Spatially organized cellular communities form the developing human heart.

The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.

Regulation of Transplanted Cell Homing by FGF1 and PDGFB after Doxorubicin Myocardial Injury.

There is no effective treatment for the total recovery of myocardial injury caused by an anticancer drug, doxorubicin (Dox). In this study, using a Dox-induced cardiac injury model, we compared the cardioprotective effects of ventricular cells harvested from 11.5-day old embryonic mice (E11.5) with those from E14.5 embryos. Our results indicate that tail-vein-infused E11.5 ventricular cells are more efficient at homing into the injured adult myocardium, and are more angiogenic, than E14.5 ventricular cells. In addition, E11.5 cells were shown to mitigate the cardiomyopathic effects of Dox. In vitro, E11.5 ventricular cells were more migratory than E14.5 cells, and RT-qPCR analysis revealed that they express significantly higher levels of cytokine receptors Fgfr1, Fgfr2, Pdgfra, Pdgfrb and Kit. Remarkably, mRNA levels for Fgf1, Fgf2, Pdgfa and Pdgfb were also found to be elevated in the Dox-injured adult heart, as were the FGF1 and PDGFB protein levels. Addition of exogenous FGF1 or PDGFB was able to enhance E11.5 ventricular cell migration in vitro, and, whereas their neutralizing antibodies decreased cell migration. These results indicate that therapies raising the levels of FGF1 and PDGFB receptors in donor cells and or corresponding ligands in an injured heart could improve the efficacy of cell-based interventions for myocardial repair.

Automated quantitative evaluation of fetal atrioventricular annular plane systolic excursion.

OBJECTIVES: The primary aim of this study was to evaluate the feasibility of automated measurement of fetal atrioventricular (AV) plane displacement (AVPD) over several cardiac cycles using myocardial velocity traces obtained by color tissue Doppler imaging (cTDI). The secondary objectives were to establish reference ranges for AVPD during the second half of normal pregnancy, to assess fetal AVPD in prolonged pregnancy in relation to adverse perinatal outcome and to evaluate AVPD in fetuses with a suspicion of intrauterine growth restriction (IUGR). METHODS: The population used to develop the reference ranges consisted of women with an uncomplicated singleton pregnancy at 18-42 weeks of gestation (n = 201). The prolonged-pregnancy group comprised women with an uncomplicated singleton pregnancy at ≥ 41 + 0 weeks of gestation (n = 107). The third study cohort comprised women with a singleton pregnancy and suspicion of IUGR, defined as an estimated fetal weight < 2.5th centile or an estimated fetal weight < 10th centile and umbilical artery pulsatility index > 97.5th centile (n = 35). Cineloops of the four-chamber view of the fetal heart were recorded using cTDI. Regions of interest were placed at the AV plane in the left and right ventricular walls and the interventricular septum, and myocardial velocity traces were integrated and analyzed using an automated algorithm developed