Thursday 12 May
10:00

Thursday 12 May

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HOS
10:00 - 12:00

Hands on session & Gastruloids Workshop

Keynote Speakers: Lucille HOUYEL (Pediatric cardiologist) (Le Plessis Robinson, France), Monique JONGBLOED (Leiden, The Netherlands), Fabienne LESCROART (PI) (Marseille, France)
Auditorium
13:00

Thursday 12 May

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WOR
13:00 - 13:15

Welcome and opening remarks

Auditorium
13:15

Thursday 12 May

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PS1
13:15 - 15:00

Session I
Early heart development

Moderators: Sigolene MEILHAC (PI) (PARIS, France), Miguel TORRES (Researcher) (Madrid, Spain)
13:15 - 15:00 #29326 - 001. Spatiotemporal sequence of endocardium and myocardium specification in the mammalian primitive heart tube.
001. Spatiotemporal sequence of endocardium and myocardium specification in the mammalian primitive heart tube.

The primitive heart tube is formed by two layers: the contractile myocardium and the endocardium that lines the inside of the chamber. Previous studies show that, at the onset of gastrulation in mouse, chicken and zebra fish, these two populations have already segregated. Nevertheless, when and how these lineages divert during embryogenesis remains unknown. Here, we use prospective and retrospective clonal analysis in mouse embryos to define the temporal sequence of cell fate specification. Our results confirm that endocardium and myocardium precursors have already segregated just after they undergo gastrulation (embryonic day E6.75). Besides, we observe that multipotent clones induced at E6.25-E.6.5 give rise to other mesoderm lineages apart from endocardium and myocardium. This shows segregation must take place just before or at gastrulation and that a bipotent progenitor exclusive to myocardium and endocardium does not exist, or it does only very transiently. To learn about the location and dynamics of these progenitors, we used long-term live imaging to track cardiovascular progenitors from the differentiated hear tube back to their positions at the primitive streak. We found that the original positions of both precursor populations are intermingled, suggesting they ingress the primitive streak simultaneously. Finding how this key fate decision takes place will help to understand cardiac congenital diseases as well as provide a tool to ambition new tissue engineering and regenerative strategies.


Miquel SENDRA (Madrid, Spain), Morena RAIOLA, Katie MCDOLE, Léo GUIGNARD, Jorge N. DOMÍNGUEZ, Miguel TORRES
13:15 - 15:00 #30489 - 002. A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors.
002. A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors.

Precise regulation of transcriptional networks governs emergence of early cardiac precursor cells (CPCs) within developing mesoderm during gastrulation. We leveraged detection of early cardiac lineage transgenes within a single cell RNA sequencing time course of whole mouse embryos to identify emerging CPCs and describe their transcriptional profiles during gastrulation prior to organogenesis.  Mesp1, a transcription factor (TF) transiently expressed in CPCs emerging from the primitive streak, has been described as an early regulator of cardiac specification. We observed a perdurance of cardiac transgene-expressing cells in Mesp1 mutants, albeit posterior-laterally located, prompting us to investigate how genetic programs for cardiogenesis and mesoderm specification potentially progress independently of Mesp1. Although mutant CPCs fail to robustly activate markers of cardiomyocyte maturity and TFs critical for heart morphogenesis, mutant CPCs express some structural myocyte genes and exhibit transcriptional profiles resembling those of cardiac mesoderm cells progressing towards cardiomyocyte fates. These results reveal Mesp1-independent aspects of early CPC specification and underscore Mesp1’s role in insuring subsequent progress through cardiogenesis. Single cell chromatin accessibility analysis of embryos at this developmental breakpoint identifies a required shift from mesendoderm transcriptional networks to cardiogenic networks in the designation of early-stage cardiac gene programs. Our investigation indicates that early CPCs are being specified distinctly from the mesoderm up until the Mesp1-deficient regulatory landscape inhibits further cardiac lineage maturation.   


Alexis Leigh KRUP (San Francisco, USA), Sarah A. B. WINCHESTER, Sanjeev S. RANADE, W. Patrick DEVINE, Deepak SRIVASTAVA, Benoit G. BRUNEAU
13:15 - 15:00 #30530 - 003. Precardiac mesoderm auto-regulates second heart field cell fate via Wnt secretion.
003. Precardiac mesoderm auto-regulates second heart field cell fate via Wnt secretion.

Congenital heart defects are often restricted to derivatives of two distinct groups of cells — the first and second heart fields (FHF & SHF). Thus, it is crucial to understand the mechanisms controlling their development. Wnt signaling has been implicated in SHF proliferation, however, the source of such Wnts remains unknown. Previously, we found upregulation of Wnts and Wnt receptor/target genes in the FHF and SHF, respectively, suggesting that cardiac progenitors may regulate SHF cell fate. To test this, we deleted Wntless (Wls), a gene required for Wnt secretion, in precardiac mesoderm. Deletion of Wls in Mesp1+ cells resulted in formation of a single-chambered heart with compromised SHF development. This phenotype was recapitulated by deleting Wls in pan-cardiac progenitors. However, no defects were observed when deleting Wls in SHF progenitors. To gain mechanistic insights, we isolated Mesp1-lineage cells and performed single-cell RNA-sequencing. We found that Wls deletion dysregulates developmental trajectories of SHF cells, marked by impaired proliferation. These results demonstrate a critical role of precardiac mesodermal Wnts in SHF fate decisions, identifying the crucial role of heart field coordinated development in chamber formation.


Matthew MIYAMOTO, Suraj KANNAN, Xihe LIU, David SUH, Myo HTET, Tejasvi KAKANI, Sean MURPHY, Emmanouil TAMPAKAKIS, Peter ANDERSEN, Hideki UOSAKI, Chulan KWON, Biyi LI (Baltimore, USA)
13:15 - 15:00 #30510 - 008. Tbx5-sensitive cues guide early progenitors for cardiac septation.
008. Tbx5-sensitive cues guide early progenitors for cardiac septation.

Many congenital heart defects (CHDs) involve incomplete septation of the atria or ventricles, in isolation or as part of more complex lesions, such as atrioventricular canal (AVC) defects. Early cardiac progenitor populations have been identified that contribute later to specific anatomical structures that are affected in CHDs. In the developing mouse heart, we found that a Tbx5+/Mef2cAHF+ progenitor lineage is intricately arranged into a band of cells at the interface of the first and second heart fields. This cohesive, coordinated population is found at a morphogenetic nexus, forming a compartment boundary bisecting the interventricular septum (IVS) and extending to the interatrial septum (IAS) via the cardiac cushions. Conditional ablation of these septal progenitors caused IVS disorganization, AVC defects and right ventricular chamber hypoplasia. Reduced dosage of the cardiac transcription factor TBX5 disrupted the integrity of the boundary, leading to lineage mixing, disrupted cell alignment and patterning defects, resulting in ventricular septal defects (VSDs), AVC defects and atrial septal defects. In the setting of reduced TBX5 dosage, we found that genes encoding guidance cues, best known for axonal guidance, were dysregulated. We further observed that loss of either guidance cue caused VSDs, suggesting that these TBX5-dependent signals are necessary for orchestrating cardiac septation. Thus, we identify essential morphogenetic cues that guide early progenitors for cardiac septation, revealing insights into the developmental origins of genetically-susceptible CHDs.


Irfan KATHIRIYA (San Francisco, USA), Martin DOMINGUEZ, W. Patrick DEVINE, Kavitha RAO, Kevin HU, Jonathon MUNCIE, Swetansu HOTA, Bayardo GARAY, Diego QUINTERO, Piyush GOYAL, Sarah WINCHESTER, Benoit BRUNEAU
13:15 - 15:00 #30629 - 005. Nkx2.7 is a novel regulator of pharyngeal arch development.
005. Nkx2.7 is a novel regulator of pharyngeal arch development.

The clinical phenotypes associated with DiGeorge Syndrome illustrate a developmental link between cardiovascular and craniofacial morphogenesis. Fate mapping studies in mice and zebrafish support this notion given the identification of a multipotent progenitor in the cardiopharyngeal field that gives rise to the heart, branchiomeric muscles, and pharyngeal arch (PA) arteries. NKX2-5 is a key cardiac transcription factor associated with human congenital heart disease and mouse models of Nkx2-5 deficiency highlight critical roles in cardiac development. nkx2.5 and nkx2.7 are two NKX2-5 homologs expressed in zebrafish cardiomyocytes and PAs. We demonstrate that Nkx2.7 serves as a previously unappreciated, crucial regulator of the first and second PA derivatives. Our previous studies show that nkx2.7 is required in the late-differentiating cardiac progenitors to maintain ventricular identity. While expression of nkx2.7 can function redundantly in this role, nkx2.5 is unable to compensate for nkx2.7 in developing PA1- and PA2-derived anterior and posterior mandibular and interhyoid muscles and cartilage elements. Taken together, these results underscore the unique function of Nkx2.7 in regulating pharyngeal arch morphogenesis. To identify targets that are exclusively regulated by Nkx2.7, we employed cardiac-specific bulk RNA-seq in wild-type, nkx2.5-/-, and nkx2.5-/-;nkx2.7-/-  embryos and single cell RNA-seq data from microdissected PA tissues in wild-type and nkx2.7-/- embryos. Our complex computational analysis uncovers barx1 and nkx3.2 as downstream effectors of Nkx2.7 in mediating muscle and cartilage development. Through examination of the tissue-specific functions and targets of Nkx2.7, we expect to reveal exciting new pathways responsible for branchiomeric muscle and craniofacial abnormalities in patients.


Caitlin FORD (New York, USA), Carmen DE SEMA TOMÁS, Cynthia GAO, Uday RANGASWAMY, Michael SEE, Hieu NIM, Remo SANGES, Mirana RAMIALISON, Kimara TARGOFF
Auditorium
15:00 Break
15:30

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PS2
15:30 - 17:15

Session II
CHD Mechanisms I

Moderators: Antonio BALDINI (Professor) (Napoli, Italy), Didier STAINIER (Director) (Bad Nauheim, Germany)
15:30 - 17:15 #30484 - 006. Nuclear envelope integrity is essential for proper cardiac development.
006. Nuclear envelope integrity is essential for proper cardiac development.

Nuclear envelope integrity is essential for compartmentalisation of nucleus and cytoplasm. Importantly, mutations in nuclear envelope-encoding genes are the second-highest cause of familial dilated cardiomyopathy. One such nuclear envelope protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in heart remains poorly understood.

We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue and cellular analyses. High resolution episcopic microscopy was used for 3D reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided physiological assessment of Lem2 iCKO adult mice. 

We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels.

Our data suggest that Lem2 is critical for integrity at the nascent nuclear envelope in fetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by Lem2 loss, perhaps owing to a more established nuclear envelope and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general.


Jacob ROSS, Matthew STROUD (London, United Kingdom)
15:30 - 17:15 #29417 - 007. Dissecting the cell type-specific roles of Hand2 during cardiac development in zebrafish.
007. Dissecting the cell type-specific roles of Hand2 during cardiac development in zebrafish.

Cardiogenesis requires the integration of diverse cardiac cell types including the myocardial and endocardial cells that form the initial layers of the heart. Differentiation, patterning, and morphogenesis of cardiomyocytes depend on the transcription factor Hand2. However, it is unclear how Hand2 regulates these early developmental events. To investigate this question, we first performed single-cell RNA sequencing on hand2 expressing cells from the cardiac cone and linear heart tube stages, and found increased expression of immature cardiomyocyte markers and concomitantly decreased expression of mature cardiomyocyte markers in hand2 mutants. In addition to these expected phenotypes, we observed an increased percentage of endothelial cells in hand2 mutants. To investigate the roles of hand2 in the myocardial and endothelial cells, we generated a floxed allele of hand2 for conditional inactivation. Knocking out hand2 in myl7 expressing cells (i.e., cardiomyocytes) led to a late phenotype whereby the trabeculae mostly fail to form. Interestingly, knocking out hand2 in kdrl expressing cells (i.e., endothelial cells) resulted in cardia bifida, recapitulating the global hand2 mutant phenotype. Transcriptional analysis of the endothelial cells revealed that the loss of Hand2 leads to a dysregulation of PDGF signaling. Notably, overexpressing pdgfra in endothelial cells could partially rescue the cardia bifida phenotype in hand2 mutants.  Together, these data indicate that during cardiac development, Hand2 serves critical functions not only in cardiomyocytes but also in endothelial cells to drive cardiomyocyte specification and also direct the migration of myocardial progenitors towards the midline. 


Yanli XU (Bad Nauheim, Germany), Stefan GUENTHER, Guilherme VALENTE, Mario LOOSO, Didier STAINIER
15:30 - 17:15 #29481 - 009. Multimodal Single Cell Analysis Reveals Regulatory Mechanisms Underlying Cell Signaling Defects in DiGeorge Syndrome.
009. Multimodal Single Cell Analysis Reveals Regulatory Mechanisms Underlying Cell Signaling Defects in DiGeorge Syndrome.

Communication between myriad cell types during development underlies proper organ morphogenesis. During cardiac development, reciprocal signaling between diverse cells is essential and disruption leads to congenital heart malformations; however, mechanistic interrogation of temporally dynamic gene networks and cis regulatory elements in signaling and receiving cells has been limited. Here, we integrated single cell chromatin accessibility (scATAC-seq) and transcriptomics (scRNA-seq) over multiple stages to provide a comprehensive epigenomic landscape of diverse cell types including mesodermal progenitors and neural crest cells in the developing mouse heart. Integrated multiomics and machine learning methods defined cis regulatory elements as enhancers for genes initiating or responding to intercellular signaling. Disruption of TBX1, a transcription factor that functions non-cell autonomously in mesodermal cardiac progenitors to affect neighboring neural crest-derived cells, causes morphogenetic defects of the cardiac outflow tract in humans and offered an opportunity to determine consequences of dysregulated signaling at single cell resolution. In mice lacking Tbx1, broad closure of chromatin regions enriched in Tbx and multiple cardiac progenitor TF motifs within a narrow subset of mesodermal progenitors correlated with diminished transcription of multiple signaling factors such as FGF, retinoic acid, Wnt, Tgfb and Semaphorins. In response, we identified subsets of cardiac and craniofacial neural crest cells containing differentially accessible chromatin regions that suggested a failure of differentiation. Lastly, we found aberrant persistence of neural crest cells with markers of multipotency within the pharyngeal region. This study provides an atlas of spatiotemporally dynamic regulatory elements in cardiogenesis and a mechanistic framework for how disruptions in cell-cell communication affect morphogenetic decisions at single cell resolution.


Sanjeev RANADE (San Francisco, USA), Sean WHALEN, Ivana ZLATANOVA, Lin YE, Benjamin VAN SOLDT, Tomohiro NISHINO, Angelo PELONERO, Langley Grace WALLACE, Pawel PRZYTYCKI, Casey GIFFORD, Brian BLACK, Katie POLLARD, Deepak SRIVASTAVA
15:30 - 17:15 #30554 - 010. Single-cell transcriptomics uncovers a Tbx1-dependent genetic program controlling cardiac neural crest cell deployment and progression.
010. Single-cell transcriptomics uncovers a Tbx1-dependent genetic program controlling cardiac neural crest cell deployment and progression.

Cardiac neural crest cells (cNCCs) are required for outflow tract (OFT) septation and in pharyngeal arch arteries (PAAs) development. Inactivation of Tbx1, the gene for 22q11.2 deletion syndrome, results in failure of cNCCs deployment leading to congenital heart disease. We aim to elucidate the cell fate transitions of the cNCCs during their deployment to the OFT and PAAs in normal development and that depends on Tbx1. We performed single-cell RNA-sequencing (scRNA-seq) of NCCs from control and Tbx1 null mouse embryos. We discovered the transcriptional signature that defines cNCC populations and investigated the gene expression dynamics that regulates cNCC fate progression into smooth muscle cells of the OFT and PAAs. We discovered three distinct cNCCs populations that emerge between embryonic day (E)9.5 and E10.5, reflecting transcriptional heterogeneity of cNCCs. Our trajectory analysis indicates that cells expressing Tbx2 and Tbx3 transition to a population that expresses Isl1 and Gata3 that then differentiates into smooth muscle of the OFT. Tbx2/3 cNCCs also form smooth muscle cells of the PAAs and we found that both Tbx2 and Tbx3 in cNCCs are necessary for aortic arch branching development. In the absence of Tbx1, multiple genes are dysregulated in the Tbx2/3 population and the Isl1/Gata3 population is strongly reduced in size, reflecting a lineage progression failure. Comparative analysis of our scRNA-seq data from control and Tbx1 null embryos indicates an abnormal upregulation of BMP signaling in Tbx1 null embryos. BMP signaling overactivation could, in part, explain failed cNCC deployment and progression in Tbx1 null embryos.


Christopher DE BONO (New York, USA), Yang LIU, Alexander FERRENA, Deyou ZHENG, Bernice MORROW
15:30 - 17:15 #30609 - 004. Dissecting Mechanisms of Chamber-Specific Cardiac Differentiation and its Perturbation Following Retinoic Acid Exposure.
004. Dissecting Mechanisms of Chamber-Specific Cardiac Differentiation and its Perturbation Following Retinoic Acid Exposure.

Proper heart development requires specification and differentiation of multiple progenitor populations, and dysregulation of these processes can lead to congenital heart defects (CHDs). Furthermore, different forms of CHDs may be driven by defects in distinct progenitor subtypes, who’s heterogeneity remains incompletely understood. To understand the transcriptomic landscape of the developing heart we performed single-cell RNA sequencing (scRNASeq) at the cardiac crescent (E8.25), primitive heart tube (E8.75) and late heart tube (E9.25) stages using Foxa2-Cre;mTmG embryos, allowing us to label atrial/ventricular fated cells prior to and during chamber morphogenesis. Through RNA velocity and lineage trajectory tools we identify heart field progenitors in multiple differentiation states, and uncover the top dynamically regulated genes for each cell type, which represent putative drivers of cell-state transitions during differentiation. We find that clustering of myocardial cell types occurs primarily based on heart field progenitor origin, and that different progenitor populations contribute to ventricular or atrial identity through separate differentiation mechanisms. Furthermore, we find that differentiation of anterior or posterior second heart field (SHF) cells occurs through deployment of separate components of the cardiac gene-regulatory network. Lastly, we show that in utero exposure to exogenous retinoic acid (RA), which plays a role in atrial chamber specification and acts as a teratogen during development, causes defects in ventricular chamber size. scRNASeq of RA-exposed embryos demonstrated dysregulation in FGF signaling in anterior SHF cells and a shunt in differentiation towards formation of head mesenchyme, and defects in cell-cycle exit in myocardial progenitors. These data demonstrate the utility of comparative scRNAseq studies for understanding lineage relationships during development and revealing cell-specific sensitivity to perturbations.


David GONZALEZ (New York, USA), Nadine SCHRODE, Tasneem A.m EBRAMIN, Nicolas BROGUIERE, Guiliana ROSSI, Lika DRAKHLIS, Robert ZWEIGERDT, Matthias P. LUTOLF, Kristin G. BEAUMONT, Robert SEBRA, Nicole DUBOIS
Auditorium
17:15

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MAG
17:15 - 17:45

Memorial Adriana Gittenberger-de Groot

Chairman: Robert KELLY (PI) (Marseille, France)
Speaker: Robert POELMANN (Leiden, The Netherlands)
Auditorium
17:45

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PO1
17:45 - 20:00

Poster session I (even number posters)

17:45 - 20:00 #29336 - 048. Conserved transcription factors promote cell fate stability and restrict reprogramming potential in differentiated cells.
048. Conserved transcription factors promote cell fate stability and restrict reprogramming potential in differentiated cells.

The characterization of novel mechanisms safeguarding cell fate identity in differentiated cells is crucial to improve our understanding of 1) how differentiation is maintained in healthy tissues or altered in a disease state and 2) the basic mechanisms governing cell fate reprogramming and our ability to use this technology for regenerative purposes. Here, we report on the first identification of a generic and transcription factor-mediated mechanism controlling cell fate stability in differentiated cells via the concomitant regulation of chromatin accessibility and transcription of genes required for large-scale phenotypic changes. Importantly, resulting knowledge could be translated in vivo, as the targeted inhibition of these new fate-stabilizers could significantly improve direct cardiac reprogramming-mediated heart repair post-myocardial infarction in adult mice.


Maria MISSINATO, Sean MURPHY, Michaela LYNOTT, Yu-Ling CHANG, Pier Lorenzo PURI, Chulan KWON, Peter ADAMS, Li QIAN, Alessandra SACCO, Peter ANDERSEN, Alexandre COLAS (LA JOLLA, USA)
17:45 - 20:00 #29443 - 050. Comparison of the regenerative capacity of seven wild-type zebrafish strains reveals inter-strain variations in the wound healing process, cardiomyocyte proliferation and apoptosis levels following ventricular cryoinjury.
050. Comparison of the regenerative capacity of seven wild-type zebrafish strains reveals inter-strain variations in the wound healing process, cardiomyocyte proliferation and apoptosis levels following ventricular cryoinjury.

