Pulmonary arterial hypertension (PAH) is a chronic condition characterized by vascular remodeling and increased vaso-reactivity. PAH is more common in females than in males (~3:1). Connexin (Cx)43 has been shown to be involved in cellular communication within the pulmonary vasculature. Therefore, we investigated the role of Cx43 in pulmonary vascular reactivity using Cx43 heterozygous (Cx43+/−) mice and 37,43Gap27, which is a pharmacological inhibitor of Cx37 and Cx43. Contraction and relaxation responses were studied in intra-lobar pulmonary arteries (IPAs) derived from normoxic mice and hypoxic mice using wire myography. IPAs from male Cx43+/− mice displayed a small but significant increase in the contractile response to endothelin-1 (but not 5-hydroxytryptamine) under both normoxic and hypoxic conditions. There was no difference in the contractile response to endothelin-1 (ET-1) or 5-hydroxytryptamine (5-HT) in IPAs derived from female Cx43+/−mice compared to wildtype mice. Relaxation responses to methacholine (MCh) were attenuated in IPAs from male and female Cx43+/− mice or by pre-incubation of IPAs with 37,43Gap27. Nω-Nitro-L-arginine methyl ester (l-NAME) fully inhibited MCh-induced relaxation. In conclusion, Cx43 is involved in nitric oxide (NO)-induced pulmonary vascular relaxation and plays a gender-specific and agonist-specific role in pulmonary vascular contractility. Therefore, reduced Cx43 signaling may contribute to pulmonary vascular dysfunction.
The heart develops in a synchronized sequence of proliferation and differentiation of cardiac progenitor cells (CPCs) from two anatomically distinct pools of cells, the first heart field (FHF) and second heart field (SHF). Congenital heart defects arise upon dysregulation of these processes, many of which are restricted to derivatives of the FHF or SHF. Of the conserved set of signaling pathways that regulate development, the Wnt signaling pathway has long been known for its importance in SHF development. The source of such Wnts has remained elusive, though it has been postulated that these Wnts are secreted from ectodermal or endodermal sources. The central question remains unanswered: Where do these Wnts come from? Here, we show that CPCs autoregulate SHF development via Wnt through genetic manipulation of a key Wnt export protein (Wls), scRNA-seq analysis of CPCs, and use of our precardiac organoid system. Through this, we identify dysregulated developmental trajectories of anterior SHF cell fate, leading to a striking single ventricle phenotype in knockout embryos. We then applied our findings to our precardiac organoid model and found that Wnt2 is sufficient to restore SHF cell fate in our model of disrupted endogenous Wnt signaling. In this study, we provide a basis for SHF cell fate decision—proliferation vs. differentiation—autoregulated by CPCs through Wnt.
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