APOE-NOTCH axis governs elastogenesis during human cardiac valve remodeling.

Valve remodeling is a process involving extracellular matrix organization and elongation of valve leaflets. Here, through single-cell RNA sequencing of human fetal valves, we identified an elastin-producing valve interstitial cell (VIC) subtype (apolipoprotein E (APOE)+, elastin-VICs) spatially located underneath valve endothelial cells (VECs) sensing unidirectional flow. APOE knockdown in fetal VICs resulted in profound elastogenesis defects. In valves with pulmonary stenosis (PS), we observed elastin fragmentation and decreased expression of APOE along with other genes regulating elastogenesis. Cell-cell interaction analysis revealed that jagged 1 (JAG1) from unidirectional VECs activates elastogenesis in elastin-VICs through NOTCH2. Similar observations were made in VICs cocultured with VECs under unidirectional flow. Notably, a drastic reduction of JAG1-NOTCH2 was also observed in PS valves. Lastly, we found that APOE controls JAG1-induced NOTCH activation and elastogenesis in VICs through the extracellular signal-regulated kinase pathway. Our study suggests important roles of both APOE and NOTCH in regulating elastogenesis during human valve remodeling.

egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

Biomechanical forces, and their molecular transducers, including key mechanosensitive transcription factor genes, such as KLF2, are required for cardiac valve morphogenesis. However, klf2 mutants fail to completely recapitulate the valveless phenotype observed under no-flow conditions. Here, we identify the transcription factor EGR3 as a conserved biomechanical force transducer critical for cardiac valve formation. We first show that egr3 null zebrafish display a complete and highly penetrant loss of valve leaflets, leading to severe blood regurgitation. Using tissue-specific loss- and gain-of-function tools, we find that during cardiac valve formation, Egr3 functions cell-autonomously in endothelial cells, and identify one of its effectors, the nuclear receptor Nr4a2b. We further find that mechanical forces up-regulate egr3/EGR3 expression in the developing zebrafish heart and in porcine valvular endothelial cells, as well as during human aortic valve remodeling. Altogether, these findings reveal that EGR3 is necessary to transduce the biomechanical cues required for zebrafish cardiac valve morphogenesis, and potentially for pathological aortic valve remodeling in humans.

Crk and Crkl Are Required in the Endocardial Lineage for Heart Valve Development.

Background Endocardial cells are a major progenitor population that gives rise to heart valves through endocardial cushion formation by endocardial to mesenchymal transformation and the subsequent endocardial cushion remodeling. Genetic variants that affect these developmental processes can lead to congenital heart valve defects. Crk and Crkl are ubiquitously expressed genes encoding cytoplasmic adaptors essential for cell signaling. This study aims to explore the specific role of Crk and Crkl in the endocardial lineage during heart valve development. Methods and Results We deleted Crk and Crkl specifically in the endocardial lineage. The resultant heart valve morphology was evaluated by histological analysis, and the underlying cellular and molecular mechanisms were investigated by immunostaining and quantitative reverse transcription polymerase chain reaction. We found that the targeted deletion of Crk and Crkl impeded the remodeling of endocardial cushions at the atrioventricular canal into the atrioventricular valves. We showed that apoptosis was temporally increased in the remodeling atrioventricular endocardial cushions, and this developmentally upregulated apoptosis was repressed by deletion of Crk and Crkl. Loss of Crk and Crkl also resulted in altered extracellular matrix production and organization in the remodeling atrioventricular endocardial cushions. These morphogenic defects were associated with altered expression of genes in BMP (bone morphogenetic protein), connective tissue growth factor, and WNT signaling pathways, and reduced extracellular signal-regulated kinase signaling activities. Conclusions Our findings support that Crk and Crkl have shared functions in the endocardial lineage that critically regulate atrioventricular valve development; together, they likely coordinate the morphogenic signals involved in the remodeling of the atrioventricular endocardial cushions.

DZIP1 regulates mammalian cardiac valve development through a Cby1-ß-catenin mechanism.

