Cardiac Development Research Articles

Membrane Physiology Symposium April 22nd-23rd, 2024, Napa California, USA.

The Membrane Physiology Symposium was created with the goal of joining basic research with technology companies, where questions and conversations are open and welcomed in a universal language. For many years, academic physiology research areas have been naturally siloed into their own niche communities, which can surely be beneficial. Linking different technological application areas with varied research sectors is an integral formula for successful scientific breakthroughs. The meeting covers a wide variety of topics related to channelopathies, neurological and cardiac disease, drug development, and therapeutic applications, with research programs represented by core academic facilities, medical science institutions, small and large pharmaceutical enterprises, as well as novel cell-based and reagent providers. For this reason, gathering the brightest minds of all relevant fields in one integrative forum is essential for new avenues of discovery, development, and process optimization to occur.

Embryonic Lethal Phenotyping to Identify Candidate Genes Related with Birth Defects.

Congenital birth defects contribute significantly to preterm birth, stillbirth, perinatal death, infant mortality, and adult disability. As a first step to exploring the mechanisms underlying this major clinical challenge, we analyzed the embryonic phenotypes of lethal strains generated by random mutagenesis. In this study, we report the gross embryonic and perinatal phenotypes of 55 lethal strains randomly picked from a collection of mutants that carry piggyBac (PB) transposon inserts. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested most of the analyzed mutations hit genes involved in heart and nervous development, or in Notch and Wnt signaling. Among them, 12 loci are known to be associated with human diseases. We confirmed 53 strains as embryonic or perinatal lethal, while others were subviable. Gross morphological phenotypes such as body size abnormality (29/55, 52.73%), growth or developmental delay (35/55, 63.64%), brain defects (9/55, 16.36%), vascular/heart development (31/55, 56.36%), and other structural defects (9/55, 16.36%) could be easily observed in the mutants, while three strains showed phenotypes similar to those of human patients. Furthermore, we detected body weight or body composition alterations in the heterozygotes of eight strains. One of them was the TGF-β signaling gene Smad2. The heterozygotes showed increased energy expenditure and a lower fat-to-body weight ratio compared to wild-type mice. This study provided new insights into mammalian embryonic development and will help understand the pathology of congenital birth defects in humans. In addition, it expanded our understanding of the etiology of obesity.

Activation of VGLL4 Suppresses Cardiomyocyte Maturational Hypertrophic Growth.

From birth to adulthood, the mammalian heart grows primarily through increasing cardiomyocyte (CM) size, which is known as maturational hypertrophic growth. The Hippo-YAP signaling pathway is well known for regulating heart development and regeneration, but its roles in CM maturational hypertrophy have not been clearly addressed. Vestigial-like 4 (VGLL4) is a crucial component of the Hippo-YAP pathway, and it functions as a suppressor of YAP/TAZ, the terminal transcriptional effectors of this signaling pathway. To develop an in vitro model for studying CM maturational hypertrophy, we compared the biological effects of T3 (triiodothyronine), Dex (dexamethasone), and T3/Dex in cultured neonatal rat ventricular myocytes (NRVMs). The T3/Dex combination treatment stimulated greater maturational hypertrophy than either the T3 or Dex single treatment. Using T3/Dex treatment of NRVMs as an in vitro model, we found that activation of VGLL4 suppressed CM maturational hypertrophy. In the postnatal heart, activation of VGLL4 suppressed heart growth, impaired heart function, and decreased CM size. On the molecular level, activation of VGLL4 inhibited the PI3K-AKT pathway, and disrupting VGLL4 and TEAD interaction abolished this inhibition. In conclusion, our data suggest that VGLL4 suppresses CM maturational hypertrophy by inhibiting the YAP/TAZ-TEAD complex and its downstream activation of the PI3K-AKT pathway.

Gata6 functions in zebrafish endoderm to regulate late differentiating arterial pole cardiogenesis.

