Mechanosensitive Notch-Dll4 and Klf2-Wnt9 signaling pathways intersect in guiding valvulogenesis in zebrafish.

In the zebrafish embryo, the onset of blood flow generates fluid shear stress on endocardial cells, which are specialized endothelial cells that line the interior of the heart. High levels of fluid shear stress activate both Notch and Klf2 signaling, which play crucial roles in atrioventricular valvulogenesis. However, it remains unclear why only individual endocardial cells ingress into the cardiac jelly and initiate valvulogenesis. Here, we show that lateral inhibition between endocardial cells, mediated by Notch, singles out Delta-like-4-positive endocardial cells. These cells ingress into the cardiac jelly, where they form an abluminal cell population. Delta-like-4-positive cells ingress in response to Wnt9a, which is produced in parallel through an Erk5-Klf2-Wnt9a signaling cascade also activated by blood flow. Hence, mechanical stimulation activates parallel mechanosensitive signaling pathways that produce binary effects by driving endocardial cells toward either luminal or abluminal fates. Ultimately, these cell fate decisions sculpt cardiac valve leaflets.

Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces.

Developing cardiovascular systems use mechanical forces to take shape, but how ubiquitous blood flow forces instruct local cardiac cell identity is still unclear. By manipulating mechanical forces in vivo, we show here that shear stress is necessary and sufficient to promote valvulogenesis. We found that valve formation is associated with the activation of an extracellular adenosine triphosphate (ATP)­dependent purinergic receptor pathway, specifically triggering calcium ion (Ca2+) pulses and nuclear factor of activated T cells 1 (Nfatc1) activation. Thus, mechanical forces are converted into discrete bioelectric signals by an ATP-Ca2+-Nfatc1­mechanosensitive pathway to generate positional information and control valve formation.

Inappropriate cathepsin K secretion promotes its enzymatic activation driving heart and valve malformation.

Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab-deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-ß-related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.

Nfatc1 Promotes Interstitial Cell Formation During Cardiac Valve Development in Zebrafish.

RATIONALE: The transcription factor NFATC1 (nuclear factor of activated T-cell 1) has been implicated in cardiac valve formation in humans and mice, but we know little about the underlying mechanisms. To gain mechanistic understanding of cardiac valve formation at single-cell resolution and insights into the role of NFATC1 in this process, we used the zebrafish model as it offers unique attributes for live imaging and facile genetics. OBJECTIVE: To understand the role of Nfatc1 in cardiac valve formation. METHODS AND RESULTS: Using the zebrafish atrioventricular valve, we focus on the valve interstitial cells (VICs), which confer biomechanical strength to the cardiac valve leaflets. We find that initially atrioventricular endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the ECM (extracellular matrix) between the 2 endocardial cell monolayers, undergo endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a promigratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. With high-speed microscopy and echocardiography, we show that reduced VIC formation correlates with valvular dysfunction and severe retrograde blood flow that persist into adulthood. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b-a well-known regulator of epithelial-to-mesenchymal transition. CONCLUSIONS: Our study sheds light on the function of Nfatc1 in zebrafish cardiac valve development and reveals its role in VIC formation. It also further establishes the zebrafish as a powerful model to carry out longitudinal studies of valve formation and function.

Development of the human heart.

In 2014, an extensive review discussing the major steps of cardiac development focusing on growth, formation of primary and chamber myocardium and the development of the cardiac electrical system, was published. Molecular genetic lineage analyses have since furthered our insight in the developmental origin of the various component parts of the heart, which currently can be unambiguously identified by their unique molecular phenotype. Moreover, genetic, molecular and cell biological analyses have driven insights into the mechanisms underlying the development of the different cardiac components. Here, we build on our previous review and provide an insight into the molecular mechanistic revelations that have forwarded the field of cardiac development. Despite the enormous advances in our knowledge over the last decade, the development of congenital cardiac malformations remains poorly understood. The challenge for the next decade will be to evaluate the different developmental processes using newly developed molecular genetic techniques to further unveil the gene regulatory networks operational during normal and abnormal cardiac development.

