Development of the cardiac conduction system

The cardiac conduction system (CCS) is a specialized tissue network that initiates and maintains a rhythmic heartbeat. The CCS consists of several functional subcomponents responsible for producing a pacemaking impulse and distributing action potentials across the heart in a coordinated manner. The formation of the distinct subcomponents of the CCS occurs within a precise temporal and spatial framework; thereby assuring that as the system matures from a tubular to a complex chambered organ, a rhythmic heartbeat is always maintained. Therefore, a defect in differentiation of any CCS component would lead to severe rhythm disturbances. Recent molecular, cell biological and physiological approaches have provided fresh and unexpected perspectives of the relationships between cell fate, gene expression and differentiation of specialized function within the developing myocardium. In particular, biomechanical forces created by the heartbeat itself have important roles in the inductive patterning and functional integration of the developing conduction system. This new understanding of the cellular origin and molecular induction of CCS tissues during embryogenesis may provide the foundation for tissue engineering, replacement and repair of these essential cardiac tissues in the future.     

Visualization and functional characterization of the developing murine cardiac conduction system

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.     

Induction and initial patterning of the nervous system - the chick embryo enters the scene

Until recently, almost everything known about the molecular controls of early neural development came from studies in amphibians. It is now possible to misexpress factors in chick embryos at relatively late stages in development, allowing careful dissection of the timing of cell interactions. This is starting to contribute significantly to our understanding of neural induction and early patterning.     

Induction and patterning of the telencephalon in Xenopus laevis

We report an analysis of the tissue and molecular interplay involved in the early specification of the forebrain, and in particular telencephalic, regions of the Xenopus embryo. In dissection/recombination experiments, different parts of the organizer region were explanted at gastrula stage and tested for their inducing/patterning activities on either naive ectoderm or on midgastrula stage dorsal ectoderm. We show that the anterior dorsal mesendoderm of the organizer region has a weak neural inducing activity compared with the presumptive anterior notochord, but is able to pattern either neuralized stage 10.5 dorsal ectoderm or animal caps injected with BMP inhibitors to a dorsal telencephalic fate. Furthermore, we found that a subset of this tissue, the anterior dorsal endoderm, still retains this patterning activity. At least part of the dorsal telencephalic inducing activities may be reproduced by the anterior endoderm secreted molecule cerberus, but not by simple BMP inhibition, and requires the N-terminal region of cerberus that includes its Wnt-binding domain. Furthermore, we show that FGF action is both necessary and sufficient for ventral forebrain marker expression in neuralized animal caps, and possibly also required for dorsal telencephalic specification. Therefore, integration of organizer secreted molecules and of FGF, may account for patterning of the more rostral part of Xenopus CNS.     

The emerging genetic landscape underlying cardiac conduction system function

Proper function of an organized Cardiac Conduction System (CCS) is vital to the survival of metazoans ranging from fly to man. The routine use of non-invasive electrocardiogram measures in the diagnosis and monitoring of cardiovascular health has established a trove of reliable CCS functional data in both normal and diseased cardiac states. Recent combination of echocardiogram (ECG) data with genome-wide association studies has identified genomic regions implicated in ECG variability which impact CCS function. In this study, we review the substantial recent progress in this area, highlighting the identification of novel loci, confirming the importance of previously implicated loci in CCS function, and exploring potential links between genes with important roles in developmental processes and variation in function of the CCS.     

Normal and abnormal development of the cardiac conduction system; implications for conduction and rhythm disorders in the child and adult

The cardiac conduction system is a specialized network that initiates and closely coordinates the heart beat. Cardiac conduction system development is intricately related to the development and maturation of the embryonic heart towards its four-chambered form, as is indicated by the fact that disturbed development of cardiac structures is often accompanied by a disturbed formation of the CCS. Electrophysiological studies have shown that selected conduction disturbances and cardiac arrhythmias do not take place randomly in the heart but rather at anatomical predilection sites. Knowledge on development of the CCS may facilitate understanding of the etiology of arrhythmogenic events. In this review we will focus on embryonic development of the CCS in relation to clinical arrhythmias, as well as on specific cardiac conduction abnormalities that are observed in patients with congenital heart disease.     

Specification of the mouse cardiac conduction system in the absence of Endothelin signaling

Coordinated contraction of the heart is essential for survival and is regulated by the cardiac conduction system. Contraction of ventricular myocytes is controlled by the terminal part of the conduction system known as the Purkinje fiber network. Lineage analyses in chickens and mice have established that the Purkinje fibers of the peripheral ventricular conduction system arise from working myocytes during cardiac development. It has been proposed, based primarily on gain-of-function studies, that Endothelin signaling is responsible for myocyte-to-Purkinje fiber transdifferentiation during avian heart development. However, the role of Endothelin signaling in mammalian conduction system development is less clear, and the development of the cardiac conduction system in mice lacking Endothelin signaling has not been previously addressed. Here, we assessed the specification of the cardiac conduction system in mouse embryos lacking all Endothelin signaling. We found that mouse embryos that were homozygous null for both ednra and ednrb, the genes encoding the two Endothelin receptors in mice, were born at predicted Mendelian frequency and had normal specification of the cardiac conduction system and apparently normal electrocardiograms with normal QRS intervals. In addition, we found that ednra expression within the heart was restricted to the myocardium while ednrb expression in the heart was restricted to the endocardium and coronary endothelium. By establishing that ednra and ednrb are expressed in distinct compartments within the developing mammalian heart and that Endothelin signaling is dispensable for specification and function of the cardiac conduction system, this work has important implications for our understanding of mammalian cardiac development.     

