 49:365–379. Lo CW. 2000. Role of gap junctions in cardiac conduction and development: insights from the connexin knockout mice. Circ Res 87:346 –348. Machida S, Matsuoka R, Noda S, Hiratsuka E, Takagaki Y, Oana S, Furutani Y, Nakajima H, Takao A, Momma K. 2000. Evidence for the expression of neonatal skeletal myosin heavy chain in primary myocardium and cardiac conduction tissue in the developing chick heart. Dev Dyn 217:37– 49. Manasek FJ. 1968. Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo. J Morphol 125:329 –365. Marban E. 2002. Cardiac channelopathies. Nature 415:213–218. Markwald RR, Wessels A. 2001. Overview of Heart Development. In: Tomanek R, Runyan R, editors. Formation of the Heart and Its Regulation. Birkhauser, pp 1–22. McCabe CF, Gourdie RG, Thompson RP, Cole GJ. 1995. Developmentally regulated neural protein EAP-300 is expressed by myocardium and cardiac neural crest during chick embryogenesis. Dev Dyn 203:51– 60. Meyer D, Birchmeier C. 1995. Multiple essential functions of neuregulin in development. Nature 378:386 –390. Mikawa T, Borisov A, Brown AMC, Fischman DA.1992. Clonal analysis of cardiac morphogenesis in the chicken embryo using a replication-defective retrovirus, I: formation of the ventricular myocardium. Dev Dyn 193:11– 23. Mikawa T, Gourdie RG, Hyer J Takebayashi-Suzuki K. 2001. Cardiac conduction system development. In: Runyan R, Tomanek R, editors. Formation of the heart and its regulation. Birkhauser. p 121–135. Mikawa T, Gourdie RG, Poma CP, Shulimovich M, Hall C, Hewett KW, Justus C, Reckova M, Sedmera D, Tobita K, Hurtado R, Pennisi, DJ Kanzawa N, Takebayashi-Suzuki K. Induction and Patterning of the Impulse Conducting Purkinje Fiber Network. In: Clark, EB, Takao A, Nakazawa M, editors. Etiology and Morphogenesis of Congenital Heart Disease, Sankei Press, Tokyo, in press. Moorman AF, de Jong F, Denyn MM, Lamers WH. 1998. Development of the cardiac conduction system. Circ Res 82:629 – 644. O’Brien TX, Edmonson AM, Rackley MS, Benson DW, Gourdie RG. 2001. Role of the cardiac transcription factor

Nkx2.5 in the cardiac conduction system. Circulation 14:II–288. Patten BM, Kramer TC. 1933. The initiation of contraction in the embryonic chicken heart. Am J Anat 53:349 – 375. Pennisi DJ, Rentschler S, Gourdie RG, Fishman GI, Mikawa T. 2002. Induction and patterning of the cardiac conduction system. Int J Dev Biol 46:765–775. Poelmann RE, Gittenberger-de Groot AC. 1999. A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development? Dev Biol 207:271–286. Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE, Jalife J, Fishman GI. 2001.Visualization and functional characterization of the developing murine cardiac conduction system. Development 128: 1785–1792. Rentschler S, Zander J, Meyers K, France D, Levine R, Porter G, Rivkees SA, Morley GE, Fishman GI. 2002. Neuregulin-1 promotes formation of the murine cardiac conduction system. Proc Natl Acad Sci USA 99: 10464 –10469. Satin J, Fujii S, de Haan RL. 1988. Development of cardiac heartbeat in early chick embryos is regulated by regional cues. Dev Biol 129:103–113. Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, Moak JP, Maron BJ, Seidman CE, Seidman JG. 1998. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281:108 – 111. Sedmera D, Reckova M, DeAlmeida A, Sedmerova M, Biermann M, Volejnik J, Sarre A, Raddatz E, McCarthy RA, Gourdie RG, Thompson RP. 2002. Functional and morphological evidence for a ventricular conduction system in the zebrafish and Xenopus heart. Am J Physiol Heart Circ Physiol, in press. Severs NJ, Rothery S, Dupont E, Coppen SR, Yeh HI, Ko YS, Matsushita T, Kaba R, Halliday D. 2002. Immunocytochemical analysis of connexin expression in the healthy and diseased cardiovascular system. Microsc Res Tech 52:301–322. Sedmera D, Reckova M, deAlmedia A, Stanlety CP, Gourdie RG, Thompson RP. Changes in mechanical loading modulate maturation of the conduction system in the embryonic chick heart. FASEB J Abstract, in press. Sperelakis N. 1990. Properties of calcium channels in cardiac muscle and vascular smooth muscle. Mol Cell Biochem 99:97–109. Stainier DY, Fishman MC. 1992. Patterning the zebrafish heart tube: acquisition of anteroposterior polarity. Dev Biol 153:91–101.