Teleost fish maintain the capacity to regenerate lost cardiac tissue throughout adulthood. However, recent reports suggest that both inter- and intra-species variations exist. Based on these studies, we hypothesised that there is strain-dependent variability in zebrafish heart regeneration. To test this, we characterised the regenerative response of seven wild-type zebrafish strains (AB, NA, SAT, TL, TU, WIK, KCL). Analysing morphology, wound size and composition, apoptosis, proliferation and gene expression, we identified significantly different characteristics between the NA, TL, TU and WIK strains during regeneration. The WIK strain showed increased proliferation in cardiomyocytes bordering the wound at 7dpci . Accordingly, RNA-seq showed upregulation of cell cycle and DNA replication-related genes at 7dpci in the WIK compared to the other strains, resulting in the strongest wound size reduction between 7- and 21dpci. However, no further reduction was observed between 21- and 90dpci, with scar remaining in all WIK hearts at 90dpci, likely caused by elevated ECM gene expression and increased collagen presence in the 7dpci WIK wound. Correlation analysis indeed identified collagen deposition at 7dpci as a predictor of the 90dpci regenerative outcome. At 90dpci, the NA line showed the highest percentage of scar-free hearts whereas 80% of the TU hearts failed to regenerate the compact wall. Comparing gene expression between the TU and NA showed a reduced activation of the metabolic genes required for regeneration in the TU after injury. In conclusion, comparing seven wild-type zebrafish strains has allowed us to identify correlations between different cellular processes occurring during regeneration.


Konstantinos LEKKOS (Oxford, United Kingdom), Zhilian HU, Jana KOTH, Madeleine LEMIEUX, Katherine BANECKI, Helen POTTS, Gennaro RUGGIERO, Mathilda MOMMERSTEEG
17:45 - 20:00 #29527 - 052. Examining the interaction between metabolism and heart regeneration in Mexican cavefish.
052. Examining the interaction between metabolism and heart regeneration in Mexican cavefish.

Unlike humans, certain species possess a robust ability to regenerate their hearts. We have identified the Astyanax mexicanus fish as a unique model for heart regeneration research. While Astyanax surface fish regenerate their heart after injury, their cave-dwelling counterparts cannot and, like humans, form a permanent scar. This model provides an opportunity for direct comparison of natural regeneration versus scarring within a single species. 

We have previously shown that there are similar levels of cardiomyocyte proliferation at the wound border zone in both surface fish and cavefish. However, BrdU pulse-chase revealed a higher number of BrdU-positive cardiomyocytes in surface fish compared to cavefish which may be a result of defective cytokinesis resulting in a failure to regenerate. Indeed, examining cavefish cardiomyocytes showed higher DNA content in comparison to the surface fish heart. 

Using RNA-seq to investigate mechanisms underlying cardiomyocyte proliferation in Astyanax hearts after injury, we identified metabolism-related genes among those most differentially expressed. Specifically, glycolytic genes were highly upregulated in surface fish border zone cardiomyocytes. Moreover, glucose levels were reduced in the cavefish heart after injury. Seahorse metabolic assays revealed a reduction in anaerobic glycolysis in the cavefish heart after injury but no difference in oxygen consumption rates or fatty acid utilisation between the fish. This was accompanied by reduced ATP in cavefish hearts compared to surface fish after injury. Inhibiting glucose metabolism in surface fish using 2-deoxy-d-glucose increased cardiomyocyte DNA content showing a similar failure in cell cycle progression to the cavefish heart.

Overall, this suggests an important role for glucose metabolism in cardiomyocyte cell cycle progression, and that inability to maintain sufficient ATP in the cavefish heart might be linked to their inability to support a regenerative response.


Rita ALONAIZAN (Oxford, United Kingdom), William STOCKDALE, Helen POTTS, Madeleine LEMIEUX, Konstantinos LEKKOS, John WALSBY-TICKLE, Mathilda MOMMERSTEEG
17:45 - 20:00 #30249 - 054. Novel Role of Proteoglycan Sulfation as a Barrier to Direct Cardiac Reprogramming.
054. Novel Role of Proteoglycan Sulfation as a Barrier to Direct Cardiac Reprogramming.

Direct cardiac reprogramming (CR) of fibroblasts into induced cardiomyocytes (iCMs) represents a promising therapeutic avenue to promote heart repair post injury. Our lab previously identified four transcription factors playing a conserved role as barriers to CR. Remarkably, knock-down of these barriers increases CR up to six-fold as compared to MGT (MEF2C; GATA4; TBX5) overexpression alone. Next, mechanistic and functional, exploration of downstream transcriptional targets led to the identification of SULF2 and CHST2 as novel regulators of CR, indicating a potential novel role for proteoglycan sulfation-modifying enzymes as regulators of cell fate reprogramming. In this context, functional screening of all genes involved in the regulation of PG modification and sulfation in CR assays, identified CHST7 as most potent and evolutionarily conserved barrier to CR. Collectively, these data confirm that PG sulfation plays a previously unrecognized role in the regulation of CR and cell fate stability in differentiated cells.


Michaela LYNOTT (San Diego, California, USA), Alexandre COLAS
17:45 - 20:00 #30320 - 056. Interleukin 4 and 13 Signaling in Macrophages Regulates Cardiac Regeneration and Capillary Morphology in Neonatal Mice.
056. Interleukin 4 and 13 Signaling in Macrophages Regulates Cardiac Regeneration and Capillary Morphology in Neonatal Mice.

Heart failure (HF) is a prevalent disease, projected to affect over 8 million Americans by 2030. Disease burden continues to be high, despite therapy. Thus, new strategies that target HF progression should be developed. Our lab employs the neonatal mouse model of heart regeneration to identify reparative pathways to apply them to HF. We previously showed that deletion of Interleukin 13 (IL13) or its receptor, IL4Rα, impair heart regeneration. However, the cell types mediating this response are not known. IL13 shares the receptor IL4Rα with IL4 and both cytokines are known to polarize macrophages into a pro-reparative phenotype. Here, we hypothesize that IL4/13 signaling to macrophages promotes neonatal heart regeneration after myocardial infarction (MI) and we explore the cell types producing IL13/IL4 during cardiac regeneration. We generated a genetic model whereby IL4Rα is depleted in macrophages by crossing IL4Rα floxed (IL4Rαfl/fl) mice with transgenic mice whereby Cre recombinase is driven by the CX3CR1 promoter (CX3CR1Cre). We performed MI on P1 mice and assessed cardiac function by ultrasonography 21 days post-injury. We found that Cre-positive mice had a lower ejection fraction compared to Cre-negative controls. Additionally, we found smaller capillaries in the myocardium of Cre-positive mice. To identify the cellular source of IL4 and IL13 in the heart we used fluorescent reporter mouse lines; IL4-green fluorescent protein and IL13-yellow fluorescent protein. In homeostasis, we found IL4 expression in innate lymphoid cells (ILCs) and T cells. Following P1 MI, IL13 was expressed primarily in ILCs and IL4 in ILCs and myeloid cells. This suggests a novel role for ILCs during cardiac regeneration. In addition, we found that lack of IL4/13 signaling in macrophages impairs cardiac function and capillary morphology after MI in neonatal mice. Future studies will be aimed to identify the downstream signaling in macrophages that mediates this reparative response.


Santiago ALVAREZ-ARGOTE (Milwaukee, USA), Caitlin C. O'MEARA
17:45 - 20:00 #30422 - 058. Parasympathetic Axon Development, Disease, and Neuroregeneration in the Cardiac Ventricles.
058. Parasympathetic Axon Development, Disease, and Neuroregeneration in the Cardiac Ventricles.

The heart relies on opposing signals from sympathetic and parasympathetic nerves to guide developmental, homeostatic, and repair functions. However, the cardiac innervation patterning that regulates these essential functions has been relatively unexplored. This is partially a result of technical limitations, as histological sectioning prevented nerve morphology to be accurately examined. In this work, we are analyzing the spatial patterning of cardiac innervation by employing organ clearing, whole-mount staining, and three-dimensional (3D) imaging techniques. 

 

We are particularly interested in mapping the parasympathetic nerves, which are thought to only innervate the atria and nodes; however, their contribution to ventricular innervation has not been analyzed in whole-mount and 3D. Excitingly, our results demonstrate for the first time that dense parasympathetic axon bundles innervate the cardiac ventricles. Moreover, preliminary data demonstrates that parasympathetic and sympathetic nerve fibers are intertwined in the ventricles through a developmental patterning mechanism that we are currently exploring. 

 

The intricate neuronal networks are susceptible to injury and fatal misfiring following an adult myocardial infarction (MI). Remarkably, an MI in the neonatal mouse results in robust regeneration and restoration of autonomic functions. Our lab discovered that neonatal heart regeneration is regulated by nerve signaling. Here, we demonstrate reinnervation of the regenerating myocardium following injury in contrast to non-regenerating hearts. Moreover, these newly formed nerves exhibit significant crossover with smooth muscle positive arteries, suggesting potential physiological restoration of neurovascular coupling. The results of our research will expand our knowledge of the development and plasticity of the cardiac nervous system during homeostasis, disease, and regeneration. 


Rebecca SALAMON (Madison, USA), Ahmed MAHMOUD
17:45 - 20:00 #30442 - 060. Cell-reprogramming and myocardial regeneration in a pig model of right-ventricular failure.
060. Cell-reprogramming and myocardial regeneration in a pig model of right-ventricular failure.

Heart failure is a major burden to our societies. Besides patients with left ventricular dysfunction following ischemic insults or hypertension, progress in pediatric surgery to repair cardiac malformations has led to a growing population of now-adult congenital heart diseases (CHD) patients. These patients with right ventricular (RV) failure are left without any efficient pharmacological therapeutic approach. Cell therapy has been an option to regenerate the myocardium although the mechanism of action of such an approach remains questionable.

Here, we used human embryonic stem cell-derived cardiac Nkx2.5+ progenitor cells seeded in a collagen-based patch covering the whole RV to regenerate failing RV of a pig model of repaired tetralogy of Fallot. We report that these cells migrate within the myocardium while reversing the interstitial fibrosis. They then engraft and fully differentiate into small clusters of fetal-like human myocytes within the myocardium. Degradation of the fibrotic extracellular matrix triggers an inflammatory reaction involving resident macrophages releasing cytokines in the neighborhood of myocytes. This leads to the activation of pig myocytes' inflammasome and their Nfkb-dependent reprogramming into Oct4+ cells. The reprogrammed myocytes redifferentiate and proliferate around human myocytes. Altogether, the graft of human CPC triggers a reprogramming of pig myocytes and in turn endogenous regeneration and improved contractility of the RV. 


Virginie LAMBERT (Paris), Ambre DELERIS, Fahd TIBOURTINE, Virginie FOUILLOUX, Pauline BRIDGE, Michel PUCEAT
17:45 - 20:00 #30445 - 062. Antigen presentation is critical for zebrafish cardiac regeneration.
062. Antigen presentation is critical for zebrafish cardiac regeneration.

Post-ischemic tissue remodeling involves the formation of a permanent scar that impairs cardiac function.  Unlike mammals, zebrafish can regenerate its heart by forming new functional tissues without a persistent scar.  The severity of tissue damage and the regenerative capacity depend on the quality, extent and temporal dynamics of the immune response.  However, how the immune system regulates regeneration, particularly how the antigen presentation-adaptive immunity axis plays a role in this process, remains to be elucidated.

In the present study, scRNA-Seq revealed a strong antigen presentation signature in the injured zebrafish heart, with distinct antigen-presenting cell clusters at different stages of the regenerative process.  Therefore, we hypothesized a role for the adaptive immune response during cardiac regeneration.  Indeed, we observed the infiltration of T-cells, especially Cd4+ T-cells, within the injured tissue at intermediate stages of regeneration.  Genetic targeting of Cd74, a key component of MHC class II antigen presentation, dampened regeneration as evidenced by impaired cardiomyocyte cell cycle re-entry and defective scar formation/resolution.  Pharmacological inhibition of T-cell activation further confirmed these data.  Importantly, these defects were accompanied by changes in T-cell populations and by an overall faulty immune response.  Further transcriptomic and immunohistochemical analyses revealed the involvement of the MAPK signaling pathway in the regulation of the regenerative response by antigen presentation.

Our work highlights the importance of antigen presentation in modulating the spectrum of regenerative/reparative outcomes post-cardiac injury.  Ultimately, these data will help identify mechanisms with the potential to ameliorate adverse remodeling and improve key aspects of mammalian heart regeneration.


João CARDEIRA-DA-SILVA (Bad Nauheim, Germany), Stephan LATTING, Stefan GÜNTHER, Michail YEKELCHYK, Bo HU, Janita MINTCHEVA, Philipp JUNKER, Didier STAINIER
17:45 - 20:00 #30469 - 064. 14-3-3 binding motif phosphorylation disrupts Hdac4 organized inhibitory condensates to stimulate cardiac reprogramming.
064. 14-3-3 binding motif phosphorylation disrupts Hdac4 organized inhibitory condensates to stimulate cardiac reprogramming.

Limited understanding of the molecular mechanisms of induced cardiomyocyte (iCM) reprogramming is a key obstacle preventing its effective clinical applications. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates iCM formation. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and epigenetic factors. Akt1 kinase and PP2A phosphatase are a key writer and eraser of the PC14-3-3 code, respectively and PC14-3-3 activation induces iCM formation with only Tbx5 but without Mef2c and Gata4. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We further discover that key PC14-3-3 embedded factors, such as Mef2c, Nrip1, and Foxo1, form inhibitory nuclear condensates with Hdac4 under hypo-phosphorylation state and PC14-3-3 activation disrupts these condensates to promote cardiac gene expression. This study provides a framework in decoding post-translational modifications for cell reprogramming and organ regeneration.


Liu LIU (Ann Arbor, USA), Zhong WANG
17:45 - 20:00 #30488 - 066. Role of endothelial Tal1 during zebrafish heart regeneration: from single cell transcriptomic analysis to a functional study.
066. Role of endothelial Tal1 during zebrafish heart regeneration: from single cell transcriptomic analysis to a functional study.

Objectives: Unlike mammals, zebrafish are able to regenerate their heart after an injury at all stages of life. It has been identified that this process involves cardiomyocyte dedifferentiation/proliferation, but the implication of interstitial cell types during heart regeneration remains poorly understood. Our study aims to describe the non-myocyte cell types present in the heart during regeneration in adult zebrafish as well as focus on the functional role of endothelial Tal1 expression in this process.

Methods: We performed single cell RNA sequencing (scRNAseq) on unamputated, 3-, 7- and 14-days post amputation (dpa) zebrafish hearts to provide a transcriptomic analysis of the zebrafish regenerating heart. We identified genes of interest from the dataset obtained. We then used a zebrafish conditional line expressing a dominant negative isoform of Tal1 (DNTal1) specifically in endothelial cells. After validating our DNTal1 line with a developmental characterization, adult fish were amputated and their hearts were extracted at different timepoints to be fixed and stained in order to assess their regenerative abilities.  

Results: Using scRNAseq, we described the different interstitial cell types present in the zebrafish heart and the changes they undergo during heart regeneration. We observed an increase in the number of Tal1 expressing endothelial cells during heart regeneration compared to unamputated controls. After validating our zebrafish DNTal1 line showing it exerts cardiac developmental defects already described in the literature, we saw DNTal1 expression inhibited heart regeneration primarily by disrupting the formation of the regenerating vascular plexus.

Conclusion: We showed Tal1 expression is essential for the endothelial regenerative response and its inactivation leads to a failure of regeneration.


Laura ROLLAND (Montpellier), Alenca HARRIGNTON, Adèle FAUCHERRE, Jourdano MANCILLA ABAROA, Thomas MOORE-MORRIS, Chris JOPLING
17:45 - 20:00 #30512 - 070. A small molecule screen identifies novel activators of epithelial to mesenchymal transition in human epicardial cells.
070. A small molecule screen identifies novel activators of epithelial to mesenchymal transition in human epicardial cells.

Upon ischemic cardiac injury, the epicardium, the outer layer of the heart which is essential for cardiac development, becomes re-activated and displays reparative potential. In this process, epicardial epithelial-to-mesenchymal transition (epiMT) is an essential step. To understand and increase epicardial activation, we aim to identify novel epiMT-inducing pathways by performing a small molecule screen.

Primary human epicardial cells were derived from human heart auricles. These epicardial derived cells (EPDCs) were cultured as epithelial-like cells maintaining a cobblestone morphology, and could be induced to undergo EMT by adding e.g. TGFbeta. Using this cell culture model, a phenotypic screen was performed on epithelial-like EPDCs using the LOPAC1280 small molecule library to identify epiMT-inducing compounds. The screen was performed 3 times to exclude patient variability. EpiMT was confirmed using αSMA-positive immunostaining as a hallmark for a phenotypic switch to a mesenchymal cell.
After validation of the positive hits, five compounds were selected that reproducibly induced epiMT, as shown by: 1) a phenotypic switch from epithelial (cobble) to mesenchymal (spindle-shaped) cells, 2) a decrease in CHD1 expression, and 3) an increase in EMT-related transcription factors (Snail, Slug) and mesenchymal markers (a-SMA, PSTN). To further establish a potential mechanism, cells were treated for 3 hours with the two most promising compounds and were subjected to RNA sequencing. These results suggest novel EMT regulators in human epicardium which are currently being validated.

In conclusion, high-throughput experiments using human primary epicardial derived cells to identify novel epiMT-inducing compounds is feasible. Using this model, we have identified several novel inducers of epiMT.
This work is funded by the DHF (2017T059, to AMS)


Esther DRONKERS, Tessa VAN HERWAARDEN, Esmee GROENEVELD, Marie-Jose GOUMANS, Anke SMITS (Leiden, The Netherlands)
17:45 - 20:00 #30535 - 072. FGF10 gene transfer therapy promotes cardiac regeneration and repair.
072. FGF10 gene transfer therapy promotes cardiac regeneration and repair.

The stimulation of terminally differentiated cardiomyocyte proliferation represents one of the main therapeutic approaches for heart regeneration and repair. We uncovered a role for the Fibroblast Growth Factor 10 (FGF10) signalling in regulating both fetal and adult cardiomyocyte proliferation. Using Fgf10 gain and loss of function mouse models together with experimental mouse model of myocardial infarction (MI) we demonstrated FGF10 regenerative potential.

Our study aims to determine the clinical relevance of FGF10 therapy for heart regeneration. Thus, using AAV strategy, we aim to evaluate Fgf10 gene transfer therapeutic impact after myocardial infarction (MI).

We first aimed to determine the most efficient route of delivery for AAV9 administration by considering short- and long-term heart expression but also side organ targeting. We thus analyzed the spatiotemporal pattern of Fgf10 expression following AAV9-Fgf10 injection (retro-orbital or tail vein) with 1011 viral particles of either AAV9-GFP or AAV9-Fgf10Our results reveal that tail-vein administration route is more appropriated for a rapid, long term and efficient cardiac FGF10 treatment, with a pic of Fgf10 expression at 3 weeks post injection and a maintained expression 10 weeks post-injection. In order to investigate the impact of AAV9-Fgf10 administration following MI on cardiac function and remodeling, mice subjected to MI were tail vein-injected with 1011 particles of either AAV9-GFP or AAV9-Fgf10 at the time of surgery. Our preliminary experiments indicate that AAV9-Fgf10 treatment promotes cardiomyocyte proliferation and prevents cardiac fibrosis infiltration. Moreover, echocardiographic measurements show that AAV9-Fgf10 treatment preserves cardiac remodeling and function post-MI.

Altogether, our results are thus of particular interest regarding the use of AAV9-Fgf10 strategy as a clinical perspective to promote heart repair following myocardial injury.


Fabien HUBERT (Marseille), Sandy PAYAN, Francesca ROCHAIS
17:45 - 20:00 #30544 - 074. Interplay between calcium cycling and sarcomeres directs cardiomyocyte redifferentiation and maturation during regeneration.
074. Interplay between calcium cycling and sarcomeres directs cardiomyocyte redifferentiation and maturation during regeneration.

A promising strategy to repair the injured mammalian heart is by the induction of cardiomyocyte dedifferentiation followed by their proliferation. Thus, there is now a focus on identifying factors and pathways that can drive the production of new cardiomyocytes through proliferation. However, a largely underappreciated concept is the redifferentiation and maturation of cardiomyocytes following proliferation.  This becomes particularly prominent as current methods to induce cardiomyocyte proliferation often result in cardiomegaly due to uncontrolled cell numbers. Unlike mammals, the zebrafish adult heart can robustly regenerate its heart following injury.  Here, we have studied zebrafish heart regeneration to discover an essential mechanism that restricts proliferation and stimulates the redifferentiation and maturation of cardiomyocytes after injury. Using an ex vivo imaging technique that we developed, we observed differences in intracellular calcium dynamics in cardiomyocytes located at the wound border zone, where cardiomyocyte proliferation is induced. These changes in calcium dynamics provided evidence that after a period of strong proliferation, cardiomyocytes redifferentiate and mature. An adaptor protein lrrc10 was identified to be able to specifically halt proliferation and promote cardiomyocyte redifferentiation. Finally, these maturation mechanisms were similarly observed in mouse and human iPSC-derived cardiomyocytes. Hence demonstrating that rather than being a passive process, redifferentiation is actively regulated and highlights the importance on also determining its underlying mechanisms in order to generate fully functional cardiomyocytes.


Phong NGUYEN (Utrecht, The Netherlands), Iris GOOIJERS, Mara BOUWMAN, Jeroen BAKKERS
17:45 - 20:00 #30547 - 076. Live imaging of zebrafish cardiac slices to study heart regeneration.
076. Live imaging of zebrafish cardiac slices to study heart regeneration.

The adult zebrafish is able to fully regenerate its heart after injury, making it an attractive model to identify and study mechanisms that regulate heart regeneration. In addition, a wide range of transgenic tools are available for zebrafish which allows the live imaging of cellular events. Thus far, live imaging in adult zebrafish have been hampered by the opacity and movement of the heart. In addition, ex vivo cultures don’t allow for deep tissue imaging and show rapid cell death. To overcome these issues, we present the use of cardiac slices of the zebrafish heart to study heart regeneration ex vivo. Cardiac slices can be cultured for several days while retaining characteristics of the in vivo heart. We have used these slices to perform live imaging on proliferating adult cardiomyocytes within their native tissue context for the first time. Currently, we are expanding the use of cardiac slice cultures to study the dynamics of calcium cycling and energy metabolism to get a better understanding of the role of these processes during heart regeneration. In conclusion, culturing cardiac slices of the adult zebrafish heart allows us to gain temporal insights of cardiomyocyte biology during physiological and pathological conditions. 