in-house to obtain mitral (MAPSE), tricuspid (TAPSE) and septal (SAPSE) annular plane systolic excursion. Gestational-age specific reference ranges were constructed and normalized for cardiac size. The correlation between AVPD measurements obtained using cTDI and those obtained by anatomic M-mode were evaluated, and agreement between these two methods was assessed using Bland-Altman analysis. The mean Z-scores of fetal AVPD in the cohort of prolonged pregnancies were compared between cases with normal and those with adverse outcome using Mann-Whitney U-test. The mean Z-scores of fetal AVPD in IUGR fetuses were compared with those in the normal reference population using Mann-Whitney U-test. Inter- and intraobserver variability for acquisition of cTDI recordings and offline analysis was assessed by calculating coefficients of variation (CV) using the root mean square method. RESULTS: Fetal MAPSE, SAPSE and TAPSE increased with gestational age but did not change significantly when normalized for cardiac size. The fitted mean was highest for TAPSE throughout the second half of gestation, followed by SAPSE and MAPSE. There was a significant correlation between MAPSE (r = 0.64; P < 0.001), SAPSE (r = 0.72; P < 0.001) and TAPSE (r = 0.84; P < 0.001) measurements obtained by M-mode and those obtained by cTDI. The geometric means of ratios between AVPD measured by cTDI and by M-mode were 1.38 (95% limits of agreement (LoA), 0.84-2.25) for MAPSE, 1.00 (95% LoA, 0.72-1.40) for SAPSE and 1.20 (95% LoA, 0.92-1.57) for TAPSE. In the prolonged-pregnancy group, the mean ± SD Z-scores for MAPSE (0.14 ± 0.97), SAPSE (0.09 ± 1.02) and TAPSE (0.15 ± 0.90) did not show any significant difference compared to the reference ranges. Twenty-one of the 107 (19.6%) prolonged pregnancies had adverse perinatal outcome. The AVPD Z-scores were not significantly different between pregnancies with normal and those with adverse outcome in the prolonged-pregnancy cohort. The mean ± SD Z-scores for SAPSE (-0.62 ± 1.07; P = 0.006) and TAPSE (-0.60 ± 0.89; P = 0.002) were significantly lower in the IUGR group compared to those in the normal reference population, but the differences were not significant when the values were corrected for cardiac size. The interobserver CVs for the automated measurement of MAPSE, SAPSE and TAPSE were 28.1%, 17.7% and 15.3%, respectively, and the respective intraobserver CVs were 33.5%, 15.0% and 17.9%. CONCLUSIONS: This study showed that fetal AVPD can be measured automatically by integrating cTDI velocities over several cardiac cycles. Automated analysis of AVPD could potentially help gather larger datasets to facilitate use of machine-learning models to study fetal cardiac function. The gestational-age associated increase in AVPD is most likely a result of increasing cardiac size, as the AVPD normalized for cardiac size did not change significantly between 18 and 42 weeks. A decrease was seen in TAPSE and SAPSE in IUGR fetuses, but not after correction for cardiac size. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells.

