Cardiovascular diseases are a significant health burden worldwide. Recent studies have shown promising results regarding the ability of single radiotherapy treatment to elicit long-term improvement in electrical propagation and lower arrhythmia burden in patients and in rodent models. While highly promising, the mechanism for these therapies is not fully understood. In this study, we aim to characterize the effect of radiotherapy using an in vitro engineered heart model based on human induced pluripotent stem cells. The dynamic engineered heart tissue (dynEHT) model is allows hiPSC cardiomyocytes to experience similar mechanical loads as they would experience within the body. Tissue-level physiology, including conduction velocity, wavelength, contractile force, and fractional shortening can be assessed. In pilot studies, dynEHTs subjected to 25 Gy radiation exhibited an increase of conduction velocity in irradiated tissue. The non-treatment tissues showed a conduction velocity of an average 9 cm/s, whereas the irradiated tissues conducted at an average of 32 cm/s. Ongoing analysis of force and fractional shortening will provide additional insight of the treatment effects. This research contributes to the understanding of the restoration of electrical propagation.

The most common form of cardiac arrhythmia is atrial fibrillation (AF), which affects 2-3% of the population and can lead to stroke and heart failure. Several lines of evidence support a genetic basis for AF. The most significant risk locus is on chromosome 4q25, a region upstream of the gene PITX2, which encodes a crucial transcriptional regulator of cardiac morphogenesis: pitx2c. Altered PITX2 expression is reported in patients with AF, yet there is an incomplete understanding of how altered PITX2 expression leads to AF. We previously reported that loss of pitx2c in zebrafish leads to cardiac arrhythmia during larval stages. Here, we characterize conduction system development and function during developmental stages. Using the Ca2+ biosensors GCaMP and Twitch-4, we observed reduced Ca2+ amplitude, rise time, and rise/decay slopes in pitx2c+/- and pitx2c-/- atria compared to wild-type siblings, with no differences in ventricular Ca2+ handling. Analysis of chamber morphogenesis revealed that pitx2c-/- have reduced atrial chamber size without changes in cell number, suggesting that altered cell morphology may drive this phenotype. To characterize sinoatrial node patterning, we performed in situ hybridization for several markers. While many of these transcripts were unchanged, we found an upregulation of tbx18 expression in the sinoatrial node region. We are currently examining other aspects of the conduction system, including the localization of Connexin-43 and the trabecular network. Together, these data suggest that loss of Pitx2c disrupts atrial morphogenesis and electrical conduction, leading to cardiac arrhythmia.

The cardiac conduction system (CCS) disorders give rise to cardiac arrhythmias, a major source of morbidity and mortality worldwide. microRNAs (miRs) are common epigenetic regulators that repress gene expression post-transcriptionally. Although miRs have been studied extensively in the other context of heart, their function in the CCS remains largely unknown. Here, we found that conditional knockout (CKO) of the miR-17-92 cluster in the CCS using Hcn4CreERT2, led to cardiac arrhythmias including abnormal P-waves, atrioventricular (AV) blocks, sinoatrial node (SAN) dysfunction, and irregular RR intervals, indicating that miR-17-92 is required for maintaining CCS homeostasis. Though there were no obvious changes in cardiac structure and function of miR-17-92 CKO compared to controls, action potential (AP) recorded from isolated miR-17-92 CKO pacemaker cells (PCs) exhibited slower and irregular spontaneous-AP firing rates compared to control PCs. Moreover, confocal Ca2+ imaging indicated that compared to PCs from control mice, miR-17-92 CKO PCs showed a greater variability in the spontaneous Ca2+ transient rates and a stronger response to the caffeine-induced Ca2+ signal. Furthermore, we identified putative targets of the miR-17-92 cluster through bioinformatics screening, including ion channels (Scn2b and Serca2) and a core kinase of Hippo signaling pathway (Lats2). Immunohistochemistry data indicated increased expression level of Scn2b, Serca2 and Lats2 in SANs of miR-17-92 CKO compared to control, suggesting that these genes were repressed by miR-17-92. Together, these results reveal a novel mechanism by which miR-17-92 regulates CCS homeostasis.

Popeye domain-containing (POPDC) proteins selectively bind cAMP and mediate cellular responses to sympathetic nervous system (SNS) stimulation. The first discovered human genetic variant, POPDC1S201F, is associated with atrioventricular (AV) block, which is exacerbated by increased SNS activity. Zebrafish carrying a homologous mutation, popdc1S191F, display a similar phenotype to human patients. To investigate the impact of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling, homozygous popdc1S191F and popdc1KO zebrafish larvae and adult isolated popdc1S191F hearts were studied by functional fluorescent analysis. In popdc1S191F and popdc1KO larvae, heart rate (HR), AV delay, action potential (AP) and calcium transient (CaT) upstroke speed, and AP duration were less than in wild-type larvae, whereas CaT duration was greater. SNS stress by β-adrenergic receptor stimulation with isoproterenol increased HR, lengthened AV delay, slowed AP and CaT upstroke speed, and shortened AP and CaT duration. In adult popdc1S191F zebrafish hearts, there was a higher incidence of AV block, slower AP upstroke speed, and longer AP duration compared to wild-type hearts. SNS stress increased AV delay and led to further AV block in popdc1S191F hearts, while decreasing AP and CaT duration. Overall, these results have revealed that arrhythmogenic effects of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling in zebrafish are varied, but already present in early development, and that AV node dysfunction may underlie SNS-induced arrhythmogenesis associated with popdc1 mutation in adults. Given that POPDC1 proteins are present in the central nervous system, we are now investigating the role of POPDC1 in neurons of the intracardiac nervous system (IcNS), which may be an upstream mediator of detrimental electrophysiological effects in cardiomyocytes with popdc1 mutation. This includes investigations of the development and neuroanatomy of the IcNS, as it may represent a possible future therapeutic target.

Cardiac morphogenesis is an intricate and highly conserved process that requires precisely controlled gene regulatory networks. GATA family transcription factors (TFs) sit near the top of the cardiac regulatory network hierarchy and are crucial for determining cardiac fate. During heart development, the mesoderm produces a bipotent population of cardiopharyngeal progenitors that gives rise to the cardiac and pharyngeal lineages. Previous work from the Scott and Wilson labs suggest GATA TFs enable cardiopharyngeal divergence through differentially modulating chromatin accessibility. In the pre-cardiac mesoderm, Gata5 and Gata6 (Gata5/6) promote open-chromatin states at cardiac enhancers while repressing accessibility of pharyngeal enhancers. In contrast, down-regulation of Gata5/6 in the pharyngeal mesoderm enables cells to adopt a pharyngeal fate. It is well established that GATA TFs are critical for cardiac development and their role in cardiopharyngeal divergence has been highlighted, yet how GATA TF expression is governed remains to be elucidated. My project focuses on the regulation of gata5 during zebrafish heart development. Using single-cell ATAC-seq data, accessible genomic regions around the gata5 gene locus were examined for candidate cis-regulatory elements (cCREs). Twenty-six cCREs have been identified based on differential chromatin accessibility between the cardiac and pharyngeal populations. So far, six cCREs are active in the heart and cCRE4 has been prioritized for further examination. cCRE4 displays robust pan-cardiac activity at 48 hours post-fertilization (hpf) and characterization at earlier time points reveals activity as early as 8 hpf, during gastrulation. In preliminary analyses of cCRE4 knock-outs, homozygous mutants appear to be viable during embryonic stages. Additionally, the conservation of cCRE4 across species has been examined and used to narrow down which TF motifs in cCRE4 may have biological relevance for regulating gata5 expression.

H9c2 cells are a female rat ventricle cardiomyoblast cell line that can be differentiated towards a “cardiomyocyte-like” phenotype. While differentiated H9c2 cells are unreported to beat like primary cardiomyocytes, they model key aspects of cardiomyocyte biology including membrane morphology, energy metabolism, and electrophysiological properties. Differentiated H9c2 cells express key cardiomyogenesis genes and undergo a simpler, shorter differentiation protocol compared to other models (e.g. iPSC-derived cardiomyoctyes). Many key differentiation marker levels have been studied, but gaps remain. Comprehensive characterization of H9c2 gene expression, morphology, and Ca2+ handling across their differentiation is needed to facilitate their optimal use as models for studying genetic and environmental factors influencing cardiomyocyte development. At 5 and 14-days post-induction of differentiation, or days in vitro (DIV), mRNA transcript and protein levels were quantified via RT-qPCR and western blotting. Cell shape was quantified using confocal and stimulated emission depletion imaging. Live Fluo-4 Ca2+ imaging studies were performed to assess excitability and synchronicity. Our results show differentiated H9c2 cells exhibit increased levels of key cardiac transcription factors, morphology markers, and ion channels. Specifically, the transcript levels of -catenin, Nkx2.5, cardiac troponin T (cTnT), Na+/K+ ATPase, and Cav1.2 were increased at DIV5 and at DIV14 approach the range of the levels found in murine neonatal heart and primary cardiomyocytes. Protein levels of -catenin, cTnT, vinculin, and -actin all increase, while vimentin decreases at both DIV5 and DIV14. Along with these biochemical changes, the morphology of H9c2 cells alters significantly during differentiation. At DIV14 the cells were elongated, with increased area and perimeter. Moreover, the proportion of multinucleated cells increases, the physical alignment of cells increases, and the cells develop actin clusters. Ca2+ handling amongst the experimental groups is currently under analysis. Our coordinated gene expression, morphology, and Ca2+ characterization of H9c2 cell-derived cardiomyocyte differentiation will provide valuable insights into utilizing this cellular model for studying factors affecting cardiomyocyte development and function.

Understanding the diversification of mammalian cell lineages is an essential to embryonic development, organ regeneration and tissue engineering. Shortly after implantation in the uterus, the pluripotent cells of the mammalian epiblast generate the three germ layers: ectoderm, mesoderm and endoderm1. Although clonal analyses suggest early specification of epiblast cells towards particular cell lineages, single-cell transcriptomes do not identify lineage-specific markers in the epiblast and thus, the molecular regulation of such specification remains unknow. Here, we studied the epigenetic landscape of single epiblast cells, which revealed lineage priming towards endoderm, ectoderm or mesoderm. Unexpectedly, epiblast cells with mesodermal priming show a strong signature for the endothelial/endocardial fate, suggesting early specification of this lineage aside from other mesoderm. Through clonal analysis and live imaging, we show that endothelial precursors show early lineage divergence from the rest of mesodermal derivatives. In particular, cardiomyocytes and endocardial cells show limited lineage relationship, despite being temporally and spatially co-recruited during gastrulation. Furthermore, analysing the live tracks of single cells through unsupervised classification of cell migratory activity, we found early behavioral divergence of endothelial precursors shortly after the onset of mesoderm migration towards the cardiogenic area. These results provide a new model for the phenotypically silent specification of mammalian cell lineages in pluripotent cells of the epiblast and modify current knowledge on the sequence and timing of cardiovascular lineages diversification.

