Molecular Analysis of Patterning of Conduction Tissues in the Developing Human Heart

Collection of human embryonic material and its preparation for histological studies was described previously.¹³ Additionally, we used 3 human embryos of about 8 weeks of development, one coming from the MRC–Wellcome Trust Human Developmental Biology Resource, managed by the Institute for Human Genetics of Newcastle University, United Kingdom, and the other 2 from the Center for anticonception, sexuality, and abortion, Leiden University Medical Center, The Netherlands. Embryos were examined under a stereomicroscope for gross anomalies and graded according to Carnegie criteria. In total, we used 3 embryos at stage 12, 5 embryos at stage 13 to 14, 6 embryos at stage 15 to 16, 4 embryos at stage 18, and 6 embryos at stages 21 to 23. We included only embryos considered normal. Because the blood was not removed from the embryos, immunohistochemical stainings with some fluorochromes resulted in erythrocyte autofluorescence. Collection and use of the human embryonic material for research were approved by the medical ethics committees of the Universities of Tartu, Estonia, Leiden, and Amsterdam, The Netherlands.

Immunofluorescent staining, in situ hybridization, and 3D reconstructions were performed as described.^13,14 Serial sections of the embryos collected at Leiden University Medical Center were processed by indirect immunohistochemistry. Detailed description of the protocols, antibodies, and riboprobe are given in the online-only Data Supplement.

Assessment of conduction requires direct measurements in the myocardium, which cannot be performed in human embryos. The assessment of expression patterns of fast-conducting gap-junctional proteins connexin40 and connexin43, which play an important role in the conduction of the myocardium of the mouse heart,¹⁵ may give an impression of the possible patterning of conduction. The nonstandardized fixation and limited numbers of available embryos did not permit complete optimization of the staining protocol for some antibodies and probes. However, immunohistochemical and in situ hybridization stainings shown in the present article proved to be reproducible. The intensity of immunofluorescent stainings had to be described qualitatively and was assessed during image acquisition: A weak signal required a 2- to 3-times longer exposure than a strong signal to reach the level displayed in the figures.

The definitive sinus node of the human heart is a comma-shaped structure located in the terminal groove at the junction of the superior caval vein and the right atrium. No such structure is seen in the early human embryos studied. The systemic venous sinus, or sinus venosus, representing the confluence of the systemic venous tributaries, is first recognizable at stage 12. Its walls are initially nonmuscular¹³ but already express HCN4 (Figure 1A). As the venous sinus shifts to the right, prominent venous valves form at its junction with the right atrium. The left superior caval vein acquires its position within the left atrioventricular groove, where it is recognizable as the precursor of the coronary sinus. The walls of the venous tributaries then muscularize, with the right lateral myocardial wall of the venous sinus becoming thickened and porous,¹³ remaining traceable along the right-sided sinoatrial junction in the oldest embryo analyzed (Figure 2 and Figure 3). We consider this structure to be the primordium of the sinus node.

Many molecular markers have been used to delineate the precursors of the definitive conduction axis in the embryonic mouse heart,¹⁸ including the transcription factor Tbx3.¹⁹ This factor represses the differentiation of chamber myocardium, permitting specific areas of the looping heart to develop into the conduction system.²⁰ Using 3D reconstructions, we assessed the myocardial expression of TBX3 relative to the primordium of the sinus node. TBX3 is expressed in the nodal primordium, in the right venous valve, and the myocardium of the atrioventricular canal, and, in late embryos, the atrioventricular junctions (Figure 2). At stages 14 through 16, at the stage of formation of the primary atrial septum, TBX3 is expressed in the left venous valve, the leading edge of the atrial septal myocardium, and the floor of the right atrium, which, in turn, are contiguous with the atrioventricular canal (Figure 2A*). The expression of TBX3 outside the atrioventricular canal is weak (not shown) and disappears by the time of completion of atrial septation at stage 18 (Figure 2B**).