Iris GOOIJERS, Hessel HONKOOP (Utrecht, The Netherlands), Phong NGUYEN, Jeroen BAKKERS
17:45 - 20:00 #30567 - 078. Distinct epicardial gene regulatory programmes drive development and regeneration of the zebrafish heart.
078. Distinct epicardial gene regulatory programmes drive development and regeneration of the zebrafish heart.

Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart can fully regenerate following injury. Reactivation of cardiac developmental programmes is considered key to successfully regenerating the heart, yet the regulatory elements underlying the response triggered upon injury and during development remain elusive. Organ-wide activation of the epicardium is essential for zebrafish heart regeneration and is considered a potential regenerative source to target in the mammalian heart. Here we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardium by integrating gene expression profiles with open chromatin ATAC-seq data. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programmes driving each of these processes, which were largely distinct. We identified wt1a, wt1b, and the AP-1 subunits junbb, fosab and fosb as central regulators of the developing network, whereas hif1ab, zbtb7a, tbx2b and nrf1 featured as putative central regulators of the regenerating epicardial network. By interrogating developmental gene regulatory networks that drive cell-specific transcriptional heterogeneity, we tested novel subpopulation-related epicardial enhancers in vivo. Taken together, our work revealed striking differences between the regulatory blueprint deployed during epicardial development and regeneration. These findings challenge the dogma that heart regeneration is essentially a reactivation of developmental programmes, and provide important insights into epicardial regulation that can assist in developing therapeutic approaches to enable tissue regeneration in the adult mammalian heart.


Michael WEINBERGER, Filipa SIMOES (Oxford, United Kingdom), Tatjana SAUKA-SPENGLER, Paul RILEY
17:45 - 20:00 #30569 - 080. The role of RUNX1 in CM cell cycle activity and its impact on cardiac regeneration.
080. The role of RUNX1 in CM cell cycle activity and its impact on cardiac regeneration.

Factors responsible for cardiomyocyte (CM) proliferation may serve as a potential therapeutic to stimulate endogenous myocardial regeneration. It is established that RUNX1 induction in CMs increases after injury. Here, we examine the effect of RUNX1 on CM cell cycle and establishment of the presumed proliferative-competent Mononuclear Diploid CM population (MNDCM) during postnatal development and cardiac regeneration using both CM-specific gain-and loss of function mouse models. We hypothesize that RUNX1 overexpression (OE) increases CM cell cycle activity with expansion of the MNDCMs, thereby extending the neonatal regenerative window and positively impacting adult cardiac remodeling post injury. 

During postnatal development, RUNX1 KO decreased postnatal CM cell cycle activity, while RUNX1 OE extended the period of cell cycle activation. This extension observed in RUNX1 OE mice is complete with cytokinesis resulting in an expansion of the MNDCMs and total CM endowment. To determine whether RUNX1 could similarly regulate CM cell cycle in a regenerative and non-regenerative model, we induce P6 and 8-week MIs to measure cell cycle activity and cardiac function post injury. RUNX1 OE neonatal mice with a P6 MI displayed no difference in CM cell cycle activity 7 days post injury compared to control littermates. However, RUNX1 OE in adult mice with an 8-week MI showed increased CM cell cycle activity with completion of cytokinesis 2 weeks post injury and limited improvement in cardiac function 28 days post injury. RUNX1 influences CM cell cycle activation in the context of normal postnatal development and adult MI. Conversely, this phenomenon does not appear to translate to the neonatal injury context. We are examining this possible discrepancy with further experiments to fully understand the role of RUNX1 in CM cell cycle activity and its impact on cardiac regeneration.


Kaelin AKINS (Milwaukee, USA), Samantha SWIFT, Mary KOLELL, Michael FLINN, PHD, Caitlin O'MEARA, PHD, Michaela PATTERSON, PHD
17:45 - 20:00 #30580 - 082. Macrophages regulate cardiomyocyte repopulation of fibrotic tissue during zebrafish heart regeneration.
082. Macrophages regulate cardiomyocyte repopulation of fibrotic tissue during zebrafish heart regeneration.

The last two decades of research have significantly increased our understanding of mechanisms regulating zebrafish heart regeneration.  However, an aspect that remains largely underexplored is how newly proliferated cardiomyocytes invade and repopulate fibrotic tissue following cryoinjury.  We have previously shown that blocking the function of AP-1 transcription factors leads to defects in regeneration, partly due to a defect in cardiomyocyte protrusion and invasion into injured tissue.

 

Here, we show that cardiomyocyte protrusion peaks at 10 days post cryoinjury, shortly following the peak in cardiomyocyte proliferation.  Furthermore, using live imaging of myocardial slices, we observe that border zone (BZ) cardiomyocytes are dynamic, extending multiple protrusions into the collagen-containing injured area.  This dynamic behavior is accompanied by collagen degradation by BZ cardiomyocytes, which may be regulated by Mmp14b, a membrane-tethered matrix metalloprotease with collagen-degrading ability.  In addition, we find that macrophages are very closely associated with protruding cardiomyocytes at the BZ.  In irf8 mutants, which lack macrophages, we observe a decrease in the length of cardiomyocyte protrusions while the number of extended protrusions remains unchanged.  Single-cell RNA-seq of BZ cells suggests that both macrophages and cardiomyocytes contribute to regulation of the BZ microenvironment to promote invasion of cardiomyocytes to replenish lost tissue following injury.

 

Altogether, our data reveal a crosstalk between BZ cardiomyocytes and macrophages to facilitate repopulation of injured tissue by proliferating cardiomyocytes.  Detailed understanding of these mechanisms may have therapeutic implications, e.g., to promote the engraftment of exogenous cardiomyocytes following myocardial infarction in the mammalian heart.


Arica BEISAW, Arica BEISAW (Heidelberg, Germany), Julia DALLMANN, Stefan GUENTHER, Till LAUTENSCHLAEGER, Didier Yr STAINIER
17:45 - 20:00 #30595 - 084. Modest upregulation of Tbx5 stimulates prenatal ventricular cardiomyocyte proliferation.
084. Modest upregulation of Tbx5 stimulates prenatal ventricular cardiomyocyte proliferation.

Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital heart defects and conduction disorders, whereas slightly increased levels of TBX5 in human heart samples have been associated with atrial fibrillation. To gain insight into the impact of slight dosage changes of Tbx5 in vivo, we deleted the mouse orthologue of a conserved atrial fibrillation-associated regulatory region in the TBX5 locus (RE(int)-/- mice). Postnatal RE(int)-/- mouse atria showed slightly increased Tbx5 expression levels (30%) and increased susceptibility to atrial arrhythmia. Strikingly, we observed increased Tbx5 (30%) in the ventricles before birth, accompanied by increased heart size. We observed an increase in fetal cardiomyocyte proliferation rates specific to the left ventricle. Transcriptional profiling of ventricles of fetal control and RE(int)-/- mice revealed induction of several cell cycle related genes and Prrx1, a transcription factor associated with atrial fibrillation. When expression of Prrx1 was reduced by introducing a Prrx1 enhancer deletion in RE(int)-/- mice, we observed a normalization of heart size. These data indicate prenatal cardiomyocyte proliferation rates are Tbx5 dosage sensitive and act in part through moderating Prrx1 levels. To examine the potential of Tbx5 to induce postnatal cardiomyocyte renewal, AAV9-TNNT2-TBX5 was retro-orbitally administered to juvenile mice. Preliminary results show cardiomyocyte-specific expression of TBX5, and induction of target genes (Gja5, Nppa) and genes involved in proliferation. Future experiments will focus on the capacity of Tbx5 to induce cardiomyocyte proliferation and the underlying mechanisms.


Fernanda M. BOSADA, Alexandra GIOVOU (Amsterdam, The Netherlands), Bastiaan BOUKENS, Gerard J.j BOINK, Monika GLADKA, Vincent M. CHRISTOFFELS
17:45 - 20:00 #30635 - 086. Investigating the role of endocardial cell heterogeneity and EndoMT in the regenerative zebrafish heart.
086. Investigating the role of endocardial cell heterogeneity and EndoMT in the regenerative zebrafish heart.

Cardiovascular diseases account for nearly 18 million deaths per year, and majority of these deaths occur due to myocardial infarction (MI).  To augment cardiac repair and regeneration following MI, mechanistic insights derived from the regenerative zebrafish heart are considered to be of high translational value.  Unlike the mammalian heart, the zebrafish heart is able to restore lost cardiomyocytes (CMs) through mechanisms that allow spared cardiomyocytes to dedifferentiate, proliferate, and repopulate the injured tissue.  This regenerative ability is attributed to a stringent spatiotemporal regulation and crosstalk between the various cardiac cell types.  Therefore, for an effective bench-to-bedside application of cardio-regenerative strategies, it is imperative that the distinct roles and spatiotemporal dynamics of the injury-responsive cardiac cell populations in zebrafish are well characterized.  

The injury-responsive endocardial cells remain poorly characterized.  Since they provide direct structural support, and may activate several signaling pathways in the CMs, we aim to further investigate their role in facilitating cardiac regeneration in adult zebrafish.  Using single-cell and bulk transcriptomic approaches on the ET(krt4:EGFPsqet33-1A) line, in which endocardial cells express GFP, we have identified the injury-specific endocardial sub-populations enriched for EndoMT (serpine1, vim), and immune-cell (lgals2a) genes. Expression of these genes was mapped to the injury area and wound-border zone interface using the available Tomo-seq data for injured hearts.  In addition, several markers of mature endothelial/endocardial cells were identified, and observed to be repressed in these clusters.  These data corroborate recent findings in mouse that show EndoMT in endothelial cells of the infarcted hearts. Thus, we will perform functional studies on a few identified candidates, aiming to identify factors that promote CM dedifferentiation and/or proliferation.


Pooja SAGVEKAR (Bad Nauheim, Hesse, Germany), Stefan GÜNTHER, Khrievono KIKHI, Didier STAINIER
17:45 - 20:00 #30564 - 088. Duty cycle of cyclic stretch induces syncytial organization and functional maturation of biomimetic fetal ventricular tissue.
088. Duty cycle of cyclic stretch induces syncytial organization and functional maturation of biomimetic fetal ventricular tissue.

Afterload, also known as resistance to contraction, and preload, also known as stretch, are key mechanical stimulation parameters that influence ventricular maturation but are difficult to test in vivo. Current in vitro mechanical stimulation bioreactors can mimic preload via cyclically stretching cardiac tissue but fail to simultaneously mimic afterload due to the usage of high afterload anchorage points. Therefore, we developed a mechanical stimulation bioreactor system that better recapitulates the developmental cardiac cycle. This system can apply duty cycles that vary the afterload and preload during the stretch regimen, which can be used to assess how these mechanical parameters affect maturation of biomimetic cardiac tissues. To determine the optimal stage to construct biomimetic cardiac tissues, we conducted single cell RNA sequencing on embryonic chicken ventricular cells spanning four key ventricular development stages. This data revealed embryonic day 7 as an important ventricular maturation transitional stage where epicardial-derived cells are actively contributing to the compacting myocardium. Embryonic day 7 ventricular biomimetic tissues were created and cultured statically for seven days followed by three different stimulation regimens for seven additional days: static, cyclic stretch without a duty cycle, or cyclic stretch with a duty cycle. Cyclically stretched biomimetic tissues maintained their overall spontaneous contractile function and led to significant increases in contractile frequency and mechanical stiffness compared to static controls. Strikingly, biomimetic tissues conditioned with a duty cycle led to the greatest improvement in cardiomyocyte elongation, alignment, and sarcomere organization. This data demonstrates duty cycles are a driver of ventricular developmental maturation.


Gaetano SCUDERI, Jonathan BUTCHER (Ithaca, USA), Brianna HOU, Kathleen CLIFFORD
17:45 - 20:00 #30711 - 090. Massive Expansion of Functional Human iPSC-derived Cardiomyocytes by Concomitant Glycogen Synthase Kinase-3 Beta Inhibition and Removal of Cell-Cell Contact.
090. Massive Expansion of Functional Human iPSC-derived Cardiomyocytes by Concomitant Glycogen Synthase Kinase-3 Beta Inhibition and Removal of Cell-Cell Contact.

Therapeutic induction of cardiomyocyte proliferation in vivo by modulating Wnt, Hippo, neuregulin, and other signaling pathways has shown significant promise to treat congenital and acquired heart diseases. However, when these signaling molecules are applied in vitro to expand cardiomyocytes (CMs) from pluripotent stem cells, the extent of proliferation has generally been modest (<5 fold), precluding the use of these cells for industrial-scale applications. Here, we demonstrate the massive expansion of beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro by glycogen synthase kinase-3 beta (GSK-3β) inhibition using CHIR99021 (CHIR), a small molecule GSK-3β antagonist, together with the removal of contact inhibition by low-density serial passaging, resulting in a total of 100-250-fold increase in CM number. Prevention of cell-cell contact stimulated hiPSC-CM cell cycle activity, providing 10 to 25-times greater expansion beyond GSK-3β inhibition alone. To disambiguate the role of Wnt/b-catenin signaling on hiPSC-CM proliferation upon CHIR-treatment and low-density passaging, we found that the maintenance of a more immature sarcomeric structure and gene expression was dependent on LEF/TCF transcriptional activity while cell cycle activation was regulated by cell contact-mediated AKT phosphorylation. To demonstrate the functionality of expanded hiPSC-CM, we engineered heart tissues and showed that the contractile performance is equivalent between expanded and unexpanded hiPSC-CMs. In summary, we uncovered a molecular interplay between GSK-3β inhibition and cell-cell contact that enables the massive expansion of functional hiPSC-CMs thereby facilitating large-scale drug screening efforts and engineering of therapeutically relevant-sized cardiac tissues.


Jan Willem BUIKEMA (Amsterdam, The Netherlands), Soah LEE, Sean M. WU
17:45 - 20:00 #29415 - 092. Building the muscular wall in the cardiac atrium involves cell elongation and reorganization of tissue polarity.
092. Building the muscular wall in the cardiac atrium involves cell elongation and reorganization of tissue polarity.

During vertebrate development, cardiomyocytes undergo cellular rearrangements to form complex myocardial structures.  Malformations of these structures lead to diseases that include cardiomyopathies and cardiac arrythmias.  Previous studies have mostly focused on the formation of the trabecular network in the ventricle; however, morphogenetic processes that drive atrial myocardial complexity, which is crucial to propagate the action potential for cardiac contraction, have largely been overlooked.  Our study uses zebrafish larvae to elucidate cardiomyocyte behaviors during atrial development, as they allow for high-resolution live imaging and are easily amenable to genetic manipulations.

Using live 3D confocal imaging of zebrafish hearts, combined with mosaic labelling and temporal tracking of individual atrial cardiomyocytes, we found that atrial myocardial morphogenesis is driven by complex cell behaviors.  Specifically, we observed that atrial cardiomyocytes in zebrafish larvae form membrane protrusions and adopt an elongated shape in a non-stochastic orientation that establishes atrial tissue-level polarity.  These shape changes lead to restricted multilayering between neighboring cardiomyocytes, and the formation of new cell contacts, resulting in populations of elongated cardiomyocytes that span the atrium in an orientation parallel to the direction of blood flow.  These cell behaviors lead to the appearance of muscle ridges on the inner surface of the atrium.  Notably, these atrial cardiomyocyte behaviors appear to be independent from factors important in ventricular morphogenesis such as Erbb2 and Notch signaling.  Altogether, these data suggest that atrial morphogenesis is driven by oriented cell elongation as well as by distinct molecular and environmental/physical factors, all of which need further investigation.

 


Marga ALBU (Bad Nauheim, Germany), Felix GUNAWAN, Rashmi PRIYA, Alessandra GENTILE, Didier STAINIER
17:45 - 20:00 #29456 - 094. Egr3 regulates atrioventricular valve formation in zebrafish.
094. Egr3 regulates atrioventricular valve formation in zebrafish.

Congenital heart disease (CHD) is the leading cause of death from congenital anomalies, with 7.4% of the cases caused by abnormal development of the atrioventricular (AV) canal. The AV canal gives rise to the atrioventricular valves and node as well as the septae in mammalian hearts, all of which are essential for heart function.  A previous transcriptomic analysis performed in our laboratory identified the transcription factor gene egr3 as a novel endocardial AV valve marker.  Here, we investigate egr3 function during AV valve development and valve leaflet formation using zebrafish as a model system.  We generated a promoter-trap line and several mutant alleles, including a full locus deletion, by Crispr/Cas9-mediated genome engineering.  We find that egr3 expression is specific to the presumptive endocardial AV canal, and that loss of Egr3 function leads to the absence of AV valve leaflets, resulting in severe retrograde blood flow.  In addition, we observed that egr3 expression in the AV endocardial cells was lost in non-beating hearts, and that it was expanded in perturbed cardiac flow conditions, suggesting that it is responsive to mechanical forces.  Altogether, this work identifies Egr3 as a critical regulator of AV valve formation, downstream of blood flow, thereby uncovering a new candidate gene for valve-related CHDs.


Agatha RIBEIRO DA SILVA (Bad Nauheim, Germany), Thomas JUAN, Felix GUNAWAN, Didier STAINIER
17:45 - 20:00 #29486 - 096. Cell-autonomous role of mesodermal Fn1 in outflow tract elongation.
096. Cell-autonomous role of mesodermal Fn1 in outflow tract elongation.

Introduction: Defective outflow tract (OFT) elongation causes congenital heart disease (CHD) resulting in newborn lethality. OFT formation involves the addition of second heart field (SHF) cells to the nascent heart tube, and regulation of SHF cell proliferation, migration, and differentiation in the OFT. Fibronectin (Fn1) is an extracellular matrix glycoprotein essential for the development of cardiac structures, including the OFT. Although, the requirement for Fn1 in the OFT elongation is clear, the cellular and molecular mechanisms underlying the role of Fn1 in OFT development remain unknown. Materials and Methods: We ablated Fn1 from the anterior mesoderm, including the SHF, using the Mesp1Cre/+ knock-in strain of mice. To study the role of mesodermal Fn1, we performed immunostainings and western blots using Fn1f/+;Mesp1Cre/+ (control) and Fn1f/-;Mesp1Cre/+ (mutant) embryos isolated at different stages of development. Results: The ablation of mesodermal Fn1 results in a shortened OFT. This defect was due to an altered SHF cell shape and polarity, defects in cell-cell adhesion, proliferation, and premature cell differentiation in the SHF. Interestingly, even when mesodermal Fn1 was depleted, Fn1 expression between the SHF and endoderm was similar in control and mutants. However, the presence of endodermal Fn1 was not sufficient to rescue SHF cell defects in mutant embryos, suggesting that mesodermal Fn1 has a cell-autonomous role in OFT elongation. Discussion: In these studies, we have gained new insights into mechanisms regulating the addition of SHF-derived cells to the primitive heart tube during the formation of the OFT. Our work will lead to a deeper understanding of mechanisms regulating normal cardiac development and alterations that cause CHD.  Future work will be concentrated to understand if SHF cells migration is affected in vivo and why mesodermal Fn1 is required in a cell-autonomous manner.


Cecilia ARRIAGADA (Newark, USA), Sophie ASTROF
17:45 - 20:00 #29515 - 098. Genomic insertion location and orientation affect Bacterial Artificial Chromosome reporter expression patterns.
098. Genomic insertion location and orientation affect Bacterial Artificial Chromosome reporter expression patterns.

Transgenic mouse models are an essential tool to understand mammalian genetics and cell biology. We used a piggyBac-on-BAC strategy to generate transgenic mice carrying a Bacterial Artificial Chromosome (BAC) containing the human ADAMTS19 enhancer and gene, with a tamoxifen-inducible Cre (CreERT2) replacing the first exon. hADAMTS19-CreERT2 mice (F0-F3) were crossed with Rosa26-tdTomato reporter mice and timed-pregnant females dosed with tamoxifen at E13.5. Surprisingly, E16.5 whole embryonic hearts showed three separate, but complementary and founder lineage-specific tdTomato expression patterns that together represent all mouse Adamts19-expressing cell types: atrial and ventricular (AV) cardiomyocytes, outflow tract (OFT) cells, and valve interstitial cells (VICs). Nanopore gDNA sequencing of a selection of founder lineages revealed that the hADAMTS19-CreERT2 BAC had uniquely integrated in a 5’ to 3’ orientation in either N-Cadherin (Cdh2) intron 14 (AV line) or Plexin A2 (Plxna2) intron 9 (OFT line). Immunofluorescence and mouse heart single cell data analyses confirmed that tdTomato expression patterns were consistent with Cdh2 or Plxna2 expression patterns, suggesting that the genomic region surrounding the BAC dominated CreERT2 expression. In contrast, analysis of the VIC subline revealed BAC integration in a reverse (3’ to 5’) orientation in intron 9 of RNA Polymerase II Associated Protein 2 (Rpap2), which is ubiquitously expressed. This reverse orientation allowed CreERT2 expression to be regulated by the hADAMTS19 BAC enhancer. We conclude that the genomic location and insertion orientqtion of BAC transgenes affects their expression pattern.


Johanna COMES (paris), Piet VAN VLIET, Marieke ROZENDAAL, Florian WUNNEMANN, Smith MARTIN, Gregor ANDELFINGER
17:45 - 20:00 #29548 - 100. Mechano-Molecular Control of Heart Formation.
100. Mechano-Molecular Control of Heart Formation.