Mutations in GATA6 are associated with congenital heart disease, most notably conotruncal structural defects. However, how GATA6 regulates cardiac morphology during embryogenesis is undefined. We used knockout and conditional mutant zebrafish alleles to investigate the spatiotemporal role of gata6 during cardiogenesis. Loss of gata6 specifically impacts atrioventricular valve formation and recruitment of epicardium, with a prominent loss of arterial pole cardiac cells, including those of the ventricle and outflow tract. However, there are no obvious defects in cardiac progenitor cell specification, proliferation or death. Conditional loss of gata6 starting at 24 h is sufficient to disrupt the addition of late differentiating cardiomyocytes at the arterial pole, with decreased expression levels of anterior secondary heart field (SHF) markers spry4 and mef2cb. Conditional loss of gata6 in the endoderm is sufficient to phenocopy the straight knockout, resulting in a significant loss of ventricular and outflow tract tissue. Exposure to a Dusp6 inhibitor largely rescues the loss of ventricular cells in gata6-/- larvae. Thus, gata6 functions in endoderm are mediated by FGF signaling to regulate the addition of anterior SHF progenitor derivatives during heart formation.

The Function of H2A Histone Variants and Their Roles in Diseases.

Epigenetic regulation, which is characterized by reversible and heritable genetic alterations without changing DNA sequences, has recently been increasingly studied in diseases. Histone variant regulation is an essential component of epigenetic regulation. The substitution of canonical histones by histone variants profoundly alters the local chromatin structure and modulates DNA accessibility to regulatory factors, thereby exerting a pivotal influence on gene regulation and DNA damage repair. Histone H2A variants, mainly including H2A.Z, H2A.B, macroH2A, and H2A.X, are the most abundant identified variants among all histone variants with the greatest sequence diversity. Harboring varied chromatin occupancy and structures, histone H2A variants perform distinct functions in gene transcription and DNA damage repair. They are implicated in multiple pathophysiological mechanisms and the emergence of different illnesses. Cancer, embryonic development abnormalities, neurological diseases, metabolic diseases, and heart diseases have all been linked to histone H2A variant alterations. This review focuses on the functions of H2A histone variants in mammals, including H2A.Z, H2A.B, macroH2A, and H2A.X, and their current roles in various diseases.

Integrated single-cell RNA-seq analysis reveals mitochondrial calcium signaling as a modulator of endothelial-to-mesenchymal transition.

Endothelial cells (ECs) are highly plastic, capable of differentiating into various cell types. Endothelial-to-mesenchymal transition (EndMT) is crucial during embryonic development and contributes substantially to vascular dysfunction in many cardiovascular diseases (CVDs). While targeting EndMT holds therapeutic promise, understanding its mechanisms and modulating its pathways remain challenging. Using single-cell RNA sequencing on three in vitro EndMT models, we identified conserved gene signatures. We validated original regulators in vitro and in vivo during embryonic heart development and peripheral artery disease. EndMT induction led to global expression changes in all EC subtypes rather than in mesenchymal clusters. We identified mitochondrial calcium uptake as a key driver of EndMT; inhibiting mitochondrial calcium uniporter (MCU) prevented EndMT in vitro, and conditional Mcu deletion in ECs blocked mesenchymal activation in a hind limb ischemia model. Tissues from patients with critical limb ischemia with EndMT features exhibited significantly elevated endothelial MCU. These findings highlight MCU as a regulator of EndMT and a potential therapeutic target.

Generative AI for Cell Type-Specific Fluorescence Image Generation of hPSC-derived Cardiac Organoid.

Human pluripotent stem cell (hPSC)-derived cardiac organoid is the most recent three-dimensional tissue structure that mimics the structure and functionality of the human heart and plays a pivotal role in modeling heart development and disease. The hPSC-derived cardiac organoids are commonly characterized by bright-field microscopic imaging for tracking daily organoid differentiation and morphology formation. Although the brightfield microscope provides essential information about hPSC-derived cardiac organoids, such as morphology, size, and general structure, it does not extend our understanding of cardiac organoids on cell type-specific distribution and structure. Then, fluorescence microscopic imaging is required to identify the specific cardiovascular cell types in the hPSC-derived cardiac organoids by fluorescence immunostaining fixed organoid samples or fluorescence reporter imaging of live organoids. Both approaches require extra steps of experiments and techniques and do not provide general information on hPSC-derived cardiac organoids from different batches of differentiation and characterization, which limits the biomedical applications of hPSC-derived cardiac organoids. This research addresses this limitation by proposing a comprehensive workflow for colorizing phase contrast images of cardiac organoids from brightfield microscopic imaging using conditional Generative Adversarial Networks (GANs) to provide cardiovascular cell type-specific information in hPSC-derived cardiac organoids. By infusing these phase contrast images with accurate fluorescence colorization, our approach aims to unlock the hidden wealth of cell type, structure, and further quantifications of fluorescence intensity and area, for better characterizing hPSC-derived cardiac organoids.