Primary cilia defects causing mitral valve prolapse.

Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, DZIP1 A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP.

Role of Periostin in Cardiac Valve Development.

Although periostin plays a significant role in adult cardiac remodeling diseases, the focus of this review is on periostin as a valvulogenic gene. Periostin is expressed throughout valvular development, initially being expressed in endocardial endothelial cells that have been activated to transform into prevalvular mesenchyme termed "cushion tissues" that sustain expression of periostin throughout their morphogenesis into mature (compacted) valve leaflets. The phenotype of periostin null indicates that periostin is not required for endocardial transformation nor the proliferation of its mesenchymal progeny but rather promotes cellular behaviors that promote migration, survival (anti-apoptotic), differentiation into fibroblastic lineages, collagen secretion and postnatal remodeling/maturation. These morphogenetic activities are promoted or coordinated by periostin signaling through integrin receptors activating downstream kinases in cushion cells that activate hyaluronan synthetase II (Akt/PI3K), collagen synthesis (Erk/MapK) and changes in cytoskeletal organization (Pak1) which regulate postnatal remodeling of cells and associated collagenous matrix into a trilaminar (zonal) histoarchitecture. Pak1 binding to filamin A is proposed as one mechanism by which periostin supports remodeling. The failure to properly remodel cushions sets up a trajectory of degenerative (myxomatous-like) changes that over time reduce biomechanical properties and increase chances for prolapse, regurgitation or calcification of the leaflets. Included in the review are considerations of lineage diversity and the role of periostin as a determinant of mesenchymal cell fate.

Genome-Wide Association Study-Driven Gene-Set Analyses, Genetic, and Functional Follow-Up Suggest GLIS1 as a Susceptibility Gene for Mitral Valve Prolapse.

Background Mitral valve prolapse (MVP) is a common heart valve disease, the most frequent indication for valve repair or replacement. MVP is characterized by excess extracellular matrix secretion and cellular disorganization, which leads to bulky valves that are unable to coapt correctly during ventricular systole resulting in mitral regurgitation, and it is associated with sudden cardiac death. Here we aim to characterize globally the biological mechanisms underlying genetic susceptibility to MVP to better characterize its triggering mechanisms. Methods We applied i-GSEA4GWAS and DEPICT, two pathway enrichment tools to MVP genome-wide association studies. We followed-up the association with MVP in an independent dataset of cases and controls. This research was conducted using the UK Biobank Resource. Immunohistochemistry staining for Glis1 (GLIS family zinc finger 1) was conducted in developing heart of mice. Knockdown of Glis1 using morpholinos was performed in zebrafish animals 72 hours postfertilization. Results We show that genes at risk loci are involved in biological functions relevant to actin filament organization, cytoskeleton biology, and cardiac development. The enrichment for positive regulation of transcription, cell proliferation, and migration motivated the follow-up of GLIS1, a transcription factor from the Krüppel-like zinc finger family. In combination with previously available data, we now report a genome-wide significant association with MVP (odds ratio, 1.20; P=4.36×10-10), indicating that Glis1 is expressed during embryonic development predominantly in nuclei of endothelial and interstitial cells of mitral valves in mouse. We also show that Glis1 knockdown causes atrioventricular regurgitation in developing hearts in zebrafish. Conclusions Our findings define globally molecular and cellular mechanisms underlying common genetic susceptibility to MVP and implicate established and unprecedented mechanisms. Through the GLIS1 association and function, we point at regulatory functions during cardiac development as common mechanisms to mitral valve degeneration.

Contractile and hemodynamic forces coordinate Notch1b-mediated outflow tract valve formation.