Suma K. 2001. Sunao Tawara: a father of modern cardiology. Pacing Clin Electrophysiol 24:88 –96. Szabo E, Viragh S, Challice CE. 1986. The structure of the atrioventricular conducting system in the avian heart. Anat Rec 215:1–9. Takebayashi-Suzuki K, Pauliks LB, Eltsefon Y, Mikawa T. 2001. Purkinje fibers of the avian heart express a myogenic transcription factor program distinct from cardiac and skeletal muscle. Dev Biol 234:390 – 401. Takebayashi-Suzuki K, Yanagisawa M, Gourdie RG, Kanzawa N, Mikawa T. 2000. In vivo induction of cardiac Purkinje fiber differentiation by coexpression of preproendothelin-1 and endothelin converting enzyme-1. Development 127:3523–3532. Thomas PS, Kasahara H, Edmonson AM, Izumo S, Yacoub MH, Barton PJ, Gourdie RG. 2001. Elevated expression of Nkx-2.5 in developing myocardial conduction cells. Anat Rec 263:307– 313. Thompson JT, Rackley MS, O’Brien TX. 1998. Upregulation of the cardiac homeobox gene Nkx2-5 (CSX) in feline right ventricular pressure overload. Am J Physiol 274:H1569 –73. Thompson RP, Lindroth JR, Wong M. 1990. Regional differences in DNA synthetic activity in the preseptation myocardium of the chick. In: Clark EB, Takao A, editors. Developmental Cardiology: Morphogenesis and Function, Mt. Kisko, New York. p 219 –234. Thompson RP, Rosenthal P, Cheng G. 2000. Origin and fate of cardiac conduction tissue. In: Clark EB, Takao A, Nakazawa M, editors. Etiology and Morphogenesis of Congenital Heart Disease, Sankei Press, Tokyo. pp 247–251. Toshimori H, Toshimori K, Oura C, Matsuo H, Matsukura S. 1988. Immunohistochemical identification of Purkinje fibers and transitional cells in a terminal portion of the impulse-conducting system of porcine heart. Cell Tissue Res 253:47–53. Veenstra RD, Wang HZ, Beyer EC, Brink PR. 1994. Selective dye and ionic permeability of gap junction channels formed by connexin45. Circ Res 75: 483– 490. Viragh S, Challice CE. 1983. The development of the early atrioventricular conduction system in the embryonic heart. Can J Physiol Pharmacol 61: 775–792. Wang GF, Nikovits W Jr, Schleinitz M, Stockdale FE. 1998. A positive GATA element and a negative vitamin D receptor-like element control atrial chamber-specific expression of a slow myosin heavy-chain gene during cardiac morphogenesis. Mol Cell Biol 18: 6023– 6034. Wessels A, Vermeulen JL, Verbeek FJ, Viragh S, Kalman F, Lamers WH, Moorman AF. 1992. Spatial distribution of “tissue-specific” antigens in the

CARDIAC CONDUCTION SYSTEM DEVELOPMENT 57 developing human heart and skeletal muscle. III. An immunohistochemical analysis of the distribution of the neural tissue antigen G1N2 in the embryonic heart; implications for the development of the atrioventricular conduction system. Anat Rec 232:97–111. Xavier-Neto J, Neville CM, Shapiro MD, Houghton L, Wang GF, Nikovits W Jr, Stockdale FE, Rosenthal N. 1999. A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart. Development 126: 2677–2687. Xu D, Emoto N, Giaid A Slaughter C, Kaw S, deWit D, Yanagisawa M. 1994. ECE-1: a membrane-bound metalloprotease that catalyzes the proteo-

lytic activation of big endothelin-1. Cell 78:473– 485. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411– 415. Yoshisue H, Suzuki K, Kawabata A, Ohya T, Zhao H, Sakurada K, Taba Y, Sasaguri T, Sakai N, Yamashita S, Matsuzawa Y, Nojima H. 2002. Large scale isolation of non-uniform shear stress-responsive genes from cultured human endothelial cells through the preparation of a subtracted cDNA library. Atherosclerosis 162:323–334. Yoshizumi M, Kurihara H, Sugiyama T, Takaku F, Yanagisawa M, Masaki T,

Yazaki Y. 1989. Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biochem Biophys Res Commun 161:859 – 864. Yutzey KE, Rhee JT, Bader D. 1994. Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart. Development 120:871– 883. Zhao YY, Sawyer DR, Baliga RR, Opel DJ, Han X, Marchionni MA, Kelly RA. 1998. Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J Biol Chem 273:10261–10269.