BACKGROUND: Mitral valve prolapse (MVP) is a common and progressive cardiovascular disease with developmental origins. How developmental errors contribute to disease pathogenesis are not well understood. RESULTS: A multimeric complex was identified that consists of the MVP gene Dzip1, Cby1, and ß-catenin. Co-expression during valve development revealed overlap at the basal body of the primary cilia. Biochemical studies revealed a DZIP1 peptide required for stabilization of the complex and suppression of ß-catenin activities. Decoy peptides generated against this interaction motif altered nuclear vs cytosolic levels of ß-catenin with effects on transcriptional activity. A mutation within this domain was identified in a family with inherited non-syndromic MVP. This novel mutation and our previously identified DZIP1S24R variant resulted in reduced DZIP1 and CBY1 stability and increased ß-catenin activities. The ß-catenin target gene, MMP2 was up-regulated in the Dzip1S14R/+ valves and correlated with loss of collagenous ECM matrix and myxomatous phenotype. CONCLUSION: Dzip1 functions to restrain ß-catenin signaling through a CBY1 linker during cardiac development. Loss of these interactions results in increased nuclear ß-catenin/Lef1 and excess MMP2 production, which correlates with developmental and postnatal changes in ECM and generation of a myxomatous phenotype.

Structural remodelling of the heart valves extracellular matrix during embryo development.

Alterations in heart valve development represent more than 20% of congenital cardiovascular malformations. Most of the functional properties of heart valves depend on extracellular matrix. Despite its relevance, little is known about fibrillar components on developing stages. Our objective is to define histological changes on valves fibrillar components in late embryonic development of Mus musculus. We found type III collagen as the predominant fibre type in the ECM in prenatal stages followed by a switch to a type I predominance for postnatal ages. The change in fibrillar components is necessary to support the normal mechanical function of adult heart valves.

PERK participates in cardiac valve development via fatty acid oxidation and endocardial-mesenchymal transformation.

Protein kinase R-like endoplasmic reticulum kinase (PERK) is one of the endoplasmic reticulum (ER) stress sensors. PERK loss-of-function mutations are known to cause Wolcott-Rallison syndrome. This disease is characterized by early-onset diabetes mellitus, skeletal dysplasia, and cardiac valve malformation. To understand the role of PERK in valve formation in vivo, we used an endothelial-specific PERK conditional knockout mice as well as in vitro PERK inhibition assays. We used ProteoStat dyes to visualize the accumulation of misfolded proteins in the endocardial cushion and valve mesenchymal cells (VMCs). Then, VMCs were isolated from E12.5 fetal mice, by fluorescence assisted cell sorting. Proteomic analysis of PERK-deleted VMCs identified the suppression of proteins related to fatty acid oxidation (FAO), especially carnitine palmitoyltransferase II (CPT2). CPT2 is a critical regulator of endocardial-mesenchymal transformation (EndoMT); however how TGF-ß downstream signaling controls CPT2 expression remains unclear. Here, we showed that PERK inhibition suppressed, not only EndoMT but also CPT2 protein expression in human umbilical vein endothelial cells (HUVECs) under TGF-ß1 stimulation. As a result, PERK inhibition suppressed mitochondrial metabolic activity. Taken together, these results demonstrate that PERK signaling is required for cardiac valve formation via FAO and EndoMT.

Mechanisms of heart valve development and disease.

The valves of the heart are crucial for ensuring that blood flows in one direction from the heart, through the lungs and back to the rest of the body. Heart valve development is regulated by complex interactions between different cardiac cell types and is subject to blood flow-driven forces. Recent work has begun to elucidate the important roles of developmental pathways, valve cell heterogeneity and hemodynamics in determining the structure and function of developing valves. Furthermore, this work has revealed that many key genetic pathways involved in cardiac valve development are also implicated in diseased valves. Here, we review recent discoveries that have furthered our understanding of the molecular, cellular and mechanosensitive mechanisms of valve development, and highlight new insights into congenital and acquired valve disease.