The influence of COVID-19 pandemic on body mass and cardiopulmonary endurance of Chinese adolescents: a longitudinal follow-up study.

With the spread and spread of COVID-19 around the world, youth's learning, lifestyle and health have been greatly affected. Based on the current research, there is no adequate analysis of the development of young people's physique and heart and lung health during COVID-19, and there is a lack of relevant targeted research. The aim of this study was to investigate the changes of BMI and Maximum Oxygen Absorption (VO2max) in 12-14 year old teenagers before and after COVID-19. The BMI, 1,000/800 m running time and associated data related to 29,813 individuals between 2019 and 2022 were collected by cluster sampling, and the changes of BMI Z and VO2max before and after the outbreak were analyzed. Moreover, the relationship between BMI and cardiovascular endurance was analyzed by means of multi-linear stepwise regression. The covariance analysis models indicated that compared with 2019, adolescent weight, BMI, and 1,000/800 m running time showed varying degrees of growth in 2020, while lung capacity decreased. All indicators achieved rapid rebound in 2021 and 2022 (p < 0.01); the one-way analysis of variance models indicated that The BMI Z score and VO2max of adolescents showed growth and decline in 2020, respectively, and achieved rapid recovery and development in 2021 and 2022 (p < 0.01). The results of the multiple linear stepwise regression analysis indicate that, after the years of BMI Z and novel coronavirus infection were included (△R2 = 0.179), adolescents' overweight and obesity were positively correlated with the maximum oxygen uptake (B = 0.643, 95%CI = 0.634 ~ 0.652); There is a negative correlation between weight loss and maximum oxygen uptake (B = -0.510, 95%CI = -0.537~-0.484); The year of novel coronavirus infection was positively correlated with the maximum oxygen uptake of adolescents (B = 0.116, 95%CI = 0.107~0.125). This study shows that the impact of COVID-19 on BMI and heart and lung health in adolescents is significant. Young people of all ages and sexes showed similar developmental trends.

Risk factors for postoperative delirium in frail elderly patients undergoing on-pump cardiac surgery and development of a prediction model-a prospective observational study.

To identify the risk factors for postoperative delirium (POD) after cardiac surgery in frail elderly patients and develop a receiver operating characteristic (ROC) prediction model to confirm the effectiveness. This was a prospective observational study, patients were assessed preoperatively according to the frailty index (FI) scale. Cerebral (SctO2) was assessed at different time points using near-infrared spectroscopy (NIRS). On the basis of the occurrence of POD within 7 days after surgery, patients were divided into POD and non-POD groups. Risk factors were analyzed using logistic regression analysis, while their predictive values were evaluated using the receiver operating characteristic curve analysis. POD was significantly associated with frailty, lower preoperative MMSE scores, hyperlipidemia, diabetes, cerebrovascular disease, lower hemoglobin level, lower albumin level, longer operation time, longer CPB time, lower SctO2 at T5, and lower SctO2baseline (P < 0.05). SrtO2 and SmtO2 did not differ significantly between groups. FI, preoperative MMSE score, and operation time as independent risk factors (P < 0.05). Significant predictive value was demonstrated in all 3 variables (P < 0.001; respectively). Among them, high sensitivity and specificity were observed with the FI (cut-off value 0.27, sensitivity 75%, specificity 73.5%) and operation time (cut-off value 237.5, sensitivity 62.5%, specificity 78.6%). The FI, preoperative MMSE score, and operation time were independent risk factors for POD in elderly patients after cardiac surgery, with high predictive value observed with the FI and operation time. Cerebral oxygen saturation was associated with POD but was not an independent risk factor. Chinese Clinical Trail Registry, No: chictr2200056038.

The New Frontiers of Fetal Imaging: MRI Insights into Cardiovascular and Thoracic Structures.

