Nav1-8, a new marker of Purkinje fibers, provides insight into defective conduction system maturation in a mouse model of congenital cardiomyopathy

Abstract

The transcription factor Nkx2-5 is known to be a key regulator of cardiac development and NKX2-5 mutations have been found in numerous cardiac diseases associated with conduction defects. This factor is required for the differentiation of Purkinje fibers (PF), which have been implicated in triggering ventricular arrhythmias. A better understanding of the development of the Purkinje system in normal and Nkx2-5 mutant hearts could provide insight into their involvement in conduction defects in cardiac diseases. However, analysis of the Purkinje network development is hampered by the lack of specific lineage tracers. Recently, the voltage-gated sodium channel Nav1-8 short transcript has been shown to be expressed in the ventricular conduction system (VCS). This study aims to characterize the expression domain of Nav1-8 in the murine VCS to determine whether it is a suitable tool for tracing conductive cells. This would also help to identify defects in conductive recruitment in congenital disease. Nav1-8-Flp mice were crossed with a GFP reporter line for genetic tracing analyses in WT and Nkx2-5 heterozygous mice. The specificity and efficiency of Nav1-8 labelling was measured from P1 to P21 based on the number of GFP-positive (Nav1-8 derivatives), Cntn2-positive (VCS) and double positive cells (Nav1-8 derivatives in the VCS). GFP reporter expression starts at birth and is restricted to the PF at the distal part of the VCS. The specificity of Nav1-8 as a marker of PF increased over time between P1 and P21, suggesting that Nav1.8 is expressed in PF progenitor cells prior to their recruitment to the conduction system. However, its efficiency is limited to 20% of PF being marked at a mature stage of development (P21). In Nkx2-5 mutant hearts, GFP labelling within the left ventricle delimits the PF scaffold and is almost absent in the complex ramifications of the PF network. This anatomical distribution is consistent with the hypoplastic Purkinje system of these mice. However, unlike in control hearts, additional GFP+ cells are found in the bundle branches at the proximal part of the VCS in Nkx2-5 mutants. Our study shows that the sodium channel Nav1-8 represents a unique and highly specific lineage tracer of the peripheral PF in mature hearts. In addition, we show an enlargement of the Nav1-8 expression domain to the bundle branches in Nkx2-5 mutants. This indicates that Nkx2-5 may influence cell fate choice within the VCS.