The heart is the first functional organ to form during vertebrate development. First, a linear heart tube is formed, followed by cardiac looping, chamber formation, and maturation. As for many organs, the heart arises from a simple epithelium with planar polarity properties. It has been established that cardiac chamber remodeling is coordinated through tissue-scale polarization of actomyosin. However, the mechanism governing the looping process is not fully understood. Here, using Zebrafish as a model, we describe the role of actomyosin to generate and distribute the tension forces necessary across the cardiac epithelium during cardiac looping and chamber formation. We found a supracellular actomyosin network required for this process to occur, which is regulated mainly by two kinases (Mylk3 and Rock2a) to achieve the proper distribution of phosphorylated myosin across the ventricular myocardium. Moreover, we also identified that the planar cell polarity pathway (non-canonical Wnt signaling) modulates the appropriate phosphorylation pattern in the cardiac epithelium by controlling the kinase activity in a spatio-temporal manner. Our findings describe a mechano-molecular mechanism necessary for proper looping and chamber formation during cardiogenesis.


Kevin Manuel MÉNDEZ-ACEVEDO (Berlin, Germany), Motahareh MOGHTADAEI, Luca HUMMEL, Anca MARGINEANU, Anne MERKS, Daniela PANÁKOVÁ
17:45 - 20:00 #29726 - 102. High throughput screen of mouse mutant lines identifies new genetic mutations implicated in congenital heart disease.
102. High throughput screen of mouse mutant lines identifies new genetic mutations implicated in congenital heart disease.

Congenital heart disease (CHD) affects 0.8-1.1% of live births each year, making it the most common birth defect globally. Although the significant role of genes in the pathogenesis of CHD has been previously established, the etiology of 60% of CHD remains unexplained. To uncover additional genetic causes of CHD, we analyzed 3D microCT images of lethal and subviable mouse mutant strains generated by the International Mouse Phenotyping Consortium  and looked for evidence of CHD at embryonic days 15.5 and 18.5. We evaluated 430 mutant lines and 2,479 idividual mutants, and scored them for the following CHDs: atrial septal defects (ASD), ventricular septal defects (VSD), patent ductus arteriosis (PDA), truncus arteriosis (TA), double outlet right ventricle (DORV), atrioventricular septal defect (AVSD), transposition of the great arteries (TGA), pulmonary atresia, dextrocardia, situs inversus totalis (SIT), and cleft palate. From our data we identified 28.5% of null mutant mouse strains that exhibited at least a 25% penetrance of CHD. 65.1% of the mutant lines meeting our penetrance threshold were novel, meaning that they have not been associated with CHD previously. Thus, we have identified 79 new genes that give rise to CHD when mutated. We therefore hold that this type of analysis is valuable to identify novel genes implicated in CHD that have not been revealed by other conventional methods. We will extend our findings by performing pathway analyses to uncover novel pathways associated with CHD. We will also use sequence data from CHD patients to uncover novel mutations associated with CHD in patients. Results will eventually be used to help to guide clinical prognoses and treatments.  

 


Monika CHUNG (Newark, USA)
17:45 - 20:00 #30419 - 106. Mechanisms of cardiac de novo sarcomerogenesis.
106. Mechanisms of cardiac de novo sarcomerogenesis.

Defective sarcomeric assembly is implicated in the pathophysiology of several inherited cardiomyopathies. While the mature sarcomere structure has been extensively studied, the molecular mechanisms that drive de novo sarcomere assembly are largely unknown. One of the earliest components in de novo sarcomere assembly is alpha-actinin 2 (ACTN2). ACTN2 localizes to the z body which fuses to form the z disc that serves as a linker for adjacent sarcomeric units.  Here we used human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), which closely follow this temporal progression of sarcomere formation, to interrogate the early ACTN2 interactome.  We performed RNAseq and mass spectrometry on hPSC-CMs and ACTN2 co-IP components at different times during cardiac differentiation. We find that ACTN2 sequentially associates with structural components such as intermediate filament and junctional proteins, and functional regulators such as calcium channel and metabolic proteins reflecting a coordinated program over time. The early cardiomyocyte coordinates translation and processing of sarcomeric proteins with their spatial organization and incorporation into developing myofibrils. ACTN2 in the early z body associates with chaperones and actin binding proteins, suggesting that the z body acts as a nidus for de novo actin filament assembly. In support of this role WNT and ROCK inhibition result in an increase of ordered sarcomeric filaments. We further identified ribosomal subunits and RNA binding proteins in the ACTN2 interactome, suggesting a role of the z body as a dynamic condensate. We confirmed this dynamic state of the z body using ACTN2 FRAP. We further show that epiblast-specific loss of Ddx3x, an early ACTN2 interactor and RNA binding protein, leads to embryonic lethality and cardiac defects in the mouse. Together these findings highlight the importance of the z body in the assembly process and provide a window to study the mechanisms of sarcomere organization.


Bhavana SHEWALE (New York, USA)
17:45 - 20:00 #30447 - 110. Ploidy reversal as a postnatal developmental program in murine hearts.
110. Ploidy reversal as a postnatal developmental program in murine hearts.

Polyploidization is a normal cellular adaptation for a variety of cell types in mammals, including cardiomyocytes (CMs); however, the development of polyploidization understudied. Recent work suggests that genetics contribute to diverse displays of ploidy in the adult murine heart. Thus, we hypothesized that the developmental progression to differing end-states may similarly be affected. Here, we assessed CM endowment, cell cycle, and ploidy temporally across two diverse inbred mouse strains, A/J and C57Bl/6J. Consistent with previous work, C57Bl/6J hearts displayed rapid cell cycle activation in the first postnatal week. Total CM numbers are relatively unchanged after P7, while final ploidy states are generally constant from P14 on. In contrast, A/J mice displayed depressed cell cycle activation in the first week of life.  Polyploidy in A/Js reaches its peak at P21, whereby only ~3.5% of CMs remained mononuclear and diploid. Interestingly, from 3-6 weeks of age, we observed an expansion of total CM numbers and of the 1x2N subpopulation to ~8%, which could not be explained by proliferation of the residual diploid population. Instead, the expanded diploid population appears to come from a polyploid CM. This finding was confirmed by analysis identifying a population of CMs which had completed cytokinesis by 6 weeks which was not present at 3 weeks. We believe this is the first report of ploidy reversal by a mammalian CM, a phenomenon first observed by the hepatocyte field. Ongoing single nuclear RNA seq is examining the transcriptomic differences between A/J and C57Bl/6J CMs at P21 to identify this unique population competent to undergo ploidy reversal. Preliminary results suggest A/J CM nuclei, regardless of their ploidy state, are more likely to be in the cell cycle in comparison to C57Bl/6J. Understanding the developmental paths to end state CM endowment and ploidy states helps us understand the biology of organ size and can be applied to stimulating regeneration.


Samantha SWIFT (Milwaukee, USA), Mary KOLELL, Alexandra PURDY, Michael FLINN, Caitlin O'MEARA, Christoph RAU, Michaela PATTERSON
17:45 - 20:00 #30467 - 112. Embryonic macrophage loss results in cardiac dysfunction and disrupts adult heart health.
112. Embryonic macrophage loss results in cardiac dysfunction and disrupts adult heart health.

Macrophages are well-characterized as sentinel immune cells that coordinate cellular responses to injury and infection. However, recent developments in cardiac macrophage biology have broadened our understanding of this critical cell type in heart development and function. Still, it is not known how loss of embryonic-derived macrophages affects heart health into adulthood. Here, we are the first group to show in larval and adult zebrafish as well as in adolescent mice that macrophages reside at the sinoatrial node, where they couple to nodal cardiomyocytes via connexin-45. Using a combination of histology, echocardiograms, electrocardiograms (ECGs), and physiological stress tests, we demonstrate that loss of embryonic macrophages significantly disrupts adult heart health and function, leading to arrhythmia and fibrosis. Adult irf8st96/st96 embryonic-macrophage knockout zebrafish have significant disruption in epicardial integrity, detachment, and expansion, known indicators of heart stress or damage. Mutant zebrafish also exhibit increased collagen deposition within the myocardium and along the atrial wall. Longitudinal analysis of echocardiographic measurements reveals significant age- and sex-specific changes in irf8 mutant heart function, including arrhythmia with notable variability in diastasis time as well as consistently prolonged isovolumic relaxation time. ECG traces of macrophage mutants following a 1-hr swim tunnel-based endurance test reveal significantly prolonged P-wave durations and PR intervals compared to wildtype. A subset of mutants also exhibited intermittent P-wave absence and potential flutter, indicating sinus or atrial dysfunction. Overall, these data reveal significant cardiac abnormalities following the specific loss of embryonic-derived macrophages and expand our knowledge of critical macrophage functions governing long-term heart health. 


Shannon PAQUETTE (Providence, USA), Cadence LEE, Alan MORRISON, Jessica PLAVICKI
17:45 - 20:00 #30473 - 114. Investigating the role of DOCK6 and EOGT in cardiac development and congenital heart disease.
114. Investigating the role of DOCK6 and EOGT in cardiac development and congenital heart disease.

Congenital Heart Defects (CHDs) are structural malformations that occur during development and affect 1% of live births worldwide. Correct heart development requires tight spatiotemporal control of both the Rac1 and Notch signalling pathways, and mutations in components or regulators of both pathways can lead to CHDs. Adams-Oliver Syndrome (AOS) is a multisystemic disorder in which 20% of patients also suffer from CHDs. All known causative mutations in AOS have been identified in Rac1 or Notch signalling pathways, including homozygous recessive mutations in DOCK6 and EOGT which regulate Rac1 and Notch signalling respectively. However, it is unknown how mutations in these genes lead to onset of this disease. We aim to understand how mutations in DOCK6 and EOGT lead to dysregulation of Rac1 and Notch signalling during heart development and cause CHDs in patients with AOS, using zebrafish as a model. 

We find that mRNA of both dock6 and eogt is expressed transiently during early zebrafish cardiovascular development. eogt mRNA expression is restricted to a subset of developing vascular structures, while dock6 exhibits slightly broader expression. Interestingly, both genes also have non-coding transcripts which are expressed in the myocardium during early heart morphogenesis. Using CRISPR mediated genome editing we have created zebrafish disease models of AOS with mutations in both dock6 and eogt, in which we are assessing cardiac morphology throughout development. In addition, we are investigating whether non-coding transcripts of dock6 and eogt play a role in heart development, or the onset of CHDs in AOS patients.


Emma ARMITAGE (Sheffield, United Kingdom), Emily NOEL
17:45 - 20:00 #30487 - 116. Sequential action of jnk genes establishes the embryonic left-right axis.
116. Sequential action of jnk genes establishes the embryonic left-right axis.

Establishment of the left-right body axis is critical for the placement, morphogenesis and function of organs, with disruption to laterality often associated with congenital heart malformations – known as heterotaxy. Left-right specification has long been proposed to be dependent on asymmetric fluid flow in the embryonic node, driven by motile (nodal) cilia. Nodal flow ultimately results in asymmetric expression of Nodal in the left lateral plate mesoderm, which directs organ morphogenesis.

Positioning of nodal cilia is governed by the highly conserved Wnt planar cell polarity (PCP) pathway, yet the exact effectors of PCP signalling remain elusive. We have examined the role of the highly conserved JNK (c-Jun N-terminal Kinase) gene family – a proposed downstream component of PCP signalling – in the development and function of the zebrafish node.

We show that together jnk1 and jnk2 function to specify length of nodal cilia, generate asymmetric flow in the node and restrict Nodal to the left lateral plate mesoderm. Crucially, loss of asymmetric Nodal expression does not result in disturbances to heart looping morphogenesis, supporting an emerging model that nodal flow may be dispensable for organ laterality.

This work uncovers multiple roles for the JNK gene family acting at different points during embryonic development. Furthermore, it reinforces observations that other node-independent pathways function in parallel to establish cardiac laterality and that genetic causes underlying heterotaxy are likely to be multi-faceted.


Christopher J DERRICK (Newcastle, UK, United Kingdom), Adrian SANTOS-LEDO, Lorraine ELEY, Isabela Andhika PARAMITA, Deborah J HENDERSON, Bill CHAUDHRY
17:45 - 20:00 #30497 - 118. Establishing the Role of the Conserved TN Domain in Tinman.
118. Establishing the Role of the Conserved TN Domain in Tinman.

Congenital heart disease (CHD) is a major factor in mortality and morbidity in children and adults. To advance our abilities to identify and manage CHD, we need to further understand the key genetic factors involved in causing these maladies. Drosophila melanogaster shares similar cardiac developmental mechanisms with humans and has been an essential model for human heart development. One similarity occurs between Tinman (Tin), a transcription factor in Drosophila vital for cardiac cell differentiation, and its mammalian ortholog NK2 Homeobox 5 (Nkx2.5). These proteins share two conserved regions: the homeobox and the tin (TN) domain. Although the TN domain is completely conserved between these two proteins, there is little known about its significance. By utilizing CRISPR/Cas9 gene editing, I established a line of Drosophila containing an in-frame deletion of the TN domain. Staining Drosophila embryos for a marker of cardial cells revealed mutant embryos generate a significantly more cardial cells than wild type (p<0.01). I also stained embryos for Tin and Svp, a Drosophila protein important for heart development, to determine if the TN domain deletion affected cardial cell specification. Mutant embryos contained more cells expressing tin and more cells expressing svp than wild type embryos. These results support the importance of the TN domain in heart development and indicate the increase in cardial cells occurs before cardial cells differentiate into cells exclusively expressing tin or svp. Characterizing the role of the conserved TN domain in Tin and Nkx2.5 can expand our understanding of cardiac formation and maturation


Cayleen BILECKYJ (San Diego, USA), Richard CRIPPS
17:45 - 20:00 #30520 - 120. Electrical organization of the first heartbeats.
120. Electrical organization of the first heartbeats.

The heart starts beating early in development, and maintains its function throughout dramatic developmental changes in electrophysiology, tissue morphology, and cell fate specification. However, little is known about the heart’s initial transition from silent to active - what are the intermediate dynamics between a quiescent tissue and a periodic and spatially coordinated heartbeat? Capturing the first heartbeats is a severe technical challenge because these sub-second events only occur once in each animal’s life, and could emerge at any point in a window of hours.  To study these initial dynamics, we developed an imaging system to observe calcium dynamics in up to 72 developing zebrafish embryos simultaneously. The first beats, occurring 20-21 hours post fertilization, were initially infrequent and irregular but from onset were coherent propagating waves that swept the heart field. We then developed genetic tools for simultaneous targeted optogenetic perturbation and voltage or calcium imaging, which revealed that the heart cone supports electrical wave propagation and becomes increasingly excitable before spontaneous activity emerges. Using spatially resolved calcium imaging, we mapped the spatial origin of the first heartbeats. This origin is highly variable between individuals, can move suddenly within a short time interval, and is not correlated with genetic markers of future pacemaker cells. Together our data suggest that broad excitability and electrical connectivity impose biophysical constraints that enforce initial spatial organization and timing of the first heartbeats, despite little specialization of cardiac cell function.


Bill JIA (Boston, USA), Sean MEGASON, Adam Ezra COHEN
17:45 - 20:00 #30527 - 122. ECM asymmetry in ‎the early heart tube: dissecting the links between morphology and ECM dynamics in ‎the developing zebrafish ‎heart.
122. ECM asymmetry in ‎the early heart tube: dissecting the links between morphology and ECM dynamics in ‎the developing zebrafish ‎heart.

During development, the heart tube comprises two cellular layers, an outer myocardium and an inner endocardium, between which lies a layer of extracellular matrix (ECM). This cardiac ECM creates specialised extracellular environments important in mediating the chemical and biomechanical signals that shape the heart tube into a three-dimensional (3D) organ. Visualising and linking ECM and tissue morphology is challenging since sample processing may impact matrix hydration and tissue structure. Using morphoHeart, a novel and in-house-developed quantitative image analysis tool that allows the 3D segmentation of the heart layers  -including the ECM- from live embryos, we have identified an early left-right axis of ECM-regionalisation linked to heart looping and orientation of chamber ballooning.

To understand if defects in heart tube lateralisation result in abnormal ECM-regionalisation and hence disrupted morphogenesis, we investigated zebrafish hearts in which spaw (zebrafish homolog of Nodal responsible for left-right laterality) is disrupted. Analysis of cardiac ECM distribution in spaw mutants identified that ECM asymmetry in the early tube is maintained but mispositioned. Irrespective of subsequent looping direction, this early ECM asymmetry translates into a highly regionalised ECM in the inner and outer curvatures of the atrium at looping and ballooning stages. Further linking laterality, ECM and heart morphogenesis, quantification of heart size in spaw mutants demonstrated a role for Nodal in timely chamber growth and chamber-specific ECM remodelling, even in dextrally-looped mutant hearts. Together we propose a model whereby laterality cues orient an early asymmetric cardiac ECM that intrinsically sets an axis for chamber growth and looping morphogenesis to occur.


Juliana SÁNCHEZ-POSADA (Sheffield, United Kingdom), Emily S. NOËL
17:45 - 20:00 #30536 - 124. Developmental origins of Lmna-related dilated cardiomyopathies and impact of FGF10 treatment.
124. Developmental origins of Lmna-related dilated cardiomyopathies and impact of FGF10 treatment.

Dilated cardiomyopathies (DCM) including ischemic- and genetic-related DCM are characterized by cardiomyocyte necrosis and fibrosis associated with impaired cardiac function leading to severe heart failure. LMNA is mutated in 10% of DCM cases in humans and is responsible for rapidly progressing DCM with severe cardiac defects. While the role of LMNA has been extensively studied in adult heart, its role during heart development remained to be determined. We recently identified the fibroblast growth factor 10 (FGF10) as a potential target to promote cardiac regeneration in ischemic DCM.

This study thus aims to uncover the role and the cell-type requirement of Lmna during heart development, maturation and homeostasis and to evaluate FGF10 as a therapeutic target in Lmna-related DCM.

Transgenic mouse lines will be used to perform temporal (development, maturation and adult) and cell-type specific (cardiomyocytes and cardiac fibroblasts) deletion of the Lmna and conditional Fgf10 overexpression.

Expression profiling revealed increased LMNA myocardial expression from embryonic to postnatal stages. Interestingly, our results revealed that LMNA preferentially accumulates in cardiac fibroblasts in fetal and postnatal heart. Cardiomyocyte specific Lmna deletion in fetal, postnatal and adult heart displays rapid cardiac function alteration with dramatic left ventricular dilatation and massive fibrosis, unveiling a critical requirement in developing cardiomyocytes. Interestingly, specific Lmna-deletion in embryonic cardiac fibroblasts leads to embryonic lethality, revealing an unsuspected role for LMNA in cardiac fibroblasts. Finally, we evaluated the impact of FGF10 treatment on Lmna-related DCM and our results suggest that FGF10 preserves cardiac fibrosis infiltration and cardiac remodelling in Lmna-related DCM.

Altogether, this study unveils the key developmental role of LMNA and identifies FGF10 as a therapeutic target for cardiac regeneration in Lmna-related DCM.


Laetitia BOUCHARD (Marseille), Antoine MUCHIR, Francesca ROCHAIS
17:45 - 20:00 #30541 - 126. Functional analysis of the novel BAV candidate gene muc4 in zebrafish cardiac valve leaflet development.
126. Functional analysis of the novel BAV candidate gene muc4 in zebrafish cardiac valve leaflet development.

Bicuspid aortic valve (BAV) is the most common congenital heart defect that affects around 1.5% of population. The majority of patients develop aortic valve stenosis, aortic root dilatation or infective endocarditis. As a consequence, BAV accounts for nearly 50% of all aortic valve replacements. The genetical heterogeneity of BAV makes identification of its genetic causes complicated, thus, to date they remain largely elusive.

Based on unpublished GWAS study of 2236 BAV patients and 11604 controls, we identified a new risk locus for BAV – MUC4. The goal of this study was to functionally characterize muc4 in zebrafish and analyze its involvement in cardiac valve morphogenesis.

We used two alternative approaches for functional analyses of muc4 in zebrafish atrioventricular (AV) valvulogenesis: transient F0 CRISPR/Cas9-mediated gene knockout and antisense oligonucleotide Morpholino- (MO)-mediated gene knockdown. The 1-cell stage embryos were injected either with a mix of Cas9 protein and muc4-gRNAs or with muc4-MOs. At 56-58 hours post fertilization (hpf) the crispant and morphant embryos were fixed, genotyped and imaged using confocal microscopy against Alcam and kdrl:GFP.

Using both approaches, we observed a delay in AV valvulogenesis. In the wild-type at 56 hpf, a few AV endocardial cells had ingressed into the extracellular matrix (ECM) to form a multilayered valve leaflet. However, in muc4 crispants or morphants, the valve remained monolayered or only one cell had ingressed into the ECM. This phenotype had a penetrance of approx. 50%.

Here, we have functionally characterized a new BAV candidate gene, MUC4. Knockout and knockdown of this gene in zebrafish revealed that muc4 may impact cardiac AV valvulogenesis by causing a developmental delay during the multilayering of valve leaflets. Our research constitutes the basis for future studies on the relevance of MUC4 in BAV as one of the candidate genes, contributing to this polygenic pathology.


Dinara SHARIPOVA (Potsdam, Germany), Federica FONTANA, Jan GEHLEN, Anja STUNDL, Radoslaw DEBIEC, Markus KRANE, Salim ABDELILAH-SEYFRIED, Nilesh SAMANI, Jaenette ERDMANN, Teresa TRENKWALDER, Johannes SCHUMACHER
17:45 - 20:00 #30545 - 128. Conditional deletion of Wt1 in Wt1Cre cells reveals a partial lethality of Wt1LoxP/GFP;Wt1Cre mice.
128. Conditional deletion of Wt1 in Wt1Cre cells reveals a partial lethality of Wt1LoxP/GFP;Wt1Cre mice.