In stage 12 to 14 embryos, the entire wall of the systemic venous sinus and common atrium is positive for HCN4 (Figure 1A and 3A). At later stages, HCN4 expression gradually declines in the atrial walls and becomes confined to the myocardium surrounding the venous tributaries (Figure 1C, 1E, 3E, and 3I). In contrast to the broad expression of HCN4, only a small part of the walls of the systemic venous tributaries is positive for TBX3 (Figure 4A). This area, representing the sinus nodal primordium, increases in size at later stages (Figure 4E and4I), and is from the outset virtually negative for expression of troponin I and α-smooth muscle actin (α-SMA) (Figure 3B and 3C). As with the definitive sinus node,²¹ the nodal primordium does not express connexin40 (Figure 3B, 3F, and 3J). No histological differences were found between the presumed nodal primordium and the right atrial wall (Figure 3D, 3H, and 3L).

In addition to TBX3, the entirety of the systemic venous sinus was positive for ISL1, expression of this gene being complementary to that of NKX2–5 (Figure 4). Unlike the strong TBX18 expression in the epicardium, we observed different levels of TBX18 protein and mRNA expression in the sinus nodal primordium. Myocardial expression of TBX18 protein was weak (Figure 4B, 4F, and 4J), but expression of mRNA was robust and broader (Figure 4M). We found fundamental molecular differences between the nodal primordium within the venous sinus wall and the myocardial sleeve of the developing coronary sinus, suggested by some investigators to form a left-sided sinus node,²² albeit that they share the same developmental origin.²³ The myocardium around the coronary sinus, unlike the developing sinus node, does not express TBX3, initiates early expression of connexin40, and is strongly positive for α-SMA. The developmental changes in gene expression at the venous pole of the heart are summarized in Figure 4N.

Stages 13 through 16, equivalent to around 28 to 38 days of development, are marked by unmistakable growth of the chambers and formation of atrial and ventricular septal structures, along with a rightward shift of the atrioventricular canal.¹¹ Previously we reported that in the early chamber-forming human heart, myocardial expression of TBX3 is first observed in the floor of the common atrium and the atrioventricular canal.¹⁴ By reconstructing the myocardial expression of TBX3 at later developmental stages, we have related the development of the atrioventricular conduction tissues to the changing cardiac morphology. At stage 16, TBX3 is expressed throughout the myocardium of the atrioventricular canal, contiguous with the TBX3 expression in the right venous valve, including the spurious septum, the leading edge of the atrial septum, and the right atrial wall located dorsal to the outflow tract (Figure 5A and 5A*). The TBX3-positive atrioventricular canal myocardium does not express connexin40 and connexin43. In contrast, the ventricular trabeculations are positive for both connexin40 and connexin43, whereas the atrial walls and atrial septum express only for connexin40 (Figure 6D, 6E, 6J, and 6K). TBX3 is not only expressed in the atrioventricular canal dorsally and ventrally but also at the dorsal part of the base of the outflow tract and along the crest of the ventricular septum (Figure 5A**). The weakly TBX3-positive myocardium on top of the ventricular septum, corresponding to the presumptive bundle of His dorsally, and the so-called septal branch ventrally, is negative for connexin40. Unlike the situation in mouse,²⁴ it does express connexin43 at this stage (Figure 6J and 6K).

Expression of the protein ID2, inhibitor of DNA binding, known to be important in the specification of the atrioventricular conduction axis in mouse,²⁵ was not limited to the developing conduction tissues in humans. We observed weak expression in the atrioventricular canal musculature and ventricular trabeculations. Strong expression, however, was present in the epicardium and mesenchyme of the epicardial grooves (Figure 6C and 6I). As with the sinus nodal primordium, the myocardium of the atrioventricular canal and ventricular septum does not express α-SMA (Figure 6F and 6L), although the ventricular and atrial myocardial walls largely remain positive at these stages. In contrast to the sinus nodal primordium, the compact location of the cardiomyocytes at the top of ventricular septum, where also TBX3 expression is present, allows identification of the precursors of the atrioventricular conduction axis using routine histological staining^5–8,10 (Figure 6A and 6G).

