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Heterotaxy syndrome is characterized by multiple congenital anomalies arising from defects in embryonic left-right patterning and loss of normal asymmetric laterality (1–3). The developing heart is particularly sensitive to left-right positional information, and abnormalities are associated with significant morbidity and mortality because of structural defects and/or arrhythmias (4). Patients with heterotaxy are often described as being “bilateral left-sided” (left isomerism) or “bilateral right-sided” (right isomerism). Cardiac conduction system (CCS) abnormalities in the human heterotaxy population are thought to arise from either duplication or absence of one or more CCS elements resulting from similar left-right patterning abnormalities. Duplication creates a risk for tachycardias, whereas absence or dysfunction of sinoatrial (SA) or atrioventricular (AV) nodal elements can lead to bradyarrhythmias or heart block (5–7) and may require mechanical pacing (8). Heterotaxy syndrome itself is a risk factor for increased mortality (4).

In normal SA nodal development, the initial electrical pacemaker emerges at the inflow region of the linear heart tube (9) and develops adjacent to the venous sinus as it shifts rightward and is incorporated into the right atrium (10). As the SA node matures, there is increasing spatial restriction within the right atrium. The acute switch from ventricular activation emanating from the AV canal to a site at the ventricular apex at mouse embryonic day 10.5-11.5 (e10.5-e11.5) signals a primitive but functioning AV node and His-Purkinje system (11). Visualization of electrical system development in the mouse has been made possible through the use of several CCS transgenic mouse lines, including the CCS-LacZ and minK-lacZ models (11–15). Functional analysis has been achieved through the use of optical mapping using voltage-sensitive dye. In the CCS-LacZ model, this functional analysis has been correlated with anatomical staining patterns of CCS tissue elements (11). These tools have made possible the study of embryonic conduction system patterning; however, little is known about early embryonic patterning and development of the CCS in heterotaxy syndrome.

In humans, mutations of ZIC3 cause the X-linked form of heterotaxy, the most common known genetic etiology of heterotaxy syndrome (2,3). Zic3 is a zinc finger transcription factor and member of the GLI superfamily, important mediators of hedgehog signaling. Analysis of Zic3 null mice has demonstrated the importance of this gene in structural cardiac patterning (16–18). Although a comprehensive examination of cardiac development is lacking, the cardiac phenotypes identified mimic those seen in the human heterotaxy population (19).

In this study, we sought to describe CCS developmental patterning abnormalities in the Zic3 murine heterotaxy model using CCS-LacZ mice for anatomical visualization. Our results indicate that lack of Zic3 significantly affects normal CCS patterning and demonstrates a spatial correlation between structural and conduction system abnormalities. Importantly, the conduction system defects seen in this model are similar to those described in the human heterotaxy population. Ultimately, understanding CCS patterning and its correlation with structural cardiac defects may enhance our ability to develop new treatment strategies and provide risk counseling.

METHODS

Mouse embryo collection and β-gal detection.

Embryos were collected following timed matings. Zic3 null and control embryos were stage matched for analyses to within 0.5 d based on somite number (early embryos) or limb bud outgrowth and digit appearance (later embryos). CCS-LacZ embryos were stained with 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal) staining solution as described previously (20). CCS-LacZ and Zic3 null/CCS-LacZ embryos were collected at e9.5-e16.5 and fixed in 4% paraformaldehyde/PBS for 15 min at 4°C and then rinsed in PBS. Embryos were stained with X-Gal solution at room temperature for 48 h and postfixed in 4% paraformaldehyde/PBS for 1 h at 4°C. This study was approved by the Cincinnati Children's Hospital Institutional Animal Care and Use Committee.

Preparation of CCS-LacZ sections and photo imaging.

Embryos were dehydrated through an increasing gradient of ethanol followed by multiple xylene washes and paraffin embedding. Sections were cut transversely at 12-14 μm using a microtome (Leica Microsystems, Wetzlar, German). Sections were deparaffinized in xylene, counterstained with eosin, dehydrated in xylene, and coverslipped using cytoseal (Electron Microscopy Sciences, Hatfield, PA). Images were captured using a Nikon DXM 1200F digital camera coupled to a Nikon Eclipse E400 microscope (Nikon Instruments Inc., Melville, NY).

Synthesis of digoxigenin-labeled riboprobes.