The Wilms tumor 1 homolog (Wt1) encodes a zinc finger protein essential for heart development. Over the last ten years, several Wt1 transgenic mouse models have been generated and used by several laboratories to study the behaviour of WT1 expressing cells during heart development and repair. In this study, we generated a new mouse model of the Wt1 gene, the Wt1LoxP/GFP;Wt1Cre mice. In these mice, one copy of the Wt1 allele is flanked by loxP sites, whereas the other copy has a GFP knock-in in exon 1, which disrupts WT1 expression. In addition, the Wt1Cre line used to generate this model is a BAC transgenic line that has been extensively used in the cardiovascular field. The genotyping of postnatal mice of our model revealed a sub-Mendelian distribution of the Wt1LoxP/GFP;Wt1Cre mice. The observed lethality of Wt1LoxP/GFP;Wt1Cre mice probably corresponds to the late embryonic stages since the proportion of this genotype was not reduced when embryos were examined on days E14.5. Interestingly, all mutant mice lacked mature gonads and displayed genital tracts containing both male and female genital structures and ambiguous genitalia. In addition, the histological examination of embryonic Wt1LoxP/GFP;Wt1Cre mice demonstrated severe heart defects, which could be the main cause of lethality of some mutant mice. Our results call for cautious interpretation of data obtained by conditional gene deletion experiments using this Wt1Cre line, as mouse models in which Wt1 has been deleted in the embryonic heart are embryonic lethal.


Rosa PORTELLA-FORTUNY (BARCELONA, Spain), Otilia GLIGA, Marina RAMIRO-PARETA, Claudia MÜLLER-SÁNCHEZ, Alejo TORRES-CANO, Manuel REINA, Francesc X. SORIANO, Ofelia M. MARTÍNEZ-ESTRADA
17:45 - 20:00 #30553 - 130. Whole genome sequencing identifies evolutionarily constrained genes with chromatin regulators and CHD genes as modifiers of conotruncal heart defects in 22q11.2DS.
130. Whole genome sequencing identifies evolutionarily constrained genes with chromatin regulators and CHD genes as modifiers of conotruncal heart defects in 22q11.2DS.

The prevalence of CHD in 22q11.2 deletion syndrome (22q11.2DS) patients is 65% as compared to the general population at ~0.7%, of which most have conotruncal heart defects (CTD).  To understand the basis of variable phenotypic expression, we analyzed whole-genome sequence data of 456 CTD cases and 537 controls with 22q11.2DS retaining the most damaging coding/splicing rare variants (MDRV), as identified by two independent variant calling pipelines with multiple annotation algorithms and examined 19 gene-sets for the association.  We found a significantly higher burden of MDRVs in three interdependent gene-sets comprising evolutionarily constrained genes (OR=3.31; P-value, 4.00E-07), chromatin regulator genes (OR=4.16; P-value, 7.00E-05) and known congenital heart disease genes (OR=6.63; P-value, 5.9E-04) based upon a weighted gene-set based test.  When removing chromatin regulators and considering constrained genes, most are neuronal synapse genes, while the CHD genes are associated with cardiac development.  Further, by focusing on recurrently affected genes, we found the chromatin regulatory genes were overrepresented in cases, but no enrichment was found in controls.  Overall, using both methods, there are a total of 46 affected chromatin genes in 42 CTD cases accounting for 9.21% of the total, with six recurrently affected chromatin genes including EP400, KAT6A, KMT2C, KMT2D, NDS1, and PHF21A, involved in histone peptidyl-lysine modification.  Although ubiquitously expressed, the chromatin genes have enriched expression in cardiac progenitor cells in the pharyngeal apparatus, where TBX1, the transcription factor gene for 22q11.2DS is expressed.  Modifier genes identified might act in the genetic and epigenetic pathways of TBX1 function.


Yingjie ZHAO (Bronx, USA), Lijie SHI, International 22Q11.2Ds Brain And Behavior CONSORTIUM, Bernice MORROW
17:45 - 20:00 #30557 - 132. Mendelian randomization reveals sex-differences in atherogenic cardiovascular disease pain perception.
132. Mendelian randomization reveals sex-differences in atherogenic cardiovascular disease pain perception.

Clinical manifestation of atherogenic cardiovascular disease differs between men and women. Men often present with classical symptoms i.e., chest pain radiating to left arm and jaw. Women, on the other hand, seem to show a wider range of symptoms that are considered less typical, including but not limited to back pain and nausea. The anatomical pathways feeding cardiac pain perception have not been properly characterized with respect to sex-related differences.

 

In our study, we explored the UK Biobank cohort to perform sex-stratified genome-wide association studies for myocardial infarction, coronary artery disease and a variety of pain types to identify instrumental variables for Mendelian randomization. This approach enables determination of sex-differences of the causality between heart disease and its clinical manifestation, thus allowing for large-sample size evidence of the differences in atherogenic cardiovascular disease manifestation between sexes.

 

We observed that a genetically-influenced chest pain was associated with a higher risk of atherogenic cardiovascular disease in men (OR: 1.49 95% CI: 1.01 – 2.19)Surprisingly, for women, this chest pain was not linked with atherogenic cardiovascular disease (OR: 1.06, 95% CI: 0.96 – 1.17), suggesting chest pain by itself is a less reliable predictor of heart disease. Furthermore, we characterized sex-stratified causal relationships of other pain types with myocardial infarction and coronary artery diseases.

 

Our study identified differences in clinical manifestation of myocardial infarction and coronary artery disease between men and women, that form the basis for our research on developmental and anatomical differences in pain perception between the sexes. 


Ruben METHORST (Leiden, The Netherlands), R NOORDAM, M.r.m. JONGBLOED, M.c DERUITER
17:45 - 20:00 #30565 - 134. Fibulin Genes Regulate Outflow Tract Caliber and Extracellular Matrix Integrity.
134. Fibulin Genes Regulate Outflow Tract Caliber and Extracellular Matrix Integrity.

Critical developmental roles for matrix molecules in proliferation and differentiation and in generating tissue structural integrity are evident by the range of clinical disorders induced by mutations in extracellular matrix (ECM) genes. In the cardiovascular system, recent data underscore the dynamic nature of ECM composition during embryogenesis. This emerging focus on the function of ECM in cardiac development and disease has advanced our understanding of the morphogenetic basis of congenital heart defects (CHD). Yet, we have limited appreciation of the cellular and biophysical mechanisms regulated by individual ECM proteins during formation of the cardiac outflow tract (OFT). Our data reveal that Fibulin molecules, a family of glycoproteins that regulate cell behaviors and structural contributions to the ECM, have previously unrecognized requirements in establishing the proper dimensions of the vertebrate OFT. Zebrafish fibulin mutants also exhibit decreased TGF-β signaling in second heart field (SHF) progenitors of the linear heart tube. Consequently, fibulin-deficient SHF progenitors supply fewer endothelial and smooth muscle cells, resulting in a narrower arterial pole. In addition to effects on SHF progenitor differentiation, we find that deposition of Elastin, a crucial ECM component of the OFT, is impaired as evidenced by decreased expression, fiber number and alignment in fibulin mutant embryos. Importantly, tissue stiffness of the OFT auxiliary chamber is impaired by disrupted ECM integrity and these factors govern biomechanical function. Our insights will yield valuable strategies to improve engineered biomaterials for surgical intervention in congenital OFT anomalies and will fuel further identification of novel disease genes in CHD.


Di YAO, Angelika ALEMAN (New York, USA), Caitlin FORD, Micah WOODARD, Carmen DE SENA TOMAS, Kimara TARGOFF
17:45 - 20:00 #30571 - 136. Estimation of tissue-scale dynamics during mouse cardiac morphogenesis based on microscopic observation.
136. Estimation of tissue-scale dynamics during mouse cardiac morphogenesis based on microscopic observation.

Diabetic pregnancy has increased risk of asymmetric septal hypertrophy in the fetus, and we aim to elucidate how the nutritional and metabolic environment of the embryo regulates cardiac morphogenesis. In a previous study, we revealed that cardiomyocyte maturation is impaired in a high glucose environment in vitro, however it remains unclear how ventricular chamber undergoes asymmetric hypertrophy at organ scale.

To understand morphogenetic mechanisms, it is essential to clarify the hierarchical relationship among organ, tissue and cellular activities. Histological analysis revealed disarrayed muscle fibers in the hypertrophic tissue at cellular scale, however little is known about the tissue scale deformation process. Based on histological observation, we hypothesized that the tissue scale morphogenetic process of hypertrophy is isotropic growth caused by disruption of proper orientation in tissue elongation. To test this hypothesis, we developed a new method to estimate the size and direction of local tissue growth during heart morphogenesis. Specifically, 3D whole organ imaging is performed to detect the cells of the same origin in a fixed heart using MADM-based tracking. Then, based on the detected spatial distribution of these cells, thickening of myocardial wall and the direction of tissue stretch at each local tissue are calculated. In this presentation, we provide a proof of concept for this method through the analysis of artificial data. We discuss how to apply this method to reveal the anisotropy of tissue growth and how to dissect the cellular mechanisms that cause tissue scale dynamics.

This new methodology for estimating the tissue scale deformation process during organogenesis will help us understand the multiscale morphogenetic mechanisms of organ development.


Naofumi KAWAHIRA (Los Angeles, USA), Haruko NAKANO, Atsushi NAKANO
17:45 - 20:00 #30576 - 138. Adaptation of the sinus venosus reveals insights into emergent CHDs and evolutionary transitions of the vertebrate heart.
138. Adaptation of the sinus venosus reveals insights into emergent CHDs and evolutionary transitions of the vertebrate heart.

Nr2f transcription factors play an integral role in vertebrate atrial development and have been linked to congenital heart defects (CHDs) in humans. Adult patients with structural CHDs affecting the atrium often present with secondary complications, such as pulmonary hypertension and vascular remodeling. Here, we report a zebrafish nr2f1a mutant allele with embryonic atrial defects that can survive to adulthood. Remarkably, despite essentially lacking an atrium, the adjacent sinus venosus (SV) in these mutants undergoes adaptive remodeling, which entails substantial thickening due to increased cell proliferation. Transcriptomic analysis of the bulbus arteriosus (BA) and SV, transgenic reporters, and genetic lineage tracing showed previously unknown molecular and cellular similarity between the wild-type BA and SV, including the presence of vascular smooth muscle (VSM) and neural crest cells. Furthermore, single cell RNA-seq revealed novel heterogeneity of VSM cell populations in the BA and SV and that VSM and endothelial cells within the adapting SV of the nr2f1a mutants take on transcriptional signatures of the BA. Echocardiography showed retrograde blood flow in adult nr2f1a mutant hearts, suggesting that aberrant blood flow contributes to the adaptive remodeling of the SV. Consistent with this hypothesis, prolonged treatment with a vasodilator mitigated thickening, while increased exercise exacerbated the adaptive thickening. Altogether, our data demonstrate an unexpected latent similarity between the large chamber-like vessels at the arterial and venous poles of the zebrafish heart and adaptive remodeling of the SV in nr2f1a mutants due to hemodynamic forces, which may provide insights into complications associated with CHDs in humans.


Jacob GAFRANEK (Cincinnati, OH, USA), Enrico D'ANIELLO, Nathan SALOMONIS, Joshua WAXMAN
17:45 - 20:00 #30585 - 140. Vascular Outflow Tract Development: A New Source of Smooth Muscle Cells in the Ascending Aorta.
140. Vascular Outflow Tract Development: A New Source of Smooth Muscle Cells in the Ascending Aorta.

The ascending aorta harbors a heterogenic smooth muscle cell (SMC) population. Embryonic lineage tracing studies have shown that extracardiac cells contributing to the vascular outflow tract (OFT), originate in the cardiac neural crest and second heart field (SHF). The cardiac neural crest is important for proper outflow tract septation and formation of the inner SMCs layer. The SHF mainly contributes to the SMC population of the pulmonary trunk and a small part of the ascending aorta. Currently, the origin of the outer medial SMC and adventitial fibroblasts is unknown. Recently we described a distinct Wilms Tumor 1 (WT1) positive cell population in the area of the OFT. We hypothesize that these cells contribute to the aortic SMC and fibroblast population. Using the Cre-LoxP recombination system driven by an epicardial specific promotor (WT1-CreER2) and the ROSAmT/mG reporter line we studied the fate of this cell populationfrom stage E9.5 till E17.5 in mouse embryos.

Immunohistochemistry revealed that this  population of cells shares other SHF markers.. GFP-labeled epicardial cells undergo -mesenchymal transformation and migrate into the arterial vessel wall of the ascending aorta. During this process,these cells lose their epicardial phenotype and contribute to the mural cells of the outer layer of the aorta and pulmonary trunk. Our results demonstrate a new embryonic origin of SMC which specifically contributes to media and adventitia of the ascending aorta but not to the aortic arch or descending aorta. Future studies will focus on their specific role in normal development and during pathological circumstances.


Tamara BORSBOOM (Leiden, The Netherlands), Esther DRONKERS, Lambertus WISSE, Conny MUNSTEREN, Bart LOEYS, Marie-Jose GOUMANS, Monique JONGBLOED, Marco DERUITER
17:45 - 20:00 #30591 - 142. A dedicated approach for transmural visualization and quantification of nerve density: application in myocardial infarction biopsies.
142. A dedicated approach for transmural visualization and quantification of nerve density: application in myocardial infarction biopsies.

Abnormal cardiac innervation plays a role in arrhythmogenicity after myocardial infarction(MI). Nerve quantification is challenging as visualization of  nerves requires high magnifications. We developed a method to analyze nerve density(ND) in large transmural biopsies.

Myocardial biopsies from 4 swine, 3 months after MI,  were stained with Sirius Red(fibrosis) and β3Tubulin(autonomic nerves). Fibrosis cut-offs classified biopsies in: MI core(>59.8%), borderzone(BZ)(14.3-59.8%) or remote zone(RZ)(<14.3%). Using a custom software pipeline, biopsies were graphically divided into 1x1mm grids. Nerve and myocardial tissues were thresholded and after  quality check and artefact removal, divided into 0.1x0.1mm squares. ND/square was calculated and classified into denervation, hypoinnervation, normal innervation and hyperinnervation according to control derived cut-offs(5th, 95th %, n biopsies=38). Squares were located back to their original biopsy position for visualization and quantification of innervation types.

All 83 MI biopsies showed variable innervation patterns. Denervation, hypo- and hyperinnervation were largest in MI core and BZ. Innervation was normal in the RZ: 94.9%(93.8-96.0%), decreased in the BZ: 87.9%(85.1-92.4%, p<0.001), and even lower in the MI core: 72.8%(66.3-75.8%, p<0.001). Denervation and hypoinnervation were highest in core biopsies, 16.7%(13.5-23.5%) and 3.8%(2.9-5.0%) resp., lower in the BZ, 2.6%(1.6-6.5%, p<0.008) and 1.2%(0.8-1.7%, p=0.015) and even lower in the RZ, 1.2%(0.7-1.8%, p<0.001), 0.6%(0.3-1.3, p<0.001). Hyperinnervation was observed mainly in the BZ, 5.3%(3.7-9.4%) and in the core, 5.3%(3.7-9.4%, p>0.999), whilst being low in the RZ 2.6%(1.7-4.3%, p=0.002 and p=0.038 respectively).

This method allows detailed visualization and quantification of ND after cardiac damage and can be broadly applied to high resolution nerve imaging. Variable innervation types within the same MI-biopsies indicate a heterogeneous arrhythmia substrate.


H.s. CHEN (Leiden, The Netherlands), J.c. VAN MUNSTEREN, L.m. VOORTMAN, L.j. WISSE, C.a. CLASHAN, M.c. DERUITER, K. ZEPPENFELD, M.r.m. JONGBLOED
17:45 - 20:00 #30594 - 144. The sex of epicardium-derived cells influences the outgrowth of cardiac sympathetic nerves in vitro.
144. The sex of epicardium-derived cells influences the outgrowth of cardiac sympathetic nerves in vitro.

In the past decades, attention on sex differences in the prevalence and outcomes of a wide range of cardiac diseases has increased. Next to overt sex differences in disease presentation and outcome, also differences in autonomic function between males and females have been exposed. After myocardial infarction, male patients have an increased risk for ventricular arrhythmias and sudden cardiac death. Part of these arrhythmias have been attributed to an increase in cardiac sympathetic nerve fibers occurring after cardiac damage. Although mechanical studies of post-myocardial infarction cardiac sympathetic hyperinnervation have raised growing awareness on the role of the autonomic nervous system in arrhythmogenesis, data on the role of sex herein are still scarce and not conclusive. We found that co-cultures of male superior cervical ganglia with male myocardium and male mesenchymal epicardium-derived cells (EPDCs) results in significant higher neurite directional outgrowth towards myocardium compared to entirely female co-cultures. Moreover, male EPDCs in a female setting enhanced the female neurite outgrowth comparable to an entire male environment. We are currently exploring by RNA sequencing of male and female EPDCs, whether  male and female EPDCs have a differential expression of axon repellent guidance cues. Our results confirm the stimulating effect of EPDCs on neurite outgrowth and also demonstrate that sympathetic nerve outgrowth and density differs between male and female in vitro. Our data underlines the potential relevance of sex differences in post-myocardial infarction cardiac hyperinnervation.


Yang GE, Janine VAN GILS (Leiden, The Netherlands), Ruben METHORST, Conny VAN MUNSTEREN, Lieke VAN ROON, Anke SMITS, Marie-José GOUMANS, Marco DERUITER
17:45 - 20:00 #30603 - 146. Role of reactive oxygen species in maternal hyperglycemia-induced congenital heart defects: a potential therapeutic target.
146. Role of reactive oxygen species in maternal hyperglycemia-induced congenital heart defects: a potential therapeutic target.

Congenital heart defects (CHD) affect 1% of all live births in the US annually. The etiology of CHD is multifactorial, that is, both pathogenic genomic variation and environmental risk factors act as CHD contributors. Among the environmental teratogens, maternal pre-gestational diabetes mellitus (matDM) is associated with up to ~5-fold increase in the risk of having an infant with CHD.  Generation of excess reactive oxygen species (ROS) due to maternal hyperglycemia is routinely observed in embryonic hearts exposed to matDM but the teratogenic effect of ROS on cardiac developmental pathways is not fully understood. The Notch and Nitric oxide (NO) signaling pathways, which are highly expressed in the endocardium, are critical for normal heart development.  Previously, we identified a reduction in NO bioavailability and Notch signaling in mouse embryonic hearts exposed to matDM and reported a gene-environment interaction between Notch1 haploinsufficiency and matDM in the development of CHD. Here, we investigated the effect of modulating intracellular levels of oxidative stress in endocardial and endocardial-derived cell lineages to determine the role of ROS in matDM-associated CHD. Endocardial-derived cells exposed to oxidative stress elicit similar responses as hyperglycemia in reduction of NO bioavailability and Notch signaling. To study the role of oxidative stress in vivo, we generated WT and Notch1+/- embryos overexpressing the antioxidant gene, SOD1, to determine whether mitigating oxidative stress can rescue matDM associated CHD. We have confirmed a reduction of oxidative stress in SOD1-tg embryonic hearts and preliminary data shows lower incidence of CHD in SOD1-tg compared to WT embryos exposed to matDM. The role of oxidative stress in matDM-associated CHD requires further investigation, as mitigation of endocardial oxidative stress may be a promising approach to lower the incidence of CHD in high-risk populations.


Talita CHOUDHURY (Columbus, USA), Sara ADAMCZAK, Emily CAMERON, Madhumita BASU, Vidu GARG
17:45 - 20:00 #30612 - 148. Probing the functional necessity of cardiac enhancers associated to key regulators of heart development.
148. Probing the functional necessity of cardiac enhancers associated to key regulators of heart development.

Mammalian heart development depends on the contributions of two major sources of cardiac progenitor cells, the first and the second heart field (FHF/SHF). While FHF cells establish the early heart tube and are major contributors to the left ventricle (LV), SHF progenitors enter the heart tube at the poles and are essential for outflow tract (OFT) and right ventricle (RV) formation. The cardiac transcription factors (TFs) Gata4 and Hand2 are linked in a key gene regulatory module orchestrating SHF progenitor specification, differentiation, proliferation and migration. Mouse embryonic hearts deficient for these TFs exhibit OFT and RV defects, pointing to essential roles of the cis-regulatory landscapes of these genes in controlling SHF progenitors and mirroring congenital heart defects. Using transgenic reporter assays, previous studies have identified sets of enhancers with overlapping activities in SHF progenitors in each locus. However, the related subregional specificities and functions of these enhancers have not yet been characterized in vivo, leaving their functional necessity and involvement in heart disease unexplored. Here, we have generated mouse lines harboring individual deletions of Hand2 and Gata4 cardiac enhancers. Our results based on qualitative and quantitative transcriptional profiling and cardiac marker analysis have revealed considerable cis-regulatory robustness in both, Gata4 and Hand2 topologically associated domains. Nevertheless, we have identified a genomic cardiac enhancer module essentially contributing to Gata4 gene dosage in a heart compartment-specific manner. Our results highlight the complexity of cardiac TF enhancer landscapes and provide a framework for assessment of heart disease-related loss-of-function effects of genomic heart enhancers in vivo.