Fetal magnetic resonance imaging (fMRI) represents a second-line imaging modality that provides multiparametric and multiplanar views that are crucial for confirming diagnoses, detecting associated pathologies, and resolving inconclusive ultrasound findings. The introduction of high-field magnets and new imaging sequences has expanded MRI's role in pregnancy management. Recent innovations in ECG-gating techniques have revolutionized the prenatal evaluation of congenital heart disease by synchronizing imaging with the fetal heartbeat, thus addressing traditional challenges in cardiac imaging. Fetal cardiac MRI (fCMR) is particularly valuable for assessing congenital heart diseases, especially when ultrasound is limited by poor imaging conditions. fCMR allows for detailed anatomical and functional evaluation of the heart and great vessels and is also useful for diagnosing additional anomalies and analyzing blood flow patterns, which can aid in understanding abnormal fetal brain growth and placental perfusion. This review emphasizes fMRI's potential in evaluating cardiac and thoracic structures, including various gating techniques like metric optimized gating, self-gating, and Doppler ultrasound gating. The review also covers the use of static and cine images for structural and functional assessments and discusses advanced techniques like 4D-flow MRI and T1 or T2 mapping for comprehensive flow quantification and tissue characterization.

How Does Listening to “Fetal Heartbeats” Affect the Feelings of Pregnant Women?

OBJECTIVES: This study was conducted to evaluate the impact of listening to fetal heartbeats (FHBs) on the feelings of normal and high-risk pregnancies (HRPs), and to learn the colors and geometrical figures representing their feelings. STUDY DESIGN: This retrospective study consisted of 291 pregnant women who applied for a non-stress test (NST). Participants were divided into two groups; 1: Normal pregnancies (Group1) and 2: HRPs (Group2). Demographic variables, feelings in response to listening to FHBs (negative, neutral, positive), grade mark (0-10) of feelings, color (warm, neutral, cold), and figure (simple, complex) preferences were retrospectively evaluated. RESULTS: Of the 291 women, 172 (59.1%) Group 1 participants were compared to 119 (40.9%) Group 2 participants. Group 1 and Group 2 had similar feelings (p&gt;0.05), however, the rate of negative feelings was higher in Group 2 (11.6% vs. 20.2%). Higher marks were observed in Group 2 with happiness and peace/relaxation after listening to FHBs. Fear was the lowest mark in women with positive/neutral feelings in both groups. Women in Group 1 preferred warm colors regardless of their feelings. Women in Group 2 who have positive feelings chose warm colors, whereas women who felt neutral or negative more commonly chose cold (55.5%) and neutral (41.7%) colors (p=0.017). Independent of feelings, simple figures were predominately preferred (p=0.04). CONCLUSION: NST is more than a perinatal surveillance method and listening to FHBs has a positive impact on the feelings of pregnancies. Warm colors were commonly related to positive feelings. Simple figures were preferred regardless of feelings.

Comparison of colostrum and milk extracellular vesicles small RNA cargo in water buffalo

Recently, much interest has been raised for the characterization of signaling molecules carried by extracellular vesicles (EVs), which are particularly enriched in milk (mEVs). Such interest is linked to the capability of EVs to cross biological barriers, resist acidification in the gastric environment, and exert modulation of the immune system, mainly through their microRNA (miRNA) content. We characterized the small-RNA cargo of colostrum EVs (colosEVs) and mEVs from Italian Mediterranean buffalo through next generation sequencing. Colostrum (first milking after birth) and milk (day 50 of lactation) were sampled from seven subjects from five farms. ColosEVs and mEVs were subjected to morphological characterization, followed by high-depth sequencing of small RNA libraries produced from total RNA. The main difference was the amount of EV in the two samples, with colostrum showing 10 to 100-fold higher content than milk. For both matrices, miRNA was the most abundant RNA species (95% for colosEVs and 96% for mEVs) and three lists were identified: colosEV-specific, mEV-specific and shared most expressed. Gene ontology (GO) enrichment analysis on miRNA targets highlighted many terms related to the epigenetic, transcriptional and translational regulations across the three lists, with a higher number of enriched terms for colosEV-specific miRNAs. Terms specific to colosEVs were related to “cell differentiation” and “microvillus assembly”, while for mEV “cardiac and blood vessel development” and “mitochondria” emergerd. Immune modulation terms were found for both sample-specific miRNAs. Overall, both matrices carry a similar molecular message in terms of biological processes potentially modulated into receiving cells, but there is significant difference in the abundance, with colostrum containing much more EVs than milk. Moreover, colosEVs carry molecules involved in signal transduction, cell cycle and immune response, as for mEVs and EVs of other previously characterized species, but with a special enrichment for miRNAs with epigenetic regulation capacities. These beneficial characteristics of colosEVs and mEVs are essential for the calf and could also be exploited for the therapeutic purposes in humans, although further studies are necessary to measure the sanitization treatment impact on EV conservation, especially in buffalo where milk is consumed almost exclusively after processing.