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

Echogenic intracardiac focus - existence in the first trimester and the role of microRNAs.

Echogenic intracardiac focus (EIF) constitutes a finding in the ultrasound study that indicates an area which is echogenically bright in the fetal heart and is as bright as the bone that moves synchronically to the atrioventricular valves. Microcalcifications of the papillary muscles or chordae tendinae are being represented by this echogenicity and are mostly present in the left ventricle (90% of cases). EIF appears usually at the ultrasound that is realized in the mid-trimester in a percentage that reaches 3.5% in euploid fetuses and 15% to 30% in fetuses with trisomy 21. In the current paper, the rare and curious case of a 21-year-old primigravida woman is described, who presented for ultrasound scan at the 12th week of gestation. The scan revealed the presence of EIF, which is very rare, as it is well-known that it usually appears in the second trimester of pregnancy. Counseling and debriefing for dismissing parents' anxiety is necessary as well as further examinations, because EIF has low sensitivity. This specific case report could constitute a beginning in the research of whether investigating EIF in the first trimester of pregnancy is possible and which are the benefits of its detection for the mother, the fetus and the whole family, in clinical practice.

Reducing lung liquid volume in fetal lambs decreases ventricular constraint.

BACKGROUND: This study evaluated whether an increased left ventricular (LV) pump function accompanying reduction of lung liquid volume in fetal lambs was related to increased LV preload, augmented LV contractility, or both. METHODS: Eleven anesthetized preterm fetal lambs (gestation 128 ± 2 days) were instrumented with (1) an LV micromanometer-conductance catheter to obtain LV end-diastolic volume (EDV) and end-diastolic pressure (EDP), the maximal rate of rise of LV pressure (dP/dtmax), LV output, LV stroke work, and LV end-systolic elastance (Ees), a relatively load-independent measure of contractility; (2) an endotracheal tube to measure mean tracheal pressure and to reduce lung liquid volume. LV transmural pressure was calculated as LV EDP minus tracheal pressure. RESULTS: Reducing lung liquid volume by 16 ± 4 ml kg-1 (1) augmented LV output (by 16%, P = 0.001) and stroke work (29%, P < 0.001), (2) increased LV EDV (12%, P < 0.001), (3) increased LV transmural pressure (2.2 mmHg, P < 0.001), (4) did not change LV dP/dtmax normalized for EDV (P > 0.7), and (5) decreased LV Ees (20%, P < 0.01). CONCLUSION: These findings suggest a rise in LV pump function evident after reduction of lung liquid volume in fetal lambs was related to increased LV preload secondary to lessening of external LV constraint, without any associated rise in LV contractility. IMPACT: This study has shown that reducing the volume of liquid filling the fetal lungs lessens the degree of external constraint on the heart. This lesser constraint permits a rise in left ventricular dimensions and thus greater cardiac filling that leads to increased left ventricular pumping performance. This study has defined a mechanism whereby a reduction in lung liquid volume results in enhanced pumping performance of the fetal heart. These findings suggest that a reduction in lung liquid volume which occurs during the birth transition contributes to increases in left ventricular dimensions and pumping performance known to occur with birth.

Fetal echocardiographic predictors of biventricular circulation in hypoplastic left heart complex.

OBJECTIVES: To determine which echocardiographic features of hypoplastic left heart complex (HLHC) in the fetal period are predictive of biventricular (BV) circulation and to evaluate the long-term outcome of patients with HLHC, including rates of mortality, reintervention and development of further cardiac disease. METHODS: Echocardiograms of fetuses with HLHC obtained at 18-26 weeks and 27-36 weeks' gestation between 2004 and 2017 were included in the analysis. The primary outcome was successful BV circulation (Group 1). Group 2 included patients with single-ventricle palliation, death or transplant. Univariate analysis was performed on data obtained at 18-26 and 27-36 weeks and multivariate logistic regression was performed on data obtained at 27-36 weeks only. RESULTS: Of the 51 included cases, 44 achieved successful BV circulation (Group 1) and seven did not (Group 2). Right-to-left/bidirectional foramen ovale (FO) flow and a higher mitral valve (MV) annulus Z-score were associated with successful BV circulation on both univariate and multivariate analysis. Bidirectional or left-to-right FO flow, left ventricular length (LVL) Z-score of < -2.4 and a MV Z-score of < -4.5 correctly predicted 80% of Group 2 cases. Late follow-up was available for 41 patients. There were two late deaths in Group 2. Thirteen patients in Group 1 required reintervention, 12 developed mitral stenosis and five developed isolated subaortic stenosis. CONCLUSIONS: BV circulation is common in fetuses with HLHC. Higher MV annulus and LVL Z-scores and right to left direction of FO flow are important predictors of BV circulation. Long-term sequelae in those with BV circulation may include mitral and subaortic stenosis. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Reference ranges for fetal cardiac, ventricular and atrial relative size, sphericity, ventricular dominance, wall asymmetry and relative wall thickness from 18 to 41 gestational weeks.