Virginia ROLAND (Bern, Switzerland), Matteo ZOIA, Julie GAMART, Marco OSTERWALDER
17:45 - 20:00 #30622 - 152. Transcriptional and epigenetic regulation of the cardiac neural crest from induction through migration.
152. Transcriptional and epigenetic regulation of the cardiac neural crest from induction through migration.

The cardiac neural crest is a transient embryonic cell population that contributes to portions of the heart, including the septum of the cardiac outflow tract. Here, we elucidate regulatory changes that may confer ectomesenchymal ability onto the cardiac neural crest, enabling them to contribute to elements of the heart. To this end, we analyzed transcriptional and epigenetic changes in the cardiac neural crest as a function of time from induction to migratory stages. Analysis of single-cell RNA-seq data reveals groups of genes with differential expression profiles, with those associated with ectomesenchymal potential expressed at premigratory stages and then up-regulated during migration. In contrast, genes associated with specification show a reciprocal profile, peaking at the premigratory stages. We next looked for transcription factors co-expressed in the premigratory cardiac but absent from the cranial neural crest at a similar developmental timepoint, identifying FoxC2, FoxP1, and Twist1 as putative regulators of ectomesenchymal fate. Targeted knockouts in the cardiac neural crest using CRISPR-Cas9 revealed that individual loss of each transcription factor results in severe persistent truncus arteriosus. Finally, to build regulatory linkages, we analyze the chromatin landscape of cardiac neural crest cells using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). The results reveal numerous enhancers activated in the cardiac crest population at premigratory stages that are further upregulated during migration, characterized by GO terms associated with heart morphogenesis. Taken together, our results suggest that cardiac crest-specific transcription factors expressed during the specification stage may be critical for mediating their ectomesenchymal differentiation at later stages of heart development.


Shashank GANDHI (Berkeley, USA), D. Ayyappa RAJA, Max EZIN, Marianne BRONNER
17:45 - 20:00 #30626 - 154. Congenital heart defects in the fetus, classifications and embryology: cladistics or phenetics?
154. Congenital heart defects in the fetus, classifications and embryology: cladistics or phenetics?

Congenital heart defects (CHD) are a common cause of fetal death and termination of pregnancy. The aim of this study was to phenotype all fetal cardiac specimens of our  collection, and to create a user-friendly database.

Each specimen was thoroughly examined according to segmental analysis. The main CHD was determined according to the 11 categories and 23 subcategories of the clinical and anatomical classification of CHD. Associated lesions were coded using IPCCC ICD-11 Nomenclature. Codes and photographs for each of the 1236 specimens were linked into the database. Among them, 120 were normal hearts (10%). The three main groups of CHD were ventricular outflow tracts anomalies (32%), functionally univentricular hearts (23%), and anomalies of atrioventricular junctions and valves (14%). The most frequent lesions were valvar anomalies (55%), ventricular septal defects (VSD, 40%), ventricular hypoplasia (35%), interatrial communications (35%). A cladistic grouping can superimpose on the anatomical classification to identify similar characteristics and raise embryological hypotheses. For example, the term “conotruncal” anomaly comprises defects supposed to have the same embryological origin/”common ancestor”. A phenetic approach explores how a segmental characteristics associates with other defects. For example, common arterial trunk is almost always associated with outlet VSD, but also with intact interventricular septum, hypoplastic ventricle, or right aortic arch. Clustering segmental cardiac characteristics without a priori developmental hypotheses may help identifying new mechanisms for CHD. Detailed anatomical phenotyping of fetal hearts should allow us to identify rare associations of malformations, and can help elaborating different approaches of describing and grouping CHD.


Manon HILY (Paris), Damien BONNET, Bettina BESSIERES, Nicolas GARCELON, Hassan FAOUR, Lucile HOUYEL
17:45 - 20:00 #30638 - 156. Induced overexpression of rnx2a in cardiovascular cells triggers ectopic calcification.
156. Induced overexpression of rnx2a in cardiovascular cells triggers ectopic calcification.

Cardiovascular calcification (CVC) is one of the most frequent forms of cardiovascular disease, with increasing prevalence showing high morbidity and mortality rates worldwide.  CVC is characterized by the progressive dysfunction of the cardiac muscle, arteries, and cardiac valves due to ectopic calcification of their extracellular matrix (ECM).  Previously considered a passive accumulation of calcified tissue, it is now recognised that these diseases result from a cell-mediated active process.  In fact, it has been suggested that before calcification cardiovascular cells differentiate to an osteoblastic (bone-cell) fate.  Previous work has shown that overexpression of Runt-related Transcription Factor 2 (Runx2) in mouse is sufficient to induce calcification in the heart and aorta.

Here we use the zebrafish to study the initiation of CVC in vivo using genetic tools and live-imaging at single-cell resolution.  We overexpressed runx2a in cardiovascular cells using cell-specific promoters and confirmed the differentiation of these cells to a pro-osteoblastic lineage. Importantly, these cells were in direct contact with ectopic calcification sites and triggered the recruitment of immune cells.  To determine the role of the immune system in the progression of the CVC phenotype we are now using cell type-specific genetic ablation tools.

Overall, we introduce the zebrafish as a unique model to study the events leading to the initiation and progression of CVC, aiming to identify new therapeutic targets.


Inês CRISTO (Lisbon, Portugal), Didier STAINIER, Anabela BENSIMON-BRITO
17:45 - 20:00 #30641 - 158. Dysmorphic sarcoplasmic lumen, altered calcium transients, and depressed contractile ability seen in a patient-specific iPSC-CM model of Ebstein’s anomaly and left ventricular noncompaction.
158. Dysmorphic sarcoplasmic lumen, altered calcium transients, and depressed contractile ability seen in a patient-specific iPSC-CM model of Ebstein’s anomaly and left ventricular noncompaction.

Patients presenting with congenital heart diseases (CHDs), Ebstein’s anomaly (EA) and left ventricular noncompaction (LVNC), suffer higher morbidity than either CHD alone. The genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. In a familial case of EA/LVNC associated with the KLHL26 (p.R237C) variant, we discovered an altered electrostatic surface profile of the variant protein, which likely decouples the CUL3-interactome and alters protein turnover. 

To investigate the underlying mechanisms, we differentiated a family trio (EA/LVNC-unaffected daughter and EA/LVNC-affected mother and daughter) of induced pluripotent stem cells into cardiomyocytes (iPSC-CMs), assessed for significant differences in CM structure and function (P<0.05), and synthesized cellular and pathway insights. We characterized morphology via immunofluorescence and electron microscopy and contractile function via biomechanical and automated video methods. We analyzed differential mRNA expression via RNA Sequencing of iPSC-CMs and cardiac tissues, and iPSC-CM protein abundances via label-free quantification.

In comparison to the unaffected, EA/LVNC-affected iPSC-CMs exhibited aberrant morphology and contractile ability, mainly a distended endo/sarcoplasmic reticulum (ER/SR), decreased beat rate, and altered calcium transients. The affected daughter iPSC-CMs further showed decreased apoptosis and increased proliferation. From pathway enrichment analyses, we saw a suppression of myocardial contraction and an activation of ER lumen and lipid metabolism. Via RT-PCR, relative to GAPDH and unaffected iPSC-CMs, we found significantly decreased expression of Sarcoplipin (SLN) and increased Junctophilin-2 (JPH2).

Together, these results from cell and developmental studies suggest that this familial EA/LVNC associated with the KLHL26 variant develops dysregulated ER/SR signaling and altered calcium transients with subsequent lesions in CM contractility and cell cycle progression.


Suma THAREJA (Milwaukee, USA), Melissa ANFINSON, Matthew CAVANAUGH, Min-Su KIM, Peter LAMBERTON, Jackson RADANDT, Ryan BROWN, Huan Ling LIANG, Karl STAMM, Muhammad Zeeshan AFZAL, Jennifer STRANDE, Michele FROMMELT, John W. LOUGH, Robert FITTS, Michael E. MITCHELL, Aoy TOMITA-MITCHELL
17:45 - 20:00 #30644 - 160. Characterization of Hippo:YAP1 and Retinoic Acid signaling pathways during atrial cardiomyocyte acquisition.
160. Characterization of Hippo:YAP1 and Retinoic Acid signaling pathways during atrial cardiomyocyte acquisition.

Abnormalities in early development of Cardiac Progenitor Pool (CPC) located in the Second Heart Field can progress to a myriad of congenital heart defeats that can contribute to neonatal mortality. Thus, understanding the regulatory mechanisms that contribute to cell lineage differentiation and fine tuning in early human development will help to prevent or cure heart defects. Specifically, we focus on Retinoic Acid since it is a key regulator in cardiogenesis where it promotes the posterior specifications of CPCs and their progression toward atrial Cardiomyocyte (CM) lineages. Despite its importance, the specific mechanisms of RA are not well understood. In Embryonic stem cells YAP functions differentially and is controlled by multiple different signaling pathways. We have identified YAP1;TEAD 4 factors as important cell fate determinants of atrial CM in cooperation with RA signaling. It has been shown that RA induces NR2F2 (Nuclear Receptor Subfamily 2 group F member 2) expression to confer atrial identity during CPC differentiation. NR2F2 is an orphan nuclear receptor that binds DNA and it forms heterodimers with the retinoid X receptor to regulate transcription of RA target genes. Our data suggest that RA recruits YAP to the chromatin through NR2F2 and that there is a collaborative effect in CM differentiation with YAP and NR2F2. By combining a range of molecular approaches we aim to establish a Link between NR2F2 and YAP;TEAD in vitro and in vivo. Overall, our ongoing studies suggest that RA signaling induces the activation of non-canonical YAP:TEAD enhancers which are integrated with the cardiac transcription factor network of CPCs essential for the specification of atrial lineages.


Conchi ESTARAS, Corrine LITTLE (Philadelphia, USA), Elizabeth ABRAHAM, Mikel ZUBILLAGA, Clara DE PABLO REVOLTOS
17:45 - 20:00 #30650 - 162. “The role of SHROOM3 in congenital heart disease”.
162. “The role of SHROOM3 in congenital heart disease”.

We implicated a novel CHD candidate, SHROOM3, in cardiac defects within mice and humans. Mechanistic studies have focused on SHROOM3 interaction with ROCK1/2 to impact the cytoskeleton. However, a recent study indicates SHROOM3 interaction with cell adhesion protein N-CADHERIN is important during neural tube development. Given the range and severity of defects attributed to SHROOM3, we hypothesize mechanism beyond ROCK1/2 are important during cardiac development. To test this hypothesis we performed complementary analysis of the transcriptome, global proteome, and phospho-proteome, in Shroom3+/+ vs Shroom3gt/gt embryo heart lysates using RNA-Seq and tandem mass tag labeling, mass spectroscopy (MS). Pathway analysis of both the transcriptome and proteome revealed altered G-Protein coupled receptor signaling. However, GO analyses of the phospho-proteome, indicate the top pathway disrupted in Shroom3gt/gt embryos is cardiac cell-cell adhesions. An in vitro cell dissociation assay confirmed SHROOM3-loss-of-function downregulates cell-cell adhesions.  Next, we generated a SHROOM3 protein interactome using co-immunoprecipitation/MS (co-IP/MS), with both full length GFP-SHROOM3 and deletion constructs. The SHROOM3 protein interactome confirms N-CADHERIN is a top binding component and the deletion constructs co-IP/MS reveal a likely interaction site, with the deletion of a.a. 286-776, disrupting SHROOM3-N-CADHERIN interaction. Finally, we interrogated exome sequencing of 283 patients with left-right patterning and cardiac defects, and identified 12 rare, potentially damaging variants in SHROOM3. Whereas no variants fell within the ROCK1/2 binding region, one third cluster to the proposed N-CADHERIN binding region, indicating these variants may interrupt N-CADHERIN interaction and cell adhesions during cardiac development, resulting in CHD. Taken together these data indicate novel role for SHROOM3 interacting with N-CADHERIN driving cell adhesions during cardiac development.


Matthew DURBIN (INDIANAPOLIS, USA), James ZWIERZYNSKI, Stephanie WARE
17:45 - 20:00 #30657 - 164. In Vivo and In Vitro Cartilage Differentiation from Embryonic Epicardial Progenitor Cells.
164. In Vivo and In Vitro Cartilage Differentiation from Embryonic Epicardial Progenitor Cells.

The presence of cartilage tissue in the embryonic and adult hearts of different vertebrate species is a well-recorded fact. However, while the embryonic neural crest has been historically considered as the main source of cardiac cartilage, recently reported results on the wide connective potential of epicardial lineage cells suggest they could also differentiate into chondrocytes. In this work, we describe the formation of cardiac cartilage clusters from proepicardial cells, both in vivo and in vitro. Our findings report, for the first time, cartilage formation from epicardial progenitor cells, and strongly support the concept of proepicardial cells as multipotent connective progenitors. These results are relevant to our understanding of cardiac cell complexity and the responses of cardiac connective tissues to pathologic stimuli.


Paul PALMQUIST-GOMES, Ernesto MARÍN-SEDEÑO, Adrián RUIZ-VILLALBA, Gustavo Adolfo RICO-LLANOS, José María PÉREZ-POMARES, Juan Antonio GUADIX (Málaga, Spain, Spain)
17:45 - 20:00 #30686 - 166. Genome-wide transcriptomics analysis of genes regulated by GATA4, 5 and 6 during cardiomyogenesis.
166. Genome-wide transcriptomics analysis of genes regulated by GATA4, 5 and 6 during cardiomyogenesis.

The transcription factors GATA4, GATA5 and GATA6 are key regulators of vertebrate heart muscle differentiation (cardiomyogenesis), but specific target genes regulated by these individual cardiogenic GATA factors remain unknown. We have identified genes that are specifically regulated by each of them, as well as those regulated by either of them using genome-wide transcriptomics analysis in Xenopus laevis. The genes regulated by gata4 are particularly interesting because GATA4 is able to induce differentiation of beating cardiomyocytes in Xenopus and in mammalian systems. Among the specifically gata4-regulated transcripts we identified two SoxF family members, sox7 and sox18. Experimental reinstatement of gata4 restores sox7 and sox18 expression, and loss of cardiomyocyte differentiation due to gata4 knockdown is partially restored by reinstating sox7 or sox18 expression, while (as previously reported) knockdown of sox7 or sox18 interferers with heart muscle formation. In order to test for conservation in mammalian cardiomyogenesis, we confirmed in mouse embryonic stem cells (ESCs) undergoing cardiomyogenesis that knockdown of Gata4 leads to reduced Sox7 (and Sox 18) expression and that Gata4 is also uniquely capable of promptly inducing Sox7 expression.  Our genome-wide transcriptomics analysis therefore identifies an important and conserved gene regulatory axis from gata4 to the SoxF paralogs sox7 and sox18 and further to heart muscle cell differentiation. Our identification of genes that are differentially regulated by each of cardiogenic gata factors also provides a platform for future investigations on the gene regulatory network underpinning embryonic cardiomyogenesis.


Boni A AFOUDA (Aberdeen, United Kingdom), Adam T LYNCH, Stefan HOPPLER
17:45 - 20:00 #30690 - 168. Neural crest cell-derived Wnt5a regulates second heart field planar cell polarity during cardiac outflow tract development.
168. Neural crest cell-derived Wnt5a regulates second heart field planar cell polarity during cardiac outflow tract development.

Wnt5a is a known regulator of planar cell polarity (PCP) signals in second heart field (SHF) progenitors. However, the exact identity of the cell source of Wnt5a has not been fully elucidated. We studied murine heart development following neural crest cell (NCC)- and SHF-specific conditional knock out of Wnt5a. Our results demonstrate that SHF-derived Wnt5a regulates PCP within the early SHF cells required for initial OFT elongation. Mef2c-cre mediated loss of SHF-derived Wnt5a leads to reduced SHF incorporation into the early OFT and a spectrum of OFT phenotypes, ranging from common arterial trunk arising from RV (25%) to double-outlet right ventricle (DORV, 25%) to normal OFT development (50%). As NCC migrate into the developing OFT, they also secrete Wnt5a. Wnt1-cre mediated loss of NCC-derived Wnt5a causes downregulation of PCP signaling and migratory arrest of more cranial, late wave SHF progenitors. The resulting foreshortened OFT is unable to align over the ventricles such that the appropriate rotation and development of the pulmonary trunk is perturbed. All NCC-mutants demonstrate DORV (100%), with additional pulmonary outflow defects observed in 62%. Neither conditional mutant demonstrates disrupted NCC migration into OFT or venous pole defects. Overall, these results demonstrate that different subsets of SHF progenitors respond to PCP signals from SHF and NCC depending on their location and timing during embryonic development. Apart from providing new insights into the source of Wnt5a during heart development, our data are also the first to demonstrate a novel role for NCC in regulating PCP in SHF cells.


Omar TOUBAT, Jongkyu CHOI, Prashan DE ZOYSA, Drayton HARVEY (Los Angeles, USA), Henry SUCOV, Jianbo WANG, Ram Kumar SUBRAMANYAN
17:45 - 20:00 #29372 - 170. Ventricular septation in wild-type and Down Syndrome mice.
170. Ventricular septation in wild-type and Down Syndrome mice.

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and is the most common cause of congenital heart defects (CHDs), with approximately half of all DS births being born with a form of CHD. Most of these arise from aberrant heart septation during development, leading to a combination of shunting between the left and right heart chambers at either the ventricular or atrial levels and incorrect valve formation. However, the developmental origins of the CHDs in DS remain poorly understood. To address this, we are using the Dp1Tyb mouse model of DS, which contains an extra copy of mouse chromosomal regions orthologous to Hsa21, and recapitulates human DS CHD phenotypes, with ~60% of Dp1Tyb embryos at embryonic day 14.5 (E14.5) showing defects in cardiac septation. Analysis of morphogenetic changes during ventricular septation in Dp1Tyb E10.5-E13.5 embryos found no difference in growth of the muscular ventricular septum but showed abnormalities in the outflow tract cushions (OFTCs), the transient developmental structures that complete the final stages of ventricular septation and separation of the pulmonary artery from the aorta. Results indicate that Dp1Tyb OFTCs have altered morphology and reduced cell density. Since most of the OFTCs are derived from cardiac neural crest cells, these results imply that the CHDs found in DS may be caused by changes in neural crest differentiation, potentially linking these defects to other DS-associated abnormalities such as craniofacial dysmorphology.


Mint HTUN (London, United Kingdom), Rifdat AOIDI, Eva LANA-ELOLA, Dorota GIBBINS, Jeremy GREEN, Victor TYBULEWICZ
17:45 - 20:00 #30515 - 172. Identifying key genes in the formation of mammalian heart valves.
172. Identifying key genes in the formation of mammalian heart valves.

Heart valve defects are the most common birth defects in new-borns worldwide. Endocardial (EC) cells are a subpopulation of endothelial (ET) cells that have a unique ability to undergo endothelial-to- mesenchymal (EndoMT) transition, a process critical for heart valve formation. To date, only one molecular maker, NFATc1, is known that uniquely labels EC cells during valve development. However, it is not the master regulator of EC cell specification since mice deleted for Nfatc1 forms underdeveloped heart valves. The ability of EC cells to undergo EndoMT makes them distinct from ET cells, despite both sharing a common precursor origin during cardio-genesis. We hypothesize that EC cells become distinct from ET cells via the expression of a sub-set of genes that are regulated differently at genetic and epigenetic levels.

The integrated analysis of bulk RNA-sequencing and whole-genome bisulphite sequencing data alongside single-cell RNA-sequencing datasets from public domain has identified candidate genes involved in EC-fate determination. These candidates have been validated at the gene expression level. Additionally, inducible shRNA knockdowns of candidate genes have been studied in an Nfatc1-mCherry mouse ES cell line using an in vitro hanging drop culture differentiation system. This has provided a functional assay for the temporal knockdown effects of these genes on the EC differentiation and specification. One of the top candidates is Zfpm1 and the effect of its knockdown on the functional ability of EC cells to undergo EndoMT is assessed using trans-well invasion and migration assays. Altogether, this will not only facilitate our understanding of the role of Zfpm1 in heart development but will also provide a basis for stem cell-based therapies for valve-related defects. 


Punkita LOHIYA (London, United Kingdom)
17:45 - 20:00 #30719 - 174. Epigenetic control of cardiac metabolism by the histone demethylase Kdm8 prevents heart failure.
174. Epigenetic control of cardiac metabolism by the histone demethylase Kdm8 prevents heart failure.

Kdm8 demethylates the di-methylated form of lysine 36 of histone H3 (H3K36me2) and prevents spurious gene expression. Kdm8 is enriched in the developing mouse heart, and its constitutive inactivation is embryonically lethal. Moreover, H3K36me distributes abnormally genome wide in the failing heart, in which metabolism shifts towards glycolysis. However, the function of Kdm8 in the heart or the contribution of metabolic imbalance to initiate the events leading to heart failure are unknown. We show that Kdm8 maintains the expression of genes controlling mitochondrial metabolism by repressing Tbx15 to prevent dilated cardiomyopathy leading to lethal heart failure. Kdm8 inactivation in cardiomyocytes by cre-mediated homologous recombination increased global levels of H3K36me2, caused adverse myocardial remodeling beginning at 4 months, and dead due to heart failure by 9 months of age. Genome wide mRNA profiling revealed that Tbx15 target genes controlling NAD+ metabolism were downregulated in Kdm8 mutant hearts before the initiation of adverse myocardial remodelling. Moreover, metabolomics showed decreased NAD+ pathway intermediates, and NAD+ treatment blunted the initiation of cardiac deterioration towards heart failure. Furthermore, Tbx15 overexpression in cardiomyocytes blunted the respiratory increase induced by NAD+. This suggests that dysregulation of a KDM8 – TBX15 axis sits high in the hierarchy of events initiating cardiac deterioration towards heart failure. Indeed, KDM8 was downregulated in human hearts affected by heart failure. Furthermore, higher expression of TBX15 tracked with stronger downregulation of genes encoding mitochondrial proteins. Our findings suggest that epigenetically controlled metabolic gene networks are dysregulated to initiate adverse myocardial remodelling towards heart failure.


