Cooperative Response to Endocardial Notch Reveals Interaction With Hippo Pathway.

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 formation and calcific aortic valve disease, but knowledge is very limited about the role played in valve development and disease by noncoding endocardial regulatory regions and upstream factors. METHODS: We manipulated Notch signaling in mouse embryonic endocardial cells by short-term and long-term coculture with OP9 stromal cells expressing Notch ligands and inhibition of Notch activity. We examined the transcriptional profile and chromatin accessibility landscape for each condition, integrated transcriptomic, transcription factor occupancy, chromatin accessibility, and proteomic datasets. We generated in vitro and in vivo models with CRISPR-Cas9-edited deletions of various noncoding regulatory elements and validated their regulatory potential. RESULTS: We identified primary and secondary transcriptional responses to Notch ligands in the mouse embryonic endocardium, and a NOTCH-dependent transcriptional signature in valve development and disease. By defining the changes in the chromatin accessibility landscape and integrating with the landscape in developing mouse endocardium and adult human valves, we identify potential noncoding regulatory elements, validated selected candidates, propose interacting cofactors, and define the timeframe of their regulatory activity. Additionally, we found cooperative transcriptional repression with Hippo pathway by inhibiting nuclear Yap (Yes-associated protein) activity in the endocardium during cardiac valve development. CONCLUSIONS: Sequential Notch-dependent transcriptional regulation in the embryonic endocardium involves multiple factors. Notch activates certain noncoding elements through these factors and simultaneously suppresses elements that could hinder cardiac valve development and homeostasis. Biorxviv: https://www.biorxiv.org/content/10.1101/2023.03.23.533882v1.full.

Midkine-a Regulates the Formation of a Fibrotic Scar During Zebrafish Heart Regeneration.

Unlike the hearts of mammals, the adult zebrafish heart regenerates after injury. Heart cryoinjury in zebrafish triggers the formation of a fibrotic scar that gradually degrades, leading to regeneration. Midkine-a (Mdka) is a multifunctional cytokine that is activated after cardiac injury. Here, we investigated the role of mdka in zebrafish heart regeneration. We show that mdka expression was induced at 1-day post-cryoinjury (dpci) throughout the epicardial layer, whereas by 7 dpci expression had become restricted to the epicardial cells covering the injured area. To study the role of mdka in heart regeneration, we generated mdka-knock out (KO) zebrafish strains. Analysis of injured hearts showed that loss of mdka decreased endothelial cell proliferation and resulted in an arrest in heart regeneration characterized by retention of a collagenous scar. Transcriptional analysis revealed increases in collagen transcription and intense TGFß signaling activity. These results reveal a critical role for mdka in fibrosis regulation during heart regeneration.

Loss of Caveolin-1 and caveolae leads to increased cardiac cell stiffness and functional decline of the adult zebrafish heart.

Caveolin-1 is the main structural protein of caveolae, small membrane invaginations involved in signal transduction and mechanoprotection. Here, we generated cav1-KO zebrafish lacking Cav1 and caveolae, and investigated the impact of this loss on adult heart function and response to cryoinjury. We found that cardiac function was impaired in adult cav1-KO fish, which showed a significantly decreased ejection fraction and heart rate. Using atomic force microscopy, we detected an increase in the stiffness of epicardial cells and cells of the cortical zone lacking Cav1/caveolae. This loss of cardiac elasticity might explain the decreased cardiac contraction and function. Surprisingly, cav1-KO mutants were able to regenerate their heart after a cryoinjury but showed a transient decrease in cardiomyocyte proliferation.

Notch signalling restricts inflammation and serpine1 expression in the dynamic endocardium of the regenerating zebrafish heart.

The zebrafish heart regenerates after ventricular damage through a process involving inflammation, fibrotic tissue deposition/removal and myocardial regeneration. Using 3D whole-mount imaging, we reveal a highly dynamic endocardium during cardiac regeneration, including changes in cell morphology, behaviour and gene expression. These events lay the foundation for an initial expansion of the endocardium that matures to form a coherent endocardial structure within the injury site. We studied two important endocardial molecules, Serpine1 and Notch, which are implicated in different aspects of endocardial regeneration. Notch signalling regulates developmental gene expression and features of endocardial maturation. Also, Notch manipulation interferes with attenuation of the inflammatory response and cardiomyocyte proliferation and dedifferentiation. serpine1 is strongly expressed very early in the wound endocardium, with decreasing expression at later time points. serpine1 expression persists in Notch-abrogated hearts, via what appears to be a conserved mechanism. Functional inhibition studies show that Serpine1 controls endocardial maturation and proliferation and cardiomyocyte proliferation. Thus, we describe a highly dynamic endocardium in the regenerating zebrafish heart, with two key endocardial players, Serpine1 and Notch signalling, regulating crucial regenerative processes.

NOTCH pathway inactivation promotes bladder cancer progression.

NOTCH signaling suppresses tumor growth and proliferation in several types of stratified epithelia. Here, we show that missense mutations in NOTCH1 and NOTCH2 found in human bladder cancers result in loss of function. In murine models, genetic ablation of the NOTCH pathway accelerated bladder tumorigenesis and promoted the formation of squamous cell carcinomas, with areas of mesenchymal features. Using bladder cancer cells, we determined that the NOTCH pathway stabilizes the epithelial phenotype through its effector HES1 and, consequently, loss of NOTCH activity favors the process of epithelial-mesenchymal transition. Evaluation of human bladder cancer samples revealed that tumors with low levels of HES1 present mesenchymal features and are more aggressive. Together, our results indicate that NOTCH serves as a tumor suppressor in the bladder and that loss of this pathway promotes mesenchymal and invasive features.