Abstract We083: A new mouse model for the study of cardiogenic TFs Tbx5 , Gata4 , and Mef2c during cardiogenesis

Congenital heart defects (CHDs) are be caused by mutations in genes that drive cardiac development, such as Tbx5 , Gata4 , and Mef2c . Cardiac development, and its transcriptional regulators are also regulated by microRNAs (miRs). These small RNAs target the transcripts of genes in numerous cardiac cell types during embryonic development. We and others have shown that the miR-200 family modulates the transcripts of cardiogenic transcription factors (TFs) Tbx5 , Gata4 , and Mef3c. However, the relationship between these miRs and cardiogenic TFs during in vivo cardiac development is poorly understood. During cardiogenesis, miR-200 family members are highly expressed (E14.5) but reduced in adults (3mo) (Ct Value: miR-200a : 24.3 ± 0.59 v 38.3 ± 0.21; miR-200c : 25.0 ± 0.64 v 32.5 ± 0.16). Using our miR-200 family inhibitor mice models (PMIS), we have found these miRs are required for cardiac development. PMIS-miR-200 embryos are found with a ventral septal defect and poor ventricle wall development, which is lethal by e16.5. At e14.5, PMIS-miR-200 hearts have a significant increase in expression of Tbx5, Gata4, and Mef2c compared to Wild-Type. This induced expression of these TFs is seen within CMs of the ventricle at E14.5. snMulti-Omics of WT and PMIS-miR-200 hearts found a population of CMs enriched in the PMIS-miR-200 hearts. These CMs are marked by expression of Tbx5 , Nppa , and Sox5 . RNA velocity analysis found these CMs to be “progenitor-like” and associated with an early pseudotime. Expression of Tbx5 , Nppa , and Sox5 correlated along the pseudotime with the “progenitor-like” CMs. ATAC-seq showed enrichment of Tbx5 and Mef2c motifs within this new CM cell state. Conclusions: The miR-200 family is a modulator of cardiogenic TFs expression and activity during development. Inhibition of miR-200 induces a CM cell state with “progenitor-like” qualities. Future directions will determine the role of miR- 200 in adult cardiac disease, such as ischemic injury. Our work provides new insights into gene dosage, modulated by miRs, that is required and necessary during cardiac development.

Abstract We146: CHD8 is Essential for Postnatal Heart Function through Preserving Mitochondrial Integrity

Objective: Epigenetic regulation plays a crucial role in heart development and disease, although its mechanisms remain elusive. Known mutations in epigenetic regulators have been linked to cardiac disease. For instance, the mutation in the chromodomain helicase DNA-binding (CHD) protein 7 gene, which leads to CHARGE syndrome, is known to results in heart defects. However, the role of CHD8, an interacting partner of CHD7, in the heart has not been investigated. This study aims to explore the function of CHD8 in the heart. Methods and Results: We generated a cardiac-specific Chd8 conditional knockout mouse line (Chd8 cKO) by breeding Chd8 fl/fl mice with aMHC Cre mice. Over 60% of Chd8 cKO mice died between 7 and 9 months of age, with increased heart weight to bodyweight ratio and reduced cardiac function, increased fibrosis, and signs of dilated cardiomyopathy. Electron microscopy analysis revealed disordered sarcomeres and escalated mitophagy in CHD8-deficient hearts. Using Genome-wide transcriptome analysis though RNA sequencing, we discovered that genes involved in mitochondrial oxidative phosphorylation and muscle contraction were down-regulated in Chd8 cKO hearts. Proteins in the electron transport chain complexes were down regulated and mitophagy marker LC3 increased in the Chd8 cKO hearts compared with the control mice. Upregulated pathways include inflammatory response suggesting that the innate immune response may contribute to dilated cardiomyopathy. Conclusions: Our findings underscore CHD8 as a key regulator of cardiac function and regeneration by influencing chromatin accessibility of genes involved in mitochondrial oxidative phosphorylation and contractility. Overall, our study provides new insights into the epigenetic regulation of cardiac function and the CHD8’s role in heart development and disease. It implies that activating CHD8 may be a promising therapeutic strategy for treating dilated cardiomyopathy and other heart-related diseases.

