Identification of a peripheral blood gene signature predicting aortic valve calcification.

Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational weeks 9, 13, and 22 and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at week 9 rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification.

Desert hedgehog-primary cilia cross talk shapes mitral valve tissue by organizing smooth muscle actin.

Non-syndromic mitral valve prolapse (MVP) is the most common heart valve disease affecting 2.4% of the population. Recent studies have identified genetic defects in primary cilia as causative to MVP, although the mechanism of their action is currently unknown. Using a series of gene inactivation approaches, we define a paracrine mechanism by which endocardially-expressed Desert Hedgehog (DHH) activates primary cilia signaling on neighboring valve interstitial cells. High-resolution imaging and functional assays show that DHH de-represses smoothened at the primary cilia, resulting in kinase activation of RAC1 through the RAC1-GEF, TIAM1. Activation of this non-canonical hedgehog pathway stimulates α-smooth actin organization and ECM remodeling. Genetic or pharmacological perturbation of this pathway results in enlarged valves that progress to a myxomatous phenotype, similar to valves seen in MVP patients. These data identify a potential molecular origin for MVP as well as establish a paracrine DHH-primary cilium cross-talk mechanism that is likely applicable across developmental tissue types.

Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis.

RATIONALE: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. OBJECTIVE: The aim of this study is to determine the functional specificity of Notch in valve development. METHODS AND RESULTS: Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype. CONCLUSIONS: During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction.

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1(Cre)) mouse. Embryonic Wt1(Cre);Alk3(fl/fl) specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1(Cre);Alk3(fl/fl) mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1(Cre);Alk3(fl/fl) specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.

Tgfß-Smad and MAPK signaling mediate scleraxis and proteoglycan expression in heart valves.

Mature heart valves are complex structures consisting of three highly organized extracellular matrix layers primarily composed of collagens, proteoglycans and elastin. Collectively, these diverse matrix components provide all the necessary biomechanical properties for valve function throughout life. In contrast to healthy valves, myxomatous valve disease is the most common cause of mitral valve prolapse in the human population and is characterized by an abnormal abundance of proteoglycans within the valve tri-laminar structure. Despite the clinical significance, the etiology of this phenotype is not known. Scleraxis (Scx) is a basic-helix-loop-helix transcription factor that we previously showed to be required for establishing heart valve structure during remodeling stages of valvulogenesis. In this study, we report that remodeling heart valves from Scx null mice express decreased levels of proteoglycans, particularly chondroitin sulfate proteoglycans (CSPGs), while overexpression in embryonic avian valve precursor cells and adult porcine valve interstitial cells increases CSPGs. Using these systems we further identify that Scx is positively regulated by canonical Tgfß2 signaling during this process and this is attenuated by MAPK activity. Finally, we show that Scx is increased in myxomatous valves from human patients and mouse models, and overexpression in human mitral valve interstitial cells modestly increases proteoglycan expression consistent with myxomatous mitral valve phenotypes. Together, these studies identify an important role for Scx in regulating proteoglycans in embryonic and mature valve cells and suggest that imbalanced regulation could influence myxomatous pathogenesis.

Developmental basis for filamin-A-associated myxomatous mitral valve disease.

AIMS: We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development. METHODS AND RESULTS: Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype. CONCLUSIONS: These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.

Avaliação da evolução da área das valvas mitral e tricúspide fetal com ultrassonografia tridimensional / Assessment of the fetal mitral and tricuspid valves areas development by three-dimensional ultrasonography

OBJETIVO: avaliar as áreas das válvulas atrioventriculares (tricúspide e mitral) de fetos normais por meio da ultrassonografia tridimensional (US3D) utilizando o método STIC (spatiotemporal image correlation). MÉTODOS: realizou-se estudo de corte transversal com 141 mulheres entre a 18ª e a 33ª semana de gestação. As medidas dos volumes cardíacos foram obtidas por um transdutor volumétrico transabdominal acoplado ao aparelho Voluson 730 Expert. Utilizou-se como referência o plano de quatro câmaras com a ROI (região de interesse) posicionada a partir dos ventrículos, sendo a área das valvas delimitada manualmente. Para conhecer a correlação das áreas valvulares com a idade gestacional, foram construídos diagramas de dispersão e calculou-se o coeficiente de correlação de Pearson (r). Foram calculadas médias, medianas, desvios padrão (DP), valores máximo e mínimo. Para se determinar intervalos de referência das áreas valvulares em função da idade gestacional, seguiu-se o modelo de regressão linear simples, utilizando o método de Altman, com nível de significância de p<0,05. Para o cálculo da reprodutibilidade intraobservador, utilizou-se o coeficiente de correlação intraclasse (CCI) e o gráfico de Bland-Altman. RESULTADOS: as áreas valvulares tricúspide e mitral se correlacionaram com a idade gestacional (r=0,80 para a tricúspide e r=0,79 para a mitral), sendo que a média aumentou da válvula tricúspide e mitral, respectivamente, de 0,22±0,10 cm² e de 0,23±0,10 cm² na 18º semana para 0,92±0,29 cm² e para 1,08±0,41 cm² na 33º semana de gestação. A reprodutibilidade intraobservador resultou em CCI=0,993 (IC95 por cento 0,987; 0,996), com diferença média de 0,01 cm² (DP±0,2 cm² e IC95 por cento±0,4 cm²). CONCLUSÃO: intervalos de referência para a área das valvares mitral e tricúspide entre a 18ªe a 33ª semana de gestação foram determinados pela US3D e se mostraram altamente reprodutíveis.

