Temporal requirement of Nkx2-5 during cardiac conduction system development

Abstract

During cardiac development, transient invaginations of the myocardium, termed trabeculae, appear at the inner surface of the ventricles, which disappeared by compaction to form a functional ventricular wall. Trabeculae also contain progenitor cells of the ventricular conduction system (VCS), a complex network of Purkinje fibers driving the rapid propagation of electrical activity in the ventricles. Defects in ventricular compaction and conduction have been observed in patients and mutant mice carrying mutations in NKX2-5, encoding a key transcriptional regulator of heart development. In order to analyze the link between trabecular fate and VCS differentiation, we carried out genetic tracing and prospective clonal analyses of trabeculae using Cx40-CreERT2 mice and Rosa26-Confetti mice. Our results show that the peripheral VCS segregates progressively during embryonic development. Cells exclusively fated to give rise to the VCS are present in the trabecular compartment as early as E9.5. Specification of new conductive myocytes within the trabecular compartment during subsequent development contributes to the formation of a complex Purkinje fiber network at birth. We performed the same lineage tracing experiments in Nkx2-5 heterozygous mice with severe hypoplasia of the Purkinje fiber network at adult stages. The number of Purkinje fibers originating from early Nkx2-5 hapolinsufficient progenitors is identical to control. In contrast, at later stages of development, we observed an increase in the number of trabecular cells that do not give rise to Purkinje fibers. This suggests that Nkx2-5 controls the progressive recruitment of trabecular cells into the VCS as well as the later maintenance of the conductive phenotype. Overall our study highlights the early segregation of the VCS lineage at the onset of trabeculation and the later role of Nkx2-5 in formation of the Purkinje fiber network.