Abstract Or122: Noncoding regulation of epicardial gene expression and epithelial-to-mesenchymal transition during heart development

During organogenesis, epicardial cells undergo epithelial-to-mesenchymal transition (EMT), contributing essential cell types and paracrine signalling to the growing heart. The epicardium is integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties in development. Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is established, the mechanisms underpinning its activation remain elusive. We identified two e volutionary c onserved r egions (ECRs) shared in mouse and human, located within intron 1 of Wt1 locus. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development. Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR or a deletion comprising both ECRs. Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation. Mendelian ratios indicated an overall underrepresentation of Wt1 ΔECR/ΔECR mutants in adulthood. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular and membranous defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal patterning of coronary artery stems in mutant hearts. Expression of Wt1 was markedly reduced in Wt1 ΔECR/ΔECR hearts but not kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue immunostaining revealed abnormal coronary vessel and innervation patterning in the subepicardium, as well as reduced EMT and myocardial non-compaction/hyper-trabeculation in Wt1 ΔECR/ΔECR . Collectively, we demonstrated a requirement for novel Wt1 intronic enhancers regulating locus activity and essential epicardial EMT-associated biological processes in normal heart development. Importantly, observation of septum and semilunar valve defects in Δ ECR hearts suggests an hitherto unrecognised role for WT1-driven EMT, opening new avenues of research to improve our understanding of congenital heart disease affecting at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.

Abstract We089: Numb Family Proteins in Epicardium Regulate Epicardial Cells Differentiation and Ventricular Patterning During Development

Background: Numb Family Proteins (NFPs), specifically two mammalian homologs Numb (denoted by Nb ) and Numblike (denoted by Nl ), have been evident to regulate essential cardiac functions during development. Previous studies reported NFPs as integral for epicardial cells (EpiCs) epithelial mesenchymal-transition (EMT). However, whether NFPs in epicardium regulate EpiCs differentiation to affect cardiac morphogenesis have not been explored. In this study, we investigated how NFPs in epicardium regulate EpiCs EMT and differentiation to modulate cardiac development and subsequently affect cardiac maintenance in adult stages. Method: Using mCherry:Numb knockin mouse model we observed enrichment of Numb in epicardium. We deleted Nb and Nl in epicardium via Tbx18 Cre/+ lineage and refer to this as EDKO. We harvested hearts at embryonic day 15.5/18.5 and assessed morphological and mechanistic differences between control and EDKOs. Conventional and Speckle tracking Echocardiography were used to examine adult heart structure and functions. Results: Our findings revealed that EDKO embryonic hearts display specific morphology with extended left ventricles. We found a surprising difference between female and male EDKOs in terms of survival to adulthood. Reduced EpiCs EMT and Cardiac Fibroblasts (CFs) migration distances, changes in CFs distribution have been observed in EDKO developing hearts. Reduced expression of smooth muscle cells (SMCs) and pericytes also was noticed in EDKOs. Strikingly, EDKO embryonic hearts show defects in ventricular compaction, ventricular wall patterning and in cell proliferation. Furthermore, when survived to adult stage, EDKO hearts revealed significant structural and functional changes over their aging. While digging into regulatory pathways, we found changes in Fibroblast growth factor receptors (Fgfrs)/p-Erk pathway and in activation of Notch1 in EDKO hearts. Conclusion: NFPs in epicardium regulate EpiCs differentiation into CFs and SMCs, and regulate ventricular wall morphogenesis during development. NFPs are also required to maintain cardiac structure and function at adult stages. Fgfrs/p-Erk pathway and Notch1 could be the possible mechanism behind cardiac morphogenetic regulation by NFPs in epicardium. Future aims: Further studies will identify downstream targets of NFPs’ regulation of EpiCs differentiation and ventricular wall patterning in embryonic hearts, and mechanistic pathways of epicardial NFPs’ role in adult hearts.