Cardiac pacemaking activity is confined to a specialized population of cardiomyocytes in the cardiac inflow tract (IFT), but the patterning processes that establish IFT dimensions remain unknown. Our data indicate that Hedgehog (Hh) signaling has a potent effect on limiting the number of IFT cells in the embryonic zebrafish heart. Using either genetic or pharmacological manipulation of the Hh pathway, loss of Hh signaling results in a significantly expanded population of IFT cardiomyocytes. Conversely, we find that Bmp signaling plays a dose-dependent role in promoting the formation of IFT cardiomyocytes: reduction of Bmp signaling diminishes the number of IFT cells, and increased Bmp signaling enhances the number of IFT cells. Temporal inhibition of each pathway demonstrates that Hh and Bmp signaling are both required in the same timeframe, prior to myocardial differentiation, to establish a normal number of IFT cardiomyocytes. Furthermore, simultaneous reduction of both Hh and Bmp signaling restores the IFT population to a relatively normal size, suggesting that these pathways act in opposition during IFT patterning. Lastly, epistasis analysis demonstrates that Bmp signaling acts upstream of canonical Wnt signaling to promote IFT cell formation, whereas Hh signaling appears to restrict IFT cell formation in a Wnt-independent manner. Together, our results support a model in which Hh signaling limits the size of the IFT progenitor pool, while BMP signaling promotes IFT progenitor specification, upstream of Wnt-directed IFT differentiation, to generate an appropriate number of pacemaker cells in the IFT.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a useful tool to investigate cellular pathways, model diseases in vitro, and regenerate tissue in vivo upon implantation into injured hearts. However, PSC-CMs, like other PSC-derived cell types remain in an immature fetal-like stage and fail to fully recapitulate adult somatic cells. Consequently, their use for in vitro studies may not accurately represent in vivo conditions, and their therapeutic applications in vivo raise safety concerns. To further understand the maturation process, we aimed to identify microRNAs (miRNAs) regulating the transcriptomic changes that occur by conducting small RNA-sequencing (sRNA-seq) at five embryonic and postnatal timepoints in mouse heart, brain, and liver. We clustered miRNAs based on their expression trend over time and compared clusters across organs. To predict target genes of miRNAs in the heart, we integrated computationally predicted target genes from the miRmap prediction tool with downregulated genes observed in transcriptomics data of the heart. From the sRNA-seq results, we identified 176 miRNAs that were upregulated over time in at least one organ and 5 in all three organs (miR-22, miR-29a, miR-29c, miR-26a-1, and miR-378c). Gene Ontology (GO) enrichment analysis revealed that the predicted target genes of the 5 conserved miRNAs correspond with various cell cycle related terms, which is a typical process of immature cells. Additional GO terms included negative regulation of both fatty acid and lipid biosynthesis. Our findings suggest that these five candidate miRNAs may be crucial for facilitating cell cycle exit and metabolic maturation in cardiomyocytes. Our study is the first to compare miRNA trends during maturation across multiple organs and provides insight on new methods to advance the maturation state of PSC-CMs and other PSC-derived cell types in vitro.

Limited maturity of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hampers appropriate development of cardiac disease models in vitro using patient-derived induced pluripotent stem cells (iPSCs). Our previous study revealed aberrant inactivation of transcriptional factors in mouse PSC-CMs compared to neonatal and adult hearts. Therefore, we hypothesized that upregulation of these transcriptional factors (TFs) improves cardiomyocyte maturation. To this end, we first developed a transcriptome-based quantitative maturation scoring method. We first performed expression screening of 92 candidate TFs in mouse PSC-CMs. After several rounds of screening, we found that top hits included PGC1α and β, and ERRα and γ, which are all known to be involved in cardiomyocyte maturation. Because they are nuclear receptors and ~20 nuclear receptors with known agonists were in the list of candidate TFs, we hypothesized that the lack of nuclear receptor agonists in culture is the cause of immaturity. Thyroid hormone (T3) was the strongest agonist among 20 agonists for cardiomyocyte maturation when cells were treated with a single agonist. Next, we combined T3 and 19 other agonists and found that SR11237 (SR) and GW0742, RXR and PPARβ/δ agonists, respectively, improved maturation. Finally, we found that the combination of T3, SR, and GW0742 was the best combination for mouse PSC-CM maturation and the treated cells reached the adolescent stage based on the maturation score. Morphological, structural, physiological, and metabolic analyses all indicate that mouse PSC-CMs with triple hormone treatment were very close to adult cardiomyocytes. To extend our findings to human PSC-CMs, we found some combinations improved maturation to the early adolescent stage. The combinations were T3, SR, and one of WY14632 (PPARα), Hydrocortisone (glucocorticoid receptor), DY131 (ERRγ and β), and 27OHC (LXR). Depending on the assessment, the better combinations were different. 27OHC was the best for transcriptional and metabolic analysis, while Hydrocortisone and DY131 were better for morphological and physiological aspects. Taken together, we demonstrated that the combinatorial activation of nuclear receptors is essential for cardiomyocyte maturation however there should be a further regulatory mechanism should exist especially in human cardiomyocytes.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with an increased risk of heart failure, stroke, and sudden cardiac death. Familial linkage and genome-wide association studies have identified >100 genetic risk loci, strongly supporting a genetic component to AF. The most significant locus associated with AF affects expression of PITX2C, a gene that encodes a transcription factor involved during cardiac morphogenesis. Yet, the precise mechanism explaining how the change in PITX2 expression leads to AF is still poorly understood. Based on our observations in a zebrafish pitx2c loss-of-function model, we are exploring the hypothesis that loss of PITX2C leads to oxidative stress that promotes cardiac arrhythmia. Here, we are investigating the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine protein kinase, as a candidate to link PITX2C-derived oxidative stress with arrhythmogenic changes. CaMKII functions to maintain cellular balance and signaling under normal conditions of activation. However, persistent elevation in CaMKII activity has been associated with compromised excitation-contraction coupling, calcium handling, inflammation and fibrosis, collectively impairing cardiac performance. Additionally, post-translational modifications of CaMKII, such as oxidation and phosphorylation, lead to its constitutive activation. Given its central mechanistic role in arrhythmias, we investigated if CaMKII signaling was affected in the pitx2c model. We show by immunostaining and confocal imaging an increase in the proportion of phospho-CaMKII that localizes to the sarcomere Z-disks in pitx2c-/- adult hearts. Interestingly, we observed the same localization pattern after treating hearts with rotenone, a complex I inhibitor that leads to ROS formation. Additionally, we did not observe an increased localization of Ox-CaMKII at the sarcomere in pitx2c-/- adult heart. We have also developed a Pitx2c knockdown approach in neonatal rat cardiomyocytes and observed that loss of PITX2C leads to oxidative stress in these cells. We hypothesize that translocation in response to increased ROS allows CaMKII to pathologically phosphorylate specific myocyte targets. We are currently exploring the mechanisms by which increased oxidative stress and sustained CaMKII activation promote chronic arrhythmia using a combination of genetic models in zebrafish and cultured neonatal rat atrial cardiomyocytes.

Congenital heart defects (CHD) affect ~1% of all births and are the leading cause of morbidity and mortality in infants. Notably, over 50% of Down Syndrome infants, the most prevalent autosomal abnormality caused by trisomy 21 (T21), are born with a CHD, with septal defects most common. The pathways that contribute to cardiac defects in T21 are poorly understood. The critical region of human chromosome 21 associated with CHD harbors genes coding for extracellular matrix (ECM) as well as cell-ECM and cell-cell adhesion proteins, among others. We suggest that disruption of ECM during heart development leads to downstream effects on cell signaling and to cytoskeletal reorganization that alters nuclear architecture and gene expression programs. Using T21 hiPSCs, our preliminary data show increased ECM deposition in iPSC derived cardiomyocytes, tissue stiffening, and altered ECM-cell interactions compared to isogenic controls. We also observe precocious differentiation including changes in cell cycle, contractility, and nuclear shape. Understanding mechanobiological regulation of heart development in T21 will fill a major knowledge gap and is expected to reveal new molecular mechanisms that contribute to CHD. More broadly, this work has the potential to identify new therapeutic modalities that minimize both T21 phenotypes as well as provide a broader solution to a range of diseases such as age-related degeneration, fibrosis, and cancer.

Maturation of cardiomyocytes (CMs) is critical to allow the adult heart to function throughout life, yet the signals controlling this process are not fully understood. Ironically, maturation leads to a trade-off whereby the heart loses the ability to regenerate in response to injury; thus, understanding the molecular mechanisms that drive CM maturation is critical to improve disease outcomes and realize the promise of regenerative medicine. Reactive oxygen species (ROS) signaling plays complex roles in CM differentiation, but the mechanisms by which ROS promotes CM maturation remain controversial. Notably, ROS is known to regulate cellular metabolism, which shifts during CM maturation from glycolysis to oxidative phosphorylation. Recent evidence has demonstrated that this metabolic shift not only facilitates increased ATP production, but also promotes CM maturation through altered gene expression. Here, we demonstrate that the NADPH oxidase Nox4, a ROS-producing enzyme, is required in human iPSC-derived CMs for maintenance of mitochondrial networks. Knockdown of Nox4 results in an increase in glycolysis and corresponding decrease in oxidative phosphorylation, effectively reversing the metabolic shift which occurs during maturation. Additionally, Nox4 knockdown impairs the development of organized sarcomeres in iPSC-derived CMs, suggesting a functionally immature cell state. Together, these data indicate that Nox4 regulates metabolism in CMs, suggesting a mechanism by which ROS signaling may act to promote CM maturation. We propose to further investigate the downstream effects of ROS signaling and metabolic maturation on the gene expression program of CMs.

During heart development, a tightly regulated cardiac gene program defines the precise timing and location of cardiac progenitor specification. While critical roles for highly evolutionarily conserved families of transcription factors in controlling the initiation of cardiac gene transcription have been firmly established, mechanisms driving mesoderm differentiation into the myocardial lineage are not yet fully understood. In this study, we discovered that mutations in Rtf1, a multifunctional transcription regulatory protein that modulates transcription elongation and histone modification, prevent cardiac progenitor formation in both zebrafish and mice. Single nuclei RNA sequencing analysis showed that while mesoderm differentiation toward the myocardial lineage was initiated in the absence of Rtf1 activity, these cells failed to transition to the cardiac progenitor state defined by the expression of cardiac markers such as nkx2.5 and tbx5a. We further showed that Rtf1’s ability to support cardiac progenitor formation depends on its Plus3 domain, which confers interaction with the pausing/elongation factor Spt5. Indeed, the occupancy of RNA Pol II at the transcription start site (TSS) of cardiac genes was reduced in rtf1 morphants, suggesting a reduction in transcriptional pausing. Intriguingly, pharmacological or morpholino antisense inhibition of pause release restored the formation of cardiac cells and improved Pol II occupancy at the TSS of key cardiac genes in rtf1 deficient embryo. Together, our findings suggest that Rtf1 potentiates myocardial differentiation from multipotent lateral plate mesoderm through promoting promoter-proximal pausing and highlight the crucial role that transcriptional pausing plays in governing cell-state transitions during cardiogenesis.

