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.S

Modelling the heterogeneity and complex inheritance of Left Ventricular Non-Compaction

The compaction or formation of a thick and smooth cardiac ventricular wall is a fundamental process for cardiac function. This process takes place during late gestation and early postnatal period, and it is regulated by intercellular signalling from endocardium that controls myocardial proliferation and maturation. During compaction, the transient trabeculae that allowed cardiac growth in early development give rise to the cardiac conduction system, and are integrated in the thick compact wall. Defects in this process of compaction give rise to left ventricular non-compaction or LVNC. This cardiomyopathy can range from asymptomatic to causing arrhythmia or heart failure, even requiring heart transplantation. In addition, LVNC has been described isolated or combined with other congenital heart diseases (CHD), such as hypertrophic cardiomyopathy (HCM) or bicuspid aortic valve (BAV). The most common genetic causes underlying this disease are mutations in sarcomeric genes, but mutations affecting MIB1 (MIB1R530X and MIB1V943F), a member of the NOTCH signalling pathway, were recently identified by our lab as causative of LVNC in two different families. LVNC was observed in mice lacking Mib1 expression in the myocardium, confirming its importance in chamber development. As defects in NOTCH signalling pathway have also been described as causing BAV, we aimed at generating mouse models with the point mutations found in MIB1 by CRISPR-Cas9 to investigate the mechanisms underlying LVNC. These mouse lines allowed us to confirm that the inactivating mutation (Mib1R530X) showed LVNC when combined with the conditional heterozygous null allele in the myocardium, while the missense mutation (Mib1V943F) did not cause LVNC, but affected valve development when combined with heterozygous NOTCH inactivating mutations. These findings and the genetic and phenotypic heterogeneity of LVNC led us to hypothesise that there were other candidate mutations masked behind the autosomal dominant inheritance pattern. We performed exome sequencing in the families carrying the mentioned MIB1 variants, and could identify polymorphisms affecting novel candidates as APCDD1 and ASXL3 in one family, and CEP192, TMX3 and BCL7A in the other. The generation of mouse lines harbouring each set of mutations allowed us to identify new genes implicated in LVNC (Mib1R530X/+ Apcdd1V150I/+ Asxl3M1416V/+ mice) or in BAV (Mib1V943F/+ Cep192T1522M/+ Tmx3-204F191X/+ mice). Further analysis of these lines and the combination of the last one with the Bcl7a mutant, could help elucidating the distinct molecular mechanisms behind the variability of the severity or the ones that cause combined with CHD or isolated LVNC

Trabeculated Myocardium in Hypertrophic Cardiomyopathy: Clinical Consequences.

Hypertrophic cardiomyopathy (HCM) is often accompanied by increased trabeculated myocardium (TM)-which clinical relevance is unknown. We aim to measure the left ventricular (LV) mass and proportion of trabeculation in an HCM population and to analyze its clinical implication. We evaluated 211 patients with HCM (mean age 47.8 ± 16.3 years, 73.0% males) with cardiac magnetic resonance (CMR) studies. LV trabecular and compacted mass were measured using dedicated software for automatic delineation of borders. Mean compacted myocardium (CM) was 160.0 ± 62.0 g and trabecular myocardium (TM) 55.5 ± 18.7 g. The percentage of trabeculated myocardium (TM%) was 26.7% ± 6.4%. Females had significantly increased TM% compared to males (29.7 ± 7.2 vs. 25.6 ± 5.8, p < 0.0001). Patients with LVEF < 50% had significantly higher values of TM% (30.2% ± 6.0% vs. 26.6% ± 6.4%, p = 0.02). Multivariable analysis showed that female gender and neutral pattern of hypertrophy were directly associated with TM%, while dynamic obstruction, maximal wall thickness and LVEF% were inversely associated with TM%. There was no association between TM% with arterial hypertension, physical activity, or symptoms. Atrial fibrillation and severity of hypertrophy were the only variables associated with cardiovascular death. Multivariable analysis failed to demonstrate any correlation between TM% and arrhythmias. Approximately 25% of myocardium appears non-compacted and can automatically be measured in HCM series. Proportion of non-compacted myocardium is increased in female, non-obstructives, and in those with lower contractility. The amount of trabeculation might help to identify HCM patients prone to systolic heart failure.This work was supported by a grant from the Foundation Marató TV3 2018/C/2015 and by the Spanish MICINN and AEI, as well as European Commission FEDER funds, under grant RTI2018-098156-B-C53. Investigators are part of the cardiovascular research network and Cell Therapy network (TerCel) of the Carlos III Health Institute (SAF2015-71863-REDT, RTI2018-095377-B-I00, CIBERCV CB16/11/00385, RD16/0011/0021, RD16/0011/0024, CB16/11/00399, RIC; RD12/0042/0049), MCIU (SAF2016-78370-R) and IMIB (Instituto Murciano de Investigación Biosanitaria). María Sabater was supported by a grant from FFIS. Investigators are part of clinical group of ERN Guard-Heart, CIBERCV, CIBERER and University of Murcia.S