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.

Reference ranges for the fetal mitral, tricuspid, and interventricular septum annular plane systolic excursions (mitral annular plane systolic excursion, tricuspid annular plane systolic excursion, and septum annular plane systolic excursion) between 20 and 36 + 6 weeks of gestation.

Objectives This study aimed to establish reference ranges for fetal mitral, tricuspid, and interventricular septum annular plane systolic excursions (MAPSE, TAPSE, and SAPSE) in normal pregnant women between 20 and 36 + 6 weeks of gestation. Methods This prospective and cross-sectional study included 360 low-risk singleton pregnancies between 20 and 36 + 6 weeks of gestation. MAPSE, TAPSE, and SAPSE were measured by M-mode in real time in an apical or basal four-chamber view through placing the cursor at the atrioventricular junction, marked by the valve rings at the tricuspid, mitral, and basal septum, respectively. A regression analysis was done to determine the appropriate polynomial equation model for both measurements and standard deviation (SD) values in relation to gestational age (GA). The intra- and inter-observer reproducibility was evaluated using the concordance correlation coefficient (CCC) and limits of agreement (LoA). Results There was a significant positive correlation between MAPSE (r=0.705, p<0.0001), TAPSE (r=0.804, p<0.0001), and SAPSE (r=0.690, p<0.0001) and GA. The mean of each parameter ranged as follows: 2.87-5.56 mm, MAPSE; 3.98-8.07 mm, TAPSE; and 2.34-4.21 mm, SAPSE. Poor/moderate intra- and inter-observer reliability (CCC between 0.70 and 0.90) and poor/moderate agreement of all the tested parameters were evaluated (LoA between 10 and 50%). Conclusions Reference values were established for the fetal MAPSE, TAPSE, and SAPSE between 20 and 36 + 6 weeks of gestation in low-risk pregnant women. These parameters showed poor/moderate reproducibility.

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.

Z-scores of early diastolic blood flow widths of mitral and tricuspid valves in normal fetuses and fetuses with dilated coronary sinus.

Objectives: To establish Z-score models for early diastolic blood flow widths of mitral and tricuspid valves in normal fetuses and compare Z-scores and other blood flow dynamic parameters between normal fetuses and fetuses with dilated coronary sinus (CS). This study seeks to assess whether dilated CS affects cardiac structure or hemodynamics.Methods: One hundred twenty-nine normal fetuses (Group I) and 15 fetuses with dilated CS connected to persistent left superior vena cava (PLSVC) (Group II) were included in this study. Noncardiac biometrical parameters, including gestation age (GA), biparietal diameter (BPD), femoral length (FL), and heart area (HA), were obtained. Hemodynamic parameters, such as the early diastolic blood flow widths of mitral and tricuspid valves (MV-CDFI-Width, TV-CDFI-Width), velocity and time integral of blood flow (VTI), peak early and late diastolic velocity of mitral and tricuspid valves [peak early diastolic velocity of the mitral valve (MVE), peak late diastolic velocity of the mitral valve (MVA), peak early diastolic velocity of the tricuspid valve (TVE), peak late diastolic velocity of the tricuspid valve (TVA)], were measured.Results: Z-score models for MV-CDFI-Width and TV-CDFI-Width against noncardiac biometrical parameters were best described by linear regression analysis. The mean equations of MV-CDFI-Width against noncardiac biometrical parameters were 0.066 + (0.025 × GA), 0.169 + (0.084 × BPD), 0.213 + (0.106 × FL) and 0.533 + (0.028 × HA). The SD for MV-CDFI-Width against FL was estimated based on values from the following equation: -0.006 + (0.02 × FL). Other SDs were constants estimated as the standard deviations of the unscaled residuals. Z-scores and other blood flow dynamic parameters exhibited no statistically significant differences between Group I and Group II.Conclusions: This study demonstrated that dilated CS did not affect fetal hemodynamics of the mitral or tricuspid valves or cardiac structures. We also suggested that the causes of diminutive left heart or coarctation of the aorta might not be associated with dilated CS in fetuses with PLSVC.

