Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling

In the clinic, most cases of congenital heart valve defects are thought to arise through errors that occur after the endothelial–mesenchymal transition (EndoMT) stage of valve development. Although mechanical forces caused by heartbeat are essential modulators of cardiovascular development, their role in these later developmental events is poorly understood. To address this question, we used the zebrafish superior atrioventricular valve (AV) as a model. We found that cellularized cushions of the superior atrioventricular canal (AVC) morph into valve leaflets via mesenchymal–endothelial transition (MEndoT) and tissue sheet delamination. Defects in delamination result in thickened, hyperplastic valves, and reduced heart function. Mechanical, chemical, and genetic perturbation of cardiac forces showed that mechanical stimuli are important regulators of valve delamination. Mechanistically, we show that forces modulate Nfatc activity to control delamination. Together, our results establish the cellular and molecular signature of cardiac valve delamination in vivo and demonstrate the continuous regulatory role of mechanical forces and blood flow during valve formation.

Differential Requirement of DICER1 Activity during Development of Mitral and Tricuspid Valves

Mitral and tricuspid valves are essential for unidirectional blood flow in the heart. They are derived from similar cell sources, and yet congenital dysplasia affecting both valves is clinically rare, suggesting the presence of differential regulatory mechanisms underlying their development. We specifically inactivated Dicer1 in the endocardium during cardiogenesis, and unexpectedly found that Dicer1-deletion caused congenital mitral valve stenosis and regurgitation, while it had no impact on other valves. We showed that hyperplastic mitral valves were caused by abnormal condensation and extracellular matrix (ECM) remodeling. Our single-cell RNA Sequencing analysis revealed impaired maturation of mesenchymal cells and abnormal expression of ECM genes in mutant mitral valves. Furthermore, expression of a set of miRNAs that target ECM genes was significantly lower in tricuspid valves compared to mitral valves, consistent with the idea that the miRNAs are differentially required for mitral and tricuspid valve development. Our study thus reveals miRNA-mediated gene regulation as a novel molecular mechanism that differentially regulates mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.

Myocardial Afterload Is a Key Biomechanical Regulator of Atrioventricular Myocyte Differentiation in Zebrafish

Heart valve development is governed by both genetic and biomechanical inputs. Prior work has demonstrated that oscillating shear stress associated with blood flow is required for normal atrioventricular (AV) valve development. Cardiac afterload is defined as the pressure the ventricle must overcome in order to pump blood throughout the circulatory system. In human patients, conditions of high afterload can cause valve pathology. Whether high afterload adversely affects embryonic valve development remains poorly understood. Here we describe a zebrafish model exhibiting increased myocardial afterload, caused by vasopressin, a vasoconstrictive drug. We show that the application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. We have found that increased afterload alters the rate of growth of the cardiac chambers and causes remodeling of cardiomyocytes. Consistent with pathology seen in patients with clinically high afterload, we see defects in both the form and the function of the valve leaflets. Our results suggest that valve defects are due to changes in atrioventricular myocyte signaling, rather than pressure directly acting on the endothelial valve leaflet cells. Cardiac afterload should therefore be considered a biomechanical factor that particularly impacts embryonic valve development.