Abstract We082: Contribution of local hematopoiesis to endocardial cushion remodeling during development

Introduction: The cells that comprise the circulatory system not only share their origins but also mutually promote each other’s differentiation during the formation of functional circulatory system. We previously reported a subset of endocardial cells is hemogenic during early embryogenesis. They are enriched in the cushion region, the primordia of the cardiac valves and septa, where active remodeling via endothelial-mesenchymal transition takes place. Hemogenic endocardial cells undergo endocardial-hematopoietic transition (EHT) via Nkx2-5-dependent manner, suggesting that Drosophila tinman-dependent cardio-hematopoietic program is conserved in mammals. Methods: In this study, we analyzed the regulatory network of Nkx2-5-dependent endocardial hematopoiesis using a scRNA-seq data from wildtype and Nkx2-5-null embryonic hearts. In vivo genetic analysis were performed to confirm the candidate signal pathways driven under Nkx2-5 transcription. Results and Conclusion: Nkx2-5-null hearts were devoid of clusters for hemogenic endocardium and cushion endocardium. Interestingly, scRNA-seq analysis further revealed that genes related to Notch signaling pathway are significantly downregulated in Nkx2-5-null endocardium. A further gene network analysis identified that Dhrs3, an enzyme that attenuates retinoic acid (RA) signal by catalyzing the reduction of all-trans-retinaldehyde to all-trans-retinol, is a signature gene of the hemogenic endocardial cells downstream of Nkx2-5. Although RA signal is known to induce the formation of multipotent progenitor, our ex vivo hematopoietic colony forming assay revealed that macrophage formation is strongly inhibited by RA signal. Notch inhibition also suppressed the formation of macrophages. Consistently, in vivo forced activation of NICD drastically increased the number of CD41 + hemogenic endocardial cells as well as macrophages in the cardiac cushion. Strikingly, impaired hematopoiesis and cushion defects in the Nkx2-5-null heart were both rescued by overexpression of NICD, suggesting that Notch signaling promotes endocardial hematopoiesis downstream of Nkx2-5. Taken together, our study demonstrated that the Nkx2-5/RA/Notch signaling axis plays a pivotal role in EHT during early embryogenesis, thereby facilitating local tissue remodeling by inducing macrophage differentiation. The talk will include the latest findings on the specific role of endocardially-derived tissue macrophages.

Abstract We081: β1 integrins regulate cellular behavior and cardiomyocyte organization during ventricular wall formation

Introduction: Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. However, the mechanisms through which these cells organize themselves to establish the tissue architecture of trabeculae and the ventricular wall remain inadequately elucidated. Integrins are the primary cell surface receptors for adhesion to the ECM and mediate both inside-out and outside-in signal transduction pathways that control various cell functions, including proliferation, survival, migration, and gene expression. We found that the β1 integrin subunit (β1), encoded by Itgb1 , is highly expressed in all cardiac cell types. Hypothesis: β1 integrins play a crucial role in regulating cardiomyocyte behavior and organization during ventricular wall morphogenesis in mice. Methods and Results: We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/β integrins and their ligands in the embryonic heart. β1 and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, and fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1 , which encodes the β1, was deleted via Nkx2.5 Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts lack a trabecular zone but a thicker compact zone. The levels of hyaluronic acid and versican, essential for trabecular initiation, were not significantly different between control and B1KO. Instead, fibronectin, a ligand of β1, was absent in the myocardium of B1KO hearts. Furthermore, B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. Mosaic clonal lineage tracing showed that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. Conclusions: β1 is asymmetrically localized in the cardiomyocytes, and some of its ECM ligands are enriched along the luminal side of the myocardium, and fibronectin surrounds cardiomyocytes. β1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1 leads to loss of β1 and fibronectin and prevents cardiomyocytes from engaging the ECM network, resulting in failure to establish tissue architecture to form trabeculae.