Abdalla AHMED, Carmina PEREZ ROMERO, Lijun CHI, Jibran SYED, Yaxu WANG, Quetzalcoatl ESCALANTE COVARRUBIAS, Dorothy LEE, Etri KOCAQI, Lorena AGUILAR ARNAL, Gerard Bryan GONZALES, Kyoung-Han KIM, Paul DELGADO OLGUIN (Toronto, Canada)
17:45 - 20:00 #30322 - 176. A digital framework to analyse, understand, and stereotypically compare early myocardium morphogenesis in the mouse model.
176. A digital framework to analyse, understand, and stereotypically compare early myocardium morphogenesis in the mouse model.

A dynamic Atlas with cellular resolution is an essential resource for understanding at a multiscale level the key mechanics of cardiac morphogenesis and assessing variability between embryos.
Reconstructing a 4D atlas of the mouse heart is a challenge; indeed, although new live-cell imaging techniques allow a crucial understanding of cellular and tissue kinetics, they do not guarantee a whole-organ acquisition at a spatiotemporal resolution necessary for a detailed multidimensional reconstruction.
To overcome this technological limitation, we designed a dedicated computational framework that, starting from a high-resolution stereotyped atlas, incorporates cellular kinetics and dynamic data of the myocardium provided by time-lapse images from a collection of specimens.
The incorporation of live-embryo data into the Atlas is performed in two fundamental steps: a temporal staging and a spatial mapping of cardiac shapes. Our framework implements a staging system that, through a morphometric feature, assigns to each frame of the time-lapse a developmental stage of the Atlas. Given the temporal matching, each cardiac shape is then mapped, by combined applications of image processing and registration techniques, in the respective Atlas shape.
This last step allows to map the individual cells tracked in the time-lapse images and to project dynamic features such as growth rate, anisotropy and strain to which the myocardium is subjected during its morphogenesis.
This computational method is intended as a novel tool to compare different live-embryos in a single time-space convention, which is necessary to investigate key processes in heart development.


Morena RAIOLA (Spain, Spain), Isaac ESTEBAN, Miguel TORRES
17:45 - 20:00 #30521 - 180. QUANTITATIVE CHARACTERISATION OF A CARDIAC PROGENITOR CELL EPITHELIUM.
180. QUANTITATIVE CHARACTERISATION OF A CARDIAC PROGENITOR CELL EPITHELIUM.

During development, cells adopt diverse strategies to sculpt epithelial tissues into characteristic 3D shapes with molecularly and mechanically distinct regions. The vertebrate heart tube is a good example, as it extends by progressive addition of second heart field (SHF) progenitor cells within an epithelial sheet in the dorsal pericardial wall. Initially contributing to the entire cardiac primordium across the dorsal mesocardium, SHF cell addition is restricted to the poles of the heart tube after dorsal meoscardial breakdown. Perturbation of SHF deployment results in a spectrum of congenital heart defects (CHD). T-box transcription factors implicated in CHD regulate the emergence of a boundary segregating progenitor cells to alternate cardiac poles. In addition, the epithelial properties of SHF cells, including cell elongation, clonal anisotropy and epithelial tension, have been identified as regulatory targets during heart tube elongation. However, the relationship between epithelial properties, mechanical tissue stress and progenitor cell patterning in the SHF remains unknown. Indeed, understanding how cellular and tissue forces contribute to growth and form during organogenesis is a major challenge in developmental biology. Here we present quantitative phenomenological analysis of epithelial cellular properties of cells throughout the dorsal pericardial wall, including cell elongation, polarity, anisotropy and cell contacts.  Patterns of epithelial stress and tension in the SHF are integrated in our dataset using force inference image analysis. We document the dynamic relationship between epithelial features and progenitor cell patterning at embryonic days 8.5 and 9.5, before and after dorsal mesocardium breaks down, and in T-box mutant embryos. By integrating findings from cell scale feature patterning, mechanical tissue stress and cell biology our project will provide new mechanistic insights into cardiac morphogenesis and the origins of CHD.


Clara GUIJARRO CALVO (Marseille), Benoît AIGOUY, Paul VILLOUTREIX, Robert G. KELLY
17:45 - 20:00 #30537 - 182. Role of the cAMP-dependent protein kinase A regulatory subunit RIα in cardiac morphogenesis.
182. Role of the cAMP-dependent protein kinase A regulatory subunit RIα in cardiac morphogenesis.

Second heart field (SHF) progenitors allow the rapid elongation of the embryonic heart tube. The precise control of SHF deployment is a prerequisite for correct heart tube elongation and any alteration results in congenital heart defects (CHD). We previously identified the transcriptional repressor Hes1 as a critical regulator of SHF deployment and preliminary results suggest that regionalized SHF expression of the cAMP-dependent protein kinase (PKA) regulatory subunit RIα (Prkar1a) may control Hes1 expression in the SHF. In the adult heart, PKA is known to regulate heart contraction nevertheless its role during heart development still remains unknown. In human, mutations in PRKAR1A gene cause haploinsufficiency which results in a rare Carney Complex syndrome, associated with benign tumors including cardiac myxomas described to be associated with congenital heart defects.

To evaluate the role of R1α during early heart morphogenesis, we developed transgenic mouse lines allowing conditional deletion of Prkar1a in diverse cardiac progenitor cell lineages.

Our results demonstrate a key role for RIα in controlling SHF deployment. Indeed, RIα deletion in SHF progenitors results in impaired SHF proliferation, associated with early embryonic lethality. Upregulated Hes1 expression was detected in RIα mutants and increased phenotype severity observed in RIα/Hes1 compound mutants strongly support RIα and Hes1 genetic interaction. The extracardiac cell population, cardiac neural crest (CNC), plays a critical role in heart morphogenesis and our results suggest key role for RIα in CNC cell deployment. Additionally, heterozygous RIα deletion in CNC leads to a 5 week-old lethality associated with cardiac hyperplasia and CNC lineage tracing reveals the existence of subendocardial CNC progenitors in the adult heart, suggesting a potential role for CNC derivatives in the development of cardiac myxomas.

Altogether our study reveals a critical role for RIα in early cardiac development.


Corentin PORADA (Marseille), Fabien HUBERT, Grégoire VANDECASTEELE, Francesca ROCHAIS
17:45 - 20:00 #30563 - 184. The lipid sensor Pparg and non-canonical Notch pathway contribute to the regional identity of the outflow tract.
184. The lipid sensor Pparg and non-canonical Notch pathway contribute to the regional identity of the outflow tract.

The arterial pole of the heart is a hotspot for life-threatening forms of congenital heart defects (CHDs). This cardiac region results from the elongation of the embryonic outflow tract (OFT) that requires the addition of Second Heart Field (SHF) progenitor cells to provide the myocardium at the base of the ascending aorta and pulmonary trunk. Investigating the transcriptional program of future sub-aortic and sub-pulmonary myocardium we found that the gene encoding the lipid sensor Pparγ is preferentially expressed in the inferior OFT wall. Here we show that TBX1 is required for the regional expression of PPARγ and its downstream target genes in the inferior OFT. Using mouse genetics and ex vivo embryo culture in the presence of PPARγ agonists or antagonists, we demonstrate that Pparg controls cardiac progenitor cell proliferation and addition of the future subpulmonary myocardium to the OFT, both critical determinants for normal arterial pole development. Moreover, we show that the non-canonical DLK1/NOTCH/HES1 pathway negatively regulates Pparγ in future subaortic myocardium. In conclusion, we present evidence that Pparg is a regulatory component of the cross-circuitry controlling regional identity at the arterial pole of the heart, thus providing new insights into gene, and potential gene-environment, interactions involved in CHD.


Magali THÉVENIAU-RUISSY (Marseille), Mayyasa RAMMAH, Rachel STURNY, Francesca ROCHAIS, Robert G KELLY
17:45 - 20:00 #30590 - 186. Uncovering the signalling cues that influence the development and differentiation of the cardiopharyngeal mesoderm.
186. Uncovering the signalling cues that influence the development and differentiation of the cardiopharyngeal mesoderm.

The heart is built by distinct progenitor populations that contribute to specific cardiac chambers, valves and vessels. These progenitors arise from different germ layers in the gastrulating embryo. The mesoderm-derived cardiac progenitors include a population called the cardiopharyngeal mesoderm (CPM), that contributes to second heart field-derived cardiac regions, but also to head skeletal muscles by populating the pharyngeal arches. While the development and differentiation of cardiac cell types has been examined closely, the developmental mechanisms regulating fate commitment of the head muscle progenitors in the CPM are unclear. I have addressed the early development of the CPM, and its differentiation into skeletal muscle. Using mouse embryos and an embryonic stem cell model, our work shows that inhibition of Wnt/beta-catenin and Nodal pathways underlies the fate specification of these progenitors. Using our findings, we directed embryonic stem cells to differentiate into the CPM and subsequently, into cardiomyocytes and skeletal muscles. Our in vitro model also suggests that there may be a distinct myogenic cue for skeletal muscles derived from the CPM, as compared to skeletal muscles derived from somitic mesoderm. By generating derivatives in vitro, specifically via the CPM, this approach can provide a tractable model of syndromes including both congenital heart diseases and muscular dystrophies.


Nitya NANDKISHORE (Marseille), Fabienne LESCROART, Ramkumar SAMBASIVAN
17:45 - 20:00 #30616 - 188. The role of the apelin receptor in cardiac lineage specification in zebrafish.
188. The role of the apelin receptor in cardiac lineage specification in zebrafish.

The existence of an early cardiac progenitor (CP) population has long been supported by fate mapping experiments which identify presumptive CPs at specific embryonic positions as gastrulation begins. These presumptive CPs then migrate to the anterior lateral plate mesoderm and engage a conserved transcriptional network to direct specification, migration, and differentiation of the cardiac lineage. While later stages of cardiac development are well characterized, initial specification remains poorly understood, due to a lack of specific molecular markers for early CPs. To address the outstanding question of CP specification our group is studying the Apelin receptor (Aplnr), a highly conserved GPCR, first shown to regulate cardiac development in zebrafish where aplnra/b mutants do not express nkx2.5, the earliest specific CP marker, following gastrulation and do not develop cardiomyocytes. Fate mapping analysis has shown that migration of presumptive CPs during gastrulation requires Aplnr. Following loss of Aplnr function Nodal signalling fails to activate correctly and this failure disrupts mesendoderm specification. scRNA-seq has identified that with loss of Aplnr function a cardiac-like mesoderm population fails to develop during gastrulation. Our results indicate that Aplnr is required for the initial commitment of mesodermal cells to the cardiac lineage. Current work is focused on characterizing this specification failure to understand both the function of Aplnr and the mechanisms regulating early CP development.


Nathan STUTT (Toronto, Canada), Ian SCOTT
17:45 - 20:00 #30685 - 190. Exploring the interface between the first and second heart fields: implications for cardiac septation.
190. Exploring the interface between the first and second heart fields: implications for cardiac septation.

The embryonic heart elongates by progressive addition of second heart field (SHF) cardiac progenitor cells to the arterial and venous poles of the early heart tube, derived from the first heart field (FHF). The SHF gives rise to the right ventricle, outflow tract and part of the atrial myocardium. Defects in SHF deployment contribute to a spectrum of congenital heart defects (CHD) affecting the cardiac poles. Septation defects account for over 50% of CHD. The primary atrial septum and dorsal mesenchymal protrusion originate in Tbx1 expressing cells in the posterior dorsal pericardial wall where they activate the FHF regulator Tbx5 in response to retinoic acid (RA) signaling. Transient coexpression of Tbx1 and Tbx5 is followed by downregulation of the SHF program, resulting in emergence of a sharp boundary between Tbx1-positive arterial pole progenitors and Tbx5-positive venous pole progenitors. A similar history of expression of both FHF and SHF regulators, followed by downregulation of the SHF program, demarcates boundary formation at the site of ventricular septation. Here we show that transient overlap between the FHF and SHF programs is observed along the dorsal mesocardium during early heart development, followed by downregulation of the FHF program in the midline of the dorsal pericardial wall. Single cell RNA-seq analysis of the SHF lineage identifies venous pole progenitor cells that switch to a FHF program and mouse genetics reveals that SHF genes are downregulated through T-box gene dependent and independent mechanisms. Finally, using a conditional dominant negative RA receptor we show that RA signaling is required at the heart field interface for morphogenesis of the muscular ventricular septum. Our results point to the importance of a transient regulatory module at the heart field interface in boundary formation during early heart morphogenesis and patterning of future septal structures, providing insights into the etiology of CHD and septum evolution.


Charlotte THELLIER (Marseille), Marcel GRUNERT, Marie COUDERC, Clara GUIJARRO-CALVO, Claudio CORTÉS, Nicolas BERTRAND, Rachel STURNY, Christopher DE BONO, Magali THÉVENIAU-RUISSY, Silke RICKERT-SPERLING, Stéphane ZAFFRAN, Robert G. KELLY
17:45 - 20:00 #30329 - 192. Development of an in vitro model of cardiomyocyte cell competition.
192. Development of an in vitro model of cardiomyocyte cell competition.

Heart regeneration after infarction has been an important goal in the last decades. The low regenerative potential of the mammalian heart has set an essential barrier to this achievement, and innovative strategies need to be developed to recover heart function. Cell competition (CC) has been described in multiple tissues, including the mammalian developmental and adult heart. In this context, fitter cardiomyocytes outcompete their neighbors, thus improving the proportion of more functional cells with no deleterious consequences. However, this process has not been thoroughly characterized yet in the mammalian heart, even though understanding it could help unlock cardiomyocytes' regenerative capacity and heal the injured heart.

We have developed a model to closely study cardiomyocyte CC based on Myc mosaic overexpressing hearts (iMOS Myc). By dissociating P1 iMOS Myc;MHC-Cre mouse hearts, we obtain highly reproducible cultures in which the winner population can be tracked over time. Using this approach we have been able to detect the expansion of Myc-overexpressing cardiomyocytes at the expense of the wildtype ones. This indicates that CC is potentially happening in our experimental setting. We are currently characterizing key features of CC, such as winner cell proliferation and loser cell death. Preliminary data suggests increased proliferation in iMOS-Myc cultures compared to controls, although further characterization is warranted.

Additionally, we have set an in vitro system to overexpress factors of our interest in our cardiomyocyte cultures, taking advantage of the specific tropism of Serotype 9 AAVs. So far, we have expressed YAP5SA, a constitutively active form of YAP that elicits LATS1/2 repressive phosphorylation. We found 2,24% proliferating cardiomyocytes at 3 dpi (compared to a 0,16% rate in controls) with clear signs of sarcomere disassembly. We plan to use this model to test for new factors capable of triggering CC.


Jorge PEÑA PEÑA (Madrid, Spain), Cristina VILLA DEL CAMPO, Miguel TORRES SÁNCHEZ
17:45 - 20:00 #30476 - 194. Generation of iPSC-derived human cardiac valve cells.
194. Generation of iPSC-derived human cardiac valve cells.

Congenital heart disease affects nearly 1% of births and includes valvular abnormalities, such as pulmonary valve stenosis, which commonly occurs in the RASopathies. During valvulogenesis, a subpopulation of endocardial cells overlaying the cardiac jelly undergoes endothelial-to-mesenchymal transition (EndMT), producing valvular interstitial cells (VICs). These resident mesenchymal cells of the cardiac valve are responsible for extracellular matrix production and homeostasis. Furthermore, a subpopulation of endocardial cells escapes EndMT and becomes valvular endothelial cells (VECs), which line the outside of cardiac valve leaflets. Critically, a differentiation strategy to generate both cell populations is needed so that human valvular disease can be modeled in vitro. Recently, a feeder-dependent embryoid body differentiation strategy utilizing BMP10 has been shown to generate an endocardial population that is distinct from vascular endothelial cells. Here, we show that iPSCs maintained without feeder cells can be differentiated as a monolayer towards mesoderm with the GSK3 inhibitor CHIR-99021 and subsequently to an endocardial lineage with BMP10 and bFGF. Importantly, these endocardial cells express high levels of endocardial markers, such as NKX2.5, GATA4, GATA5, and NRG1. EndMT can then be induced on this endocardial population. Following six days of EndMT, some of these endocardial cells become VICs and upregulate key mesenchymal genes, such as SOX9, CDH11, and COL1A1. A subpopulation of endocardial cells escapes EndMT to become VECs and maintain high expression of endothelial genes, such as PECAM1, CDH5, and EMCN. These cell populations can be utilized for in vitro modeling of human cardiac valves.


Clifford LIU (New York, USA), Bruce GELB
17:45 - 20:00 #30501 - 196. Human embryonic stem cell derived cardioids as a model of human development.
196. Human embryonic stem cell derived cardioids as a model of human development.

Chamber-like cardiac organoids or « cardioids » constitute a promising in vitro model to study human physio-pathological cardiac development. A WNT-BMP modulation-based protocol for the generation of self-organizing cardioids recently published (Hofbauer & al., Cell 2021) was optimized using several pluripotent stem cell lines. Cardioids featured expected Ca2+ spiking and contractions from differentiation day 14 when chambers were formed. The physiological significance of cardioids was further challenged.

Both the rate and the amplitude of beating cardioids were challenged using several pharmacological agents including the pacemaker channel inhibitor ivabradine, or the positive inotropic agents phenylephrine and epinephrine, a1-adrenergic (Phenylephrine) or b-adrenergic agonist, respectively. Contractility rate of cardioids was not impaired by a pacemaker current inhibitor (Ivabradine) pointing to a myocyte cell-autonomous generation of beating rate. This observation was confirmed by the presence of functional inositol 1,4,5-trisphosphate receptors (IP3R) within cardiomyocytes, revealed by the inhibition of spontaneous beating rate by Xestospongin.

We identified the early cell aggregation step as a key process which determines both the fate and position of cells in the self-generating cardioids. To improve cell aggregation  and reproducibility  of cardioids over time, micro-patterning used within a Sound Induced Morphogenesis (SIM) device (CymatiX MimiX Biotherapeutics), should turn out to be an extremely powerful technology.

Regardless of their still immature states, we report the functional and physiological properties of chamber-like cardioids and thus their relevance as a potent platform to study cardiac development and physiopathology.


Valentin AZEMARD (Marseille), Michel PUCEAT
17:45 - 20:00 #30518 - 198. Deciphering transcription factor regulatory networks during human cardiac development.
198. Deciphering transcription factor regulatory networks during human cardiac development.

Introduction – Transcription factors (TFs) are key regulators that control the temporal extensive variations of gene expression, the hallmarks of sequential stages of human cardiac development. Genetic alterations of these TFs cause human cardiac defects. TFs act within regulatory networks which have been only partially described. Using cardiac differentiation of human induced pluripotent stem cells (hiPSCs), this study aims at unravelling the global TF regulatory network that govern human cardiac development.

Methods & Results – Transcriptomic data were generated daily, from day 0 to day 30, in triplicates, during directed cardiac differentiations of three hiPSC lines reprogrammed from healthy donor somatic cells. The top 3,000 differentially expressed genes (DEGs) along cardiac differentiation were pooled into 12 gene clusters, illustrating successive temporal alterations of gene expression. Functional annotation of these gene clusters and comparison of their expression profiles to publicly available transcriptomic data of murine cardiac development, demonstrated that hiPSC cardiac differentiation accurately recapitulates transcriptomic processes governing cardiac development. Next, a regulatory network that includes all 216 TFs from the 3,000 DEGs was investigated using an expression-based correlation score involving time delay. Overall, 6,817 activation-type and 6,497 inhibition-type relationships were inferred between these 216 TFs. Compared to the literature-based STRING database, our regulatory network was 3.3 fold denser and could also inform about directions, types and time delays of each interaction. Identified sub-networks included well-known interactions, such as between FOXC1, ISL1, MEF2C and HAND2, but also new candidate sub-networks.

Conclusion – This work shows that hiPSC cardiac differentiation is relevant for developmental studies, and provides a valuable database to explore new TF regulation networks during human cardiac development.


Bastien CIMAROSTI (Nantes), Robin CANAC, Aurore GIRARDEAU, Virginie FOREST, Lemarchand PATRICIA, Redon RICHARD, Nathalie GABORIT, Guillaume LAMIRAULT
17:45 - 20:00 #30558 - 200. The Role of YAP1 in the most important event of everyone’s life: Gastrulation.
200. The Role of YAP1 in the most important event of everyone’s life: Gastrulation.

Long-standing studies in embryos have shown that polarized extraembryonic signals generate a NODAL:SMAD2/3 gradient in the epiblast that regulates gastrulation patterning and the establishment of the anterior-posterior (A-P) axis. However, epiblast-like cells (hESCs and mESC) organize in 2D and 3D gastruloid structures in vitro in the absence of extraembryonic tissues. These observations highlight the importance of intrinsic self-organizing mechanisms in hESCs, independent of polarized extraembryonic signals. Indeed, human 3D-gastruloids display features of Carnegie-stage-9 embryos, with a high degree of organization in gene expression along the A-P axis, including a posterior-to-anterior signature of somitogenesis. The A-P pattern correlates with the organization of signaling components along the length of the gastruloid. In elongated gastruloids, BMP signals are predominantly anterior, while the expression of WNT3 and NODAL genes are restricted to the posterior end, consistent with a role of the latter in the mammalian tailbud at the onset of gastrulation. However, how the regionalization of the signaling components is achieved in the 3D-gastruloids is unknown. Our ongoing analysis suggest that the Hippo-effector YAP1 regulates the antero-posterior organization of the 3D-gastruloids by restricting the posterior expression of Nodal signaling components. Our data show that YAP1 represses Nodal activity through the recruitment of the Polycomb-repressor complex to the chromatin of Nodal genes in hESCs. Hence, in the 3D-gastruloids, YAP1 deletion lead to the anterior expansion of Nodal:Smad2/3 activity. As a consequence, the YAP1 KO-derived 3D-structures are longer than controls, suggestive of an increase in Nodal-induced mesoderm derivatives. Our findings highlight a crucial interaction between YAP1 and NODAL signaling essential for the germ-layer formation and morphogenetic events occurring during the establishment of the body plan.  


