A cooperative response to endocardial NOTCH signaling stimulation regulates transcriptional activity during cardiac valve development and disease

Abstract

Background: The endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve (BAV) formation and/or calcific aortic valve disease (CAVD), but knowledge is very limited about the role played in valve development and disease by non-coding endocardial regulatory regions and upstream factors. Methods: We manipulated the NOTCH signaling pathway in mouse embryonic endocardial cells, defining the transcriptional profile associated to each condition. The endocardial chromatin accessibility landscape for each condition was defined by high-throughput sequencing (ATAC-seq) determination of transposase-accessible chromatin. In vitro and in vivo models carrying deletions of different non-coding regulatory elements were generated by CRISPR-Cas9 gene editing. Results: We identified primary and secondary transcriptional responses to NOTCH ligands in the mouse embryonic endocardium. By integrating our gene expression data with data from developing valves of mice with NOTCH loss-of-function and from human valve calcification samples, we identified a NOTCH-dependent transcriptional signature in valve development and disease. Further, by defining the endocardial chromatin accessibility landscape after NOTCH pathway manipulation and integrating with in vivo data from developing mouse endocardium and adult human valves, we were able to identify a set of potential non-coding regulatory elements, validate representative candidates, propose co-factors interacting with them, and define the timeframe of their regulatory activity. Analysis of the transcriptional repression driven by NOTCH activation revealed cooperation between the NOTCH and HIPPO pathways in the endocardium during cardiac valve development. Conclusions: Transcriptional regulation in the embryonic endocardium after NOTCH pathway stimulation occurs in a sequential manner and requires the participation of several factors. NOTCH not only triggers the transcriptional activity of the non-coding elements recognized by these factors, but also represses those elements whose activity negatively affects the development and homeostasis of the cardiac valves.