Cardiac conduction system malformations in heterotaxy result from dysregulated Pitx2 expression

The cardiac conduction system (CCS) develops asymmetrically along the body axes. In heterotaxy syndrome - resulting from aberrant left-right axis formation - atrial and atrioventricular conduction defects can cause life-threatening arrhythmias. However, the developmental mechanisms regulating the atrioventricular conduction system (AVCS) disposition and integrity remain unclear. To investigate the etiology of AVCS malformations in laterality defects, we analyzed CCS development and function in mouse mutants for Cryptic and Lefty1, which are key regulators of Pitx2 in the left-right axis formation. Cryptic-/- embryos exhibited bilateral sinoatrial nodes and an ectopic anterior AV node and bundle accompanied by reduced Pitx2 expression. In contrast, Lefty1-/- embryos showed a hypoplastic sinoatrial node and AV node-bundle dissociation with ectopic Pitx2 expression. Single-cell transcriptomic analysis of Pitx2-/- hearts revealed expansion of AV node and bundle populations, consistent with a repressive role of Pitx2 in AVCS specification. Genetic lineage tracing indicated that Pitx2-expressing cells from the left lateral plate mesoderm populate cranioventral cardiac regions, where AVCS development is suppressed. Together, these findings clarify how global left-right axis information is locally integrated to shape AVCS disposition and integrity, providing a mechanistic model for AVCS abnormalities in laterality-associated congenital heart disease.