Myocardial Differentiation is Dependent on Endocardial Signaling During Early Cardiogenesis in vitro

Abstract

Nearly 1% of all newborns are diagnosed with congenital heart defects (CHDs), which is the leading cause of morbidity and mortality among infants. Thus, it is essential that we continue efforts to expand our knowledge regarding the cellular and molecular biology of normal heart development. Current studies have revealed morphological stages during heart development, such as valvulogenesis and trabeculation, rely on cell-cell interactions between endocardial and myocardial cells. However, it is unclear whether such interactions are required during the initial stages of cardiogenesis. Our laboratory has addressed this dearth in knowledge regarding heart development by generating an in vitro model that permits the ablation of endocardial cells with the use of NFATc1, an endocardial-specific marker, and the diphtheria toxin receptor (DTR). Treatment of differentiated NFATc1-DTR ESCs with diphtheria toxin efficiently ablated endocardial cells and significantly reduced the number of contracting cardiomyocytes. Additionally, endocardial ablation decreased the expression of early and late markers of myocardial differentiation and maturation. In an effort to further elucidate the role of endocardial cells during myocardial differentiation, we utilized data from an RNAseq analysis of sorted endocardial and vascular endothelial cells that revealed Bmp2 was preferentially expressed in endocardium relative to vascular endothelium. The treatment of NFATc1-DTR embryoid bodies (EBs) with Bmp2 during endocardial ablation partially rescued beating by 50% to 60% of that observed in control EBs. Furthermore, Bmp2 treatment increased the differentiation and maturation of cardiomyocytes in the absence of endocardial cells. Overall, our results suggest that the differentiation, maturation, and function of myocardial cells during the early stages of cardiogenesis is dependent on a paracrine interaction with the endocardium that is mediated at least partially by the Bmp signaling pathway.