The cDNA sequence corresponding to amino acids 400-690 of mouse Hcn4 (base pairs 1198-2068 of NM_001081192) was amplified from mouse heart RNA with the primers 5′-ATATCTAGACAGTG GGAAGAGATCTTCCA-3′ and 5′-ATACTCGAGTGAAGTTGTCCACGCTCAGT-3′ and subcloned into the XbaI-XhoI sites of pBluescript II SK(-). This region of mHcn4 effectively labels specific components of the adult mouse CNS (21) and has been identified as a marker of the SA node. The mNkx2-5 riboprobe plasmid was kindly provided by Dr. Yutzey (22). Antisense riboprobes were generated by in vitro transcription with the appropriate RNA polymerase in the presence of digoxigenin (DIG) RNA labeling mix.

In situ hybridization.

In situ hybridization was performed as described previously (23). Paraffin-embedded embryos were sectioned transversely, and 12-14 μm sections were mounted on Superfrost Plus microscope slides (Thermo Fisher Scientific, Waltham, MA). Sections were hybridized with 200 μL of digoxigenin-labeled riboprobe at 1 μg/mL in incubation chambers (22 × 40 × 0.2 mm, Electron Microscopy Sciences, Hatfield, PA). Color development was performed using BM Purple (Roche Applied Science, Indianapolis, IN) for 8-12 h.

RESULTS

Development of structural cardiac abnormalities in Zic3 null mice.

Patients with heterotaxy have a diverse spectrum of cardiovascular malformations (CVMs). To evaluate the cardiac anomalies in a mouse model of heterotaxy, embryos were evaluated from e9.5 to e16.5. Structural cardiac abnormalities decreased at later embryonic stages (40% of nulls had structurally abnormal hearts at e10.5, 32% at e11.5, and 25% at e12.5). A reduction in litter size after e10.5 was consistent with embryonic lethality caused by complex CVMs. The cardiac phenotypes in Zic3 null embryos were variable and recapitulated the spectrum of phenotypes seen in human heterotaxy syndrome. Gross examination of the hearts at e10.5-e12.5 demonstrated a range of phenotypes including univentricular morphology and conotruncal abnormalities (Fig. 1 and data not shown). By e10.5, the normal heart has undergone the initial rightward bending of the linear heart tube (d-looping), and the common outflow tract has looped as a precursor to development of normal great artery relationship. The developing atrial chambers lie posterior to two well-developed ventricular chambers, and the outflow tract arises from the right ventricle (Fig. 1A). Figure 1C demonstrates conotruncal malposition in a Zic3 null embryo, with the common outflow arising vertically and centrally from the most superior aspect of the ventricular chambers. The atria are abnormally positioned inferior to the ventricular chambers. Figure 1C demonstrates l-looped malposition of the outflow tract and inferior displacement of the atria. Although wild-type embryos have two developing ventricles at e10.5, a subset of Zic3 null embryos demonstrate univentricular morphology (Fig. 1C and D), with a single ventricular chamber giving rise to a single outlet. Conotruncal abnormalities were common and always associated with additional structural abnormalities. The range of defects in cardiac looping observed during cardiac development in these null mice provides insight into the variable phenotypic presentations of patients with heterotaxy.

Figure 1
figure 1

Abnormal cardiac development in Zic3 embryos. A, Wild-type embryo at e10.5 with typical d-looped ventricular configuration. B, Zic3 null embryo with primitive ventricular configuration. C, Univentricular morphology with l-looping. The outflow tract arises from the leftward aspect of the ventricular chamber. There is dextrocardia with a rightward ventricular apex. D, Univentricular morphology with d-looping. The outflow tract arises from the rightward aspect of the ventricular chamber, and the ventricular apex is directed leftward. X-gal staining illustrates the developing CCS. Scale bars are 300 μm. oft, outflow tract; LV, left ventricle; RV, right ventricle; V, ventricle.

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SA nodal development.

CCS development in patients with heterotaxy syndrome has been associated with loss of the normal asymmetric restriction of the SA node within the right atrium. Both inversion of the SA node with a single pacemaker in the left atrium and duplication or “twinning” of the SA node with both right and left atrial intrinsic pacemakers have been described. Genetic and molecular methods were used to investigate SA node development in Zic3 null mice. Mating Zic3 mice with CCS-LacZ mice allowed for visualization of CCS development in Zic3 null embryos based on the staining pattern. Abnormalities in SA nodal development in Zic3 null embryos ranged from apparently normal to severely abnormal. There was strong correlation between the presence of structural defects and abnormal SA node patterning: Zic3 null embryos with normal structural anatomy had normal SA nodal staining patterns, whereas embryos with abnormal structure displayed either normal or abnormal SA nodal patterning.