FRS2α-dependent cell fate transition during endocardial cushion morphogenesis.

Atrioventricular valve development requires endothelial-to-mesenchymal transition (EndMT) that induces cushion endocardial cells to give rise to mesenchymal cells crucial to valve formation. In the adult endothelium, deletion of the docking protein FRS2α induces EndMT by activating TGFß signaling in a miRNA let-7-dependent manner. To study the role of endothelial FRS2α during embryonic development, we generated mice with an inducible endothelial-specific deletion of Frs2α (FRS2αiECKO). Analysis of the FRS2αiECKO embryos uncovered a combination of impaired EndMT in AV cushions and defective maturation of AV valves leading to development of thickened, abnormal valves when Frs2α was deleted early (E7.5) in development. At the same time, no AV valve developmental abnormalities were observed after late (E10.5) deletion. These observations identify FRS2α as a pivotal controller of cell fate transition during both EndMT and post-EndMT valvulogenesis.

Longitudinal Reference Ranges for Tricuspid Annular Plane Systolic Excursion and Mitral Annular Plane Systolic Excursion in Normally Grown Fetuses.

OBJECTIVES: The aim of this study was to construct reference ranges for fetal tricuspid annular plane systolic excursion (TAPSE) and mitral annular plane systolic excursion (MAPSE) using conventional M-mode ultrasound (US) in the second half of pregnancy. METHODS: Participants underwent US scans every 4 weeks from 18 weeks' gestation until delivery. The TAPSE and MAPSE were measured by conventional M-mode US at each examination. The relationships between TAPSE and MAPSE and gestational age and estimated fetal weight were modeled by Bayesian mixed effects linear regression. RESULTS: Positive linear relationships were observed between both MAPSE and TAPSE and gestational age and estimated fetal weight. Reference centiles for TAPSE and MAPSE were developed. CONCLUSIONS: This simple technique is a useful tool for assessing cardiac function and could be used for quantitative assessments of fetal cardiac function, particularly in high-risk pregnancies such as those complicated by maternal diabetes.

Agreement between anatomical M-mode and tissue Doppler imaging in the assessment of fetal atrioventricular annular plane displacement in uncomplicated pregnancies: A prospective longitudinal study.

AIM: To evaluate the level of agreement between M-mode and pulsed-wave tissue Doppler imaging (PW-TDI) techniques in assessing fetal mitral annular plane systolic excursion (MAPSE), tricuspid annular plane systolic excursion (TAPSE) and septal annular plane systolic excursion (SAPSE) in a low-risk population. METHODS: This prospective longitudinal study included healthy fetuses assessed from 18 to 40 weeks of gestation. Tricuspid annular plane systolic excursion, MAPSE and SAPSE were measured using anatomical M-mode and PW-TDI. The agreement between the two diagnostic tests was assessed using Bland-Altman analysis. RESULTS: Fifty fetuses were included in the final analysis. Mean values of TASPE were higher than that of MAPSE. There was a progressive increase of TAPSE, MAPSE and SAPSE values with advancing gestation. For each parameter assessed, there was an overall good agreement between the measurements obtained with M-mode and PW-TDI techniques. However, the measurements made with M-mode were slightly higher than those obtained with PW-TDI (mean differences: 0.03, 0.05 and 0.03 cm for TAPSE, MAPSE and SAPSE, respectively). When stratifying the analyses by gestational age, the mean values of TAPSE, MAPSE and SAPSE measured with M-Mode were higher compared to those obtained with PW-TDI, although the mean differences between the two techniques tended to narrow with increasing gestation. Tricuspid annular plane systolic excursion, MAPSE and SAPSE measurements were all significantly, positively associated with gestational age (all P < 0.001). CONCLUSION: Fetal atrioventricular annular plane displacement can be assessed with M-mode technique, or with PW-TDI as the velocity-time integral of the myocardial systolic waveform. Atrioventricular annular plane displacement values obtained with M-mode technique are slightly higher than those obtained with PW-TDI.