Antagonistic Activities of Vegfr3/Flt4 and Notch1b Fine-tune Mechanosensitive Signaling during Zebrafish Cardiac Valvulogenesis.

The formation of cardiac valves depends on mechanical forces exerted by blood flow. Endocardial cells lining the interior of the heart are sensitive to these stimuli and respond by rearranging into luminal cells subjected to shear stress and abluminal cells not exposed to it. The mechanisms by which endocardial cells sense these dynamic biomechanical stimuli and how they evoke different cellular responses are largely unknown. Here, we show that blood flow activates two parallel mechanosensitive pathways, one mediated by Notch and the other by Klf2a. Both pathways negatively regulate the angiogenesis receptor Vegfr3/Flt4, which becomes restricted to abluminal endocardial cells. Its loss disrupts valve morphogenesis and results in the occurrence of Notch signaling within abluminal endocardial cells. Our work explains how antagonistic activities by Vegfr3/Flt4 on the abluminal side and by Notch on the luminal side shape cardiac valve leaflets by triggering unique differences in the fates of endocardial cells.

[HYPOPLASTIC LEFT HEART SYNDROME: MORPHOGENESIS OF PATOMORPHOLOGICAL TYPES OF THE LEFT VENTRICLE].

The purpose of the study was to investigate the morphogenesis of the left ventricle in the hypoplastic left heart syndrome (HLHS). There are five types of hypoplastic left ventricles were identified: with a slit-like shape and hypoplasia of LV wall, with a slit-like cavity shape and wall hypertrophy and types with endocardial fibroelastosis (with a cylindrical cavity shape, with lacunar cavities and lacunar-cylindrical cavity of the left ventricle), as a result of differences in the wall structure, cavity shape, presence or absence of endocardial fibroelastosis. The analysis of morphometric data of pathomorphological types of the left ventricle in the HLHS revealed the possible ways of their morphogenesis. Left displacement of interventricular septum in embryogenesis at 4-5 weeks of intrauterine development is associated with the occurrence of atresia of the left atrioventricular orifice and aortic valve and the appearance of a slit-like shape and hypoplasia of LV wall in the HLHS. The displacement of only the conotruncus septum leads to the appearance of a slit-like shape of cavity and hypertrophy of LV wall in the HLHS. The pathomorphological types with endocardial fibroelastosis in the HLHS depends on the stage of embryogenesis of myocardium at which fibroelastosis appears: before the myocardial compaction (up to 4th week of gestation) - the lacunar shape of LV cavity with thin compact layer of myocardium; during the compaction of myocardium (5-6th week of gestation) - the lacunar-cylindrical shape of LV cavity and after compaction (after 7-8th week of fetal development) - a cylindrical shape of LV cavity.

The Endocardium and Heart Valves.

Endocardial cells are specialized endothelial cells that, during embryogenesis, form a lining on the inside of the developing heart, which is maintained throughout life. Endocardial cells are an essential source for several lineages of the cardiovascular system including coronary endothelium, endocardial cushion mesenchyme, cardiomyocytes, mural cells, fibroblasts, liver vasculature, adipocytes, and hematopoietic cells. Alterations in the differentiation programs that give rise to these lineages has detrimental effects, including premature lethality or significant structural malformations present at birth. Here, we will review the literature pertaining to the contribution of endocardial cells to valvular, and nonvalvular lineages and highlight critical pathways required for these processes. The lineage differentiation potential of embryonic, and possibly adult, endocardial cells has therapeutic potential in the regeneration of damaged cardiac tissue or treatment of cardiovascular diseases.

Impact of prospective measurement of outflow tracts in prediction of coarctation of the aorta.

