Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells.

Oriol Bover,João Facucho-Oliveira,Tiago Justo,Ibrahim J Domian,Paulo N G Pereira,José M Inácio,José S Ramalho,José António Belo,Michael Schubert

doi:10.1371/journal.pone.0205108

Abstract

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.

Highlights

Identification of genes and the study of their role in cardiogenesis are important to elucidate the molecular events regulating cardiomyocyte lineage commitment
To evaluate calcium-binding EGF domain-1 (Ccbe1) expression during cardiac differentiation, we exploited the double transgenic RG embryonic stem cell (ESC) line, wherein the red fluorescent protein dsRed is under the control of the second heart field (SHF) enhancer of Mef2C, and the enhanced green fluorescent protein (GFP) is under the control of the cardiac-specific enhancer of Nkx2.5 [14]
Like mouse and chick embryonic cardiac development [2, 3], Ccbe1 is upregulated in secondary heart field (SHF) cardiac progenitors derived from ESC differentiating in vitro

Summary

Introduction

Identification of genes and the study of their role in cardiogenesis are important to elucidate the molecular events regulating cardiomyocyte lineage commitment. This is critical for the control of cardiac commitment from different stem cell sources and the use of mature cardiac cells in the context of regenerative medicine. In a differential screen designed to identify novel genes required for the correct development of the heart precursor lineages [1], we identified CCBE1, a gene coding for a secreted protein that contains collagen domains and a calcium.

Methods

Results

Conclusion