Why do we have Purkinje fibers deep in our heart?

Purkinje fibers were the first discovered component of the cardiac conduction system. Originally described in sheep in 1839 as pale subendocardial cells, they were found to be present, although with different morphology, in all mammalian and avian hearts. Here we review differences in their appearance and extent in different species, summarize the current state of knowledge of their function, and provide an update on markers for these cells. Special emphasis is given to popular model species and human anatomy.

Elevated expression of Nkx-2.5 in developing myocardial conduction cells.

A number of different phenotypes emerge from the mesoderm-derived cardiomyogenic cells of the embryonic tubular heart, including those comprising the cardiac conduction system. The transcriptional regulation of this phenotypic divergence within the cardiomyogenic lineage remains poorly characterized. A relationship between expression of the transcription factor Nkx-2.5 and patterning to form cardiogenic mesoderm subsequent to gastrulation is well established. Nkx-2.5 mRNA continues to be expressed in myocardium beyond the looped, tubular heart stage. To investigate the role of Nkx-2.5 in later development, we have determined the expression pattern of Nkx-2.5 mRNA by in situ hybridization in embryonic chick, fetal mouse, and human hearts, and of Nkx-2.5 protein by immunolocalization in the embryonic chick heart. As development progresses, significant nonuniformities emerge in Nkx-2.5 expression levels. Relative to surrounding force-generating ("working") myocardium, elevated Nkx-2.5 mRNA signal becomes apparent in the specialized cells of the conduction system. Similar differences are found in developing chick, human, and mouse fetal hearts, and nuclear-localized Nkx-2.5 protein is prominently expressed in differentiating chick conduction cells relative to adjacent working myocytes. This tissue-restricted expression of Nkx-2.5 is transient and correlates with the timing of spatio-temporal recruitment of cells to the central and the peripheral conduction system. Our data represent the first report of a transcription factor showing a stage-dependent restriction to different parts of the developing conduction system, and suggest some commonality in this development between birds and mammals. This dynamic pattern of expression is consistent with the hypothesis that Nkx-2.5, and its level of expression, have a role in regulation and/or maintenance of specialized fate selection by embryonic myocardial cells.

Connexin45 is the first connexin to be expressed in the central conduction system of the mouse heart.

OBJECTIVE: To examine the spatial pattern of labelling for the gap junctional protein, connexin45, in relation to that of the other two cardiac connexins, connexin40 and connexin43, during the development of the central conduction system in mouse heart. ANIMALS AND METHODS: Hearts from Balb-c mice at stages from embryonic day (E) 12.5 to adult were frozen and sectioned. The sections were immunolabelled for connexins 45, 40 and 43 using fully characterized connexin-specific antibodies. Labelled sections were observed using confocal microscopy. Single, double and triple labelling were employed with sequential scanning to record images from multiple-labelled sections for the analysis of the spatial distribution of the three connexin types in relation to each other. RESULTS: High levels of connexin45 label were detected in specific regions within the developing mouse heart. These regions corresponded to the conus myocardium, developing interatrial septum and other developing conduction tissues of the heart. Connexin40 label was initially absent from these tissues but by E15.5 was present in the more distal regions of the conduction system. However, by E17.5, connexin45 and 40 labelling was similar to the pattern observed in the adult heart, with both connexins present in most regions of the conduction system, though they were not completely colocalized. Connexin43 label was not observed in the regions of high connexin45 labelling. CONCLUSIONS: These results show connexin45 to be the earliest detectable connexin in the central conduction system and to be the only connexin present throughout the whole conduction system. A distinct temporal pattern of connexin expression was also shown to occur during the development of the conduction tissues of the mouse heart.

Increased co-localization of connexin43 and ZO-1 in dissociated adult myocytes.

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium--a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immunoprecipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.

A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

In vivo induction of cardiac Purkinje fiber differentiation by coexpression of preproendothelin-1 and endothelin converting enzyme-1.