Abnormalities in left-right patterning were identified based on loss of normal SA node lateralization and restriction within the right atrium (Fig. 2). These findings are similar to those found in clinical descriptions of SA nodal development in the human heterotaxy population. Normal SA node staining is restricted to the right atrium and right sinus valve (Fig. 2A–C), and the pattern of staining is somewhat diffuse at developmental stages younger than e10.5. As development continues, the staining pattern becomes progressively more restricted to the superior right atrium adjacent to the superior vena cava and the venous valve, the general location of the mature SA node. In Zic3 null embryos, two patterns of SA nodal development were seen. Examples of SA node inversus were found, both with SA nodal development restricted to the left atrium and bilateralization of SA node development. Failure of proper lateralization of the SA node indicates ambiguous patterning consistent with right atrial isomerism (Fig. 2D–F). Figure 2G–I demonstrates a left-sided SA node in a null embryo with l-looping.

Figure 2
figure 2

Abnormal cardiac conduction system development in structurally abnormal embryos. Wild-type (A-C) and Zic3 null (D-I) cardiac morphology and conduction system staining at e10.5. A, Wild-type cardiac morphology with typical d-looping. B and C, Transverse sections at the levels indicated in (A) demonstrate the distal conduction system is a single linear band positioned rightward within the RV (arrow), and a normal SA nodal staining pattern restricted to the right atrium (*). D, Severe cardiac structural abnormalities with univentricular morphology and abnormal bilateralization of atrial staining. E and F, Sections at the levels indicated in (D) demonstrate abnormal atrial and ventricular development with conduction system staining in both the left and right aspects of the common atrium. G, Mirror image dextrocardia with l-looped ventricular morphology. H and I, Mirror image SA nodal patterning (*) with restriction of staining in the left atrium. The AV node and His bundle are leftward (arrow). Scale bars in A, D, and G are 100 μm. Scale bars in B, C, E, F, H, and I are 50 μm. Oft, outflow track; v, atrial venous valve.

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SA node development in Zic3 null embryos was evaluated further using molecular methods. Previous studies have elaborated a molecular pathway for localized formation of the SA node (24–26). The atrial myocardium is characterized by expression of myocardial markers such as Nppa and Nkx2.5, whereas the nonmyocardial SA node region expresses markers such as the pacemaker channel gene product, Hcn4. During heart tube maturation, Nkx2.5 progressively represses expression of Hcn4, resulting in molecular delineation of a distinct, compact SA node region in the right atrium. To determine whether specification of the SA node occurs properly in Zic3 null embryos, the expression of Nkx2.5 and Hcn4 was determined by in situ hybridization. In e11.5 wild-type embryos, the SA node is a triangular wedge of tissue, which is Hcn4 positive and Nkx2.5 negative (Fig. 3A–C). At these stages, Hcn4 expression is localized to the same anatomic structures identified by X-gal staining in CCS-LacZ mice but is more spatially restricted in its expression profile. Zic3 null embryos (Fig. 3D–I) retain the mutually exclusive expression pattern of Hcn4 and Nkx2.5 identified in wild-type embryos; however, the positioning and morphology of the SA node tissue are abnormal. There is a diffuse appearance to some SA nodal structures, as well as abnormal shape and positioning (Fig. 3E, F, H, and I). The expression pattern in embryos with bilateral staining (Fig. 3G, bilateral SA nodes) tended to be more diffuse and heterogeneous compared with the wild-type patterning. These results imply that Zic3 is not required for SA node specification but plays a central role in proper localization.

Figure 3
figure 3

SA node patterning is abnormal in e11.5 Zic3 null embryos. A-C, Molecular characterization in a wild-type embryo. In (A), Hcn4 expression delineates the SA node, positioned above the right atrium. B, High-power view of SA node visualized in (A), demonstrating Hcn4 staining of a wedge-shaped area of tissue. Nkx2.5 is excluded from this region (arrow) but is expressed in the surrounding myocardium in (C). D-F, Zic3 null embryo with abnormal anteroposterior positioning of the ventricles and misalignment of the atria. The abnormally elongated SA node region is indicated by presence of Hcn4 staining (D, E, arrow) and lack of Nkx2.5 staining (F). G-I. Zic3 null embryo with dextrocardia and common ventricle. Strong bilateral Hcn4 staining was observed above the atria (G and H), and Nkx2.5 staining was absent (I). Scale bars in A, D, and G are 0.5 mm. Scale bars in B, C, E, F, H, and I are 100 μm.