OBJECTIVE: To construct nomograms for fetal cardiac, ventricular and atrial relative size and geometry parameters from 18 to 41 weeks' gestation using a low-risk population of singleton pregnancies. METHODS: This was a prospective cohort study of 602 low-risk singleton pregnancies undergoing comprehensive fetal echocardiography, from 18 to 41 weeks of gestation, to assess fetal cardiac, atrial and ventricular relative size and sphericity, ventricular dominance, wall asymmetry and relative wall thickness. Intra- and interobserver measurement reproducibility was evaluated using intraclass correlation coefficients (ICC). In order to construct reference ranges across pregnancy, parametric regressions were tested to model each measurement against gestational age and estimated fetal weight. The measurements evaluated were: cardiothoracic ratio; atrial-to-heart area ratios; ventricular-to-heart area ratios; cardiac, ventricular and atrial sphericity indices; right-to-left basal and midventricular ratios; septal-to-free wall thickness ratios; and relative wall thickness. RESULTS: Fetal cardiac, ventricular and atrial morphometry for assessing relative size and geometry could be successfully performed in > 95% of the population, with moderate-to-excellent interobserver reproducibility (ICC, 0.623-0.907) and good-to-excellent intraobserver reproducibility (ICC, 0.787-0.938). Cardiothoracic ratio and ventricular right-to-left ratio showed a modest increase throughout gestation. Atrial-to-heart and ventricular-to-heart area ratios, atrial sphericity indices and septal-to-free wall thickness ratios were constant with gestational age. Left and right ventricular basal sphericity indices showed a tendency to decrease at the end of gestation, while left and right midventricular sphericity indices tended to decrease in the second trimester. The cardiac sphericity index and left and right relative wall thickness showed a modest decrease with gestational age. Nomograms across gestation were constructed for all echocardiographic parameters described. CONCLUSIONS: The assessment of cardiac, ventricular and atrial relative size and geometry is feasible and reproducible in the fetus. We provide standardized reference ranges for these parameters throughout gestation, enabling the accurate assessment of cardiac remodeling patterns during fetal life. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Fetal cardiac filling and ejection time fractions by pulsed-wave Doppler: reference ranges and potential clinical application.

OBJECTIVES: Fetal cardiac function can be evaluated using a variety of parameters. Among these, cardiac cycle time-related parameters, such as filling time fraction (FTF) and ejection time fraction (ETF), are promising but rarely studied. We aimed to report the feasibility and reproducibility of fetal FTF and ETF measurements using pulsed-wave Doppler, to provide reference ranges for fetal FTF and ETF, after evaluating their relationship with heart rate (HR), gestational age (GA) and estimated fetal weight (EFW), and to evaluate their potential clinical utility in selected fetal conditions. METHODS: This study included a low-risk prospective cohort of singleton pregnancies and a high-risk population of fetuses with severe twin-twin transfusion syndrome (TTTS), aortic stenosis (AoS) or aortic coarctation (CoA), from 18 to 41 weeks' gestation. Left ventricular (LV) and right ventricular inflow and outflow pulsed-wave Doppler signals were analyzed, using valve clicks as landmarks. FTF was calculated as: (filling time/cycle time) × 100. ETF was calculated as: (ejection time/cycle time) × 100. Intraclass correlation coefficients (ICC) were used to evaluate the intra- and interobserver reproducibility of FTF and ETF measurements in low-risk fetuses. The relationships of FTF and ETF with HR, GA and EFW were evaluated using multivariate regression analysis. Reference ranges for FTF and ETF were then constructed using the low-risk population. Z-scores of FTF and ETF in the high-risk fetuses were calculated and analyzed. RESULTS: In total, 602 low-risk singleton pregnancies and 54 high-risk fetuses (nine pairs of monochorionic twins with severe TTTS, 16 fetuses with AoS and 20 fetuses with CoA) were included. Adequate Doppler traces for FTF and ETF could be obtained in 95% of low-risk cases. Intraobserver reproducibility was good to excellent (ICC, 0.831-0.905) and interobserver reproducibility was good (ICC, 0.801-0.837) for measurements of all timing parameters analyzed. Multivariate analysis of FTF and ETF in relation to HR, GA and EFW in low-risk fetuses identified HR as the only variable predictive of FTF, while ETF was dependent on both HR and GA. FTF increased with decreasing HR in low-risk fetuses, while ETF showed the opposite behavior, decreasing with decreasing HR. Most recipient twins with severe TTTS showed reduced FTF and preserved ETF. AoS was associated with decreased FTF and increased ETF in the LV, with seemingly different patterns associated with univentricular vs biventricular postnatal outcome. The majority of fetuses with CoA had FTF and ETF within the normal range in both ventricles. CONCLUSIONS: Measurement of FTF and ETF using pulsed-wave Doppler is feasible and reproducible in the fetus. The presented reference ranges account for associations of FTF with HR and of ETF with HR and GA. These time fractions are potentially useful for clinical monitoring of cardiac function in severe TTTS, AoS and other fetal conditions overloading the heart. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Cardiac Na+-Ca2+ exchanger 1 (ncx1h) is critical for the ventricular cardiomyocyte formation via regulating the expression levels of gata4 and hand2 in zebrafish.