Abstract MP235: PROX1 Contributes To Cardiac Valve Disease

Background: Heart valves regulate the unidirectional forward flow and prevent retrograde backflow of blood during the cardiac cycle. Cardiac valve disease (CVD) is observed in approximately 2.5% of the general population and the incidence increases to ~10% in elderly people. Patients with severe CVD require surgery and effective pharmacological treatments are currently not available. PROX1 is a transcription factor that regulates the development of lymphatic, venous, and lymphovenous valves (vascular valves). We identified that PROX1 is also expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 regulates cardiac valve development and disease is not known. Method and Results: We have discovered that mice lacking Prox1 in their VECs ( Prox1 ΔVEC ) develop enlarged aortic and mitral valves in which the expression of proteoglycans is increased (control, N=10; Prox1 ΔVEC , N=9, p <0.05). Echocardiography revealed moderate to severe stenosis of aortic valves of Prox1 ΔVEC mice (control, N=5; Prox1 ΔVEC , N=9, p <0.05). PROX1 regulates the expression of the transcription factor FOXC2 in the vascular valves. Similarly, we have found that the expression of FOXC2 is downregulated in the VECs of Prox1 ΔVEC mice. Specific knockdown of FOXC2 in VECs results in the thickening of aortic valves (control, N=10; shFoxc2 ΔVEC , N=8, p <0.05). Furthermore, restoration of FOXC2 expression in VECs ( Foxc2 OE-VEC ) ameliorates the thickening of the aortic valves of Prox1 ΔVEC mice ( Prox1 ΔVEC , N=9; Foxc2 OE-VEC ; Prox1 ΔVEC , N=8, p <0.05). We have also determined that the expression of platelet-derived growth factor-B ( Pdgfb ) is increased in the valve tissue of Prox1 ΔVEC mice and in PROX1 deficient sheep mitral valve VECs (MVECs) (siCtrl , N=4; siProx1 , N=4, p <0.05). Additionally, hyperactivation of PDGF-B signaling in mice results in a phenotype that is similar to Prox1 ΔVEC mice (control , N=4; Pdgfb GOF , N=3, p <0.05). Conclusion: Together these data suggest that PROX1 maintains the extracellular matrix composition of cardiac valves by regulating the expressions of FOXC2 and PDGF-B in VECs.

Abstract P316: Role Of Scleraxis In Cardiac Valve Development And Disease

Valvular heart disease is one of the major causes of cardiac-related deaths in the US and effective treatment is currently limited to surgical repair or replacement. Mature heart valves are composed of highly organized and stratified layers of extracellular matrix (ECM) regulated by valve interstitial cells (VICs). This stratification that is initiated during embryogenesis and completed during valve maturation after birth, needs to be maintained throughout life for normal valve function. Myxomatous degeneration is a common disease histologically characterized by imbalance in ECM composition and organization resulting in valve biomechanical failure. Yet, key regulators of ECM homeostasis are not well characterized. Scleraxis (Scx) is a bHLH transcription factor that we previously showed to be critical for heart valve development and its loss of function leads to defective VIC maturation and aberrant ECM organization. However, due to lack of efficient tools, the mechanistic function of Scx in valve development and disease in vivo remains to be understood. Herein, we performed temporal analysis of Scx transcript levels during development of murine homeostatic valves and identified that Scx is predominantly expressed in the VICs between E13.5 and P14. We also assessed Scx levels and ECM abnormalities in valve tissues derived from patients with cardiac valve diseases and identified several distinct populations of VICs with high Scx levels that partially colocalized with elevated collagen fragmentation and proteoglycan (PG) deposition in the neighboring ECM. Scx levels were also elevated in valves of Fbn1 C1039G/+ and osteogenesis imperfecta murine ( OIM ) mice that develop myxomatous valve abnormalities signifying a potential role in pathogenesis. To further study the function of Scx in valve development and disease in vivo , we have generated a conditional Scx-transgenic model (Scx-TG) that will allow for overexpression in targeted cell lineages upon Cre recombination. Additionally, we will employ Scx-Cre mouse model expressing Scx-promoter driven Cre -recombinase to perform Scx-expressing cell lineage analyses and high-throughput sequencing analyses to identify direct gene targets and protein-interaction partners of Scx to better understand its mechanistic role in regulating valve ECM homeostasis. Together these in vivo approaches will provide novel insights into the function of Scx in heart development and disease.