Cardiovascular disease is the leading cause of morbidity and mortality with myocardial infarction being the greatest risk factor. While regenerative approaches aimed to drive proliferation of cardiomyocytes (CMs) have shown great promise in recent years, there is currently poor understanding of how CM cytoarchitecture changes are regulated to promote cell cycle re-entry, and importantly the factors that facilitate re-integration of CMs into the functional myocardium. Considering the importance of the intercalated disc (ICD) and CM cytoskeletal structure in regulating cell signaling and maintaining cardiac function, we aim to understand the proteins that control CM cytoarchitecture and re-establishment of ICD during cardiac development, regeneration, and repair. In prior studies, we generated a panel of zebrafish loss-of-function mutants for candidate genes reported to interact with the Hippo-Yap pathway; a highly conserved signaling pathway with profound influence on cardiomyocyte regeneration. From this screen, we identified Lethal giant larvae protein homolog 1 (Llgl1) as a regulator of CM shape, size, and ICD integrity in the zebrafish heart during development. Here, we generated a cardiomyocyte specific conditional knockout of Llgl1 to explore its role in mediating mammalian CMs intercalated disc composition, cell cycle activity, and the cardiac injury response. Llgl1CM-KO mice displayed impaired development of intercalated discs with a reduction of intercalated disc proteins N-cadherin, alpha-catenin, beta-catenin, and connexin 43. Impaired development of intercalated discs was associated with delayed electrical conduction and increased cardiomyocyte cytokinesis in juvenile mice. While Llgl1CM-KO mice showed minimal changes in cardiac physiology, following myocardial infarction Llgl1CM-KO Adult mice displayed significant increase in cardiomyocyte proliferation, reduced scar size, attenuated left ventricular dilation, and improved ejection fraction. Conclusions: Llgl1 depletion in cardiomyocytes impaired intercalated disc composition and increased CM proliferation in uninjured juvenile and in adult animals post MI. These results demonstrate a novel role for the polarity protein, Llgl1, in terminal differentiation and cytokinetic potential of both immature and mature mammalian CMs.

Atrial Natriuretic Peptide (ANP) plays a crucial role in maintaining cardiovascular homeostasis by signaling via its high-affinity receptor, NPRA. Cardiac dysfunction and sudden death were reported in newborn children with homozygous loss of function NPRA mutations, and the causes are unknown. We previously reported that ANP plays a regulatory role in in the ventricular conduction system (VCS) development and mice lacking NPRA revealed a hypoplastic VCS phenotype. Additional studies on NPRA-knockout (NPRA-KO) mice revealed perinatal mortality and significant reductions in litter size at weaning. We hypothesized that abnormal cardiac development in NPRA-KO mice contributes to perinatal deaths. Variations in cardiac development were noted in NPRA-KO compared to wild-type embryos at the E11.5 stage. Immunohistochemistry analysis demonstrated altered expression of sarcomeric myosin heavy chain (MHC) and Cx40 in the hearts of E11.5 NPRA-KO embryos, along with differences in ventricular wall thickness. Trans-abdominal ultrasound analysis of pregnant NPRA-KO mice revealed fetal death in late-stage development (E18). Fetal echocardiography revealed a significant increase in the thickness of the left ventricular posterior wall and a decrease in the left ventricular internal diameter. Additionally, NPRA-KO fetuses displayed lower ejection fraction and fractional shortening. Moreover, the hearts of Day 1 NPRA-KO neonates were found to be hypertrophied. The NPRA-KO neonatal electrocardiograms (ECG) demonstrated altered ECG profiles with frequent arrhythmic events. Metabolic events in Day 1 NPRA-KO neonatal hearts were also altered, indicating an inefficient transition to fatty acid metabolism. Collectively, these findings suggest that abnormalities in cardiac development in the absence of NPRA signaling can lead to perinatal mortality. Future studies will focus on pathophysiological mechanisms responsible for the transition of congenital heart defects to cardiac dysfunction in surviving adult NPRA-KO mice.

The wall of the ascending aorta harbors different smooth muscle cell (SMC) populations. Lineage tracing studies have shown that the media of the developing ascending aorta originate from the cardiac neural crest (CNC) and second heart field (SHF). The origins of the remaining outer layers of epicardium and subepicardium are unknown. We hypothesize that these cells arise from the anterior part of the SHF and contribute to the outer SMC layers via epithelial-to-mesenchymal transition (EMT). Furthermore, impaired arterial epicardial (aEP) EMT is hypothesized to cause outflow tract (OFT) malformations and aortic disease. Immunohistochemistry in E10.5 mouse embryos revealed for the first time that aEP cells originate from the SHF. GFP-labeled aEP cells in the developing OFT of WT1creERT2;RosamTmG embryos did lose their epithelial morphology, migrated into the arterial wall of the ascending aorta and aortic root and will differentiate into SMC or fibroblasts. The importance of the cellular contribution of aEP to the OFT was determined by epicardial-specific deletion of β-catenin. The number of WT1-expressing cells contributing to the developing heart was reduced in E8.5-9.5 WT1CreERT2;βcateninfl/fl;RosamTmG embryos (N=3), resulting in a significantly thinner wall of the ascending aorta. One-third of the mutant embryos showed subepicardial hemorrhage as a result of poorly developed coronary arteries. While all embryos developed a ventricular septal defect and hardly any compact myocardium was observed, 33% of the mutant embryos also developed a double outlet right ventricle. Our results show that the arterial pro-epicardial organ harbors a populations of cells that, via EMT, has a significant cellular contribution towards the aortic wall essential for proper mural development and OFT rotation of the embryonic heart. Epicardial-specific deletion of βcatenin at E8.5-E9.5 contributed to decreased EMT in the OFT and OFT malformations.

Haploinsufficient mutations resulting in altered dosage of transcriptional regulators, such as the cardiac transcription factor TBX5, are a critical underlying cause of congenital heart defects (CHDs). The haploinsufficiency of TBX5 mutations suggests that TBX5 regulates the expression of its target genes in a dose-dependent manner. Differentiation to cardiomyocytes of a human iPSC allelic series including TBX5 WT, heterozygous and null lines, has allowed us to understand the dose-dependent changes in gene expression that may underlie TBX5-dependent CHDs. However, we still don’t understand mechanistically how and why some genes are sensitive to changes in TBX5 dose. We are testing the hypothesis that target genes are sensitive to reduced TBX5 levels due to differential binding of TBX5, and the assembly of transcription-regulating chromatin-associated proteins around dose-sensitive genes. To uncover these molecular mechanisms, we generated a biotin-tagged TBX5 allelic series and performed quantitative ChIP-seq in cardiomyocytes to understand how haploinsufficiency impacts TBX5 binding. Many regions bound by TBX5 showed a graded reduction in binding in haploinsufficient conditions. To understand the transcriptional machinery that may influence TBX5 dosage-sensitive gene expression, we have optimized a protocol called CAPTURE to isolate locus-associating proteins using biotinylated dCas9 pull-down followed by mass spectrometry. Our initial work targeting the SOX2 promoter in iPSCs has demonstrated that we can isolate transcriptionally-relevant regulatory proteins, including chromatin remodeling complexes and transcription factors. Current work at the TBX5 dosage-sensitive target gene NPPA promoter and enhancer in iPSC-derived cardiomyocytes will allow us to determine the changes in transcriptional machinery that may underlie altered gene dosage in CHDs.

Many congenital heart defects (CHDs) have multifactorial etiologies where genetic susceptibility increases CHD risk with environmental exposures. Understanding how gene x environmental interactions (GxE) lead to CHDs is imperative for elucidating their causes. The developing embryo possesses a remarkable capability to buffer genetic and environmental stressors to ensure normal cardiac development. However, CHDs can occur when these insults exceed the disease threshold. Work in the Zohn laboratory aims to understand how these buffering mechanisms mediate GxE interactions and how their malfunction contributes to CHDs. Maternal diet is an important environmental factor influencing CHDs. For instance, intake of either too much or too little vitamin A can cause conotruncal defects that impact the outflow tract, ventricular septum, and aortic arch. Depending on diet and supplement usage, pregnant women may ingest varying amounts of vitamin A that the embryo must buffer to ensure normal cardiac development. Regulated expression of RA synthesis and degradation enzymes locally transforms vitamin A into retinoic acid (RA), creating a precise gradient critical for proper specification and development of cardiac progenitors. Our studies demonstrate that genetic mutations and the maternal diet interact to alter this RA gradient and the development of cardiac progenitors. Specifically, distinct genetic mutations interact differentially with maternal diet, preferentially altering the anterior (aSHF) or posterior (pSHF) second heart field, affecting outflow tract or aortic arch development, respectively. 22q11.2 deletion syndrome (22q11DS) is associated with variable penetrance and expressivity of aortic arch and outflow tract defects. Our data studying a mouse model of 22q11DS indicates that this variability may be due to impaired buffering of RA exposures. Conversely, the Hectd1 ubiquitin ligase is required to fully activate RA signaling. We demonstrate that mutation of Hectd1 interacts with mild vitamin A deficiency to cause CHDs, preferentially impacting the aSHF and outflow tract development. Thus, our establishment of new mouse models of GxE interactions provides a powerful platform to elucidate how genetic mutations can interact with changes in the maternal diet. Moreover, our results suggest differential sensitivity of aSHF and pSHF progenitors to maternal diet-induced changes in RA gradients depending on the genetic mutation involved.

Congenital heart disease occurs in ~60% of patients with 22q11.2 deletion syndrome (22q11.2DS). TBX1, encoding a T-box transcription factor, is a major gene for congenital heart disease in patients with 22q11.2DS. In the mouse embryo, Tbx1 is expressed in all three germ layers of the pharyngeal apparatus and is required for cardiac development. Many studies have been conducted on the role of Tbx1 in the pharyngeal endoderm (PE) and a few key genes, such as Nkx2-6 , Pax9, Fgf8, and Fgf10, have been noted as important. However, a comprehensive investigation of the transcriptome downstream of Tbx1 in the PE is still needed. We have employed 10X Genomics single-cell RNA-sequencing (scRNA-seq) in control versus conditional Tbx1 mutant embryos using Sox17-2A-iCre and Tbx1-Cre mice, as well as global Tbx1 mutant embryos at E9.5. Two of these lines were FACS purified and enriched for PE cells. The scRNA-seq datasets were individually subject to quality control analysis using Seurat software followed by integration via RISC software included in our streamlined single-cell Differential Analysis and Processing Pipeline (scDAPP). Then we performed subclustering analysis on the PE cells isolated from each of the three lines using a curated list of PE specific marker genes. Reads for all the PE cells in individual samples were summed to generate pseudobulking data for determining differentially expressed genes (DEGs). From this we identified 366 DEGs (nominal p-value < 0.05), including the known genes listed above. STRING software analysis revealed downregulation of biological processes that included cell communication (Gene Ontology: GO:0010646, FDR P-value 6.70E-13) associated with 51 of the downregulated DEGs. This data suggests that endodermal Tbx1 is mainly involved in growth factor signaling, but also retinoic acid signaling, calcium dependent cell-cell adhesion, and non-canonical Wnt signaling. We are currently validating these expression changes at the RNA level with whole mount in situ hybridization in these embryos.

