The process of valve formation is a complex process that involves intricate interplay between various pathways at precise times. Although we have not completely elucidated the molecular pathways that lead to normal valve formation, we have identified a few major players in this process. We are now able to implicate TGF-ß, BMP, and NOTCH as suspects in tricuspid atresia (TA), as well as their downstream targets: NKX2-5, TBX5, NFATC1, GATA4, and SOX9. We know that the TGF-ß and the BMP pathways converge on the SMAD4 molecule, and we believe that this molecule plays a very important role to tie both pathways to TA. Similarly, we look at the NOTCH pathway and identify the HEY2 as a potential link between this pathway and TA. Another transcription factor that has been implicated in TA is NFATC1. While several mouse models exist that include part of the TA abnormality as their phenotype, no true mouse model can be said to represent TA. Bridging this gap will surely shed light on this complex molecular pathway and allow for better understanding of the disease process.

Tricuspid atresia (TA) is a rare congenital heart condition that presents with a complete absence of the right atrioventricular valve. Because of the rarity of familial and/or isolated cases of TA, little is known about the potential genetic abnormalities contributing to this condition. Potential responsible chromosomal abnormalities were identified in exploratory studies and include deletions in 22q11, 4q31, 8p23, and 3p as well as trisomies 13 and 18. In parallel, potential culprit genes include the ZFPM2, HEY2, NFATC1, NKX2-5, MYH6, and KLF13 genes. The aim of this chapter is to expose the genetic components that are potentially involved in the pathogenesis of TA in humans. The large variability in phenotypes and genotypes among cases of TA suggests a genetic network that involves many components yet to be unraveled.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

BACKGROUND: Metastasis is the leading cause of death in patients with colorectal cancer (CRC). The 5-year survival rate of CRC patients in whom the cancer has spread to distant sites is 13.5%. The most common sites of CRC metastasis are liver and lung. The principal therapies for CRC metastatic disease are surgery, but its benefits are limited.
OBJECTIVE: This study aimed to reveal the regulatory mechanism of berberine on secondary homing of CRC cells to form metastatic focus. This was more valuable than the previous direct study of the migration and metastasis characteristics of CRC cells.
METHODS: In this study, we used the functional enrichment analysis of differentially expressed genes after berberine treatment and investigated co-expression modules related with CRC metastasis by WGCNA. PPI and survival analyses of significant modules were also conducted. The biological functions of berberine in CRC lung and liver metastasis were investigated by a series of in vitro and in vivo experiments: MTT, colony formation and mouse tail vein injection. And we scanned through the entire extracellular domain of HEY2 protein for autodocking analysis with berberine.
RESULTS: We found the differentially expressed genes (DEGs) after berberine treatment were related with cancer progression and metastasis related pathways. Through WGCNA analysis, four cancer progression and metastasis related modules were detected. After PPI and survival analysis, we identified and validated HEY2 as a hub gene, high expression and poor survival at the metastatic stage. Functionally, berberine inhibited the survival, invasion and migration of CRC cells in vitro and in vivo. Mechanistically, berberine treatment down-regulated the expression of HEY2, metastasis related protein E-cadherin, β-catenin and Cyclin D1 during Mesenchymal epithelial transformation (MET). Berberine and HEY2 showed a significant interaction, and berberine binded to HEY2 protein at the residue HIS-99 interface with a hydrogen-bond distance of 1.9A.
CONCLUSIONS: We revealed that berberine could significantly inhibit the expression of hub gene HEY2 and metastasis related proteins E-cadherin and β-catenin and Cyclin D1 during MET in CRC lung and liver metastases. In total, HEY2 was a promising candidate biomarker for prognosis and molecular characteristics in CRC metastasis.

A basic helix-loop-helix transcription factor Hey2 is expressed in the ventricular myocardium and endocardium of mouse embryos, and Hey2 null mice die perinatally showing ventricular septal defect, dysplastic tricuspid valve and hypoplastic right ventricle. In order to understand region-specific roles of Hey2 during cardiac morphogenesis, we generated Hey2 conditional knockout (cKO) mice using Mef2c-AHF-Cre, which was active in the anterior part of the second heart field and the right ventricle and outflow tract of the heart. Hey2 cKO neonates reproduced three anomalies commonly observed in Hey2 null mice. An earliest morphological defect was the lack of right ventricular extension along the apico-basal axis at midgestational stages. Underdevelopment of the right ventricle was present in all cKO neonates including those without apparent atresia of right-sided atrioventricular connection. RNA sequencing analysis of cKO embryos identified that the gene expression of a non-chamber T-box factor Tbx2 was ectopically induced in the chamber myocardium of the right ventricle. Consistently, mRNA expression of the Mycn transcription factor, which was a cell cycle regulator transcriptionally repressed by Tbx2, was down regulated, and the number of S-phase cells was significantly decreased in the right ventricle of cKO heart. These results suggest that Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2-dependent inhibition of Mycn expression.

Previous works characterized a novel cell population from adult human peripheral blood, designated peripheral blood insulin-producing cells (PB-IPC). PB-IPC displayed the pluripotent potential of differentiations after the treatment with platelet-derived mitochondria and gave rise to three germ layer-derived cells such as the mitochondrion-induced CD34+ hematopoietic stem cells (HSC)-like cells (miCD34+ HSC). To determine the molecular mechanism underlying the differentiation of miCD34+ cells, mechanistic studies established that MitoTracker Deep Red-labeled mitochondria could enter into the PB-IPC in a dose-dependent manner. Blocking Notch signaling pathway with a γ-secretase inhibitor, DAPT, markedly inhibited the proliferation of PB-IPC and improved the differentiation of miCD34+ HSC. Additionally, treatment with platelet-derived mitochondria can reprogram the differentiation of PB-IPC into miCD34+ HSC through inhibition of the Notch/HEY2 signaling pathway, as demonstrated by blocking experiments with HEY2 small interfering RNA (siRNA). The data indicated that Notch signaling pathway contributes to the miCD34+ HSC differentiation, thus advancing our understanding of the mitochondrial reprogramming and the potential treatment of human hematopoietic disease.

Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart regeneration have been identified, transcriptional programs that restrain injury-induced CM renewal are incompletely understood. Here, we report that mutations in gridlock (grl; also known as hey2), encoding a Hairy-related basic helix-loop-helix transcriptional repressor, enhance CM proliferation and reduce fibrosis following damage. In contrast, myocardial grl induction blunts CM dedifferentiation and regenerative responses to heart injury. RNA sequencing analyses uncover Smyd2 lysine methyltransferase (KMT) as a key transcriptional target repressed by Grl. Reduction in Grl protein levels triggered by injury induces smyd2 expression at the wound myocardium, enhancing CM proliferation. We show that Smyd2 functions as a methyltransferase and modulates the Stat3 methylation and phosphorylation activity. Inhibition of the KMT activity of Smyd2 reduces phosphorylated Stat3 at cardiac wounds, suppressing the elevated CM proliferation in injured grl mutant hearts. Our findings establish an injury-specific transcriptional repression program in governing CM renewal during heart regeneration, providing a potential strategy whereby silencing Grl repression at local regions might empower regeneration capacity to the injured mammalian heart.

In this study we aimed to establish the genetic cause of a myriad of cardiovascular defects prevalent in individuals from a genetically isolated population, who were found to share a common ancestor in 1728.
Trio genome sequencing was carried out in an index patient with critical congenital heart disease (CHD); family members had either exome or Sanger sequencing. To confirm enrichment, we performed a gene-based association test and meta-analysis in two independent validation cohorts: one with 2685 CHD cases versus 4370 . These controls were also ancestry-matched (same as FTAA controls), and the other with 326 cases with familial thoracic aortic aneurysms (FTAA) and dissections versus 570 ancestry-matched controls. Functional consequences of identified variants were evaluated using expression studies.
We identified a loss-of-function variant in the Notch target transcription factor-encoding gene HEY2. The homozygous state (n = 3) causes life-threatening congenital heart defects, while 80% of heterozygous carriers (n = 20) had cardiovascular defects, mainly CHD and FTAA of the ascending aorta. We confirm enrichment of rare risk variants in HEY2 functional domains after meta-analysis (MetaSKAT p = 0.018). Furthermore, we show that several identified variants lead to dysregulation of repression by HEY2.
A homozygous germline loss-of-function variant in HEY2 leads to critical CHD. The majority of heterozygotes show a myriad of cardiovascular defects.

Development of multi-chambered heart is associated with spatio-temporal regulation of gene expression. A basic helix-loop-helix transcription factor Hey2 is specifically expressed in the embryonic mouse ventricles and is indispensable for ventricular myocyte differentiation, compartment identity and morphogenesis of the heart. However, how Hey2 transcription is precisely regulated in the heart remains unclear. In this study, we identified a distal Hey2 enhancer conserved in the mouse and human to possess specific transcriptional activity in ventricular free wall myocytes at the looping stage of cardiac development. Deletion of the enhancer significantly decreased endogenous Hey2 expression in the ventricular myocardium but not in other tissues of mouse embryos. Mutation/deletion of the conserved binding sites for T-box and Gata proteins, but not NK-2 proteins, abolished the enhancer activity, and Tbx20 null mice completely lost the enhancer activity in the embryonic ventricles. Luciferase reporter analysis suggested that the ventricular enhancer activity was controlled by Tbx20 through its DNA binding and cooperative function with cardiac Gata proteins. These results delineate a regulatory mechanism of ventricular Hey2 expression and help fully understand molecular cascades in myocardial cell differentiation and cardiac morphogenesis during embryonic development.

Although papillary thyroid carcinoma (PTC) has a favorable prognosis after surgical or medical treatment, its survival rate is still very low. Therefore, finding more reliable therapy methods to limit PTC is a necessity. Compelling evidence has implicated the role of microRNAs (miRNAs or miRs) in PTC. This study aims at investigating the possible effect of microRNA-599 (miR-599) on proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) of PTC cells by targeting Hey2 gene. Differentially expressed genes/miRNAs associated with PTC were screened based on microarray analysis. Then, the expression of the candidate gene, as well as, the regulatory miRNA were detected in PTC cells, the related signaling pathway was verified. Afterward, the relationship between the miR and the candidate gene was verified by dual-luciferase reporter gene assay. Subsequently, the effects of overexpressed miR and silenced candidate gene on cell proliferation, cell apoptosis, EMT, migration, and invasion were detected. In PTC tissues and cells, miR-599 was downregulated while Hey2 expressed highly. Hey2 is a target gene of miR-559. In addition, the expression of Bax and E-cadherin was elevated while that of Hey2, Notch1, Delta-like1, Hes1, N1ICD, Jagged1, Snail, Slug, N-cadherin and Vimentin, and Bcl-2 was reduced in cells treated with upregulated miR-599 or downregulated Hey2. Moreover, miR-599 overexpression or Hey2 silencing inhibited cell proliferation, migration, invasion, along with EMT but promoted apoptosis. This study verified that miR-599 promotes apoptosis and represses proliferation, EMT of PTC cells through inactivating the Notch signaling pathway by downregulating Hey2, which has great clinical significance for PTC treatment.