Lies, Damned Lies, and Simulation Results: How to Change the Conversation and Build a Thriving Simulation Ecosystem

Engineering simulation has become the pivotal tool for research and development in industries including offshore oil & gas, aerospace, automotive, mobile/off-highway, health care, and others. This case study will explore the financial and time-based savings achieved through detailed simulations and a system-based design approach in two hydraulic valve development projects. The applications in this scope include subsea blowout preventer and off-highway mobile equipment controls. Tools like 1D system simulation, computational fluid dynamics, and finite element analysis are widely accepted; verification and validation (V&V) of these models is imperative in building confidence in simulation. Some V&V reference standards have been developed by groups like ASME and API, but they do not encompass all aspects of simulation regularly utilized by the modern analyst. This places the onus for the creation of V&V guidelines onto individual analysts and their respective employers. Lack of detail in these guidelines can lead to flawed interpretations of results and a corresponding loss of trust in analytical methods. Interdisciplinary organizations can provide forums to help bridge these gaps and create more comprehensive V&V guidelines. Through a study of the development cycles of a subsea valve and an off-highway mobile valve, examples will be outlined which illustrate the benefit of extensive upfront simulation validated by physical testing. Simulation work serves as a cost avoidance measure against many cycles of building and testing prototypes beyond what is truly required in the early stages of design. Accurate simulation is a key component of successful product development, but another often neglected factor is the collaboration between subject matter experts from the component suppliers and the OEM or system integrator. High performance teams comprised of seasoned designers, analysts, and market experts can collaborate to create devices that excel when integrated into a final product. Component designers may wish to isolate the design problem to the component in question, but critical engineering detail will be missed by avoiding a system approach. Expanding the scope of the design analysis to include as much of the application as possible as well as utilizing V&V techniques (beyond minimum industry standards) is key to ensuring that laboratory test data is representative of how a product will perform in its intended application. As the industry continues to evolve, powerful digital twins of systems like blowout preventers can be used for OEM validation of new technology proposed for these systems. However, the fidelity of these digital twins is contingent upon the inputs from thoroughly validated analytical models of the components that comprise the system. By collaborating across the customer-supplier value chain and investing heavily in simulation, offshore manufacturers can strategically position themselves to win in times when both customer expectations and the costs of failure are at an all-time high.

Accelerated Development of Subsurface Safety Valve Technologies

Described is a methodology for accelerating the development of innovative and high-risk technologies, specifically, subsurface safety valve technologies. Focus is on methods of mitigating technical and commercial risks that can delay or prevent successful development of new technologies. Example risk assessment and risk mitigation strategies are provided from a recent subsurface safety valve technology development project. Mitigation strategies include fixture level testing, design changes, and deep client collaboration. In the example project, it is estimated that the total development time was reduced by as much as 50% by implementing these strategies. While a subsurface safety valve development is used in this example, it is believed that many strategies are applicable to other domains.

AN AORTIC VALVE-SPARING OPERATION: INDICATIONS, TECHNICAL ASPECTS AND RESULTS

Reconstructive valve-sparing procedures on the aortic valve are one of the most dynamically developing directions in the cardiac surgery. Today cardiac surgeons all over the world prefer the aortic valve sparingoperation using autologous tissues instead biological and mechanical prosthetics. The Ross, Yakub, David, Ozaki procedures have proved their effectives, and their indicators of long-term freedom from reoperations are not inferior to classical prosthetics. In this review the authors describe the key points of the native aortic valve reconstruction. Especially, from the surgical point of view the issues of anatomy of the aortic root and the determination of the optimal «patients» for the valve-sparing procedure are discussed. The principles of reconstruction of various variants of valve development, such as uni-, bi-, three-, and quadricuspid valve anatomy, are presented in details. The approaches to aortic valve repair are described step by step, including a description of the aortic root exposure technique, options for correcting prolapse, eliminating fenestration, and annuloplasty. The evaluation of literature data showed that the overall risks of aortic valve repair in isolation or as a component of a combined intervention are nothigher than in patients with biological or mechanical prosthetics. The violation of the orientation of the commissures, the use of a pericardialcatheter, long-term prolapse, as well as expansion of the annulus fibrous are considred as an independent risk factors of significant regurgitation and reoperations in the long term after reconstruction.