Biomechanical forces and endothelial-to-mesenchymal transition (EndoMT) are known to mediate valvulogenesis. However, the relative contributions of myocardial contractile and hemodynamic shear forces remain poorly understood. We integrated 4-D light-sheet imaging of transgenic zebrafish models with moving-domain computational fluid dynamics to determine effects of changes in contractile forces and fluid wall shear stress (WSS) on ventriculobulbar (VB) valve development. Augmentation of myocardial contractility with isoproterenol increased both WSS and Notch1b activity in the developing outflow tract (OFT) and resulted in VB valve hyperplasia. Increasing WSS in the OFT, achieved by increasing blood viscosity through EPO mRNA injection, also resulted in VB valve hyperplasia. Conversely, decreasing myocardial contractility by Tnnt2a morpholino oligonucleotide (MO) administration, 2,3-butanedione monoxime treatment, or Plcγ1 inhibition completely blocked VB valve formation, which could not be rescued by increasing WSS or activating Notch. Decreasing WSS in the OFT, achieved by slowing heart rate with metoprolol or reducing viscosity with Gata1a MO, did not affect VB valve formation. Immunofluorescent staining with the mesenchymal marker, DM-GRASP, revealed that biomechanical force-mediated Notch1b activity is implicated in EndoMT to modulate valve morphology. Altogether, increases in WSS result in Notch1b- EndoMT-mediated VB valve hyperplasia, whereas decreases in contractility result in reduced Notch1b activity, absence of EndoMT, and VB valve underdevelopment. Thus, we provide developmental mechanotransduction mechanisms underlying Notch1b-mediated EndoMT in the OFT.

VGLL4 plays a critical role in heart valve development and homeostasis.

Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease.

Non-pathological Chondrogenic Features of Valve Interstitial Cells in Normal Adult Zebrafish.

In the heart, unidirectional blood flow depends on proper heart valve function. As, in mammals, regulatory mechanisms of early heart valve and bone development are shown to contribute to adult heart valve pathologies, we used the animal model zebrafish (ZF, Danio rerio) to investigate the microarchitecture and differentiation of cardiac valve interstitial cells in the transition from juvenile (35 days) to end of adult breeding (2.5 years) stages. Of note, light microscopy and immunohistochemistry revealed major differences in ZF heart valve microarchitecture when compared with adult mice. We demonstrate evidence for rather chondrogenic features of valvular interstitial cells by histological staining and immunodetection of SOX-9, aggrecan, and type 2a1 collagen. Collagen depositions are enriched in a thin layer at the atrial aspect of atrioventricular valves and the ventricular aspect of bulboventricular valves, respectively. At the ultrastructural level, the collagen fibrils are lacking obvious periodicity and orientation throughout the entire valve.

Mediator complex subunit Med12 regulates cardiac jelly development and AV valve formation in zebrafish.

The molecular mechanism essential for the formation of heart valves involves complex interactions of signaling molecules and transcription factors. The Mediator Complex (MC) functions as multi-subunit machinery to orchestrate gene transcription, especially for tissue-specific fine-tuning of transcriptional processes during development, also in the heart. Here, we analyzed the role of the MC subunit Med12 during atrioventricular canal (AVC) development and endocardial cushion formation, using the Med12-deficient zebrafish mutant trapped (tpd). Whereas primary heart formation was only slightly affected in tpd, we identified defects in AVC development and cardiac jelly formation. We found that although misexpression of bmp4 and versican in tpd hearts can be restored by overexpression of a modified version of the Sox9b transcription factor (harboring VP16 transactivation domain) that functions independent of its co-activator Med12, endocardial cushion development in tpd was not reconstituted. Interestingly, expression of tbx2b and its target hyaluronan synthase 2 (has2) - the synthase of hyaluronan (HA) in the heart - was absent in both uninjected and Sox9b-VP16 overexpressing tpd hearts. HA is a major ECM component of the cardiac jelly and required for endocardial cushion formation. Furthermore, we found secreted phosphoprotein 1 (spp1), an endocardial marker of activated AV endocardial cells, completely absent in tpd hearts, suggesting that crucial steps of the transformation of AV endocardial cells into endocardial cushions is blocked. We demonstrate that Med12 controls cardiac jelly formation Sox9-independently by regulating tbx2b and has2 expression and therefore the production of the glycosaminoglycan HA at the AVC to guarantee proper endocardial cushion development.