Congenital heart defects (CHDs), the most common type of birth defect, affect one in every 100 newborns. Cardiac neural crest cells (cNCCs), a migratory and multipotent cell population, are vital for proper heart formation and have been clinically linked to CHDs. Patient data indicates that CHDs can be associated with alterations of the fundamental Hippo signaling pathway, yet its role in cNCC-derived heart development remains largely unknown. Conditional knockout of Yap/Taz (Yap+/-;Taz-/-), the downstream effectors of the canonical Hippo pathway, resulted in mouse embryos with various CHDs, including cardiac outflow tract (OFT) and ventricular septum defects. Notably, Yap+/-;Taz-/- hearts also had ectopic cartilage tissue, an atypical cell population to be found in the embryonic cardiac system, suggesting a cNCC fate alternation. In addition, ex vivo cardiac OFT culture and in vitro NCC migration assays reduced migration capabilities due to Yap/Taz reduction. Furthermore, ultra-low bulk RNA-sequencing of cardiac OFTs from control and Yap-/-;Taz-/- E10.5 mouse embryos indicated that compared to controls, Yap-/-;Taz-/- OFTs had altered expression of genes regulating cell movement, extracellular matrix organization, stress response, and differentiation, all critical components of mechanical signaling regulation. Ongoing studies aim to validate novel candidate sequencing results using in vivo and human embryonic stem cell-derived NCCs to investigate cNCC fate mechanisms throughout development. Together, our data indicate that Yap and Taz are required for proper cNCC-derived heart formation.

Advances in genomics research has rapidly increased our ability to identify the genetic basis of disease, but a major bottleneck remains in understanding the biological significance of these genes and the pathogenic mechanisms by which they cause disease. Genetic variants occurring within the coding region of a gene may disrupt gene function, while variants in the non-coding genome may impact levels of gene expression. In many cases, the effect of a particular variant is challenging to predict. Lack of functional data results in classification of these as variants of unknown significance (VUS). Whether VUS are relevant to a disease, or how they may impact protein function, is challenging to assess in the absence of functional data. We have identified a family with aggressive neonatal arrhythmias who are followed by the Pediatric Cardiology Department at the Jim Pattison Children’s Hospital in Saskatoon. This family has multiple family members spanning two generations who present in fetal life or in the early neonatal period with multifocal atrial tachycardias, including atrial flutter and ectopic atrial tachycardia, which are very refractory to conventional treatment. We have first performed whole-exome sequencing on a proband and unaffected mother and identified 607 rare variants, of which 94 were synonymous, 124 were non-synonymous, and 1 was a stopgain mutation. Based on bioinformatics prediction of variant effects using FATHMM, SIFT4G, ClinVar, and ClinPred, we prioritized three candidates: NES, HPS6, and GATA6. We are currently developing zebrafish and cell models to identify the causative variant and define the molecular mechanism downstream of the VUS.

Congenital heart disease (CHD) affects 1.7% of live births. 30% of CHD cases can be attributed solely to genetic causes, but the causes of the other 70% are less clear. Some of these cases arise from maternal exposure to environmental teratogens. One such teratogen is the anti-epileptic drug valproic acid (VPA), a histone deacetylase inhibitor, which is known to cause specific heart and craniofacial defects. However, the molecular mechanism by which VPA perturbs development is unknown. We have created a mouse model of maternal VPA exposure. Phenotyping of VPA-exposed mouse embryos was achieved by High-Resolution Episcopic Microscopy and 3D modelling. Mechanisms were investigated using single-cell transcriptomics, ATAC-seq, and validated by RNAscope and immunofluorescent protein staining. We found that mouse embryos exposed to maternal VPA have a high incidence of ventricular septal and aortic arch defects indicative of perturbed cardiac neural crest cells. Single-cell transcriptomics of whole embryos revealed significantly fewer migratory neural crest cells (NCCs) in E8.5 mouse embryos from VPA-treated mothers compared to untreated controls. At E8.5, migratory NCCs from mouse embryos with maternal VPA exposure had altered expression of pro-migratory genes. To determine spatially how NCC migration was perturbed, VPA-exposed E9.5 embryos, RNAscope was used to define influxing and differentiating NCCs. VPA-treated E9.5 embryos had an increased gap size between influxing NCCs in the proximal and differentiating NCCs at the distal tip of brachial arch 1, indicative of perturbed migration. In addition, elevated SOX10 protein expression was seen in the frontonasal prominence, suggesting delayed NCC migration and/or differentiation. Here we have demonstrated that embryos with maternal VPA exposure develop craniofacial and heart defects by altered neural crest migration and/or differentiation. Further work will aim to determine if migration and differentiation are delayed in VPA-treated embryos compared to controls by examining different timepoints. These results may lead to the development of preventive approaches or the identification of novel therapeutic targets to reduce the incidence of CHD in humans.

Bicuspid aortic valve (BAV) is a common congenital cardiovascular defect characterized by the formation of two, rather than three, leaflets. Data shows that more than 50% of BAV patients prematurely develop calcification and aortic stenosis (AS), which is most severe in Type 1 (with raphe) compared to Type 0 BAV (no raphe). The remaining 50% of patients exhibit normal aortic valve function throughout life. Despite this, the factors that contribute to early onset AS in a subset of BAV patients are unknown. Therefore, our objective is to determine the contribution of congenital Type I and Type 0 malformations on abnormal mechanical stresses, and the temporal activation of pro-calcific signaling within the bicuspid valve. In our hands, Nfatc1Cre(+);Exoc5fl/+ mice develop Type 0 (no raphe) with mild AS by 18 weeks, while Nfatc1enCre(+);Notch1fl/fl mice develop Type 1 with a raphe conjoining the right and left coronary leaflets, and moderate to severe AS as early as 5 weeks. Preliminary computational modeling shows diverse mechanical stress patterns in these BAV subtypes, with Type 0 generating higher mechanical stress in regions of coaptation and Type 1 at attachment sites. To determine if regionalized mechanical stress patterns are associated with pro-calcific signaling, spatial transcriptomics was performed on Type 0 mice, and validated in the Type 1 model. Compared to tri-leaflet aortic valve genotype controls, we identified enrichment of ‘osteogenic signaling’ associated mRNAs, including Spp1, Sparc, and Comp in high mechanical stress regions that were temporally and spatially dependent on the model. Type 0 mice showed early calcific changes in coaptation zones at 18 weeks, however, similar changes were detected in Type 1 by 10 weeks and restricted to areas of cusp attachment. Together, these data suggest that aortic valve cusp configuration at birth significantly influences overall valve structure performance, which we hypothesize drives mechanosensitive molecular communications between valve endothelial, and valve interstitial cells to promote premature calcification, that is unique to the bicuspid anatomies.

TBX1, encoding a T-box transcription factor, is a key gene contributing to congenital heart disease and branchiomeric muscle (BrM) development in patients with 22q11.2 deletion syndrome (22q11.2DS). The cells comprising the cardiopharyngeal mesoderm (CPM) within early embryos are needed to form these structures affected in 22q11.2DS. Inactivation of Tbx1 in the CPM in mice results in neonatal lethality with a persistent truncus arteriosus and failed formation of BrMs. We performed single cell RNA sequencing using Mesp1Cre/+ and Tbx1Cre/+ mouse lines at E9.5. We previously identified a multilineage progenitor (MLP) population within the CPM that was affected by loss of Tbx1. Genes specifically enriched in MLPs and reduced in conditional mutant embryos included Aplnr and Nrg1. Gene ontology analysis revealed that downregulated genes function in cell proliferation as well as cardiac (anterior second heart field; aSHF) and BrM cell fate commitment, promoting MLP cell maturation. Some of the genes that were upregulated after Tbx1 inactivation, were normally not expressed in MLP cells including neuronal genes and other non-mesodermal genes, such as Pax8 and Bdnf. To further characterize MLP cell heterogeneity and corresponding potential for differentiation of distinct subpopulations, we performed bioinformatic subcluster analysis. Upon examination of the cell subtypes and proportions within MLPs, we found that there is a switch in cell state when Tbx1 is inactivated. During the switch, defined by expression of genes, the cells that promote maturation of the aSHF and BrM populations, were replaced by others that express atypical neuronal and non-mesodermal genes. The molecular mechanism for this switch remains to be established. In conclusion, Tbx1 is required to promote expression of genes needed for cardiac and BrM cell state progression while restricting expression of non-mesodermal genes in MLPs, thereby contributing to cardiac and BrM developmental defects that occur in mouse models and patients with 22q11.2DS.

Determining the genetic factors that contribute to Congenital Heart Disease (CHD) has been challenging. This is partly due to complex inheritance patterns marked by incomplete penetrance, which often results from gene-gene and gene-environment interactions. Additionally, many single-gene animal models of CHD do not translate to human disease states. Therefore, to understand the pathogenesis of CHDs, it is crucial to investigate environmental contributors. A recent epidemiologic study has demonstrated clear connections between the development of CHDs and the occurrence of both maternal diabetes, and maternal obesity. Interestingly, the results suggest different risk profiles for diabetes and obesity, suggesting distinct underlying mechanisms (1.) Recent studies have shed light on CHD mechanism due to maternal diabetes (2, 3). However, the mechanism of CHD pathogenesis due to obesity is mostly unexplored. Since obesity is an epidemic, with rates rising rapidly, and disproportionately impacting lower socioeconomic groups and minority populations, , it is essential to investigate its impact on CHD. Here, we generated a model of maternal obesity without diabetes via 2 months of feeding wild-type C57Bl6-J mice dams a high-fat- diet (60 kcal% fat, Research Diets). We performed transcriptomics and proteomics with complementary bulk RNA sequencing, single nuclei RNA sequencing and tandem mass tag mass spectroscopy of the developing E12.5 hearts, analyzed and integrated the datasets. We observed disruption of pathways important for cardiac development, including downregulation of reactive oxygen species scavenging enzymes, oxidative phosphorylation processes, Rho-kinase signaling, RAC GTPase signaling and second heart field markers. These findings provide important insights into pathways disrupted during cardiogenesis due to in-utero maternal obesity exposure.