The rhythmic heart beat is coordinated by electrical impulses transmitted from Purkinje fibers of the cardiac conduction system. During embryogenesis, the impulse-conducting cells differentiate from cardiac myocytes in direct association with the developing endocardium and coronary arteries, but not with the venous system. This conversion of myocytes into Purkinje fibers requires a paracrine interaction with blood vessels in vivo, and can be induced in vitro by exposing embryonic myocytes to endothelin-1 (ET-1), an endothelial cell-associated paracrine factor. These results suggest that an endothelial cell-derived signal is capable of inducing juxtaposed myocytes to differentiate into Purkinje fibers. It remains unexplained how Purkinje fiber recruitment is restricted to subendocardial and periarterial sites but not those juxtaposed to veins. Here we show that while the ET-receptor is expressed throughout the embryonic myocardium, introduction of the ET-1 precursor (preproET-1) in the embryonic myocardium is not sufficient to induce myocytes to differentiate into conducting cells. ET converting enzyme-1 (ECE-1), however, is expressed preferentially in endothelial cells of the endocardium and coronary arteries where Purkinje fiber recruitment takes place. Retroviral-mediated coexpression of both preproET-1 and ECE-1 in the embryonic myocardium induces myocytes to express Purkinje fiber markers ectopically and precociously. These results suggest that expression of ECE-1 plays a key role in defining an active site of ET signaling in the heart, thereby determining the timing and location of Purkinje fiber differentiation within the embryonic myocardium.

The rate and anisotropy of impulse propagation in the postnatal terminal crest are correlated with remodeling of Cx43 gap junction pattern.

BACKGROUND: Disruptions to intermyocyte coupling have been implicated in arrhythmogenesis and development of conduction disturbances. At present, understanding of the relationship between the microscopic organization of intercellular coupling and the macroscopic spread of impulse in the normal and diseased heart is largely confined to theoretical analyses. METHODS AND RESULTS: The abundance and arrangement of gap junctions, as well as conduction properties, were assessed in terminal crest preparations isolated from the atria of neonate, weanling, and adult rabbits. We report that the connexin composition of terminal crest was uncomplicated, with Cx43 being the most prominent isoform detectable by Western blotting and immunostaining. Terminal crest myocytes showed little change in total Cx43-gap junction per cell during postnatal growth as assessed by stereology. However, marked non-uniformities emerged in the sarcolemmal distribution of Cx43-gap junctions. Cx43-gap junction area at myocyte termini increased 3.5-fold from birth to adulthood. Correlated with this change in Cx43, impulse propagation velocity parallel to the myofiber axis, as assessed by multi-site optical mapping using voltage-sensitive dye (di-4-ANEPPS), increased 2.4-fold. Conversely, the amount of Cx43-gap junctions on myocyte sides, and the conduction velocity transverse to the myofiber axis, remained relatively invariant during maturation. Hence, the increasing electrical anisotropy of maturing terminal crest was wholly accounted for by increases in conductance velocity along the bundle. This increase in longitudinal conduction velocity was correlated with changes in the sarcolemmal pattern, but not the overall density, of Cx43-gap junctions. CONCLUSIONS: This study provides the first correlative structure/function analysis of the relationship between the macroscopic conduction of impulse and the microscopic cellular organization of gap junctions in a differentiating cardiac bundle. Confirmation is provided for theoretical predictions which emphasize the importance of the cell-to-cell geometry of coupling in determining the spread and pattern of myocardial activation.

Induction of Purkinje fiber differentiation by coronary arterialization.

A synchronized heart beat is controlled by pacemaking impulses conducted through Purkinje fibers. In chicks, these impulse-conducting cells are recruited during embryogenesis from myocytes in direct association with developing coronary arteries. In culture, the vascular cytokine endothelin converts embryonic myocytes to Purkinje cells, implying that selection of conduction phenotype may be mediated by an instructive cue from arteries. To investigate this hypothesis, coronary arterial development in the chicken embryo was either inhibited by neural crest ablation or activated by ectopic expression of fibroblast growth factor (FGF). Ablation of cardiac neural crest resulted in approximately 70% reductions (P < 0.01) in the density of intramural coronary arteries and associated Purkinje fibers. Activation of coronary arterial branching was induced by retrovirus-mediated overexpression of FGF. At sites of FGF-induced hypervascularization, ectopic Purkinje fibers differentiated adjacent to newly induced coronary arteries. Our data indicate the necessity and sufficiency of developing arterial bed for converting a juxtaposed myocyte into a Purkinje fiber cell and provide evidence for an inductive function for arteriogenesis in heart development distinct from its role in establishing coronary blood circulation.

Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage.