Abraham ELIZABETH, Stronati ELEONORA, Zubillaga MIKEL, Conchi ESTARAS (Philadelphia, USA)
17:45 - 20:00 #30608 - 202. Functional characterization of human pluripotent stem cell-derived left ventricle-like cardiomyocytes.
202. Functional characterization of human pluripotent stem cell-derived left ventricle-like cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer an attractive experimental platform to model cardiovascular diseases and advance potential regenerative therapies. However, hPSC-CM cultures exhibit heterogeneity and immature characteristics.

 

In our laboratory, we have developed a “Left Ventricle” (LV) differentiation protocol which efficiently generates LV-like cardiomyocytes (hPSC-LV-CMs), characterised by higher maturity and improved functionality than those generated using the “Standard” WNT-On/WNT-Off differentiation protocol. The CytoCypher MultiCell High Throughput System was used to prove improved contractility, calcium dynamics and increased dependence of β2-adrenergic receptor signalling as well as a positive force-frequency relationship as indicators of superior maturity at 30 days of differentiation. To address if hPSC-LV-like CMs could be further functionally matured, we grew cells from day 20-30 in the presence of fatty acid containing maturation media. Results show this media was unable to improve the cells’ contractility and overall cells loss the ability to display a positive force-frequency response. However, a trend for a higher contraction amplitude was noted. This media also led to improved calcium dynamics, but the calcium amplitude of these cells was lower, suggesting that fatty acid supplementation per se is insufficient to improve functional maturity of LV-like CMs.

 

Maturity can be measured in multiple ways and not all media supplements improve cellular maturity in the same way. Some might lead to metabolic maturity (fatty acids) and others may help cytoarchitecture maturity (mechanical loading and pacing) or functional maturity, thus raising the need to develop a media to more holistically improve hPSC-CM maturity.


Elisa FERRARO (London, United Kingdom), Wenjun LI, Lorenza Iolanda TSANSIZI, Marie-Victoire COSSON, Jose SANCHEZ ALONSO-MARDONES, Julia GORELIK, Andreia Sofia BERNARDO
17:45 - 20:00 #30614 - 204. Fast generation of left ventricle-like cardiomyocytes with mature properties from human pluripotent stem cells.
204. Fast generation of left ventricle-like cardiomyocytes with mature properties from human pluripotent stem cells.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV-cardiomyocytes (LV-CMs) are, therefore, a desirable therapeutical target. Here we made use of developmental-cues to devise a protocol for generating LV-cardiomyocytes from human pluripotent stem cells (hPSC). Voltage clamping confirmed hPSC-LV-CMs display a ventricular action potential shape with hallmarks of maturity, including increased expression of IK1. Moreover, hPSC-LV-CMs have an elongated shape, exhibit well-defined sarcomere structures with a length typical of adult cardiomyocytes, express mature cytoarchitecture markers such as TCAP and have a better respiratory capacity. Functionally, LV-cardiomyocytes display more mature calcium transients, supported by higher RYR expression. Engineered heart tissues (EHTs) generated with hPSC-LV-CMs consist of longitudinally organised cardiomyocyte bundles whereas those generated using the standard WNT-On/WNT-Off protocol (hPSC-Std-CMs) are mostly populated by clumps of cardiomyocytes. LV-EHTs produce more force, have faster contraction dynamics and a slow beating rate but can be paced, confirming LV-CMs are losing the pacemaker potentials typical of immature cardiomyocytes. Collectively, we show that: 1) it is possible to rapidly obtain LV-CMs with mature properties, even before they are exposed to reported maturation regimes; and 2) the generation of EHTs per se is unable to rescue the lag in maturation observed between hPSC-Std-CMs and hPSC-LV-CMs. This work demonstrates that hPSC-LV-CMs are a suitable model to study LV development and disease and could enable more faithful LV-specific cardiotoxicity screens. Moreover, it opens the possibility of like-for-like cell replacement therapy becoming an accessible therapy to treat heart failure patients.


Bernardo ANDREIA SOFIA (London, United Kingdom), Nicola DARK, Marie-Victoire COSSON, Thomas OWEN, Lorenza I TSANSIZI, Elisa FERRARO, Alice J FRANCIS, Selina TSAI, Anne WESTON, Lucy COLLINSON, Ken T MACLEOD, Elisabeth EHLER, Sian HARDING, Jim C SMITH
17:45 - 20:00 #30632 - 206. Defining the downstream genetic networks regulated by GATA6 during human cardiogenesis using hESC and iPSC models.
206. Defining the downstream genetic networks regulated by GATA6 during human cardiogenesis using hESC and iPSC models.

Haploinsufficiency for GATA6 is associated with various forms of congenital heart disease (CHD) including septal and conotruncal defects. Genetic loss-of-function studies in model organisms confirmed that GATA6 regulates critical aspects of heart morphogenesis but have not been useful to model human haploinsufficiency. The phenotypic diversity of CHD in patients containing GATA6 mutations is likely due to an unknown combination of interacting gene variants that converge to influence the cardiac phenotype. A greater understanding of the GATA6 genetic regulatory network that controls human heart development is thus essential for better understanding the pathophysiology and ultimately developing therapies. In the present study, we used cardiac directed differentiation with human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a platform to study GATA6 function. GATA6-/- hESCs failed to generate cardiomyocytes (CMs) or cardiac progenitor cells (CPCs) during cardiac-directed differentiation. The expression of markers for cardiac mesoderm were markedly reduced in GATA6-/- cells compared to controls, indicating a defect in mesoderm patterning. Profiling by RNA-seq and CUT&RUN at the mesoderm patterning stage identified genes of the BMP and WNT pathways as being regulated by GATA6, including LGR5, a GPCR associated with WNT signaling and a putative key target. In contrast to the homozygous mutants, GATA6+/- hESCs generated CPCs and CMs but did so less efficiently than controls. Analysis of an iPSC line containing a heterozygous mutation in GATA6 (c.1071delG) derived from a patient with CHD had similar defects in CM differentiation efficiency, suggesting that it can be used to study in vitro the human disease phenotype. Together, this study provides evidence for a regulatory function for GATA6 during human pre-cardiac mesoderm patterning and describes a system for examining GATA6 haploinsufficiency in vitro


Joseph BISSON (New York, USA), Ritu KUMAR, Kelly BANKS, Ellen YANG, Zhong-Dong SHI, Kihyun LEE, Danwei HUANGFU, Todd EVANS
17:45 - 20:00 #30688 - 208. Cardioids unravel mechanisms of compartment-specific cardiac defects.
208. Cardioids unravel mechanisms of compartment-specific cardiac defects.

The number one cause of fetal death are defects in heart development. Determining the underlying causes faces many challenges, including the complexity and inaccessibility of the embryonic heart, the unclear impact of drugs and environmental factors during pregnancy, and the lack of in vitro models representing all the compartments of the human heart. Here, we established a cardioid organoid platform recapitulating the development of the major compartments of the human embryonic heart, including the right and left ventricles, the atria, the outflow tract, and the atrioventricular canal. These cardioids have the compartment-specific in vivo-like gene expression profile, morphology, and functionality. We use this platform to unravel the developmental electrochemical signal propagation between interacting heart chambers and dissect how genetic and environmental factors cause specific defects in different regions of the developing human heart.


Clara SCHMIDT (Vienna, Austria), Alison DEYETT, Tobias ILMER, Aranxa TORRES, Simon HAENDELER, Lavinia CECI GINISTRELLI, Lokesh PIMPALE, Sasha MENDJAN
17:45 - 20:00 #30468 - 210. Cell lineage tracing and local gene ablation based on Cre recombinase microinjection.
210. Cell lineage tracing and local gene ablation based on Cre recombinase microinjection.

Microinjection of lipophilic dyes, iontophoresis and infection by modified viruses allow tracing the embryonic cell-lineages prospectively.  One the one hand, dyes are easy to use but fail to label single cells. On the other hand, iontophoresis and viral vectors require specialized equipment, unavailable in most laboratories. Here, we developed a new method for cell lineage tracing based on the microinjection of the TAT-Cre recombinase protein in embryos carrying floxed reporters. This approach allows the prospective monitoring of cell progenitors and their descendants from primitive streak stages in mouse (E6.5) to organogenesis (E8.5). Using a low titer of TAT-Cre allows single-cell labelling, which makes this method suitable for prospective clonal analysis. Moreover, TAT-Cre microinjection in a floxed targeted gene embryo yields local genetic ablation at a defined region and developmental stage. In summary, this new method is accessible and efficient for cell lineage tracing system, enabling fate mapping, clonal analysis and targeted genetic perturbation in vivo.

 


Miquel SENDRA, Juan De Dios HOURCADE, Antonio José SARABIA, Oscar Horacio OCAÑA, Miguel TORRES, Jorge N DOMINGUEZ (Jaén, Spain)
17:45 - 20:00 #30586 - 212. Retinoic-acid dependent cis-regulatory elements and mechanisms of non-coding genetic disease in heart and human cardioids.
212. Retinoic-acid dependent cis-regulatory elements and mechanisms of non-coding genetic disease in heart and human cardioids.

Congenital heart defects (CHDs) are the most common form of birth defects, occurring in nearly 1% of newborns. Retinoic acid (RA) is a signaling molecule synthesized from dietary vitamin A. An excess of RA has dramatic effects on human embryonic development. This can occur in either the offspring of women undergoing therapeutic treatment with the synthetic retinoid isotretinoin (13-cis-retinoic acid), or in the offspring of women with excess dietary Vitamin A supplementation. 13-cis-RA, known as isotretinoin (INN) and used for the treatment of acne, is correlated with a high risk of birth defects. Administration of INN during pregnancy causes multiple types of malformations in infants, including cardiovascular defects. There is also evidence that reduced maternal levels of Vitamin A can also increase the risk of CHD in her offspring. Despite the tremendous progress in deciphering the implication of the retinoic acid signaling in heart development, many gaps regarding the underlying mechanisms of RA-mediated gene regulation remains to be unraveled. Here, we are aiming at generating atlases of retinoic acid cis-regulatory elements in the developing heart of a Vitamin A deficient mouse model as in human cardioids through the use of multiomic approaches. Our pilot results indicate that retinoic acid signaling regulates a highly conserved, disease-associated candidate enhancer regulating sino-atrial development.


Sonia STEFANOVIC, Stephanie IBRAHIM (Marseille)
17:45 - 20:00 #30651 - 214. The role of Folic Acid in congenital heart disease.
214. The role of Folic Acid in congenital heart disease.

Supplementation of Folic Acid (FA) during pregnancy prevents neural tube defects (NTD). Recent studies indicate FA supplementation may similarly prevent heart defects, though the relationship remains unclear. Proposed etiologies for FA prevention of birth defects include a role in methylation, cell proliferation/differentiation and G-Protein signaling, with disruption affecting susceptible cell populations, including neural crest cells. However, the mechanism remains undetermined. To determine the mechanistic role of FA supplementation during cardiac development, we utilized an established model of high and low FA supplementation (2ppm versus 10ppm) in pregnant wild type mice, and in the resulting embryo hearts at day E12.5, we performed complementary analysis of the cardiac transcriptome, global proteome, and phospho-proteome utilizing RNA-Seq and tandem mass tag mass spectroscopy (TMT/MS). Ingenuity pathway analysis of differentially expressed genes and proteins indicates altered protein translation, with eiF2 signaling topping the list in the transcriptome and global proteome. Within the phospho-proteome, pathway analysis revealed alteration in DNA methylation, cell cycle control, tight junction signaling and RHOA signaling. The protein SHROOM3 is similarly associated with DNA methylation, cell proliferation, tight junctions and RHOA signaling and is expressed int the neural crest. SHROOM3 loss-of-function leads to NTDs that can be reversed with high FA supplementation. We recently demonstrate SHROOM3-loss-of-function leads to cardiac defects. We utilized short term FA supplementation in SHROOM3-loss-of-function mice, and preliminary analysis of 43 embryos reveals reduced penetrance of membranous VSDs from 36% to 11% with high folic acid supplementation. Overall, these findings suggest a mechanistic role for FA during cardiac development that includes DNA methylation, cell proliferation, tight junction and RHOA signaling within susceptible cell populations.


Matthew DURBIN (INDIANAPOLIS, USA), Korre FAIRMAN, James ZWIERZYNSKI, Laura HANELINE, Stephanie WARE
17:45 - 20:00 #29431 - 216. Single cell transcriptomic analysis of murine binucleated and mononucleated cardiomyocytes reveals heterogeneity within ventricular heart muscle.
216. Single cell transcriptomic analysis of murine binucleated and mononucleated cardiomyocytes reveals heterogeneity within ventricular heart muscle.

Mammalian heart regeneration has been the focus of study in the past decades, aiming to promote recovery after ischemic injury. However, mammalian cardiomyocytes have a notoriously slow turnover, incapable of compensating for the loss of contractile muscle.

 

In regenerative models (zebrafish, neonatal mammalian heart), new cardiomyocytes arise form preexisting ones; suggesting the presence of a cardiomyocyte pool able to reenter cell cycle. One hallmark of naïve, proliferative cardiomyocytes is their ploidy levels. Therefore, it has been proposed that mononucleated cardiomyocytes in mice and diploid cardiomyocytes in zebrafish and human can pose a potential population with the ability to replace lost myocardium.

 

However, defining a trancriptomic signature of these myocytes in order to understand and promote a proliferative phenotype has remained elusive due, in part, to the nature and sensitivity of the cardiac muscle cell.

 

Here we describe a method that has enabled us to perform single-cell RNA Sequencing in binucleated and mononucleated cardiomyocytes from murine ventricles. This method allows for a very high-quality RNA purification and depth of sequencing, enabling us to define several cardiomyocyte clusters with distinct transcriptomic profiles, both among binucleated and mononucleated populations, suggesting a higher heterogeneity among cardiomyocytes than what was previously described.

 


Cristina VILLA DEL CAMPO, Cristina VILLA DEL CAMPO (Madrid, Spain), Rocío SIERRA MUÑOZ, Sergio CALLEJAS ALONSO, Ana DOPAZO GONZÁLEZ, Miguel TORRES
17:45 - 20:00 #29462 - 218. Single cell characterization of valve development.
218. Single cell characterization of valve development.

Valvular heart disease affects 1-2% of the population, often requiring surgery. However, current replacement valves do not respond normally to biological signals and cannot undergo growth or proper remodeling. An alternative is the use of valves generated via differentiation of human pluripotent stem cells into valve cells, recapitulating in-vivo development. Valve leaflets are composed of 3 layers of extracellular matrix containing valve interstitial cells (VICs), ensheathed by valve endothelial cells (VECs). Valve development involves the formation of endocardial cushions populated by cells undergoing endothelial-to-mesenchymal transition (EndoMT), followed by elongation and layer stratification. The transcriptional programs driving these changes remain insufficiently defined, and little is known regarding VIC subtypes. Moreover, current differentiation protocols generate heterogeneous rather than specific valve cell populations. Our study aims to identify novel factors driving EndoMT and mechanisms driving specification of VIC subpopulations.

We performed scRNA-seq on E8.0-P1 whole mouse hearts and computationally generated cell type-specific clusters. Endocardial, VEC and VIC markers were identified. Transcriptomic changes corresponding to VICs were noted by E9.0, and two distinct VIC clusters were found at E10.25. Increased EndoMT marker expression was noted at E11.5. Early endothelial and VIC cells were analyzed using URD (Farrell et al 2018), which reconstructs differentiation trajectories in the form of a branching tree. Cells at branch points were subject to gene regulatory network analysis using SCENIC (Aibar et al 2017), and top hits included key transcription factors for valve development. CellPhoneDB (Efremova et al 2020) was then used to identify important cell-cell signalling interactions. The knowledge gained is expected to assist in optimization of differentiation protocols for VICs, and ultimately generation of “biological” stem cell-derived valves.


Lara FEULNER (Montréal, Québec, Canada), Piet VAN VLIET, Hicham AFFIA, Severine LECLERC, Florian WÜNNEMANN, Guy WOLF, Gregor ANDELFINGER
17:45 - 20:00 #30513 - 222. Single-cell RNA sequencing of the human fetal epicardium reveals novel markers and regulators of epithelial-to-mesenchymal transition.
222. Single-cell RNA sequencing of the human fetal epicardium reveals novel markers and regulators of epithelial-to-mesenchymal transition.

The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to cardiac development. Direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response. Epicardial layers were isolated from human fetal hearts (14-15 weeks gestation) and digested into single cells. Single-cell RNA sequencing was used to determine the cellular composition of human fetal epicardium, and data were further explored to identify regulators of epicardial EMT. Analysis of 2073 cells reveals an enrichment of epicardial derived populations and displays a clear clustering of the epicardial epithelial and mesenchymal populations. Importantly, we found that in humans ‘classical’ markers, such as Wilms’ Tumor 1, Transcription factor 21 and T-box transcription factor 18, are not specific enough to reliably classify the epicardium. Our analysis has provided markers that do allow for robust identification of the epicardium validated by immunohistochemistry. To establish the regulation of epicardial activation we focus on the process of EMT within our dataset. Here we present a novel pathway involved in epicardial EMT and several promising candidates that influence key developmental processes.

This work was funded by a Senior Researcher Dekker grant by the Dutch Heart Foundation (2017T059)


Tom STREEF (Leiden, The Netherlands), Tessa VAN HERWAARDEN, Esmee GROENEVELD, Marie-Jose GOUMANS, Anke SMITS
17:45 - 20:00 #30533 - 224. Single-cell analysis implicates early dysregulation of Nanog in the production of syndromic heart defects.
224. Single-cell analysis implicates early dysregulation of Nanog in the production of syndromic heart defects.

Approximately 30% of individuals with Cornelia de Lange Syndrome (CdLS) exhibit congenital heart defects (CHDs). The most common form of CdLS is caused by haploinsufficiency for NIPBL, which encodes a cohesin-associated protein. In Nipbl+/- mice, NIPBL-haploinsufficiency causes hundreds of small gene expression changes in every cell and CHDs similar to those in CdLS. Using the Nipbl+/- mouse as a model system for discovering new causal factors for CHDs, we documented abnormal heart development by cardiac crescent (CC) stage, suggesting that CHDs originate as early as gastrulation. To investigate this, we performed single-cell RNA sequencing on both CC- and gastrulation-stage wildtype and Nipbl+/- mouse embryos. Strikingly, we found that Nipbl+/- embryos dramatically overexpressed NanogNanog encodes a transcriptional repressor required for pluripotency in pre-implantation embryos and is normally transiently re-expressed during gastrulation. In Nipbl+/- mice, however, Nanog fails to shut off appropriately post-gastrulation, so that by CC-stage its levels are many-fold above normal. Interestingly, numerous gene expression changes detected in Nipbl+/-mice involve post-implantation-stage targets of Nanog. These include genes associated with pluripotency (Pou5f1/Oct4), left-right patterning (Tdgf1Lefty2Nodal), anterior-posterior patterning (Hox genes), and primitive erythropoiesis (Tal1Lmo2Hbb-bh1). Accompanying these changes are changes in the allocation of cells to clusters representing Mesp1-expressing cardiac progenitors, the first and second heart fields, and neural crest. These results suggest that a failure to downregulate Nanog expression after gastrulation, and the transcriptional dysregulation that ensues, lead to misallocation and/or dysfunction of cardiogenic cell populations. Funded by NIH-NHBLI.


Stephenson CHEA (Irvine, USA), Arianna G FAVELA, Arthur D LANDER, Anne L CALOF
17:45 - 20:00 #30611 - 228. Refinement of the four-dimensional human heart atlas during the first trimester of gestation with rare and diverse cell states.
228. Refinement of the four-dimensional human heart atlas during the first trimester of gestation with rare and diverse cell states.

Congenital heart defects are frequent and collectively represent a significant burden for human health. Understanding the developmental origins of such anomalies is key to improving diagnoses, prognoses and therapies. The Human Developmental Cell Atlas (HuDeCA) consortium in France gathers as much data and metadata as possible about normal human embryonic and early fetal tissues donated to research, complete with strict quality control procedures and use of standardized annotations, as a complement to the international Human Cell Atlas and a baseline for the scientific community worldwide. Here, we contribute new data about the identities and trajectories of cells in human male and female hearts from 8 to 12 post-conceptional weeks through judicious comparisons of individual hearts assessed through single-nucleus and spatial transcriptomics and advanced imaging approaches. We integrate and validate this information both across our datasets and with existing atlases, using multiple technical platforms to provide four-dimensional analyses at varying levels of resolution. Delineating the gene expression networks deployed within individual cells at specific positions has revealed an unsuspected diversity of cellular states. This data enables new hypotheses about the paracrine influences exerted by and on the many lineages necessary for the complex functions of the first and longest-lived vital organ.

* These authors contributed equally to this work and are joint first authors

** These authors share joint last authorship; correspondence may be addressed to both (stephane.zaffran@inserm.fr, heather.etchevers@inserm.fr).