The congenital cardiac disorder left ventricular noncompaction (LVNC) is characterized by excessive trabeculation and myocardial thinning, which leads to increased risk of heart failure, arrhythmias, and sudden cardiac death. Loss of Plxnd1, a class-3 Semaphorin receptor, within endocardial endothelial cells (EECs) causes hypertrabeculation and thin myocardium similar to that seen in patients with LVNC [1]. Plxnd1 negatively regulates vascular endothelial growth factor (VEGF) during angiogenesis [2, 3]. Interestingly, excess VEGF signaling likewise causes an LVNC-like phenotype [4, 5]. We hypothesized that hypertrabeculation and noncompaction in Plxnd1 mutants may stem from dysregulated EEC VEGF signaling. Plxnd1 and the VEGF receptors Nrp1 and Kdr are coexpressed in EECs (E12.5-E15.5). Whole mount and histological analyses revealed that hypertrabeculation and noncompaction in developing Plxnd1-null hearts is associated with anomalous sub-pericardial expansion of EMCN+ EECs, and a concomitant decrease in FABP4+ coronary endothelial cell growth. Interestingly, Plxnd1-/- endocardial ECs also exhibit strong VEGF binding affinity and upregulation of the VEGF-response gene Kcne3. To directly test whether the Plxnd1 mutant hypertrabeculation and noncompaction phenotype is due to excess VEGF signaling, we developed a method to effectively attenuate developmental angiogenesis through gestational delivery of VEGF-Trap, a potent neutralizer of VEGFA, VEGFB, and PLGF. Wild-type hearts treated with VEGF-Trap (E11.5-E14.5) exhibited localized necrosis and diminished ventricular proliferation, likely due to angiogenic arrest. However, VEGF-Trap administration to Plxnd1 loss-of-function embryos rescued expression of coronary endothelial genes and of the proliferation marker mKi67. Additionally, VEGF-blockade normalized aberrant EEC gene expression and restored the hypertrabeculation and noncompaction characteristics of Plxnd1 mutants. These findings provide evidence that endocardial angiogenesis, critically regulated by PLXND1-VEGF pathway interactions, influences ventricular myocardium development. Of clinical relevance, this study implicates exacerbated VEGF signaling in LVNC etiology and suggest that in utero VEGF-blockade may provide a therapeutic avenue to alleviate this disease. 1. Sandireddy, R., et al., Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction. JCI Insight, 2019. 4(16). 2. Moriya, J., et al., Inhibition of semaphorin as a novel strategy for therapeutic angiogenesis. Circ Res, 2010. 106(2): p. 391-8. 3. Zygmunt, T., et al., Semaphorin-PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1. Developmental cell, 2011. 21(2): p. 301-14. 4. Miquerol, L., B.L. Langille, and A. Nagy, Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development, 2000. 127(18): p. 3941-6. 5. Zhang, Z. and B. Zhou, Accelerated coronary angiogenesis by vegfr1-knockout endocardial cells. PLoS One, 2013. 8(7): p. e70570.

The outflow tract (OFT) initially develops from anterior heart field (AHF) progenitors as a single vessel connecting the right ventricle to the aortic sac. Subsequently, the OFT is septated into the aorta and pulmonary artery to establish and maintain systemic and pulmonary circulation, respectively. Although several transcription factors, including Isl1, MEF2C, and Tbx1, are known to regulate AHF and OFT development, few direct targets of these factors in the OFT have been identified. We previously identified the Tdgf1 gene, encoding the Nodal co-receptor Cripto, as a direct MEF2C target in the OFT at E10.5. Tdgf1 is initially expressed in the mesendoderm during gastrulation and becomes specifically restricted to the cardiac crescent and the outflow tract between E7.5 and E9.5. Human congenital heart defects have been associated with mutations in TDGF1. While the role of Tdgf1 in left-right patterning during gastrulation has been well established, its requirement in OFT development and the gene regulatory networks controlling its highly specific spatiotemporal expression in the OFT remain unknown. Using transgenic analyses in the mouse and zebrafish, we identified early and late enhancers of Tdgf1 that regulate its expression in the mesendoderm and cardiac fields, respectively. We further identified a 375-bp cardiac-specific enhancer of Tdgf1 that is bound by GATA4. Mutation of the GATA binding sites in this enhancer abolished enhancer activity in transgenic zebrafish and mice. Additionally, we observed a loss of Tdgf1 expression in the outflow tracts of Gata4 Mesp1-conditional knockout embryos. Collectively, these data indicate that Tdgf1 is a direct target of Gata4 during heart development via this enhancer. Surprisingly, conditional deletion of Tdgf1 from the Mef2c-AHF, Nkx2-5, or Mesp1 expression domains in mice did not affect viability or development, demonstrating that Tdgf1 is dispensable for heart and OFT development and suggesting that the expression of another Nodal co-receptor gene Cfc1 might be sufficient to support heart development in mice.

Tbx1 is required in the development of the pharyngeal apparatus and second heart field. However, the genetic elements and molecular mechanisms that regulate the expression of the gene are incompletely understood. We used single cell biology, in vitro differentiation, and bioinformatic tools to identify and validate regulatory elements of the gene. We used single cells RNA-seq and ATAC-seq data from mouse ES cells (mESCs) differentiating into precardiac organoids; on these, we correlated chromatin accessibility and Tbx1 gene expression in distinct cell clusters and identified differentially accessible regions. We applied a machine-learning approach to score the probability of being enhancers using logistic regression. Finally, we manipulated putative enhancers by CRISPR-Cas9 to test their requirement for Tbx1 gene expression. Integrating scRNAseq with scATACseq datasets, we identified 14 putative regulatory sequences (PRS) on the Tbx1 locus, and focused on a cluster of them approximately 10kb-long; the cluster included two PRS with positive predictive score, and another that we found accessible only in cells that did not express Tbx1. Using CRISPR-Cas9, we generated mESC lines deleted for the entire cluster and of for the 3 individual PRSs. We then tested Tbx1 gene expression in precardiac organoids from the engineered clones. Loss of the entire cluster resulted on strong, significant reduction of Tbx1 expression compared to the parental WT line, demonstrating the requirement of the enhancer cluster. Gene expression analyses of clones lacking individual PRS is in progress and will be presented at the meeting.

Heart development is a tightly regulated process orchestrated by well-characterized transcription factors and signaling pathways. Major effort has been dedicated to characterizing the development of cardiac progenitor cells (CPCs). During gastrulation and mesoderm establishment, these cells are driven by unique transcriptional programs and signaling pathways to undergo a series of differentiation and migration events. While the regulatory networks governing later heart development are well understood, how CPC fate is initially established and later diversified is poorly characterized. There is evidence to suggest that cardiac fate specification in progenitor populations occurs earlier than previously thought, however identifying and describing these populations has been a challenge due to the lack of markers for progenitors early in development. As such, I will investigate the earliest signs of cell fate specification in CPC-enriched populations using the zebrafish model, with the goal of identifying distinct CPC populations at these timepoints and their characteristic markers. Single-cell mRNA sequencing (scRNA-seq) data have been collected by current and previous members from the Scott and Wilson labs, investigating the role of GATA4/5/6 transcription factors in CPC specification spanning their emergence from the mesoderm (Song et al., Sci Adv, 2022). Gata5 is an established early regulator of mesendoderm-derived lineages, making gata5:GFP+ cells appropriate for investigation of CPCs. I have analyzed these data using Seurat, along with other R packages such as Clustree and UCell, to effectively annotate the data. From this, I have identified several mesoderm populations that display expression profiles indicative of cardiac progenitors, including cardiac, cardiopharyngeal, and mixed mesoderm populations. I will perform additional analyses on these data, namely subclustering of the identified populations of interest, pseudotime analyses, and simulation of transcription factor KO using Cell Oracle. Finally, I have begun functional validation for select potential markers using RNA in situ hybridization (ISH) and will also be using hybridization chain reaction RNA fluorescence ISH (HCR RNA-FISH) to validate several markers at a time. I will be performing these in both wild-type and GATA5/6 morpholino backgrounds. These experiments will provide insight on the expression profiles of these marker genes as well as their potential role in heart development.

The mouse Cre-loxP system is extensively utilized for conditional gene targeting, allowing the investigation of the cell-type-specific function of genes. However, off-target recombination can be an issue with some Cre strains. The myosin light chain-2 ventricular isoform (Mlc2v), encoded by the Myl2 gene, is a major sarcomeric protein in ventricular cardiomyocytes. Due to its specificity, the Mlc2v-Cre mice (Myl2tm1(cre)Krc/AchakJ) have been employed for ventricle-specific gene targeting to study heart development and function. Unexpectedly, we observed deletion of the floxed allele in non-muscular tissues, such as tail and ear-notch tissues, in offspring from breeders carrying Myl2Cre and floxed alleles, suggesting a possible off-target Cre expression. To investigate the prevalence and sex- dependency of off-target recombination in Mlc2v-Cre mice, we generated male and female mice carrying both the Myl2Cre allele and Rosa26tdTomato allele (Mlc2v- Cre;Rosa26tdTomato) and subsequently crossed them with CD1 wild-type mice to examine the fluorescent signal in E10.5 embryos. We noted that when a female breeder carried both the Myl2Cre allele and Rosa26tdTomato allele, 88% (22 out of 25) of fluorescent- expressing embryos, derived from 7 different females, exhibited whole-body fluorescence. When a male breeder carried the Myl2Cre allele and Rosa26tdTomato allele, approximately 22% of embryos (4 out of 18) from 6 different females crossed with 5 different males, displayed whole-body fluorescence. Importantly, among these whole- body fluorescent embryos from female and male Mlc2v-Cre;Rosa26tdTomato breeders, ~45% (10 out of 22) and 50% (2 out of 4), respectively, did not inherit the Cre gene, indicating the germline recombination. Our results suggest that Mlc2v-Cre mice can induce off-target germline recombination in both sexes, albeit with a higher prevalence in females. Careful experimental design and rigorous verification of gene recombination specificity are essential to circumvent potential problems and ensure data interpretation.

Congenital heart disease (CHD) includes structural and functional heart defects, affecting ~1% of live births. Currently, most familial CHD cases have no identifiable genetic origin. Variation in the non-coding genome can contribute to CHD, as mutations in regulatory elements such as enhancers may lead to improper spatiotemporal expression of cardiac or developmental genes. Previously, we leveraged chromatin accessibility and deep evolutionary conservation to identify 8866 putative developmental enhancers, termed accessible conserved non-coding elements (aCNEs). Through collaboration with the SickKids Cardiac Genome Clinic, 615 ultra-rare single-nucleotide variants (SNVs) from CHD patients were found within our aCNEs. To further prioritize, we focused on a subset of these aCNEs whose chromatin accessibility is dependent upon Gata transcription factors (TFs), as these are critical regulators of cardiac development. This pipeline yielded a list of 11 prioritized aCNEs. Of these, aCNE20—which is predicted to regulate TBX20 expression based on proximity and chromatin looping—has two SNVs identified in one CHD patient. TBX20 is a TF with essential roles in early heart development, and coding mutations in this gene can cause CHD. Accordingly, variation in aCNE20 may result in misregulation of TBX20 and subsequent cardiac defects. To test this hypothesis, I used a zebrafish transgenic reporter assay to demonstrate that one of the patient mutations in aCNE20 significantly weakens its cardiac enhancer activity. This SNV perturbs a conserved putative binding site for GATA, a key cardiac TF. I also generated aCNE20 deletion zebrafish mutants and I will assess these for abnormalities in heart morphology or function. Preliminary analysis suggests that deletion of aCNE20 can recapitulate the tbx20 loss-of-function phenotype, with variable penetrance. Overall, this project investigates the role of regulatory element variation in cardiac development and CHD. Broadly, it will provide a framework for the prioritization and functional validation of non-coding mutations.