The cardiac pacemaking and conduction system sets and maintains the rhythmic pumping action of the heart. Previously, we have shown that peripheral cells of the conduction network in chick (periarterial Purkinje fibers) are selected within a cardiomyogenic lineage and that this recruitment occurs as a result of paracrine cues from coronary arteries. At present, the cellular derivation of other elements of this specialized system (e.g. the nodes and bundles of the central conduction system) are controversial, with some proposing that the evidence supports a neurogenic and others a myogenic origin for these tissues. While such ontological questions remain, it is unlikely that progress can be made on the molecular mechanisms governing patterning and induction of the central conduction system. Here, we have undertaken lineage-tracing strategies based on the distinct properties of replication-incompetent adenoviral and retroviral lacZ-expressing constructs. Using these complementary approaches, it is shown that cells constituting both peripheral and central conduction tissues originate from cardiomyogenic progenitors present in the looped, tubular heart with no detectable contribution by migratory neuroectoderm-derived populations. Moreover, clonal analyses of retrovirally infected cells incorporated within any part of the conduction system suggest that such cells share closer lineage relationships with nearby contractive myocytes than with other, more distal elements of the conduction system. Differentiation birthdating by label dilution using [(3)H]thymidine also demonstrates the occurrence of ongoing myocyte conscription to conductive specialization and provides a time course for this active and localized selection process in different parts of the system. Together, these data suggest that the cardiac conduction system does not develop by outgrowth from a prespecified pool of 'primary' myogenic progenitors. Rather, its assembly and elaboration occur via processes that include progressive and localized recruitment of multipotent cardiomyogenic cells to the developing network of specialized cardiac tissues.

Conducting the embryonic heart: orchestrating development of specialized cardiac tissues.

The heterogeneous tissues of the pacemaking and conduction system comprise the "smart components" of the heart, responsible for setting, maintaining, and coordinating the rhythmic pumping of cardiac muscle. Over the last few years, a wealth of new information has been collected about the unique genetic and phenotypic characteristics expressed by these tissues during cardiac morphogenesis. More recently, genetically modified viruses, mutational analysis, and targeted transgenesis have enabled even more precise resolution of the relationships between cell fate, gene expression, and differentiation of specialized function within developing myocardium. While some information provided by these newer approaches has supported conventional wisdom, some fresh and unexpected perspectives have also emerged. In particular, there is mounting evidence that extracardiac populations of cells migrating into the tubular heart have important morphogenetic roles in the inductive pattering and functional integration of the developing conduction system.

Connexin45 (alpha 6) expression delineates an extended conduction system in the embryonic and mature rodent heart.

We previously demonstrated that alpha 6 (Cx45), one of the three connexins of the mammalian myocardium, is preferentially expressed in the peripheral portion of the ventricular conduction system in rats and mice. Here we report that alpha 6 is also prominently immunolocalized in the atrioventricular node and His bundle of these species. The distribution of immunolocalized alpha 6 reveals that the node and bundle form part of an extended central conductive network circumscribing the AV and outflow junctional regions of the fetal, and less continuously, the adult heart. Of the three cardiac connexins, alpha 6 is the isoform most continuously expressed by conduction tissues, and may thus account for the recently reported viability of the alpha 5 (Cx40) knockout mouse. It is concluded that alpha 6 expression is a defining feature of the heterogenous tissues comprising the atrioventricular conduction system of the rodent heart.

Endothelin-induced conversion of embryonic heart muscle cells into impulse-conducting Purkinje fibers.

A regular heart beat is dependent on a specialized network of pacemaking and conductive cells. There has been a longstanding controversy regarding the developmental origin of these cardiac tissues which also manifest neural-like properties. Recently, we have shown conclusively that during chicken embryogenesis, impulse-conducting Purkinje cells are recruited from myocytes in spatial association with developing coronary arteries. Here, we report that cultured embryonic myocytes convert to a Purkinje cell phenotype after exposure to the vascular cytokine, endothelin. This inductive response declined gradually during development. These results yield further evidence for a role of arteriogenesis in the induction of impulse-conducting Purkinje cells within the heart muscle lineage and also may provide a basis for tissue engineering of cardiac pacemaking and conductive cells.

Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions.