Endothelial committed oral stem cells as modelling in the relationship between periodontal and cardiovascular disease.

In the present study we have mimicked, in vitro, an inflammatory process using Lipopolysaccharide derived from Porphyromonas Gingivalis (LPS-G) and human Periodontal Ligament Stem Cells induced to endothelial differentiation (e-hPDLSCs). The research project has been organized into the three following steps: i) induction of hPDLSCs toward endothelial differentiation; ii) evaluation of the molecular signaling pathway involved in the response to the LPS-G, and iii) functional response evaluation of the living construct constituted by porcine decellularized valve/e-hPDLSCs treated with LPS-G. Obtained results showed that 5 µg/ml LPS-G stimulus provokes: a slowdown of cell growth starting from 24 hr and the release of IL6, IL8, and MCP1 molecules. Signaling network analyzed showed the activation of TLR4/ NFkB/ERK1/2/p-ERK1/2 signaling mediated by MyD88 in LPS-G stimulated e-hPDLSCs, moreover a time course put in evidence a nuclear traslocation of ERK1/2 and p-ERK1/2 in differentiated samples. Following, the ability of e-hPDLSCs to expand and colonize the decellularized porcine heart valves was appraised at ultrastructural level. Considering that, the Reactive Oxygen Species (ROS) play an important role in the progression and development of cardiovascular disease (CVD), in LPS-G living construct model e-hPDLSCs/decellularized porcine heart valves (dPHV), ROS production was assessed. Time lapse experiments evidenced that LPS-G provokes in e-hPDLSCs a rapid and sustained increase in ROS generation, negligible on undifferentiated cells. From obtained data, by multiparametric analyses, a reasonable conclusion may be that the inflammation process activated by LPS-G can affect endothelial cells and could represent in vivo a possible pathological and predictor state of CVD.

Growth and remodeling play opposing roles during postnatal human heart valve development.

Tissue growth and remodeling are known to govern mechanical homeostasis in biological tissue, but their relative contributions to homeostasis remain unclear. Here, we use mechanical models, fueled by experimental findings, to demonstrate that growth and remodeling have different effects on heart valve stretch homeostasis during physiological postnatal development. Two developmental stages were considered: early-stage (from infant to adolescent) and late-stage (from adolescent to adult) development. Our models indicated that growth and remodeling play opposing roles in preserving tissue stretch and with time. During early-stage development, excessive tissue stretch was decreased by tissue growth and increased by remodeling. In contrast, during late-stage development tissue stretch was decreased by remodeling and increased by growth. Our findings contribute to an improved understanding of native heart valve adaptation throughout life, and are highly relevant for the development of tissue-engineered heart valves.

Hemodynamic Forces Sculpt Developing Heart Valves through a KLF2-WNT9B Paracrine Signaling Axis.

Hemodynamic forces play an essential epigenetic role in heart valve development, but how they do so is not known. Here, we show that the shear-responsive transcription factor KLF2 is required in endocardial cells to regulate the mesenchymal cell responses that remodel cardiac cushions to mature valves. Endocardial Klf2 deficiency results in defective valve formation associated with loss of Wnt9b expression and reduced canonical WNT signaling in neighboring mesenchymal cells, a phenotype reproduced by endocardial-specific loss of Wnt9b. Studies in zebrafish embryos reveal that wnt9b expression is similarly restricted to the endocardial cells overlying the developing heart valves and is dependent upon both hemodynamic shear forces and klf2a expression. These studies identify KLF2-WNT9B signaling as a conserved molecular mechanism by which fluid forces sensed by endothelial cells direct the complex cellular process of heart valve development and suggest that congenital valve defects may arise due to subtle defects in this mechanotransduction pathway.

Micro and nanotechnologies in heart valve tissue engineering.