Cardiac metabolism is stereotypically deranged at end-stage heart failure. Dilated cardiomyopathy (DCM) is a leading cause of heart failure and heart transplantation in children. DCM is more severe in children than adults, leading to higher rates of heart transplantation and lethality. Yet the molecular causes of pediatric DCM remain poorly understood. Here, we found that the chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked) maintains cardiac metabolism. Cardiomyocyte-specific knock out of Atrx in the mouse strikingly recapitulated the rapid progression to lethal heart failure observed in children with DCM. Atrx mutant mice developed left ventricle dilation as early as two weeks of age and reached endpoint due to heart failure by six weeks. Well before ventricle dilation at postnatal day 7, Atrx mutant cardiomyocytes had decreased contractile function, and accumulated oxidative damage and altered mitochondria with decreased electron transport complexes. Genome wide gene expression profiling revealed that Atrx inactivation led to de-repression of the T-box transcriptional repressor Tbx15, which broadly suppressed a program controlling cardiac metabolism. Intriguingly, Tbx15 expression was activated earlier and to much higher levels in Atrx mutant hearts, than in mouse hearts with adult-onset DCM. This suggests metabolic derangement as a cause of DCM and that a distinct mechanism drives swift deterioration of the heart in pediatric DCM, leading to early-onset heart failure. Specifically, earlier, and more pronounced Tbx15 de-repression leading to stronger suppression of cardiac metabolism reveals a mechanism for pediatric DCM.

The heart performs a vital function, the main one being as a mechanical pump to circulate oxygen-carrying blood and nutrients throughout the body. Specialized cell types constitute various essential components of the heart that work in synchrony to ensure its life-sustaining activity. The core genetic program and stepwise morphogenesis involved in development of the heart is largely conserved across metazoans and aberrations to this process could result in congenital heart disease. Many key insights into heart development and function have been derived from analysis using the zebrafish as a model organism. With the growing use of the zebrafish to model human heart biology, a deeper insight into its complex cellular composition is critical for a better understanding of heart function, development, and associated malformations. Here we present a high resolution atlas of zebrafish heart single cells transcriptomics. We utilized the transgenic lines Tg(myl7:mRFP), sqet33mi59BEt and sqet31Et to mark the cells of the myocardium and cardiac conduction system. We obtained the transcriptome profiles of over 50 000 cells representing the building blocks of the zebrafish heart at 48 and 72 hpf, and define at least 18 discrete cell populations comprising major cell lineages and sublineages of the developing heart. Taking advantage of the endogenous egfp expression in the transgenic lines and well-established gene signatures, we pinpointed a population of cells likely to be the primary pacemaker and identified the transcriptome profile defining this critical cell type. Our study constitutes a valuable resource for further investigations into the cellular and molecular mechanisms of this organ.

The mammalian heart is formed predominantly from two progenitor fields, the first heart field (FHF) and the second heart field (SHF). FHF progenitors differentiate early to form the cardiac crescent and generate the left ventricle (LV). In contrast, SHF progenitors are maintained in a progenitor state and differentiate days later into structures essential for cardiopulmonary circulation, including the right ventricle (RV), atrioventricular and atrial septa and pulmonary outflow tract. Although the FHF and SHF derived from distinct population in the primitive streak, it is unknown whether FHF and SHF progenitors follow the same transcriptomic trajectory for cardiac differentiation. Most prevailing studies infer that the FHF and SHF utilize distinct differentiation trajectories, based only on post-hoc analysis of trajectories from single time-points - no studies have directly compared FHF versus SHF-derived cardiomyocyte differentiation trajectories utilizing appropriately time-controlled data. We hypothesized that FHF and SHF progenitors utilize the same cardiac differentiation trajectory, but that Hedghog signaling establishes a delayed SHF that when released allows cardiac progenitors to re-enter a FHF-like differentiation trajectory. To test this hypothesis, we built and compared the FHF and SHF transcriptomic trajectories using available time-course single-cell RNA-seq data from E6.0 to E7.75. In total, we combined analysis of 8 distinct time points comprising 60,332 cells. We found that FHF and SHF trajectories are highly overlapping at early steps, from an early progenitor pool present at E6.0. We have defined a subsequent branch point, from which one branch differentiates into the cardiac crescent (FHF) and the second enters a delayed progenitor pool (SHF). Gene expression and genomic comparisons between these earliest distinct trajectory-based cell pools defines the onset of SHF-specific pathways versus FHF specific pathways as candidates for their dinstinct cell biology. This study will help define the mechanistic basis of cardiac progenitor differentiation timing control, offering a better understanding of cardiac development and perspectives in the fields of congenital heart disease and cardiac regeneration.

The coiled-coil domain-containing protein141 (CCDC141), also called coiled-coil protein Associated with Myosin-II DISC-I (CAMDI), has been reported to colocalize with DISC-I around the centrosome in neurons. It was posited that CCDC141 stabilizes the microtubule structure and promotes radial migration in neurons. Mutation in CCDC141 has been linked to hypogonadotropic hypogonadism. Notably, we have found that ccdc141 is abundantly expressed in the hearts of zebrafish and Xenopus embryos, yet its role in cardiac development remains largely unknown. To characterize ccdc141 during heart development, morpholino antisense oligonucleotides (MO) targeting ccdc141, and ccdc141 mRNA were used to knock down and overexpress ccdc141 respectively in zebrafish embryos. We found that morpholino-induced knockdown of ccdc141 in zebrafish embryos resulted in dose-dependent pericardial oedema from two days post-fertilization. The knockdown also impaired heart looping and reduced the heart rate dose-dependently. Spatial expression of key sarcomeric cardiac markers in morphant showed reduced expression of cmlc2 and myh6 with no significant change in myh7 expression. Intriguingly, the expression of Nkx2.5, a cardiac differentiation transcription factor, was increased in the first heart field and was prematurely expressed in the second heart field in response to ccdc141 knockdown. Morphants also showed increased expression of second heart field markers (tbx1 and Isl1) during the early stage of heart development. Additionally, ccdc141 overexpression resulted in dose-dependent pericardial oedema and cardia bifida in zebrafish embryos. Like the knockdown experiment, the overexpression of ccdc141 also resulted in reduced cmlc2 and normal myh7 expressions. The disparity in expression between atrium-specific myh6 and ventricle-specific myh7 in the morphants suggests a potential involvement of Ccdc141 in chamber specification. The increased expression of second heart field transcription factors suggests that Ccdc141 plays a crucial role in the development of the second heart field. The study on the underlying mechanism is ongoing.

The mammalian heart is predominantly generated from two progenitor fields, the early differentiating first heart field and the delayed differentiating second heart field (SHF). The timing of cardiac differentiation is critical to heart formation, with dysregulation resulting in congenital heart disase, a leading cause of neonatal death. We have identified Hedgehog signaling as a heterochronic regulator of SHF developmentrequired to prevent precocious cardiomyocyte differentiation of the SHF (Rowton et al., Dev Cell, 2022). We show the forkhead transcription factor FOXF1 mediates Hedgehog-dependent heterochronic control of differentiation in the SHF. Removal of Foxf1 from cardiac progenitors causes loss of SHF cardiac progenitors, inappropriate myocardialization, and midgestational death. To explore the mechanism by which FOXF1 controls developmental timing, we generated mESCs harboring inducible Foxf1. Deployment of FOXF1 in cardiac progenitors delays cardiomyocyte differentiation, blocking cardiac gene expression and prolonging progenitor gene expression. After removal of FOXF1, cardiac progenitors resume differentiation into cardiomyocytes. Integration of FOXF1 ChIP-seq, ATAC-seq, and H3K27Ac ChIP-seq suggests FOXF1 acts as a pioneer factor to regulate cardiac differentiation genes: FOXF1 establishes chromatin accessibility at enhancers of cardiac differentiation genes that subsequently gain H3K27Ac after FOXF1 removal. These studies suggest FOXF1 mediates Hedgehog signaling-dependent heterochronic control of SHF differentiation by priming, but delaying, the onset of cardiomyocyte differentiation gene expression. Hedgehog-dependent expression of FOXF1 in the SHF establishes a paradigm for temporal control of progenitor differentiation that may be essential for complex morphogenesis in many mammalian organs. Funding sources: AHA/CHF Postdoctoral Fellowship NIH NHLBI R01HL14757

Cardiac trabeculae are ridge-like muscular columns that project from the myocardium towards the chambers and are crucial for cardiac functions in embryonic hearts1,2,3. In developing hearts, trabeculae emerge, trabeculate, and undergo compaction to increase ventricular wall thickness, in which the mesh-like interconnected trabeculae gradually integrate into the compact myocardium2,4,5. However, the cellular mechanisms and collective cellular interaction underlying cardiac trabeculation and compaction are still unclear. Here, we adapted organotypic slice culture to investigate cell and extracellular matrix (ECM) kinematics during tissue morphogenesis. Day 6-8 chicken hearts were sliced 150-300 µm along the long axis. These three-dimensional (3D) live slices enable long-term culture and imaging in vitro while maintaining its in vivo structural integrity. Slice culture preserves cardiac contractility. We observed that heartbeats persisted for more than 48 hours post-slicing, with a heart rate of 2-3 Hz. The size of the slices was found to increase by approximately 10% over 24 hours, with the thickness of the outer compact layer in the ventricular wall increasing while the thickness of the inner trabeculated layer decreasing. Further, time-lapse live imaging of Cell Tracker and collagen-binding adhesion protein 35 (CNA35) revealed the collective cellular behavior and ECM dynamics over 18 hours. Extended trabeculae strands were found to gradually approach the ventricular septum, leading to the compaction of the septum. In contrast, the base of the trabeculae was consolidated and gradually coalesced with the ventricular wall. In summary, our study employed a 3D live slice culture technique to reveal cell and ECM kinematics during trabecular morphogenesis, providing insights into this dynamic remodeling process. 1. Sedmera, D., Pexieder, T., Vuillemin, M., Thompson, R. P., & Anderson, R. H. (2000). Developmental patterning of the myocardium. The Anatomical record, 258(4), 319–337. 2. Wittig, J. G., & Münsterberg, A. (2020). The Chicken as a Model Organism to Study Heart Development. Cold Spring Harbor perspectives in biology, 12(8), a037218. 3. Captur, G., Wilson, R., Bennett, M. F., Luxán, G., Nasis, A., de la Pompa, J. L., Moon, J. C., & Mohun, T. J. (2016). Morphogenesis of myocardial trabeculae in the mouse embryo. Journal of anatomy, 229(2), 314–325. 4. López-Unzu, M. A., Durán, A. C., Rodríguez, C., Soto-Navarrete, M. T., Sans-Coma, V., & Fernández, B. (2020). Development of the ventricular myocardial trabeculae in Scyliorhinus canicula (Chondrichthyes): evolutionary implications. Scientific reports, 10(1), 14434. 5. Choquet, C., Kelly, R. G., & Miquerol, L. (2019). Defects in Trabecular Development Contribute to Left Ventricular Noncompaction. Pediatric cardiology, 40(7), 1331–1338.