Abstract We091: Maternal hyperglycemia disrupts human cardiac cell lineage differentiation

Maternal diabetes is associated with a 4-fold increased risk of offspring developing congenital heart defects (CHDs). It remains unknown how maternal diabetes interferes with cardiac cell lineage determination and leads to malformations in the heart. In this study, we aim to elucidate the cellular mechanisms by which maternal hyperglycemia causes high risk of CHDs in newborns. Here we leverage an in vitro hyperglycemic model using human induced pluripotent stem cells (iPSCs), which could recapture cardiac differentiation and cell lineage commitment during embryonic heart development. We collected differentiating cells at D5 (cardiac mesoderm), D10 (cardiac progenitors), and D14 (early cardiomyocytes) during cardiac differentiation under normal and hyperglycemic conditions, and performed single-cell transcriptomic analysis. We found that hyperglycemia significantly impedes cardiac differentiation of human iPSCs as robust cardiac differentiation is rarely observed in multiple iPSC lines (n=10) under hyperglycemia. At the cellular level, hyperglycemia interferes with cardiac differentiation of human iPSCs in response to WNT signaling activation, which is manifested by reduced number of cardiac mesoderm (MESP1+ PDGFRA+) cells at D5 of differentiation. In contrast, neural differentiation is enhanced under hyperglycemia, with a high proportion of neural cell lineage (SOX2+ PAX6+). At D10, differentiated neural cell lineage dominates the cell population under hyperglycemia at the expense of cardiac progenitors and early cardiomyocytes. At D14, the prevalence of neural lineage persist whereas early cardiomyocytes only accounts for a small portion of cell population under hyperglycemia. Moreover, we treated D30 iPSC-derived cardiomyocytes (iPSC-CMs) with high glucose concentration (25 mM) for 7 days and found that iPSC-CMs show reduced mitochondria respiration and ATP production, but elevated apoptosis and reactive oxygen species (ROS) generation. Together, our data suggest that maternal hyperglycemia could interrupt human embryonic heart morphogenesis through overriding WNT-medicated cardiac differentiation and promoting neural cell lineage determination by default.

Abstract We085: Origin and fate of CX3CR1 + cells in the heart: their unique contribution to cardiovascular cells

Introduction: During development, various cells intricately collaborate to form embryonic and fetal hearts. We can harness this naturally occurring generative mechanism to advance regenerative medicine for cardiac repair. Studies reported that C-X3-C Motif Chemokine Receptor 1 (CX3CR1) + cells play vital roles in cardiac development and disease. However, the origin and the fate of CX3CR1 + cells during cardiogenesis remain unclear. Specifically, the existence of CX3CR1 + cells before E8.5 and their commitment other than to macrophages have not been investigated. Methods: We investigated the origin and fate of CX3CR1 + cells using various knock-in reporter mice with CX3CR1 drivers for spatiotemporal genetic lineage tracing of CX3CR1 + cells. In addition, we developed a method to generate CX3CR1 + cells from differentiating mouse embryonic stem cells (mESCs). mESC-derived CX3CR1 + cells were characterized in vitro, in the fetal mouse heart ex vivo , and in the adult heart in vivo . Their heterogeneity and trajectory were analyzed via single cell RNA sequencing (scRNA-seq). Results: Genetic fate mapping of CX3CR1 + cells identified epiblasts at E6.5, the parietal endoderm at E7.0, yolk sac cells at E8.0, and cardiomyocytes at E9.5. Temporal genetic labeling (Cx3cr1-CreERT2;R26-tdTomato) showed that CX3CR1 + cells at E6.5 can contribute to cardiomyocytes (CMs) and endothelial cells (ECs) during prenatal development via both de novo differentiation and fusion with pre-existing CMs or ECs, respectively. In the adult heart, cells that have ever expressed Cx3cr1 comprised 13.5 ± 0.2% of CMs and 30.9 ± 1.3% of ECs. In addition, CX3CR1 + cells, which were differentiated from mESCs generated CMs, ECs and macrophages in vitro, in the fetal mouse heart ex vivo , and in the adult heart in vivo . Single cell RNA sequencing showed that Cx3cr1 + cells represent an intermediate cell population differentiating into the mesoderm from pluripotent stem cells. Conclusions: Our data demonstrate that CX3CR1 serves as a marker for a unique subset of progenitors that contribute to the formation of CMs and ECs as well as macrophages, providing new insights into the versatile role of CX3CR1 + cells in cardiogenesis.