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Organization of CCS after completion of cardiac looping.

By e12.5, the final phases of cardiac looping are complete, and restriction and maturation of CCS patterning occur. In Zic3 null embryos, structural defects are present along with abnormal SA node morphogenesis and abnormal development of the distal conduction system (Fig. 4). SA node development at e12.5 is characterized by further restriction and organization of the staining pattern within the right atrium (Fig. 4). In contrast, SA node tissue in Zic3 null embryos at e12.5 demonstrates diffuse, disorganized X-gal staining patterns within the atrial chamber, in addition to the bilateralization of the staining (Fig. 4F, J, and K). These results provide evidence of failure to properly pattern the SA node. Additionally, the diffuse and disorganized appearance of SA node staining suggests a possible temporal disruption, with retention of primitive patterning features. Absence of SA nodal staining, suggestive of left atrial isomerism, was also identified (data not shown).

Figure 4
figure 4

Distal conduction system patterning is abnormal in Zic3 null embryos at e12.5. A-D, Normal patterning with a single staining band (arrows) from the crest of the developing interventricular septum to the AV node. E-H, Zic3 null embryo with accessory pathway connection (arrow) separate from the normal tract connecting the bundle branches and AV node. This tract resides lateral to the normal linear band connecting the right ventricular myocardium with the right aspect of the AV valve and right atrium. I-L, Zic3 null embryo with abnormal AV valve septation and positioning relative to the ventricular chambers with retention of the common AV valve and loss of normal septation between the mitral and tricuspid valves. The embryo has a primitive circumferential AV canal staining pattern giving rise to two separate tracts (arrows) from the developing AV node to the ventricular myocardium. Scale bars are 500 μm. RA, right atrium; LA, left atrium.

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Distal conduction system abnormalities, involving the AV node, His bundle, or Purkinje system, in patients with heterotaxy can result in both abnormal positioning and duplication of CCS elements. “Twinning” or duplication of the AV node in humans may lead to a form of reentry tachycardia. Distal conduction system patterning demonstrated a relationship between structural and CCS patterning defects similar to that observed in more proximal SA nodal development. Again, Zic3 null embryos with normal cardiac structure demonstrated universally normal distal conduction system patterning. In hearts with structural abnormalities, the distal conduction system was abnormal to varying degrees in all embryos analyzed (Table 1). Before e10.5, the entire AV canal functions as AV nodal tissue and the staining pattern is circumferential, encompassing the entire periphery of the AV canal region. Following a timeline similar to SA node maturation, the AV node becomes restricted to its adult position and initiates compaction and organization by e12.5 (Fig. 4A–D). Distal to the AV node, the compacted CCS continues as a linear band of tissue extending to the distal Purkinje system through the right posterior aspect of the AV canal and right ventricular region (Fig. 4D).

Table 1 Correlation of structural and CCS patterning abnormalities
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In abnormal Zic3 null embryos, patterning was disrupted at the level of the AV node, His bundle, and bundle branches. Abnormalities of the AV node included disorganization with loss of normal compaction characterized by diffuse staining (Fig. 4G). Rarely, duplication of AV nodal tissue was visualized. His bundle and bundle branch patterning were also significantly affected. The staining around the periphery of the AV canal in Figure 4I–L illustrates the failure to restrict CCS patterning distal to the AV node. The typical single linear band extending from the AV node to the distal Purkinje system is replaced by two tracts. Staining patterns consistent with accessory connections from the atrium and ventricle were also identified in a subset of embryos, possibly indicating future phenotypic accessory pathway development (Fig. 4F and K). These findings indicate that deficiency of Zic3 can severely affect embryonic patterning of the distal conduction system and imply a strong relationship between abnormalities in cardiac structure and the distal conduction system. Importantly, these abnormalities are similar to those commonly seen in the human heterotaxy population (Table 2).