Effect of altered haemodynamics on the developing mitral valve in chick embryonic heart.

Intracardiac haemodynamics is crucial for normal cardiogenesis, with recent evidence showing valvulogenesis is haemodynamically dependent and inextricably linked with shear stress. Although valve anomalies have been associated with genetic mutations, often the cause is unknown. However, altered haemodynamics have been suggested as a pathogenic contributor to bicuspid aortic valve disease. Conversely, how abnormal haemodynamics impacts mitral valve development is still poorly understood. In order to analyse altered blood flow, the outflow tract of the chick heart was constricted using a ligature to increase cardiac pressure overload. Outflow tract-banding was performed at HH21, with harvesting at crucial valve development stages (HH26, HH29 and HH35). Although normal valve morphology was found in HH26 outflow tract banded hearts, smaller and dysmorphic mitral valve primordia were seen upon altered haemodynamics in histological and stereological analysis at HH29 and HH35. A decrease in apoptosis, and aberrant expression of a shear stress responsive gene and extracellular matrix markers in the endocardial cushions were seen in the chick HH29 outflow tract banded hearts. In addition, dysregulation of extracellular matrix (ECM) proteins fibrillin-2, type III collagen and tenascin were further demonstrated in more mature primordial mitral valve leaflets at HH35, with a concomitant decrease of ECM cross-linking enzyme, transglutaminase-2. These data provide compelling evidence that normal haemodynamics are a prerequisite for normal mitral valve morphogenesis, and abnormal blood flow could be a contributing factor in mitral valve defects, with differentiation as a possible underlying mechanism.

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.

Wnt/ß-catenin signaling enables developmental transitions during valvulogenesis.

Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector ß-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/ß-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/ß-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/ß-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects.

Cyclic Mechanical Loading Is Essential for Rac1-Mediated Elongation and Remodeling of the Embryonic Mitral Valve.

During valvulogenesis, globular endocardial cushions elongate and remodel into highly organized thin fibrous leaflets. Proper regulation of this dynamic process is essential to maintain unidirectional blood flow as the embryonic heart matures. In this study, we tested how mechanosensitive small GTPases, RhoA and Rac1, coordinate atrioventricular valve (AV) differentiation and morphogenesis. RhoA activity and its regulated GTPase-activating protein FilGAP are elevated during early cushion formation but decreased considerably during valve remodeling. In contrast, Rac1 activity was nearly absent in the early cushions but increased substantially as the valve matured. Using gain- and loss-of-function assays, we determined that the RhoA pathway was essential for the contractile myofibroblastic phenotype present in early cushion formation but was surprisingly insufficient to drive matrix compaction during valve maturation. The Rac1 pathway was necessary to induce matrix compaction in vitro through increased cell adhesion, elongation, and stress fiber alignment. Facilitating this process, we found that acute cyclic stretch was a potent activator of RhoA and subsequently downregulated Rac1 activity via FilGAP. On the other hand, chronic cyclic stretch reduced active RhoA and downstream FilGAP, which enabled Rac1 activation. Finally, we used partial atrial ligation experiments to confirm in vivo that altered cyclic mechanical loading augmented or restricted cushion elongation and thinning, directly through potentiation of active Rac1 and active RhoA, respectively. Together, these results demonstrate that cyclic mechanical signaling coordinates the RhoA to Rac1 signaling transition essential for proper embryonic mitral valve remodeling.

Pilot study of chronic maternal hyperoxygenation and effect on aortic and mitral valve annular dimensions in fetuses with left heart hypoplasia.