OBJECTIVES: Prenatal diagnosis of coarctation of the aorta (CoA) is associated with reduced mortality and morbidity, however, accurate prenatal prediction remains challenging. To date, studies have used retrospective measurements of the outflow tracts to evaluate their potential to predict CoA. Our primary objective was to evaluate prospectively acquired measurements of the outflow tracts in fetuses with prenatally suspected CoA. A secondary aim was to report the postnatal prevalence of bicuspid aortic valve in this cohort. METHODS: Pregnancies with suspicion of isolated CoA and with a minimum of 6 months' postnatal follow-up available were identified from the cardiac database of a tertiary fetal cardiology center in the UK, between January 2002 and December 2017. Measurement of the aortic valve, pulmonary valve, distal transverse aortic arch (DTAA) and arterial duct (AD) diameters were undertaken routinely in fetuses with suspected CoA during the study period. Z-scores were computed using published reference ranges based on > 7000 fetuses from our own unit. RESULTS: Of 149 pregnancies with prenatally suspected CoA included in the study, CoA was confirmed within 6 months after birth in 77/149 (51.7%) cases. DTAA diameter Z-score and the Z-score of second-trimester DTAA/AD diameter ratio were smaller in fetuses with postnatally confirmed CoA than those in false-positive cases (-2.8 vs -1.9; P = 0.039 and -3.13 vs -2.61; P = 0.005, respectively). Multiple regression analysis demonstrated that the Z-scores of DTAA and AD diameters were the only significant predictors of postnatal CoA (P = 0.001). Bicuspid aortic valve was identified in 30% of the false-positive cases. CONCLUSIONS: Measurement of DTAA and AD diameter Z-scores can be used to ascertain risk for postnatal CoA in a selected cohort. The high incidence of bicuspid aortic valve in false-positive cases merits further study with respect to both etiology and longer-term significance. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Mechanically activated piezo channels modulate outflow tract valve development through the Yap1 and Klf2-Notch signaling axis.

Mechanical forces are well known for modulating heart valve developmental programs. Yet, it is still unclear how genetic programs and mechanosensation interact during heart valve development. Here, we assessed the mechanosensitive pathways involved during zebrafish outflow tract (OFT) valve development in vivo. Our results show that the hippo effector Yap1, Klf2, and the Notch signaling pathway are all essential for OFT valve morphogenesis in response to mechanical forces, albeit active in different cell layers. Furthermore, we show that Piezo and TRP mechanosensitive channels are important factors modulating these pathways. In addition, live reporters reveal that Piezo controls Klf2 and Notch activity in the endothelium and Yap1 localization in the smooth muscle progenitors to coordinate OFT valve morphogenesis. Together, this work identifies a unique morphogenetic program during OFT valve formation and places Piezo as a central modulator of the cell response to forces in this process.

A cellular atlas of Pitx2-dependent cardiac development.

The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation; however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2 null embryos. We found that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field-derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2 Our findings offer insight into Pitx2 function and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease and arrhythmogenesis.

NOTCH Activation Promotes Valve Formation by Regulating the Endocardial Secretome.

The endocardium is a specialized endothelium that lines the inner surface of the heart. Functional studies in mice and zebrafish have established that the endocardium is a source of instructive signals for the development of cardiac structures, including the heart valves and chambers. Here, we characterized the NOTCH-dependent endocardial secretome by manipulating NOTCH activity in mouse embryonic endocardial cells (MEEC) followed by mass spectrometry-based proteomics. We profiled different sets of soluble factors whose secretion not only responds to NOTCH activation but also shows differential ligand specificity, suggesting that ligand-specific inputs may regulate the expression of secreted proteins involved in different cardiac development processes. NOTCH signaling activation correlates with a transforming growth factor-ß2 (TGFß2)-rich secretome and the delivery of paracrine signals involved in focal adhesion and extracellular matrix (ECM) deposition and remodeling. In contrast, NOTCH inhibition is accompanied by the up-regulation of specific semaphorins that may modulate cell migration. The secretome protein expression data showed a good correlation with gene profiling of RNA expression in embryonic endocardial cells. Additional characterization by in situ hybridization in mouse embryos revealed expression of various NOTCH candidate effector genes (Tgfß2, Loxl2, Ptx3, Timp3, Fbln2, and Dcn) in heart valve endocardium and/or mesenchyme. Validating these results, mice with conditional Dll4 or Jag1 loss-of-function mutations showed gene expression alterations similar to those observed at the protein level in vitro These results provide the first description of the NOTCH-dependent endocardial secretome and validate MEEC as a tool for assaying the endocardial secretome response to a variety of stimuli and the potential use of this system for drug screening.