Ca2+ signaling is critical for heart development; however, the precise roles and regulatory pathways of Ca2+ transport proteins in cardiogenesis remain largely unknown. Sodium-calcium exchanger 1 (Ncx1) is responsible for Ca2+ efflux in cardiomyocytes. It is involved in cardiogenesis, while the mechanism is unclear. Here, using the forward genetic screening in zebrafish, we identified a novel mutation at a highly-conserved leucine residue in ncx1 gene (mutantLDD353/ncx1hL154P) that led to smaller hearts with reduced heart rate and weak contraction. Mechanistically, the number of ventricular but not atrial cardiomyocytes was reduced in ncx1hL154P zebrafish. These defects were mimicked by knockdown or knockout of ncx1h. Moreover, ncx1hL154P had cytosolic and mitochondrial Ca2+ overloading and Ca2+ transient suppression in cardiomyocytes. Furthermore, ncx1hL154P and ncx1h morphants downregulated cardiac transcription factors hand2 and gata4 in the cardiac regions, while overexpression of hand2 and gata4 partially rescued cardiac defects including the number of ventricular myocytes. These findings demonstrate an essential role of the novel 154th leucine residue in the maintenance of Ncx1 function in zebrafish, and reveal previous unrecognized critical roles of the 154th leucine residue and Ncx1 in the formation of ventricular cardiomyocytes by at least partially regulating the expression levels of gata4 and hand2.

Impact of gestational diabetes mellitus on fetal cardiac morphology and function: cohort comparison of second- and third-trimester fetuses.

OBJECTIVES: To assess differences in cardiac morphology and function in fetuses of mothers with gestational diabetes mellitus (GDM) compared to controls, and to assess whether, in women with GDM, fetal cardiac changes are accentuated with advancing gestational age. METHODS: We studied 112 women with GDM and 224 women with uncomplicated pregnancy at 24-40 weeks' gestation. In all fetuses, a standard four-chamber oblique view was obtained and offline speckle-tracking analysis was performed to measure right and left endocardial global longitudinal strain (GLS) and tricuspid and mitral annular plane systolic excursion. Global sphericity index was also calculated. Echocardiographic parameters were compared between GDM fetuses and controls at two gestational time periods of 24 + 0 to 32 + 0 weeks and 32 + 1 to 40 + 1 weeks. RESULTS: At 24 + 0 to 32 + 0 weeks, we phenotyped 43 fetuses from mothers with GDM and 71 from uncomplicated pregnancies, and, at 32 + 1 to 40 + 1 weeks, we phenotyped 69 fetuses from mothers with GDM and 153 from women with uncomplicated pregnancy. In fetuses of mothers with GDM, compared to controls, right ventricular functional indices were consistently lower both at 24 + 0 to 32 + 0 weeks and at 32 + 1 to 40 + 1 weeks. Right ventricular GLS was reduced in the GDM group at 24 + 0 to 32 + 0 weeks (adjusted mean difference, 0.7%; 95% CI, 0.3-1.1%) and at 32 + 1 to 40 + 1 weeks (adjusted mean difference, 0.9%; 95% CI, 0.6-1.1%). Fetal left ventricular global longitudinal function was similar in GDM pregnancies compared with controls, with the exception of the contractility of the left ventricular basal segment, which was reduced. Global sphericity index was reduced in GDM pregnancies only at 32 + 1 to 40 + 1 weeks (adjusted mean difference, -0.4; 95% CI, -0.7 to 0.1). CONCLUSIONS: The offspring of women with GDM are at high risk for development of cardiovascular disease in childhood and early adulthood. Our study demonstrates that GDM is associated with a reduction mainly in fetal right ventricular function, compared to controls, and this response is not exaggerated with increasing gestational age. Further studies are needed to determine whether fetuses with the observed alterations in cardiac function are those at highest risk for subsequent development of cardiovascular disease. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Mef2c factors are required for early but not late addition of cardiomyocytes to the ventricle.

