Characterizing the role of atrial natriuretic peptide signaling in the development of embryonic ventricular conduction system

Arun Govindapillai,Nobuyo Maeda,Robert A Rose,Mark Baguma-Nibasheka,Kishore B S Pasumarthi,Adam Hotchkiss,Lucile Miquerol,Oliver Smithies

doi:10.1038/s41598-018-25292-0

Abstract

Patients born with congenital heart defects frequently encounter arrhythmias due to defects in the ventricular conduction system (VCS) development. Although recent studies identified transcriptional networks essential for the heart development, there is scant information on the mechanisms regulating VCS development. Based on the association of atrial natriuretic peptide (ANP) expression with VCS forming regions, it was reasoned that ANP could play a critical role in differentiation of cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) toward a VCS cell lineage. The present study showed that treatment of embryonic ventricular cells with ANP or cell permeable 8-Br-cGMP can induce gene expression of important VCS markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40). Inhibition of protein kinase G (PKG) via Rp-8-pCPT-cGMPS further confirmed the role of ANP/NPRA/cGMP/PKG pathway in the regulation of HCN4 and Cx40 gene expression. Additional experiments indicated that ANP may regulate VCS marker gene expression by modulating levels of miRNAs that are known to control the stability of transcripts encoding HCN4 and Cx40. Genetic ablation of NPRA revealed significant decreases in VCS marker gene expression and defects in Purkinje fiber arborisation. These results provide mechanistic insights into the role of ANP/NPRA signaling in VCS formation.

Highlights

The cardiac conduction system (CCS) is a complex network of cells within the heart that generates and conducts electrical impulses to enable rhythmic, coordinated contraction of the heart[1]
Our results revealed that Atrial natriuretic peptide (ANP) induces gene expression of important ventricular conduction system (VCS) markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40) in E11.5 ventricular cell cultures which are known to harbor both cardiac progenitor cells (CPCs) and CMs5,16,17, through the natriuretic peptide receptor-A (NPRA) signaling pathway
To determine whether ANP plays any role in the induction of HCN4 and Cx40 expression, exogenous ANP was added at varying concentrations (0–1000 ng/ml) to primary cultures prepared from E11.5 CD1 ventricles at 12 hr intervals for 48hrs

Summary

Introduction

The cardiac conduction system (CCS) is a complex network of cells within the heart that generates and conducts electrical impulses to enable rhythmic, coordinated contraction of the heart[1]. Additional studies demonstrated that Nrg-1 can induce embryonic mouse CMs to differentiate into cells of the conduction system[3]. While these studies suggest conversion of CMs into VCS cells, other studies suggest the existence of a common progenitor cell for working CM and VCS cells[4,5,6]. Inability of Nrg-1 to increase reporter gene expression in E10.5/11.5 hearts[3] suggests that additional factors may be involved in the development and/or maturation of VCS network in later stages of heart development. Our results revealed that ANP induces gene expression of important VCS markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40) in E11.5 ventricular cell cultures which are known to harbor both CPCs and CMs5,16,17, through the natriuretic peptide receptor-A (NPRA) signaling pathway. Pharmacological inhibition as well as genetic ablation of NPRA revealed significant decreases in VCS marker gene expression and defects in Purkinje fiber arborisation

Methods

Results

Conclusion