PURPOSE: to evaluate the areas of the atrioventricular valves (tricuspid and mitral) of normal fetuses by the use of three-dimensional ultrasound (3DUS) and the spatiotemporal image correlation (STIC) method. METHODS: a cross-sectional study was conducted on 141 women between the 18th and the 33rd week of pregnancy. Cardiac volumes were measured with a volumetric transabdominal transducer attached to the Voluson 730 Expert equipment. The four chamber plane was used as reference, with the region of interest (ROI) positioned from the ventricles, and the area of the valves was obtained manually. To determine the correlation of the areas with gestational age, scatter plots were constructed and the Pearson correlation coefficient (r) was calculated. Means, medians, standard deviations (SD) and maximum and minimum values were calculated. The simple linear regression model was used to determine reference ranges of valve areas according to the gestational age by the Altman method, with the level of significance set at p<0.05. To calculate the intraobserver reproducibility, we used the intraclass correlation coefficient (ICC) and the Bland-Altman graph. RESULTS: the mitral and tricuspid valve areas were correlated to the gestational age (r=0.80 for the tricuspid and r=0.79 for the mitral valve) and the mean value of the tricuspid and mitral valves increased from 0.22±0.10 cm² and 0.23±0.10 cm² on the 18th week to 0.92±0.29 cm² and 1.08±0.41 cm² on the 33rd of pregnancy, respectively. The intraobserver reproducibility resulted in an ICC=0.993 (95 percentCI 0.987; 0.996) and the mean difference was 0.01 cm² (SD±0.2 cm² and CI95 percent±0.4 cm²). CONCLUSION: reference intervals for the areas of the mitral and tricuspid valve between the 18th and the 33rd week of gestation were determined and proved to be highly reproducible.

Reduced sox9 function promotes heart valve calcification phenotypes in vivo.

RATIONALE: Calcification of heart valve structures is the most common form of valvular disease and is characterized by the appearance of bone-like phenotypes within affected structures. Despite the clinical significance, the underlying etiology of disease onset and progression is largely unknown and valve replacement remains the most effective treatment. The SRY-related transcription factor Sox9 is expressed in developing and mature heart valves, and its function is required for expression of cartilage-associated proteins, similar to its role in chondrogenesis. In addition to cartilage-associated defects, mice with reduced sox9 function develop skeletal bone prematurely; however, the ability of sox9 deficiency to promote ectopic osteogenic phenotypes in heart valves has not been examined. OBJECTIVE: This study aims to determine the role of Sox9 in maintaining connective tissue homeostasis in mature heart valves using in vivo and in vitro approaches. METHODS AND RESULTS: Using histological and molecular analyses, we report that, from 3 months of age, Sox9(fl/+);Col2a1-cre mice develop calcific lesions in heart valve leaflets associated with increased expression of bone-related genes and activation of inflammation and matrix remodeling processes. Consistently, ectopic calcification is also observed following direct knockdown of Sox9 in heart valves in vitro. Furthermore, we show that retinoic acid treatment in mature heart valves is sufficient to promote calcific processes in vitro, which can be attenuated by Sox9 overexpression. CONCLUSIONS: This study provides insight into the molecular mechanisms of heart valve calcification and identifies reduced Sox9 function as a potential genetic basis for calcific valvular disease.

Fetal cardiac dysfunction in preterm premature rupture of membranes.

BACKGROUND: Preterm premature rupture of membranes (PROM) is associated with one-third of preterm births. In about 50% of preterm PROM cases, the fetuses will elicit a fetal inflammatory response syndrome (FIRS). FIRS is associated with the impending onset of preterm labor, periventricular leukomalacia, neonatal sepsis, and long-term handicap, including the development of bronchopulmonary dysplasia and cerebral palsy. The fetal myocardium is a potential target organ of proinflammatory cytokines released during FIRS. The objective of this study was to determine whether preterm PROM is associated with functional changes in the fetal heart, as determined by fetal echocardiography. METHODS: A retrospective study was conducted to assess the diastolic function of fetuses with preterm PROM with documented microbial invasion of the amniotic cavity (n = 25), preterm PROM without microbial invasion of the amniotic cavity (n = 42), and fetuses from normal pregnancies (control group = 150). Pregnancies with multiple gestation, fetal distress, fetuses that were small for gestational age, and major congenital anomalies were excluded. Fetal echocardiography studies were performed with two-dimensional ultrasound, color Doppler imaging and pulsed Doppler ultrasound. Non-parametric statistics were used for comparisons. A p value of < 0.05 was considered significant. RESULTS: The prevalence of positive amniotic fluid cultures for micro-organisms in patients with preterm PROM was 35.8% (24/67). Ureaplasma urealyticum was the most frequent isolate, either alone (41.7%; 10/24) or with other micro-organisms (29.2%; 7/24). Fetuses with preterm PROM had a higher delta early diastolic filling/atrial contraction (E/A) peak velocity ratio, a higher delta E/A velocity-time integral (VTI) ratio, a lower delta A peak velocity, a lower delta A VTI, and a lower A VTI/total VTI ratio in the mitral valve compared to those with uncomplicated pregnancies. The delta E/A peak velocity ratio was significantly higher and the delta A VTI significantly lower in fetuses with preterm PROM and microbial invasion of the amniotic cavity than in those with preterm PROM without microbial invasion of the amniotic cavity. CONCLUSIONS: Preterm PROM is associated with changes in fetal cardiac function consistent with increased left ventricular compliance. These observations were also noted in fetuses with microbial invasion of the amniotic cavity. Our findings suggest that fetal cardiac function is altered in preterm PROM and, in particular, in cases with intra-amniotic infection.