Flap valve of the heart foramen ovale revisited: macroscopic and histologic observations of human near-term fetuses.

We assessed the flap valve of the foramen ovale (FO valve) by examining 30 hearts from human fetuses of gestational age 30-40 weeks. We dissected the hearts, examined their macroscopic morphology, and then prepared semiserial sagittal sections across the valve. Although the primary septum is expected to extend along the left atrial face, eight hearts had a superior rim of the fossa ovalis on the left atrial face that was too thick and high, so there was no smooth continuation with the valve. Moreover, three of these eight hearts each had a flap valve that was fused with a long and narrow plate arising from the caval orifice. Histological analysis indicated that 21 specimens each had a candidate primary septum that contained myocardium, although the left sinuatrial valve (LSAV) contained fibrous tissue, but little or no myocardium. In each of 17 hearts, a candidate primary septum was attached to the left atrial face of the fossa, and parts of the LSAV extended to and approached the right atrial face. However, seven of these 17 hearts each had a folded small primary septum. Another four of these 17 hearts each had an LSAV that extended widely to the fossa, and a candidate primary septum (which might be a remnant) attached to the left atrial side of the LSAV. These variations suggest that the LSAV makes a major contribution to the FO valve in some fetal hearts. Consequently, the fetal FO valve appears to have heterogeneous morphology and origin.

TAMM41 is required for heart valve differentiation via regulation of PINK-PARK2 dependent mitophagy.

TAMM41, located within the congenital heart diseases (CHD) sensitive region of 3p25 deletion syndrome, is a mitochondrial membrane maintenance protein critical for yeast survival, but its function in higher vertebrates remains unknown. Via in vivo zebrafish model, we found that tamm41 is highly expressed in the developing heart and deficiency of which led to heart valve abnormalities. Molecular mechanistic studies revealed that TAMM41 interacts and modulates the PINK1-PARK2 dependent mitophagy pathway, thereby implicating TAMM41 in heart valve development during zebrafish embryonic cardiogenesis. Furthermore, through screening of the congenital heart diseases (CHD) sensitive region of 3p25 deletion syndrome among 118 sporadic atrioventricular septal defect (AVSD) patients, we identified three cases carrying heterozygous pathogenic intronic variants of TAMM41. All three cases lacked normal full-length TAMM41 transcripts, most likely due to specific expression of the mutant allele. Collectively, our studies highlight essential roles for TAMM41-dependent mitophagy in development of the heart and provide novel insights into the etiology of AVSD.

Focal adhesions are essential to drive zebrafish heart valve morphogenesis.

Elucidating the morphogenetic events that shape vertebrate heart valves, complex structures that prevent retrograde blood flow, is critical to understanding valvular development and aberrations. Here, we used the zebrafish atrioventricular (AV) valve to investigate these events in real time and at single-cell resolution. We report the initial events of collective migration of AV endocardial cells (ECs) into the extracellular matrix (ECM), and their subsequent rearrangements to form the leaflets. We functionally characterize integrin-based focal adhesions (FAs), critical mediators of cell-ECM interactions, during valve morphogenesis. Using transgenes to block FA signaling specifically in AV ECs as well as loss-of-function approaches, we show that FA signaling mediated by Integrin α5ß1 and Talin1 promotes AV EC migration and overall shaping of the valve leaflets. Altogether, our investigation reveals the critical processes driving cardiac valve morphogenesis in vivo and establishes the zebrafish AV valve as a vertebrate model to study FA-regulated tissue morphogenesis.

Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of the myocardial wnt signaling pathway.

