In humans, heterotaxy syndrome is associated with severe cardiac structural and conduction system abnormalities and is an independent risk factor for post-operative mortality following surgical palliation (3
). 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
), 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
). 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
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, left atrial isomerism leads to brady-arrhythmias secondary to SA node dysfunction, while right atrial isomerism is associated with sinus propagation alternating between the right and left sided intrinsic pacemakers (8
). Understanding how SA node patterning affects function is therefore clinically relevant. The current studies cannot rule out the possibility that Zic3 deficient mice with normal appearing staining patterns have inherent functional abnormalities. Future studies assessing functional characteristics via 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 deficient embryos also demonstrate abnormal AV nodal patterning. Prior to 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 upon 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
Zic3 null mice exhibit mixed morphologies (eg. right or left isomerism) indicating that they encompass the full range of hetertoaxy 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
). 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 though 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 as well as elucidation of human rhythm abnormalities.