The epicardium and dorsal mesocardium are known to be the source of structures that form the wall of the coronary vessels. Because mouse knockout studies have shown that proper epicardial formation is also essential for myocardial development, we have studied in detail the migration and differentiation of epicardium-derived cells (EPDCs) within the developing heart. We constructed chicken-quail chimeras by grafting the quail epicardial organ, including a piece of primordial liver, at essentially stages 16 and 17. The embryos were studied at stages 25 to 43. To detect quail-derived EPDCs, an anti-quail nucleus antibody was used in combination with several differentiation markers, eg, for muscle actin, for vascular smooth muscle cells, for procollagen-I, for quail endothelium, and for Purkinje fibers. At stages 25 to 31, EPDCs are encountered in the myocardial wall and the subendocardial region. The latter deposition is spatially facilitated as the endocardium protrudes through transient discontinuities in the myocardium to contact the subepicardial layer. Later on, at stages 32 to 43, EPDCs invaded, by way of the atrioventricular sulcus, the atrioventricular cushion tissue. The localization is apparent at the interface with the myocardium, as well as subendocardially, but never within the endocardial lining. The origin of endothelium, smooth muscle cells, and fibroblasts of the coronary vessel wall from the epicardial graft were confirmed in accordance with already published data. The functional role of the novel EPDCs in the subendocardium, myocardium, and atrioventricular cushions remains to be investigated. A close positional relationship is found with the differentiating Purkinje fibers. Furthermore, a regulatory role is postulated in the process of endocardial-mesenchymal transformation. The ultimate fate of EPDCs seems to be a cardiac fibroblast cell line involved in the formation of the fibrous heart skeleton.

Identification of two further gap-junctional proteins, connexin40 and connexin45, in human myometrial smooth muscle cells at term.

The powerful synchronous contractions of the uterus in labor depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In humans and other mammals, gap junctions are scarce in the myometrium of the non-pregnant uterus, but become abundant at term and/or with the onset of labor. Previous work has shown that the gap-junctional protein (connexin) expressed by human myometrial smooth muscle cells is connexin43, the same connexin type that predominates in cardiac muscle. Here we show that two further gap junctional proteins, connexin40 and connexin45, are expressed by the myometrial smooth muscle cells of the human uterus at term. Transcripts encoding the human isoforms of these connexins were demonstrated by Northern blot analysis, and immunoconfocal microscopy enabled precise localization of the corresponding proteins to punctate contact points (i.e., gap junctions) between interacting smooth muscle cells. Double labeling demonstrated that, while some fluorescent spots comprise only connexin43, both connexin40 and connexin45 predominantly colocalize to connexin43-positive fluorescent spots. Triple labeling revealed that where all three connexin types were expressed, they frequently localized to the same gap junction spot. As gap-junctional channels composed of different connexin types have been demonstrated in vitro to have different functional properties, multiple connexin expression may contribute to modulation of gap junction function in human myometrial smooth muscle cells in vivo.

Expression of major gap junction connexin types in the working myocardium of eight chordates.

The alpha1 connexin (connexin43) is regarded as the major gap junction protein of the myocardium because it predominates there in mammals. Here, we show that it is not the major connexin of the working myocardium in non-mammalian vertebrates, which instead express beta1-like connexins homologous to mammalian connexin32. A phylogenetic series of hearts was immunostained with seven antibodies raised against peptide sequences specific for three distinct members of the gap junction connexin family: alpha1, beta1 and alpha5 (mammalian connexin40/avian connexin42). Working myocardium from two ascidian chordates (Ciona and Mogula), a teleost (Carassius), a frog (Xenopus) and two reptiles (Anolis and Alligator) was found to express a beta1-like connexin, rather than an alpha1-like connexin. An alpha1-like connexin was nevertheless often detected in other cardiac tissues. In the chicken (by ancestry a reptile), the developing myocardium expressed a beta1-like connexin strongly on embryonic day 6 but less strongly at hatching, and minimally in the adult. Myocardial expression of alpha5 connexin increased during development, but remained strongest in the coronary vascular endothelial and cardiac conduction tissues. The arteriolar smooth muscle of the chicken expressed alpha1 connexin throughout development, but its myocardium did not. In contrast, the working myocardium of a marsupial mammal (the opossum Trichosurus) strongly expressed an alpha1 connexin just like placental mammals. These results imply that a shift from beta1 to alpha1 connexin expression in the heart occurred prior to the evolution of the opossums. The beta and alpha connexin subfamilies have different permeabilities and gating properties, and we discuss factors that might have made this shift beneficial.

Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene.

Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25-31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194-2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 transgenic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.

Wnt-11 is expressed in early avian mesoderm and required for the differentiation of the quail mesoderm cell line QCE-6.

The beginning of mesodermal development involves the aggregation of newly gastrulated cells into epithelial fields, as a prelude to organ formation. To analyze the molecular regulation of this initial patterning, we have focused on the Wnt family of secreted signaling proteins, molecules which have been shown to promote embryonic patterning by regulating cell-cell associations. In this study, we show that the Wnt-11 gene is expressed by newly gastrulated mesoderm cells within avian embryos. The expression pattern of Wnt-11 also suggests that it may be involved in formation of the cardiogenic fields and somites. Subsequently, we utilized the quail mesoderm cell line QCE-6 as a culture model for examining the influence of Wnt-11 on early mesoderm cell differentiation. This cell line has been shown to be representative of early nondifferentiated mesoderm cells and has the potential to differentiate into cardiomyocytes, endothelial or red blood cells. Similar to early mesoderm cells, QCE-6 cells express Wnt-11. We have engineered stable transfectants of these cells that produce either diminished or enhanced levels of Wnt-11 protein. Our studies show that Wnt-11 regulates cellular interactions of QCE-6 cells, as demonstrated by alterations in contact-inhibited growth, tight and gap junction formation and plakoglobin expression. Both the morphology and growth factor-induced differentiation of QCE-6 cells are regulated in a cooperative fashion by Wnt-11 and fibronectin. These results, described in detail below, demonstrate the uniqueness of QCE-6 cells as a culture system for analyzing Wnt activity. In particular, QCE-6 cells are the first cell line that has demonstrated: (1) Wnt-dependent differentiation; (2) concentration-variable responses to Wnt protein; and (3) altered cell phenotypes as a direct response to Wnt-5a class proteins (e.g. Wnt-4 and Wnt-11).

Dissociated spatial patterning of gap junctions and cell adhesion junctions during postnatal differentiation of ventricular myocardium.

Nonuniformity in the spatial patterning of gap junctions between heart muscle cells is now recognized as an important determinant of electromechanical function in working myocardium. Breakdown of the normal geometry of electrical intercellular connectivity in diseased myocardium correlates with reentry, arrhythmia, and conduction disturbance. The developmental mechanism(s) that determines this precise spatial order in gap junction organization in normal myocardium is at present unknown. To examine this question, we have used immunoelectron and immunoconfocal microscopy to analyze the spatial distributions of gap junctional (connexin43), desmosomal (desmoplakin), and adherens junctional (N-cadherin) components during maturation of rodent and canine left ventricular myocardium. In rats, a striking divergence in the distribution of gap junctions and cell adhesion junctions emerged within the first 20 days of postnatal life. It was found that although gap junctions initially demonstrated dispersed distributions across myocyte cell membranes, desmosomes and adherens junctions showed more rapid polarization toward cell termini (ie, nascent intercalated disks) after birth. Over subsequent postnatal development (20 to 90 postnatal days), gap junctions became progressively concentrated in these cell adhesion junction-rich zones of membrane. Quantitative analyses of this process in a series of rats aged 15 embryonic and 1, 5, 10, 20, 40, 70, and 90 postnatal days indicated that significantly higher levels (P < .01) of N-cadherin and desmoplakin than of connexin43 were immunolocalized to cell termini by as early as postnatal day 5. Although all three junctions types showed increasing polarization to myocyte termini with development, variation between junctions remained significant (P < .05) at all times points between 5 and 70 postnatal days. Only at 90 postnatal days, when the animals were nearly full grown, did the proportions of gap junction, desmosome, and adherens junction at intercalated disks become statistically similar (P > .05). Examination of myocardium from 1- and 3-month-old canines revealed that related differential changes to the spatiotemporal distribution of intercellular junctions occurred during postnatal maturation of the dog heart, suggesting that the process was not rodent specific. It is concluded that this progressive change in the organization and pattern of association between gap junctions and cell adhesion junctions is likely to be an important factor in maturation of electromechanical function within the mammalian heart.