Table 2 Representative cardiac abnormalities in Zic3 null embryos
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DISCUSSION

In humans, heterotaxy syndrome is associated with severe cardiac structural and conduction system abnormalities and is an independent risk factor for postoperative mortality after surgical palliation (3,5). The clinical impact of these conduction system abnormalities has been based on postnatal functional assessment with bradycardia or heart block. Although the molecular cues important for development of conduction system lineage are beginning to be elaborated (24–26), the embryonic events controlling conduction system pattern formation are largely unknown. In this study, we examined conduction system development and its association with structural development in the Zic3 heterotaxy mouse model.

Loss of normal left-right differentiation affects CCS patterning. All structurally abnormal Zic3 null hearts exhibited CCS abnormalities. However, even at these early developmental stages, there were no embryos with completely identical cardiac abnormalities in our series, consistent with the clinical heterogeneity of heterotaxy. In the proximal CCS, SA node duplication was the most common finding (right isomerism, n = 7/18 sectioned embryos). In addition, lack of SA nodal development (left isomerism, n = 4/18) and exclusive left atrial staining associated with complete reversal of myocardial and CCS structures (situs inversus, n = 3/18) were identified. These abnormalities of duplicated or absent CCS elements mimic those seen in the human heterotaxy population (27–29). Hypoplasia of the SA node has been associated with aberrant lineage specification, as manifest by ectopic expression of Nkx2.5, in mouse models such as Shox2 null mice (30). In contrast, Zic3 null mice form a normal boundary of Hcn4 and Nkx2.5 expression in the atria, indicating that Zic3 deficiency does not impair lineage specification or the molecular signature of the SA node and will, therefore, be a useful tool to dissect the secondary consequences of abnormal left-right patterning on SA node patterning, structural malformation, and ultimately CCS function.

Clinically, the left atrial isomerism leads to bradyarrhythmias secondary to SA node dysfunction, whereas the right atrial isomerism is associated with sinus propagation alternating between the right- and left-sided intrinsic pacemakers (8,29). Understanding how SA node patterning affects function is therefore clinically relevant. The current studies cannot rule out the possibility that Zic3 null mice with normal-appearing staining patterns have inherent functional abnormalities. Future studies assessing functional characteristics by voltage-gated visualization embryonically (11) or intracardiac electrophysiology testing postnatally could help resolve this question (31).

In addition to the SA nodal abnormalities described, Zic3 null embryos also demonstrate abnormal AV nodal patterning. Before e10.5, staining circumferentially around the AV canal appears normal in Zic3 null embryos. After e10.5, AV node abnormalities including disorganization, malpositioning, or mislocalization are identified (n = 6/9 d12.5 sectioned embryos), similar to abnormalities seen in SA nodal patterning. This pattern is consistent with human pathology studies in which both AV nodal structures are displaced and can be dysfunctional (32). Similar inhibition of distal CCS compaction including alterations in AV node and His-purkinje system disorganization is seen on neural crest ablation (33).

In Zic3 null embryos, there was evidence of additional AV pathways consistent with conventional accessory pathways or Mahaim connections (n = 2/9), a common finding in patients with heterotaxy syndrome and l-looped ventricular configuration that can result in the typical form of AV reciprocating tachycardia (34). The CCS-LacZ model has been used previously to describe CCS abnormalities such as Mahaim fiber development and delineation of embryonic cardiac regions with future arrhythmogenic potential (13,14).

Zic3 null mice exhibit mixed morphologies (e.g. right or left isomerism) indicating that they encompass the full range of heterotaxy spectrum defects. Mouse models with exclusive right or left isomeric patterns, such as Pitx2 or Lefty-1 knockouts, have been described anatomically but have not been analyzed in depth with regard to CCS development (35,36). Clinically, understanding how conduction system patterning correlates with congenital heart disease is important for management.

In summary, proper patterning of the CCS requires a complex interplay of embryonic left-right differentiation. CCS abnormalities in the Zic3 heterotaxy model are closely linked with associated abnormalities of cardiac structure. Loss of Zic3 leads to abnormal CCS structure and maturation that coincides with the severity and location of associated structural heart disease. Potential mechanisms for this disruption include ambiguous patterning, duplication of tissue patterning, and/or failure of progressive development with retention of earlier embryonic patterns. The congruent relationship of structural and CCS patterning abnormalities seem to indicate shared regulatory programs directing developmental patterning, although future lineage studies will be needed to more clearly delineate this relationship. This mouse model provides a novel tool to dissect the genetic regulatory hierarchy linking left-right patterning with conduction system-specific gene expression and pattern formation and elucidation of human rhythm abnormalities.