Due to the increased morbidity and mortality resulting from heart valve diseases, there is a growing demand for off-the-shelf implantable tissue engineered heart valves (TEHVs). Despite the significant progress in recent years in improving the design and performance of TEHV constructs, viable and functional human implantable TEHV constructs have remained elusive. The recent advances in micro and nanoscale technologies including the microfabrication, nano-microfiber based scaffolds preparation, 3D cell encapsulated hydrogels preparation, microfluidic, micro-bioreactors, nano-microscale biosensors as well as the computational methods and models for simulation of biological tissues have increased the potential for realizing viable, functional and implantable TEHV constructs. In this review, we aim to present an overview of the importance and recent advances in micro and nano-scale technologies for the development of TEHV constructs.

Hemodynamics driven cardiac valve morphogenesis.

Mechanical forces are instrumental to cardiovascular development and physiology. The heart beats approximately 2.6 billion times in a human lifetime and heart valves ensure that these contractions result in an efficient, unidirectional flow of the blood. Composed of endocardial cells (EdCs) and extracellular matrix (ECM), cardiac valves are among the most mechanically challenged structures of the body both during and after their development. Understanding how hemodynamic forces modulate cardiovascular function and morphogenesis is key to unraveling the relationship between normal and pathological cardiovascular development and physiology. Most valve diseases have their origins in embryogenesis, either as signs of abnormal developmental processes or the aberrant re-expression of fetal gene programs normally quiescent in adulthood. Here we review recent discoveries in the mechanobiology of cardiac valve development and introduce the latest technologies being developed in the zebrafish, including live cell imaging and optical technologies, as well as modeling approaches that are currently transforming this field. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Age-dependent changes of stress and strain in the human heart valve and their relation with collagen remodeling.

In order to create tissue-engineered heart valves with long-term functionality, it is essential to fully understand collagen remodeling during neo-tissue formation. Collagen remodeling is thought to maintain mechanical tissue homeostasis. Yet, the driving factor of collagen remodeling remains unidentified. In this study, we determined the collagen architecture and the geometric and mechanical properties of human native semilunar heart valves of fetal to adult age using confocal microscopy, micro-indentation and inverse finite element analysis. The outcomes were used to predict age-dependent changes in stress and stretch in the heart valves via finite element modeling. The results indicated that the circumferential stresses are different between the aortic and pulmonary valve, and, moreover, that the stress increases considerably over time in the aortic valve. Strikingly, relatively small differences were found in stretch with time and between the aortic and pulmonary valve, particularly in the circumferential direction, which is the main determinant of the collagen fiber stretch. Therefore, we suggest that collagen remodeling in the human heart valve maintains a stretch-driven homeostasis. Next to these novel insights, the unique human data set created in this study provides valuable input for the development of numerical models of collagen remodeling and optimization of tissue engineering. STATEMENT OF SIGNIFICANCE: Annually, over 280,000 heart valve replacements are performed worldwide. Tissue engineering has the potential to provide valvular disease patients with living valve substitutes that can last a lifetime. Valve functionality is mainly determined by the collagen architecture. Hence, understanding collagen remodeling is crucial for creating tissue-engineered valves with long-term functionality. In this study, we determined the structural and material properties of human native heart valves of fetal to adult age to gain insight into the mechanical stimuli responsible for collagen remodeling. The age-dependent evolutionary changes in mechanical state of the native valve suggest that collagen remodeling in heart valves is a stretch-driven process.

The chick embryo as an expanding experimental model for cancer and cardiovascular research.

A long and productive history in biomedical research defines the chick as a model for human biology. Fundamental discoveries, including the description of directional circulation propelled by the heart and the link between oncogenes and the formation of cancer, indicate its utility in cardiac biology and cancer. Despite the more recent arrival of several vertebrate and invertebrate animal models during the last century, the chick embryo remains a commonly used model for vertebrate biology and provides a tractable biological template. With new molecular and genetic tools applied to the avian genome, the chick embryo is accelerating the discovery of normal development and elusive disease processes. Moreover, progress in imaging and chick culture technologies is advancing real-time visualization of dynamic biological events, such as tissue morphogenesis, angiogenesis, and cancer metastasis. A rich background of information, coupled with new technologies and relative ease of maintenance, suggest an expanding utility for the chick embryo in cardiac biology and cancer research.