The proper development of atrioventricular (AV) valves is critical for heart morphogenesis and for the formation of the cardiac conduction system. Defects in AV valve development are the most common type of congenital heart defect. Cardiac troponin I ( ctnni), a structural and regulatory protein involved in cardiac muscle contraction, is a subunit of the troponin complex, but the functions and molecular mechanisms of ctnni during early heart development remain unclear. We created a knockout zebrafish model in which troponin I type 1b ( tnni1b) ( Tnni-HC, heart and craniofacial) was deleted using the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein system. In the homozygous mutant, the embryos showed severe pericardial edema, malformation of the heart tube, reduction of heart rate without contraction and with almost no blood flow, heart cavity congestion, and lack of an endocardial ring or valve leaflet, resulting in 88.8 ± 6.0% lethality at 7 d postfertilization. Deletion of tnni1b caused the abnormal expression of several markers involved in AV valve development, including bmp4, cspg2, has2, notch1b, spp1, and Alcam. Myocardial re-expression of tnni1b in mutants partially rescued the pericardial edema phenotype and AV canal (AVC) developmental defects. We further showed that tnni1b knockout in zebrafish and ctnni knockdown in rat h9c2 myocardial cells inhibited cardiac wnt signaling and that myocardial reactivation of wnt signaling partially rescued the abnormal expression of AVC markers caused by the tnni1b deletion. Taken together, our data suggest that tnni1b plays a vital role in zebrafish AV valve development by regulating the myocardial wnt signaling pathway.-Cai, C., Sang, C., Du, J., Jia, H., Tu, J., Wan, Q., Bao, B., Xie, S., Huang, Y., Li, A., Li, J., Yang, K., Wang, S., Lu, Q. Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of myocardial wnt signaling pathway.

[Role of Tbx20 gene in the development of cardiac valves].

Cardiac valves are highly organized yet delicate structures that ensure unidirectional blood flow through the cardiac chambers and large vessels. Disturbed development of cardiac valves can lead to aberrant heart formation and function which account for approximately one third of congenital heart diseases. The formation of cardiac valves is a dynamic process accomplished by a series of complex events including lineage determination and cell proliferation, differentiation and migration. This paper reviews current knowledge about the role of Tbx20 gene in the development of cardiac valves, which include functional diversities of Tbx20 at various stages of cardiac valve development, its interaction with other signaling pathways, and genetic network involved in endocardial development.

Predicting and understanding collagen remodeling in human native heart valves during early development.

The hemodynamic functionality of heart valves strongly depends on the distribution of collagen fibers, which are their main load-bearing constituents. It is known that collagen networks remodel in response to mechanical stimuli. Yet, the complex interplay between external load and collagen remodeling is poorly understood. In this study, we adopted a computational approach to simulate collagen remodeling occurring in native fetal and pediatric heart valves. The computational model accounted for several biological phenomena: cellular (re)orientation in response to both mechanical stimuli and topographical cues provided by collagen fibers; collagen deposition and traction forces along the main cellular direction; collagen degradation decreasing with stretch; and cell-mediated collagen prestretch. Importantly, the computational results were well in agreement with previous experimental data for all simulated heart valves. Simulations performed by varying some of the computational parameters suggest that cellular forces and (re)orientation in response to mechanical stimuli may be fundamental mechanisms for the emergence of the circumferential collagen alignment usually observed in native heart valves. On the other hand, the tendency of cells to coalign with collagen fibers is essential to maintain and reinforce that circumferential alignment during development. STATEMENT OF SIGNIFICANCE: The hemodynamic functionality of heart valves is strongly influenced by the alignment of load-bearing collagen fibers. Currently, the mechanisms that are responsible for the development of the circumferential collagen alignment in native heart valves are not fully understood. In the present study, cell-mediated remodeling of native human heart valves during early development was computationally simulated to understand the impact of individual mechanisms on collagen alignment. Our simulations successfully predicted the degree of collagen alignment observed in native fetal and pediatric semilunar valves. The computational results suggest that the circumferential collagen alignment arises from cell traction and cellular (re)orientation in response to mechanical stimuli, and with increasing age is reinforced by the tendency of cells to co-align with pre-existing collagen fibers.