Lineage and morphogenetic analysis of the cardiac valves.

We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.

Development of the papillary muscles of the mitral valve: morphogenetic background of parachute-like asymmetric mitral valves and other mitral valve anomalies.

OBJECTIVES: To understand papillary muscle malformations, such as in parachute mitral valves or parachute-like asymmetric mitral valves, we studied the development of papillary muscles. METHODS: Normal human hearts at between 5 and 19 weeks of development were studied with immunohistochemistry, three-dimensional reconstructions, and gross inspection. Scanning electron microscopy was used to study human and rat hearts. RESULTS: In embryonic hearts a prominent horseshoe-shaped myocardial ridge runs from the anterior wall through the apex to the posterior wall of the left ventricle. In the atrioventricular region this ridge is continuous with atrial myocardium and covered with cushion tissue. The anterior and posterior parts of the trabecular ridge enlarge and loosen their connections with the atrial myocardium. Their lateral sides gradually delaminate from the left ventricular wall, and the continuity between the two parts is incorporated in the apical trabecular network. In this way the anterior and posterior parts of the ridge transform into the anterolateral and the posteromedial papillary muscles, respectively. Simultaneously, the cushions remodel into valve leaflets and chordae. Only the chordal part of the cushions remains attached to the developing papillary muscles. CONCLUSIONS: Disturbed delamination of the anterior or posterior part of the trabecular ridge from the ventricular wall, combined with underdevelopment of chordae, seems to be the cause of asymmetric mitral valves. Parachute valves, however, develop when the connection between the posterior and anterior part of the ridge condenses to form one single papillary muscle. Thus parachute valves and parachute-like asymmetric mitral valves originate in different ways.

Immunohistochemical evaluation of the developing outflow tract in the rat: achieving aortic to mitral fibrous continuity.

OBJECTIVES: To study the development of aortic to mitral fibrous continuity in the normal rat heart. METHODS: The hearts and great vessels of normally developed rat embryos and fetuses aged between 13.25 and 19.75 days of gestation were studied in conjunction with those of newborns aged 2 and 7 days post-partum. Standard histological methods and monoclonal antibodies raised against alpha smooth muscle actin (clone 1A4) and ventricular beta myosin heavy chain were used to demonstrate the ventricular outlets, ventriculo-arterial junction, inner heart curve and aortic infundibulum from the early stages of aortopulmonary septation to attainment of their definitive morphology. RESULTS: The two antibodies demonstrated temporal specificity (actin specificity increased post-partum; myosin specificity maximal during fetal period) in the labelling of their intended structures which correlated with their known developmental profile. Full-thickness fibrous continuity between aortic and mitral valves was not complete until 1 week after birth. After ventricular septation was complete, and thereafter towards the end of fetal life and beyond, separation was maintained by a muscular structure histologically identical to the vestigial netro-aortic root branch of the conduction tissue, a structure known to be derived from the primitive ventricular myocardium within the environs of the inner heart curve. CONCLUSIONS: Ventricular septation (occurring relatively early) and the attainment of fibrous continuity (occurring relatively late in development) are two independent processes. Muscular tissue separating left-sided arterial and atrioventricular valves is not derived from the aortic infundibulum but from the inner heart curve. Persistence of this structure is a feature of normal rat heart development and needs to be recognised when working with rodent-based animal models of congenital heart disease aimed at studying the disruption of the development of the ventricular outflow tracts.

Abnormal accessory mitral leaflet simulating left ventricular outflow tract tumor.

This is a case report of an uncommon form of subaortic stenosis caused by an abnormal accessory mitral leaflet simulating a left ventricular outflow tract tumor. A 5-year-old boy with a systolic heart murmur underwent cardiac catheterization which demonstrated a subaortic stenosis with a 90 mmHg peak systolic pressure gradient. Angiography showed a tumor-like structure in the left ventricular outflow tract. At operation this mass was found to be an abnormal bulky accessory mitral leaflet. Resection of this deformed leaflet was carried out successfully.