One key aspect of cardiovascular development is the development of mesenchymal structures within the outflow tract (OFT) and atrioventricular (AV) junction. The OFT valves develop from the outflow tract cushions (OFTCs) and intercalated ridges (ICRs), whereas the AV valves develop from the major and lateral AV cushions (AVCs). These mesenchymal structures that give rise to the formation of the valves rely heavily on the contribution of various cell types. While the OFTCs are composed of Second Heart Field (SHF)-derived cells, cardiac neural crest derived cells (CNCCs), and endocardial-derived cells (ENDCs), the ICRs are derived only from SHF cells. AVC development involves ENDCs and epicardial-derived cells (EPDCs). The events that lead to these cellular contributions require many transcriptional regulatory mechanisms, one of which includes the transcription factor, SOX9. The exploration of the role of SOX9 both in the SHF and EPDCs have directed our studies toward a better understanding of the lineage-specific distribution of other members within the SOX family, specifically SOX6 and SOX7. To gain an understanding of the expression of our SOX transcription factors of interest in these cell types, we have used lineage-specific cre-recombinase mouse models. Immunofluorescent staining with antibodies for SOX6, SOX7, and SOX9 indicates spatiotemporal differences in their expression in the various cell types contributing to valvuloseptal development. These spatiotemporal differences suggest that each SOX family member could be playing its own cell-lineage associated role in cardiac morphogenesis.

Tbx1 is the major gene involved in 22q11.2 deletion syndrome (22q11.2DS), the most common known genetic cause of congenital heart disease (CHD). Rare variants of the VEGFR3 gene cause cardiac OFT abnormalities, including Tetralogy of Fallot, the most common cardiac defect found in 22q11.2DS patients. We have shown that in mice, TBX1 regulates Vegfr3 in endothelial cells and the two genes interact strongly in brain vascularization and during cardiac lymphangiogenesis. We hypothesize that a similar genetic interaction in endothelial cells is required for cardiac OFT development. To test for a genetic interaction, we intercrossed Tbx1 and Vegfr3 heterozygous mice and analyzed the cardiac phenotype in coronal sections of embryonic hearts at E18.5. Then, to refine the critical Vegfr3-expressing domain for OFT development, we inactivated the gene conditionally using two secondary heart field Cre drivers, namely Tbx1Cre and Mef2C-AHF-Cre, and analyzed the hearts of conditional Vegfr3 homozygous embryos at E18.5. Our preliminary results support our hypothesis of a genetic interaction between TBX1 and Vegfr3 in OFT development and confirm the importance of VEGFR3 for cardiac morphogenesis.

Introduction: Loeys-Dietz Syndrome (LDS) is an autosomal dominant connective tissue disorder presenting with thoracic aortic aneurysm and dissection (TAAD). Remarkably, some LDS patients remain cardiovascularly unaffected throughout life, while others harbouring the exact same genetic variant face the daunting risk of aortic dissection. We hypothesize that genetic modifiers are at the basis of this observation. Therefore, the objective of the current work is to underpin the genetic modifiers that explain the variability in LDS-related aortopathy. Materials and Methods: We have access to a large LDS family (30 variant harbouring individuals) segregating a pathogenic SMAD3 (p.Arg287Trp) variant. Identification of candidate modifiers in this family encompassed WGS of a selection of patients: 5 affected (AMC, TAA(D) at a young age) and 2 unaffected mutation carriers (UMC, no TAA(D) > 40y). Functional validation of modifier candidate variants was performed using a TGFβ pathway-responsive reporter luciferase assay. Results: No potential genetic modifiers that fully segregated with either the AMC or UMC phenotype were identified upon a genome-wide exonic/intronic WGS data search. We then looked for incompletely segregating variants in the already known TAAD causative genes that could modulate the primary SMAD3 causative mutation. The analysis resulted in the identification of a predicted likely benign variant in TGFB2 c.272G>A (p.Arg91His) that is present in 10/21 AMCs and absent in the four UMCs. To assess the effect of the TGFB2 variant on TGFβ signalling we overexpressed the mutated protein together with the SMAD3 p.R287W in HEK293T cells harboring a luciferase-expressing vector under the control of a SMAD3-binding element. The TGFB2 p.Arg91His variant tends to negatively influence TGFβ signalling as compared to WT TGFB2, though less strongly in comparison to a proven pathogenic TGFB2 variant (positive control). Currently, we are validating the role of this variant as a potential modifier in the presence of the pathogenic SMAD3 variant with additional luciferase reporter gene analyses.

With recent advances in the field of tissue engineering, cardiac patches have shown great promise to restore the heart function and structure following myocardial infarction (MI). Cardiac patches can provide mechanical support to the MI tissue, while enabling sustained and targeted delivery of regenerative factors to the infarct site to promote healing. Despite the significant progress made towards fabrication of functional cardiac patches, many of current patch devices face challenges, such as the lack of vasculature and insufficient biomolecular cues. This study aims to develop a new generation of cardiac patches with perfusable vasculature and multicellular components. Patch constructs are loaded with pro-proliferative molecules, follistatin-like 1 (FSTL1) and CHIR 99021, to promote the proliferation of both endogenous and exogenous cardiomyocytes (CMs), improve angiogenesis, and hence, enhance synergistic therapeutic capacity of the patch. 3D bioprinting is utilized to manufacture cardiac patches with hollow channels. Human endothelial cells (ECs) and human induced pluripotent stem cell-derived CMs (hiPSC-CMs) are embedded within the patch to create multicellular patches with endothelialized vasculature. The in vitro culture of bioprinted patch constructs shows adequate EC-CM integration and cardiac function in both static and dynamic flow conditions. Therapeutic impact of the vascular patches is assessed in a rat model of acute MI. Echocardiography, histology, and immunohistochemistry analyses demonstrate significant enhancement of cardiac function, enhance angiogenesis, as well as reduced adverse remodeling of the myocardium in the patch-treated hearts. Through a novel integration of robust exogenous and endogenous regenerative means, the cardiac patch devices engineered in this study offer a superb therapeutic potential to treat ischemic heart injuries, while also establishing a highly tunable and high-throughput in vitro platform to study cardiovascular diseases and therapies.

Regenerative medicine approaches to treating single ventricle heart defects, including pulsatile conduits made from autologous hiPSC, are promising. However, the functional immaturity in contractility and electrical conduction of hiPSC-derived cardiomyocytes remains a significant obstacle for regenerative medicine applications. To overcome this challenge, we are using hiPSC-derived engineered micro-heart tissues (µEHTs) to screen for the best approaches to induce functionally mature cardiomyocytes from hiPSC. The developmental lineage of hiPSC-cardiomyocytes has been hypothesized to affect the long-term maturation of these cells. Furthermore, if bio-printing conduits or patches, printing cells at earlier stages of differentiation may be more feasible. Thus, we investigated the impact of cardiac differentiation approach and time of formation into µEHTs on the long-term maturation of the tissue. We formed µEHTs with hiPSC-derived cardiac progenitors (differentiation day 5) or from committed cardiomyocytes (differentiation day 15) obtained from monolayer differentiation guided by small molecule control over Wnt signaling. We assessed µEHTs formation and electrophysiological properties at day 30 of cardiac differentiation. Interestingly, µEHTs seeded with cardiac progenitors had similar morphology and calcium handling properties as compared to those seeded with committed cardiomyocytes. Point stimulation-based optical mapping likewise suggested similar conduction velocity regardless of the differentiation stage at which µEHTs were formed (respectively 3.10±0.77 and 3.00±0.90cm/s). Single cell RNAseq showed comparable cell composition between the two conditions at the end of the study. Overall, although there is no benefit in terms of eventual cardiomyocyte maturation within tissues, these findings suggest that it is feasible to form engineered heart tissues from cardiac progenitors rather than differentiated cardiomyocytes, thereby opening the door to a streamlined process for tissue formation and bioprinting at an earlier timepoint of differentiation.

Proper heart development depends on the tightly controlled interplay of cell fate changes and tissue morphogenesis. During early cardiac development progenitor cells migrate to the anterior of the embryo and fuse medially to form a primitive heart tube, which is the first functional organ to form. While fulfilling the purpose of a simple linear pump, the early heart is continuously remodeled to eventually form a four-chamber structure with a tightly controlled cardiac conduction system and directional fluid flow provided by valves. As a result, cardiac tissue is subjected to constant changes in mechanical forces exerted by neighboring tissue, ECM and blood flow. To date we still have a very poor understanding of how cells of the early heart react to surrounding mechanical forces and how this affects the first heart beats. Recent advances in human pluripotent stem cell technology have made it possible to generate three-dimensional self-organizing cardiac organoids (cardioids) that mimic aspects of early human heart development. Cardioids form large, fluid-filled cavities that resemble the lumina of heart chambers and start beating at around four days of differentiation. Using a cardioid model of the left ventricle we found that these cavities are filled with hyaluronic acid (HA), a glycosaminoglycan that can form a hydrogel. Interestingly, in-vivo HA is a major component of cardiac jelly – a specialized cardiac ECM located in the myoendocardial space that aids in the directional pumping of the early valveless heart and is later important during remodeling of the heart tube and the formation of valves. By genetically perturbing the function of the HA-secreting enzyme HAS2 and enzymatically degrading the glycosaminoglycan polymer we show that the formation of cardioid cavities is dependent on HA and that once its function is disrupted, cavities do not form. This provides us with a powerful tool to study the effect of mechanical cues on the early heart development as well as its role during contraction. Initial results indicate that the internal pressure exerted by the HA hydrogel influences the fate of cardiac progenitor cells as well as the contractile properties of cardiomyocytes. This allows us to elucidate cardiac mechanobiology in a physically accessible, high-throughput model of human cardiac development.