At the end of the embryonic period, at stages 18 through 23, the atrioventricular junction undergoes major rearrangements, with septation being completed through fusion of several mesenchymal structures, and the atrial myocardium becoming insulated from the ventricular musculature by formation of epicardially derived fibrous tissue.²⁶ With these changes, the expression of TBX3 in the atrioventricular canal becomes restricted to a figure-of-eight configuration in the lower rims of the atrial chambers (Figure 5B* and 5C*). These left and right atrioventricular ring bundles²⁷ converge dorsally in the developing primordium of the atrioventricular node close to the orifice of the coronary sinus and ventrally behind the left ventricular outflow tract in the so-called retro-aortic “node” (Figure 5C, 5C*, and 5C**). By stage 23, the atrioventricular myocardial continuity is interrupted round the greater majority of the atrioventricular junctions, albeit in incomplete fashion (Figure 7D and 7F). At the dorsal aspect of the atrioventricular junctions, in contrast, the developing atrioventricular conduction axis continues to provide muscular atrioventricular continuity (Figure 5B** and 5C**). By this stage, the primordium of the atrioventricular node, along with the lower rims of the atrial chambers, and the muscular strands that continue to cross the forming plane of atrioventricular insulation, are negative for connexin40 and connexin43 (Figure 7D, 7E, 7J, and 7K), representing slow-conducting tissues analogous to the atrioventricular canal musculature at earlier stages. The atrioventricular nodal precursors at this stage are directly contiguous with the TBX3-negative myocardium of the atrial and ventricular septa (Figure 7B). The base of the atrial septum, formed by myocardialization of the vestibular spine,²⁸ shows a gradient of decreasing levels of connexin40 toward the site of the nodal primordium (asterisk in Figure 7D) derived from the atrioventricular canal myocardium.²⁹ The ventricular septal myocytes, in contrast, express neither connexin40 nor connexin43 (Figure 7D and 7E). The TBX3-positive area of the atrioventricular nodal primordium expresses HCN4 in relatively weak heterogeneous fashion, with the part adjacent to the atrial septum being more positive than the part contiguous with the ventricular septum (Figure 7C). The atrioventricular ring bundles and retro-aortic “node” are also weakly positive for HCN4 (Figure 7C and 7I), as has been reported for experimental animals.²⁷ As with the primordium of the sinus node, the forming atrioventricular node shows a low level of troponin I (Figure 7D) and is recognizable as a lightly-stained mesh of cells by routine histology (Figure 7A), albeit that it was not yet possible at these stages to distinguish the transitional and compact portions characteristic of the definitive atrioventricular node.

When traced ventrocranially, the developing atrioventricular conduction axis enters the forming plane of fibrous insulation as the developing bundle of His, recognizable histologically on the crest of the ventricular septum (Figure 5C, 5C**, and 7G). The slightly darker stained cells of the developing conduction axis (Figure 7G) also express TBX3 and HCN4. After completion of ventricular septation, the developing bundle of His becomes negative for both connexin40 and connexin43 (Figure 7G through 7K). On both sides of the ventricular septum, the connexin40- and connexin43-positive trabeculations form the myocardial strands originating from the bundle of His (Figure 7G through 7K), corresponding to the future bundle branches. A higher expression of NKX2–5 was observed in the developing bundle (Figure 7L), as described previously for the early human fetal heart.³⁰ In terms of expression of troponin I, the myocytes of the atrioventricular conduction axis did not differ much from the ventricular myocardium (Figure 7J) and were not yet insulated from the ventricular septal myocardium (Figure 7J).