OBJECTIVE: Acute maternal hyperoxygenation (AMH) results in increased fetal left heart blood flow. Our aim was to perform a pilot study to determine the safety, feasibility and direction and magnitude of effect of chronic maternal hyperoxygenation (CMH) on mitral and aortic valve annular dimensions in fetuses with left heart hypoplasia (LHH) after CMH. METHODS: Gravidae with fetal LHH were eligible for inclusion in a prospective evaluation of CMH. LHH was defined as: sum of aortic and mitral valve annuli Z-scores < -4.5, arch flow reversal and left-to-right or bidirectional atrial level shunting without hypoplastic left heart syndrome or severe aortic stenosis. Gravidae with an affected fetus and with ≥ 10% increase in aortic/combined cardiac output flow after 10 min of AMH at 8 L/min 100% fraction of inspired oxygen were offered enrollment. Nine gravidae were enrolled from February 2014 to January 2015. The goal therapy was ≥ 8 h daily CMH from enrollment until delivery. Gravidae who were cared for from July 2012 to October 2014 with fetal LHH and no CMH were identified as historical controls (n = 9). Rates of growth in aortic and mitral annuli over the final trimester were compared between groups using longitudinal regression. RESULTS: There were no significant maternal or fetal complications in the CMH cohort. Mean gestational age at study initiation was 29.6 ± 3.2 weeks for the intervention group and 28.4 ± 1.8 weeks for controls (P = 0.35). Mean relative increase in aortic/combined cardiac output after AMH was 35.3% (range, 18.1-47.9%). Median number of hours per day on CMH therapy was 9.3 (range, 6.5-14.6) and median duration of CMH was 48 (range, 33-84) days. Mean mitral annular growth was 0.19 ± 0.05 mm/week compared with 0.14 ± 0.05 mm/week in CMH vs controls (mean difference 0.05 ± 0.05 mm/week, P = 0.33). Mean aortic annular growth was 0.14 ± 0.03 mm/week compared with 0.13 ± 0.03 mm/week in CMH vs controls (mean difference 0.01 ± 0.03 mm/week, P = 0.75). More than 9 h CMH daily (n = 6) was associated with better growth of the aortic annulus in intervention fetuses (0.16 ± 0.03 vs 0.08 ± 0.02 mm/week, P = 0.014). CONCLUSIONS: CMH is both safe and feasible for continued research. In this pilot study, the effect estimates of annular growth, using the studied method of delivery and dose of oxygen, were small. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

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.

Prenatal examination of the area and morphology of the atrioventricular valves using four-dimensional ultrasound in normal and abnormal hearts.

OBJECTIVE: Our aim is to evaluate the feasibility to examine the morphology and area of the atrioventricular (AV) valves in normal fetuses and fetuses with cardiac defects using spatiotemporal image correlation (STIC). METHODS: Atrioventricular valves were analyzed longitudinally in STIC volumes of 74 normal fetuses between the 15th and 36th week of pregnancy. The valve area was measured in a rendered view in diastole, the number of valve leaflets in systole. Longitudinal data analysis was performed using linear mixed models. Fifty fetuses with cardiac defects were examined. RESULTS: Examination of 355 STIC volumes of normal fetuses showed in 82.5% sufficient quality. The tricuspid valve leaflets were seen in 200 (68.3%) volumes and the mitral valve leaflets in 219 (74.7%) volumes. The tricuspid valve showed in 61.1% a round, 29.0% rectangle, and 8.9% elliptical shape and the mitral valve in 60.1% round, 28.0% rectangle, and 10.9% elliptical. Regression analysis revealed a positive relationship of the valve area with gestational age (p < 0.0001). Most heart defects with stenosis showed an area below the 5th percentile. CONCLUSION: Prenatal examination of the morphology and area of the AV valves using four-dimensional ultrasound is feasible. A rectangular valve opening is normal, which was visualized in about one third of the normal fetuses.

