A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaking cell fate in the pulmonary vein myocardium and sinoatrial node

Abstract

In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. The genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-like myocardial identity in the PV, is lacking in the SAN. Here we present evidence that in mice Shox2 antagonizes the transcriptional output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5 + domain within the SAN to determine cell fate. Shox2 deletion in the Nkx2-5 + domain of the SAN caused sick sinus syndrome, associated with the loss of the pacemaker program. Explanted Shox2 + cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2 − cells. Shox2 deletion led to Hcn4 ablation in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid bodies. We found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genomewide co-occupancy of Shox2, Nkx2-5 and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development.

The formation and function of the cardiac conduction system

“…Deletion of Shox2 leads to a hypoplastic SV, upregulation of Nkx2-5 and, consequently, downregulation of Hcn4 and Tbx3 in the SAN primordium and in ESCs (Blaschke et al, 2007;Espinoza-Lewis et al, 2009;Hashem et al, 2013). Consistently, the loss of the pacemaker gene programme leads to conduction defects in Shox2-deficient mice (Blaschke et al, 2007;Espinoza-Lewis et al, 2009;Ye et al, 2015).…”

A unique stylopod patterning mechanism by Shox2 controlled osteogenesis

“…Cre/− ;Rosa mTmG mice were analyzed at E13.0, the latest stage the Shox2 deficient embryos appear normal prior to severe complications caused by cardiac defects (Espinoza-Lewis et al, 2009;Ye et al, 2015b). In these embryos, the Shox2 + lineage is labeled by mGFP, and the expression pattern of mGFP reporter did not change significantly compared with controls ( Fig.…”

“…To unravel the functional mechanism of Shox2 in stylopod patterning, we first sought to comprehensively document and analyze Shox2 expression patterns and cell populations that are derived from Shox2 + cells. We took advantage of our recently generated Shox2 HA/+ Ye et al, 2015b) and Shox2 Cre/+ alleles for protein expression analysis and fate mapping.…”

“…Flox , Col2a1-Cre and Col3.6-Cre alleles used in this study were described previously (Cobb et al, 2006;Liu et al, 2004;Ovchinnikov et al, 2000;Sun et al, 2013;Wang et al, 2014;Ye et al, 2015b;Yu et al, 2005). The animal experiments in this study were approved by The Tulane University Institutional Animal Care and Use Committee.…”

“…Immunoprecipitation (IP), co-immunoprecipitation (Co-IP), chromatinimmunoprecipitation (ChIP) and western blotting were performed as described previously (Ye et al, 2015b). ChIP-Seq and associated analysis are described in Supplemental Information.…”