Working myocardium is characterized by its efficient contractile function and low automaticity, whereas the opposite is true for the conduction system. In the normal postnatal heart, spontaneous generation of the impulse is confined to the sinus node. In pathological situations, arrhythmogenic activity can originate from the atrioventricular conduction axis, along with diverse myocardial structures such as the pulmonary venous sleeves, the coronary sinus, the lower atrial rims, the right ventricular outflow tract, and even the atrial appendages.³¹ Despite their distinct developmental origins, all these structures share expression of the pacemaker channel HCN4 during development. At the intermediate stages, corresponding to around 6 to 7 weeks of development, we observed relatively high levels of HCN4 expression in these structures but very low levels, if any, in the ventricular chambers. Weak expression of HCN4 is also detectable initially in the myocardium of the developing cardiac outflow tract, increasingly recognized also as a source of arrhythmogenesis in the postnatal heart.³² During later stages, expression levels of HCN4 decline in the differentiating working myocardium at the venous pole of the heart, reflecting its ongoing maturation. In mouse, the transcription factor Nkx2–5 has been identified as an important factor in the formation of the sinoatrial boundary, suppressing Tbx3 and Hcn4 in the atrial working myocardium.³³ In contrast, we observed HCN4 expression in NKX2–5–positive areas at the venous pole of the developing human heart, suggesting a difference in the regulation of HCN4 expression between mouse and human. Maintenance of HCN4 expression in these structures during maturation, along with dysregulation of the expression of fast-conducting connexins, may provide a mechanism of the initiation of certain tachyarrhythmias.

The definitive sinus node in humans has a distinct molecular architecture, which underlies its pacemaker function and differs significantly from that of adjacent atrial muscle.^34,35 Several transcription factors, including Tbx3, Tbx18, and Nkx2–5, play a crucial role in the formation of the sinus node at the right-sided sinoatrial junction in the mouse heart.^33,36 We observed similar expression patterns of these genes in the developing human heart, suggesting conservation of the molecular mechanisms governing its development. We observed almost strict complementarity between the expression domains of the transcription factors ISL1 and NKX2–5 at the venous pole (Figure 4). ISL1 is known to play a crucial role in the maintenance of the precursor state of the cardiac progenitor cells within the heart-forming regions. Its expression is switched off in the majority of the cardiomyocytes on their differentiation.^14,37 As in the mouse,³⁸ the myocytes of the systemic venous tributaries, including the primordium of the sinus node, continue to express ISL1, a finding deserving further investigation.

We have endorsed previous morphological studies^5,6,9 showing that the thickening of the venous sinus wall is the first morphological evidence of the formation of the TBX3-positive nodal primordium in the embryonic human heart. However, the myocytes of the nodal primordium and the atrial wall display no obvious histological differences. Published electrophysiological data also fail to support strict localization of the pacemaker within the developing sinus node during the early stages of development,³⁹ which correlates well with our findings of extensive expression of HCN4 at the venous pole of the early human embryonic heart.

The specialized cardiomyocytes of the postnatal sinus node contain poorly developed myofibrils,⁴⁰ resembling the phenotype of the primary myocardium. On the other hand, the initiation of expression of fast-conducting connexins and atrial natriuretic factor, along with the gradual disappearance of smooth muscle actin in the chamber myocardium, is a sign of progressive differentiation and maturation of the working myocardium.⁴¹ When first identified, the thickened right wall of the venous sinus in the developing human heart already contained a small subset of myocytes expressing very low levels of troponin I and α-SMA, both proteins being strongly expressed in the atrial and ventricular chamber myocardium at these stages (Figures 3 and 6). This troponin I– and α-SMA–negative area within the wall of the venous sinus does express TBX3, known to control the genetic programming of the sinus node development in mouse.³⁶ This suggests that TBX3 also controls early specification of the myocytes of the systemic venous sinus into the pacemaker lineage in humans.

Human hearts with right atrial isomerism,⁴² as well as mouse hearts with complex malformations resembling right atrial isomerism caused by knockout of the left-right axis regulator Pitx2c,³³ display bilateral sinus nodes. In normal human embryonic hearts, we did not observe in the myocardial wall of the coronary sinus the molecular phenotype characteristic of the developing sinus nodal primordium. Our observations, along with the absence of reports about bilateral sinus nodes in the setting of the normally arranged laterality, lend no support to the hypothesis of the presence of bilateral sinus nodal primordia²² in the normal developing heart.