During heart formation, the heart grows and undergoes dramatic morphogenesis to achieve efficient embryonic function. Both in fish and amniotes, much of the growth occurring after initial heart tube formation arises from second heart field (SHF)-derived progenitor cell addition to the arterial pole, allowing chamber formation. In zebrafish, this process has been extensively studied during embryonic life, but it is unclear how larval cardiac growth occurs beyond 3 days post-fertilisation (dpf). By quantifying zebrafish myocardial growth using live imaging of GFP-labelled myocardium we show that the heart grows extensively between 3 and 5 dpf. Using methods to assess cell division, cellular development timing assay and Kaede photoconversion, we demonstrate that proliferation, CM addition, and hypertrophy contribute to ventricle growth. Mechanistically, we show that reduction in Mef2c activity (mef2ca+/-;mef2cb-/-), downstream or in parallel with Nkx2.5 and upstream of Ltbp3, prevents some CM addition and differentiation, resulting in a significantly smaller ventricle by 3 dpf. After 3 dpf, however, CM addition in mef2ca+/-;mef2cb-/- mutants recovers to a normal pace, and the heart size gap between mutants and their siblings diminishes into adulthood. Thus, as in mice, there is an early time window when SHF contribution to the myocardium is particularly sensitive to loss of Mef2c activity.

Notch dimerization and gene dosage are important for normal heart development, intestinal stem cell maintenance, and splenic marginal zone B-cell homeostasis during mite infestation.

Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.

Development of Helical Myofiber Tracts in the Human Fetal Heart: Analysis of Myocardial Fiber Formation in the Left Ventricle From the Late Human Embryonic Period Using Diffusion Tensor Magnetic Resonance Imaging.

Background Detection of the fiber orientation pattern of the myocardium using diffusion tensor magnetic resonance imaging lags ≈12 weeks of gestational age (WGA) behind fetal myocardial remodeling with invasion by the developing coronary vasculature (8 WGA). We aimed to use diffusion tensor magnetic resonance imaging tractography to characterize the evolution of fiber architecture in the developing human heart from the later embryonic period. Methods and Results Twenty human specimens (8-24 WGA) from the Kyoto Collection of Human Embryos and Fetuses, including specimens from the embryonic period (Carnegie stages 20-23), were used. Diffusion tensor magnetic resonance imaging data were acquired with a 7T magnetic resonance system. Fractional anisotropy and helix angle were calculated using standard definitions. In all samples, the fibers ran helically in an organized pattern in both the left and right ventricles. A smooth transmural change in helix angle values (from positive to negative) was detected in all 16 directions of the ventricles. This feature was observed in almost all small (Carnegie stage 23) and large samples. A higher fractional anisotropy value was detected at the outer side of the anterior wall and septum at Carnegie stage 20 to 22, which spread around the ventricular wall at Carnegie stage 23 and in the early fetal samples (11-12 WGA). The fractional anisotropy value of the left ventricular walls decreased in samples with ≥13 WGA, which remained low (≈0.09) in larger samples. Conclusions From the human late embryonic period (from 8 WGA), the helix angle arrangement of the myocardium is comparable to that of the adult, indicating that the myocardial structure blueprint, organization, and integrity are already formed.

Development of the ventricular myocardial trabeculae in Scyliorhinus canicula (Chondrichthyes): evolutionary implications.