Remodeling of the peripheral cardiac conduction system in response to pressure overload

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.     

Alpha actin isoforms expression in human and rat adult cardiac conduction system

In the adult heart, cardiac muscle comprises the working myocardium and the conduction system (CS). The latter includes the sinoatrial node (SAN), the internodal tract or bundle (IB), the atrioventricular node (AVN), the atrioventricular bundle (AVB), the bundle branches (BB) and the peripheral Purkinje fibers (PF). Most of the information concerning the phenotypic features of CS tissue derives from the characterization of avian and rodent developing hearts; data concerning the expression of actin isoforms in adult CS cardiomyocytes are scarce. Using specific antibodies, we investigated the distribution of α-skeletal (α-SKA), α-cardiac (α-CA), α-smooth muscle (α-SMA) actin isoforms and other muscle-typical proteins in the CS of human and rat hearts at different ages. SAN and IB cardiomyocytes were characterized by the presence of α-SMA, α-CA, calponin and caldesmon, whereas α-SKA and vimentin were absent. Double immunofluorescence demonstrated the co-localisation of α-SMA and α-CA in I-bands of SAN cardiomyocytes. AVN, AVB, BB and PF cardiomyocytes were α-SMA, calponin, caldesmon and vimentin negative, and α-CA and α-SKA positive. No substantial differences in actin isoform distribution were observed in human and rat hearts, except for the presence of isolated subendocardial α-SMA positive cardiomyocytes co-expressing α-CA in the ventricular septum of the rat. Aging did not influence CS cardiomyocyte actin isoform expression profile. These findings support the concept that cardiomyocytes of SAN retain the phenotype of a developing myogenic cell throughout the entire life span.     

Induction and axial patterning of the neural plate: Planar and vertical signals

In this review I summarize recent findings on the contributions of different cell groups to the formation of the basic plan of the nervous system of vertebrate embryos. Midline cells of the mesoderm—the organizer, notochord, and prechordal plate—and midline cells of the neural ectoderm—the notoplate and floor plate—appear to have a fundamental role in the induction and patterning of the neural plate. Vertical signals acting across tissue layers and planar signals acting through the neural epithelium have distinct roles and cooperate in induction and pattern formation. Whereas the prechordal plate and notochord have distinct vertical signaling properties, the initial anteroposterior (A-P) pattern of the neural plate may be induced by planar signals originating from the organizer region. Planar signals from the notoplate may also contribute to the mediolateral (M-L) patterning of the neural plate. These and other findings suggest a general view of neural induction and axial patterning. © 1993 John Wiley & Sons, Inc.     

Subcellular localization of dystrophin and vinculin in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle

The subcellular localization of dystrophin and vinculin was investigated in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle by immunofluorescence confocal microscopy. In ventricular cardiac muscle fibers, strong staining with antibody against dystrophin appeared as regularly arranged transverse striations at the sarcolemmal surface, and faint but uniform staining was seen in narrow strips between these striations. In fibers of the ventricular conduction system, the sarcolemma was stained uniformly with this antibody, but strong staining was found as regular striations in many areas and as scattered patches in other areas of the sarcolemma. These intensely stained striations and scattered patches of dystrophin were colocalized with those of vinculin. Because dystrophin striations were located at the level of Z bands of the underlying myofibrils, they were regarded as the concentration of this protein at costameres together with vinculin. In fibers of the conduction system, myofibrils were close to the sarcolemma where dystrophin and vinculin assumed a striated pattern, at some distance from the cell membrane where these proteins exhibited a patchy distribution, and distant from the sarcolemma where dystrophin was uniformly distributed. These data suggest that the distribution patterns of dystrophin reflect the degree of association between the sarcolemma and underlying myofibrils.     

Embryonic stem cells form an organized, functional cardiac conduction system in vitro

A functional pacemaking-conduction system is essential for maintaining normal cardiac function. However, no reproducible model system exists for studying the specialized cardiac pacemaking-conduction system in vitro. Although several molecular markers have been shown to delineate components of the cardiac conduction system in vivo, the functional characteristics of the cells expressing these markers remain unknown. The ability to accurately identify cells that function as cardiac pacemaking cells is crucial for being able to study their molecular phenotype. In differentiating murine embryonic stem cells, we demonstrate the development of an organized cardiac pacemaking-conduction system in vitro using the coexpression of the minK-lacZ transgene and the chicken GATA6 (cGATA6) enhancer. These markers identify clusters of pacemaking "nodes" that are functionally coupled with adjacent contracting regions. cGATA6-positive cell clusters spontaneously depolarize, emitting calcium signals to surrounding contracting regions. Physically separating cGATA6-positive cells from nearby contracting regions reduces the rate of spontaneous contraction or abolishes them altogether. cGATA6/minK copositive cells isolated from embryoid cells display characteristics of specialized pacemaking-conducting cardiac myocytes with regard to morphology, action potential waveform, and expression of a hyperpolarization-activated depolarizing current. Using the cGATA6 enhancer, we have isolated cells that exhibit electrophysiological and genetic properties of cardiac pacemaking myocytes. Using molecular markers, we have generated a novel model system that can be used to study the functional properties of an organized pacemaking-conducting contracting system in vitro. Moreover, we have used a molecular marker to isolate a renewable population of cells that exhibit characteristics of cardiac pacemaking myocytes.