To date, morphological studies on serial sections of human embryos have resulted in contradictory conclusions concerning the development of the atrioventricular conduction axis in humans.^{5–8,10–12} Thus, it has been suggested that the atrioventricular node develops through fusion of the ventral and dorsal parts of the atrioventricular ring of “histologically specialized” cardiomyocytes.^8,12 We have found no evidence to support this hypothesis. At the intermediate stages of development, the entire atrioventricular canal is positive for the conduction system marker TBX3,¹⁹ with somewhat thicker portions dorsally and ventrally. The lumen of the canal, along with the atrioventricular endocardial cushions, separate these parts of the TBX3-positive atrioventricular canal myocardium. Subsequently, the mesenchymal tissues of the forming central fibrous body remain interposed between the developing atrioventricular node and the so-called retro-aortic “node,” the latter structure regressing in normal hearts but persisting in certain complex cardiac malformations.⁴³

Recent lineage studies in mice have shown that, albeit atrioventricular canal musculature and the myocardializing vestibular spine both originate from a Tbx2-positive lineage, the compact part of the atrioventricular node develops in its entirety from the atrioventricular canal musculature,²⁹ without contributions from the coronary sinus musculature, specifically expressing transcription factor Tbx18,²⁴ or vestibular spine-derived myocardium, which is, unlike the atrioventricular node, marked by the MEF2c-AHF-Cre transgene.²⁹ In the human embryonic heart, we failed to observe TBX18 expression in the TBX3-positive atrioventricular nodal primordium (data not shown).

In the developing mouse heart, the expression of the connexins, which ensure rapid myocardial conduction, is suppressed in the atrioventricular canal by the transcription factors Tbx3 and Tbx2.²⁰ The cardiomyocytes on the crest of the ventricular septum, which form the atrioventricular conduction axis in mice, also express Tbx3 at early stages.^19,24 The developing and maturing bundle of His in the mouse heart remains negative for connexin40 and connexin43.²⁴ In the late embryonic human heart, we found a similar arrangement, where the cells of the developing axis, after completion of ventricular septation, remained negative for both connexin40 and connexin43 (Figure 7J and 7K), corroborating that at these stages the developing bundle of His is a slow-conducting entity as in early fetal hearts.⁴⁴ In contrast to the situation in mice, we found connexin43 expression on top of the ventricular septum (Figure 6E and 6K), the functional significance of which is unclear. Despite such differences in the timing of expression of connexin43 and in the intensity of ID2 expression, the similar patterns of expression of key regulatory and functional proteins in the developing human atrioventricular conduction system suggest evolutionary conservation of the general embryonic blueprint for the formation of atrioventricular conduction axis in humans and mice.

In the normal human postnatal heart, the atrioventricular conduction axis is the only muscular connection between the atrial and ventricular muscle masses. Defective formation of the plane of atrioventricular insulation in the mouse, particularly in the absence of transcription factor Tbx2,⁴⁵ and Bmp-receptor 1a,⁴⁶ has been shown to be associated with the persistence of accessory muscular bundles, which are capable of producing ventricular preexcitation. In humans, the connections, which are responsible for the Wolff-Parkinson-White syndrome, have fast-conducting properties and express connexin43.⁴⁷ Even up to the neonatal period, when formation of the atrioventricular insulation plane is relatively complete, multiple accessory myocardial strands are still to be found crossing the fibrous plane of insulation.⁴⁸ These remnants of the atrioventricular canal myocardium, however, are negative for connexins40 and connexin43 and would thus constitute slowly conducting pathways. The failure of disappearance of these strands, therefore, cannot be the sole mechanism for ventricular preexcitation. An additional requirement is the further differentiation of this primary myocardium into fast-conducting working myocardium.⁴⁵

In contrast to the postnatal situation, in which the atrioventricular node and the bundle of His are isolated from the myocardium of the ventricular septal crest by fibrous tissue, the TBX3-positive precursors of these structures seen at late embryonic stages are not yet isolated from the ventricular myocardium by fibrous tissue (Figure 7). In these stages, the cardiomyocytes making up the atrioventricular node and the penetrating atrioventricular bundle do not express fast-conducting connexins, thus producing an effective functional isolation in the setting of absent fibrous insulation. Persistence of the original muscular continuity between the myocytes of the atrioventricular conduction axis and the crest of the ventricular septum accounts for the so-called Mahaim connections, characterized by slow conduction.