Viewing rate and reproducibility of papillary muscle areas in foetal atrioventricular valves using spatio-temporal image correlation in the rendering mode in congenital heart disease.

OBJECTIVE: To assess the viewing rate and reproducibility of areas of the papillary muscles (PMs) of foetal atrioventricular valves using spatio-temporal image correlation (STIC) in the rendering mode in congenital heart disease (CHD). METHODS: We retrospectively reviewed 40 4D-STIC volume datasets from different foetal CHD cases at a gestational age of 18w6d­35w6d. The following papillary muscles (PMs) were assessed: antero-lateral (MPAL) and postero-medial (MPPM) to the mitral valve and antero-superior (MPAS), inferior (MPI) and septal (MPS) to the tricuspid valve. To assess the valve viewing rate, percentages (%) were used. The concordance correlation coefficient (CCC) was used to assess inter-observer reliability. RESULTS: Two independent observers concluded that a complete examination of the PMs was impossible in 11 cases, yielding a viewing rate of 72.5%. The complete examination of the PMs of the tricuspid and mitral valves was possible by both examiners in 33/40 (82.5%) and 32/40 (80.0%) cases, respectively. We observed moderate to good inter-observer reliability with CCCs of 0.95, 0.92, 0.97, 0.96 and 0.97 for MPS, MPI, MPAS, MPAL and MPPM, respectively. CONCLUSION: The viewing rate of PM areas in different CHDs using STIC in the rendering mode was moderate. The inter-observer reproducibility was moderate to good for all PM areas.

Characteristic differences and reference ranges for mitral, tricuspid, aortic, and pulmonary Doppler velocity waveforms during fetal life.

OBJECTIVES: We aimed to construct reference ranges for time intervals of each component of cardiac flow velocity waveforms in normal fetuses, comparing those variables between right and left ventricles. METHODS: In 359 fetuses at the gestational age of 17-38 weeks, the durations of atrioventricular (AV) valve opening (AVVO), AV valve closure (AVVC), total E- (total-E) and A- (total-A) waves, total ejection time (total-ET), acceleration time (acc-E for E-wave, acc-A for A-wave, and acc-ET for ejection time), and deceleration time (dec-E for E-wave, dec-A for A-wave, and dec-ET for ejection time) were studied cross sectionally. RESULTS: Both right and left acc-E showed the strongest correlations with gestational age (r = 0.478 and r = 0.519, respectively). Left AVVO showed a stronger correlation (r = 0.474) than right AVVO (r = 0.282) and, conversely, right AVVC showed a stronger correlation (r = 0.399) than left AVVC (r = 0.195) with gestational age. Significant differences (all P values <0.001) were observed for all right and left parameters other than total-A and acc-E. CONCLUSIONS: Characteristic differences between right and left ventricles were found in the reference ranges, suggesting the developmental properties of the fetal heart. © 2014 John Wiley & Sons, Ltd.

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.

Age-related analysis of structural, biochemical and mechanical properties of the porcine mitral heart valve leaflets.

The aim of this study was to assess structural and biochemical differences in the extracellular matrix of the fetal and adult porcine mitral heart valves in relation to their mechanical characteristics. Using tensile tests it was demonstrated that the material properties of porcine mitral heart valves progressively change with age. The collagen content of the adult heart valve, as estimated by hydroxyproline assay, increases three times as compared with fetal heart valves. Transmission electron microscopy demonstrated that the diameter of collagen fibrils increased in adult heart valves compared with fetal heart valves. The level of collagen cross-linking is lower in the fetal heart valve than the adult heart valve. The reported age differences in the material properties of fetal and adult porcine heart valves were associated with increases in collagen content, the diameter of collagen fibrils and the level of collagen cross-linking. These data lay a foundation for systematic elucidation of the structural determinants of material properties of heart valves during embryonic and postnatal valvulogenesis. They are also essential to define the desirable level of tissue maturation in heart valve tissue engineering.