The development of the ventricular myocardial trabeculae occurs in three steps: emergence, trabeculation and remodeling. The whole process has been described in vertebrates with two different myocardial structural types, spongy (zebrafish) and compact (chicken and mouse). In this context, two alternative mechanisms of myocardial trabeculae emergence have been identified: (1) in chicken and mouse, the endocardial cells invade the two-layered myocardium; (2) in zebrafish, cardiomyocytes from the monolayered myocardium invaginate towards the endocardium. Currently, the process has not been studied in detail in vertebrates having a mixed type of ventricular myocardium, with an inner trabecular and an outer compact layer, which is presumptively the most primitive morphology in gnathostomes. We studied the formation of the mixed ventricular myocardium in the lesser spotted dogfish (Scyliorhinus canicula, Elasmobranchii), using light, scanning and transmission electron microscopy. Our results show that early formation of the mixed ventricular myocardium, specifically the emergence and the trabeculation steps, is driven by an endocardial invasion of the myocardium. The mechanism of trabeculation of the mixed ventricular myocardium in chondrichthyans is the one that best reproduces how this developmental process has been established from the beginning of the gnathostome radiation. The process has been apparently preserved throughout the entire group of sarcopterygians, including birds and mammals. In contrast, teleosts, at least those possessing a mostly spongy ventricular myocardium, seem to have introduced notable changes in their myocardial trabeculae development.

Three-dimensional alignment of microvasculature and cardiomyocytes in the developing ventricle.

While major coronary artery development and pathologies affecting them have been extensively studied, understanding the development and organization of the coronary microvasculature beyond the earliest developmental stages requires new tools. Without techniques to image the coronary microvasculature over the whole heart, it is likely we are underestimating the microvasculature's impact on normal development and diseases. We present a new imaging and analysis toolset to visualize the coronary microvasculature in intact embryonic hearts and quantify vessel organization. The fluorescent dyes DiI and DAPI were used to stain the coronary vasculature and cardiomyocyte nuclei in quail embryo hearts during rapid growth and morphogenesis of the left ventricular wall. Vessel and cardiomyocytes orientation were automatically extracted and quantified, and vessel density was calculated. The coronary microvasculature was found to follow the known helical organization of cardiomyocytes in the ventricular wall. Vessel density in the left ventricle did not change during and after compaction. This quantitative and automated approach will enable future cohort studies to understand the microvasculature's role in diseases such as hypertrophic cardiomyopathy where misalignment of cardiomyocytes has been observed in utero.

Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression.

The role played by the Notch pathway in cardiac progenitor cell biology remains to be elucidated. Delta-like ligand 4 (Dll4), the arterial-specific Notch ligand, is expressed by second heart field (SHF) progenitors at time-points that are crucial in SHF biology. Dll4-mediated Notch signaling is required for maintaining an adequate pool of SHF progenitors, such that Dll4 knockout results in a reduction in proliferation and an increase in apoptosis. A reduced SHF progenitor pool leads to an underdeveloped right ventricle (RV) and outflow tract (OFT). In its most severe form, there is severe RV hypoplasia and poorly developed OFT resulting in early embryonic lethality. In its milder form, the OFT is foreshortened and misaligned, resulting in a double outlet right ventricle. Dll4-mediated Notch signaling maintains Fgf8 expression by transcriptional regulation at the promoter level. Combined heterozygous knockout of Dll4 and Fgf8 demonstrates genetic synergy in OFT alignment. Exogenous supplemental Fgf8 rescues proliferation in Dll4 mutants in ex-vivo culture. Our results establish a novel role for Dll4-mediated Notch signaling in SHF biology. More broadly, our model provides a platform for understanding oligogenic inheritance that results in clinically relevant OFT malformations.

Alternative splicing of jnk1a in zebrafish determines first heart field ventricular cardiomyocyte numbers through modulation of hand2 expression.

The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.

In Vitro Model of Coronary Angiogenesis.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.