Understanding human coronary vasculature development is crucial for advancing our knowledge of cardiac development, unravelling the complexities of cardiovascular development and diseases, and providing insights into regenerative medicine strategies. However, due to the lack of experimental models, studying human coronary vasculature development is a challenge. To address this gap, our study uses cardioids to model the early steps of human coronary vascularization by recapitulating key developmental processes. Initially, we developed a protocol to differentiate human induced pluripotent stem cells (hiPSC) into sinus venosus-derived endothelial cells, a primary cell source of coronary endothelial cells during heart development. Then, we employed a co-culture protocol that mimics developmental steps by incorporating the differentiated sinus venosus cells into our cardioid model. This co-culture system involves the manipulation of signaling pathways associated with coronary vascularization to drive the formation of initial vascular plexuses and their posterior maturation in cardioids. By combining cardioids with sinus venosus-derived endothelial cells, our model offers a promising strategy to study and manipulate coronary vasculature formation in a controlled in vitro environment to advance our understanding of normal and pathogenic cardiovascular processes, ultimately helping with the development of novel therapeutic strategies.

Cell fate conversion is associated with extensive epigenetic and post translational modifications (PTMs) and architectural changes of sub-organelles and organelles, yet how these events are interconnected remains unknown. We report the identification of a phosphorylationcode in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 has been 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. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3 embedded factors, such as Hdac4, Mef2c, Nrip1, and Foxo1, form Hdac4 organized inhibitory nuclear condensates. Notably, PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a post-translational modification code could be a general mechanism for stimulating cell reprogramming and organ regeneration.

Zebrafish have a high capacity to regenerate their hearts. Several studies have surveyed transcriptional enhancers to understand how the dynamics of gene expression are controlled during heart regeneration. We have identified a cardiac transcriptional enhancer that is activated at the site of injury called the runx1 enhancer or REN since it regulates the expression of nearby gene runx1. REN is usually active in cardiomyocytes and epicardial tissues surrounding the cardiac valves of uninjured zebrafish hearts. However, when REN is activated in regenerating CM and epicardial tissues at the injury site, it is concurrently downregulated around the distal heart valve. The reason for anticorrelated behavior of REN between cardiac domains is unknown. Deletion of REN (ΔREN) results in excess collagen deposition around uninjured zebrafish cardiac valves. This ΔREN collagen phenotype is rescued with a runx1 deletion (Δrunx1), suggesting that REN and runx1 function in different genetic pathways in uninjured hearts. Interestingly, CM proliferation at the site of injury is enhanced in both ΔREN and Δrunx1 mutants, suggesting that during regeneration the REN-runx1 regulatory axis acts as a rheostat of tissue proliferation. There are two previous descriptions for how enhancers are activated during regeneration. First, cis-regulatory sequences are reactivated from embryogenesis and second adult enhancers that are already active are further invigorated during regeneration. Our data point to a third and previously unappreciated mechanism for gene control during zebrafish heart regeneration. We report that an enhancer from one cardiac domain is repurposed to activate a different nearby gene in regenerating cardiac tissue.

Cardiovascular diseases are the leading causes of morbidity and mortality worldwide, notably driven by myocardial infarction (MI) and heart failure (HF). The adult mammalian heart has limited regenerative capacity and is unable to regrow the loss of cardiomyocytes (CM) after injury, thereby leading to HF and ultimately, death. In contrast to adult mammalian hearts, neonatal hearts are able to regenerate fully following cardiac injury, although this regenerative capacity declines sharply after birth. Macrophages have been shown to play a pivotal role in cardiac regeneration. The underlying cellular and molecular mechanisms, however, remain poorly understood. To understand how macrophages contribute to cardiac repair/regeneration, single-cell RNA sequencing was performed on macrophages isolated from neonatal (P1-P3) and adult (12-week) mouse hearts 10 days post-MI injury, which revealed distinct macrophage populations in regenerative neonatal vs. non-regenerative adult mouse myocardium. Among the macrophage clusters enriched in post-injury neonatal mouse hearts, one macrophage population expresses high levels of S100A8 and S100A9 (S100A8/A9+ MAC) and exhibits immune modulatory properties. S100A8/9 belongs to the S100 calcium-binding family of alarmins and preferentially forms S100A8/A9 heterodimer complexes. Flow cytometry analyses revealed a dynamic expansion of S100A8/9+ macrophages, but not neutrophils, in neonatal mouse myocardium following MI injury, coinciding with the regenerative responses observed in neonates. In addition, pharmacological inhibition of S100A8/9 with Paquinimod and ablation of S100A8/9+ cells using MRP8-Cre;iDTR transgenic mice completely abolished post-MI-induced CM proliferation/regeneration in neonatal mouse hearts, resulting in excessive scar formation and cardiac dysfunction. Taken together, our findings revealed a critical yet previously unrecognized role of S100A8/A9+ cardiac macrophages in promoting myocardial regeneration. Targeting S100A8/A9+ cardiac macrophages, therefore, could be a potential new approach to enhance CM self-renewal and preserve cardiac function in patients with MI or HF.

Unlike adult mammals, which have limited cardiac regeneration ability, adult zebrafish can regrow lost cardiac tissue and restore heart function. Dissecting the innate regenerative capacity of zebrafish can help illuminate methods to awaken the latent capacity in humans and guide therapeutic approaches for cardiovascular diseases. To understand zebrafish heart regeneration at a holistic level, we generated and assessed transcriptomes for factors that could act either locally at the cardiac injury site or from tissues distant from the heart in adult animals. Bulk and single-cell transcriptomic profiles of regenerating zebrafish hearts revealed that the EGF receptor ligand heparin binding epidermal growth factor is induced in epicardial cells after cardiac injury. Experiments using a panel of molecular genetic tools indicated a model in which Hb-egf is required for heart regeneration in zebrafish and can potently enhance heart regeneration – first, by promoting epicardial expansion, followed by cardiomyocyte proliferation. To understand the role of distant organs in heart regeneration, we explored the responses of the brain and the kidney to cardiac injury. We describe organ-specific transcriptional and epigenetic responses of these tissues during cardiac regeneration, and we identify a transcription factor gene cebpd that is involved in tissue repair proximate to an injury event, as well as in the physiological sequalae like fluid regulation mediated by remote tissues. Our experiments indicate that corticosteroids released upon heart injury bind their cognate receptor transcription factors in remote tissues and upregulate cebpd expression, mediated through an essential hormone-responsive enhancer CEN. Our assessments of brains at the single-cell level during heart regeneration identified parallel gene expression responses in multiple cell types. Collectively, our studies illustrate how cardiac regeneration involves the coordination of multiple interacting cell types as well as the long-distance efforts of remote tissues.

Myocardial infarction (MI) occurs when a coronary artery is occluded. As a consequence, tissues downstream of the occlusion die and are replaced by a fibrotic scar. In contrast to humans, zebrafish possess a remarkable capacity to regenerate their hearts after injury. Rapidly regenerating coronaries provide structural support and secrete regenerative factors that signal neighboring tissues. The crosstalk between regenerating coronaries and epicardial cells is essential to support heart regeneration. However, how this interaction is regulated remains largely unknown. Here, we find that regenerating coronary endothelial cells (cECs) and epicardium-derived cells upregulate fbln2, which encodes the ECM glycoprotein Fibulin-2. Fibulin-2 is a matricellular protein expressed in tissues undergoing epithelial-mesenchymal transition (EMT) during development and tissue remodelling with unknown function in heart regeneration. We generated loss-of-function mutant zebrafish lines using CRISPR-Cas9 and analyzed processes key to regeneration. Injured fbln2 mutant ventricles display defects in EC and cardiomyocyte (CM) proliferation and dedifferentiation. To better understand the extent of the regenerative defects in these mutants, we performed single-cell transcriptomic analyses and found that fbln2 mutants have impaired epicardial activation after injury. The epicardium is a major contributor to heart regeneration as a source of paracrine signalling, mural cells and fibroblasts via EMT. Finally, in vitro analyses show that FBLN2 regulates cell-cycle progression in mammalian cardiac cells. Our results indicate that Fibulin-2 is a pro-regenerative factor required for cardiac regeneration and involved in epicardial activation. Identifying pro-regenerative factors might stimulate innovative therapeutic approaches for the treatment of MI patients.

Zebrafish have a profound ability to regenerate heart muscle through sustained cardiomyocyte proliferation. Much research has been devoted to identifying transcriptional components that play a central role in regeneration. While some of these factors appear to support a dedifferentiation-repair model, the specific molecular, cellular, and tissue progressions remain elusive. Post-transcriptional studies during regeneration are sparse but necessary to clarify the gene expression landscape. Previously, we have characterized eif4e1c, a homolog of EIF4E1, the 5’-cap binding protein that initiates translation of most mRNAs. Canonical EIF4E1 is essential from yeast to humans and is so deeply conserved that the human gene rescues yeast viability. Aquatic vertebrates have an additional family, called Eif4e1c, that is more highly conserved than the canonical form. Eif4e1c is present in all fish but is absent in all terrestrial vertebrate. We have shown that zebrafish eif4e1c mutants have impaired survival, growth deficits, and changed mRNA translation. Importantly, Δeif4e1c have impaired growth and regeneration of their hearts. Zebrafish also have two paralogs for the canonical protein, eif4ea and eif4eb. Whether the phenotypes observed in eif4e1c mutants are unique to the fish-specific pathway or are general features of mRNA cap binding factors remains unknown. Here, we report zebrafish deletion mutants for canonical factors eif4ea and eif4eb. We find that knockout of a single paralog leads to normal viability and regular size unlike with eif4e1c mutants. Moreover, double mutants for eif4e1c and eif4eb had potentiated eif4e1c phenotypes suggesting that the canonical factors partially compensate for the loss of eif4e1c. We will further test different combinations of our three mutants to uncover how heart growth during development and regeneration is regulated by mRNA cap-binding proteins. Our hypothesis is that heart specific phenotypes will be unique to Δeif4e1c.

Factors responsible for cardiomyocyte proliferation could serve as a potential therapeutic to stimulate endogenous myocardial regeneration following insult, such as ischemic injury. A previously published forward genetics approach on cardiomyocyte cell cycle and ploidy led us to the transcription factor, RUNX1. Here, we examine the effect of Runx1 on cardiomyocyte cell cycle during postnatal development and cardiac regeneration using cardiomyocyte-specific gain- and loss-of-function mouse models. RUNX1 is expressed in cardiomyocytes during early postnatal life, decrease to negligible levels by 3 weeks of age, consistent with a decline in cardiomyocyte cell cycle activity, however, RUNX1 expression increase in myocytes upon injury. Loss of Runx1 stymied cardiomyocyte cell cycle activity during normal postnatal development but had no effect in the context of neonatal heart regeneration. On the other hand, cardiomyocyte-specific Runx1 overexpression resulted in an expansion of diploid cardiomyocytes in uninjured hearts and expansion of 4N cardiomyocytes in the context of neonatal cardiac injury, suggesting Runx1 overexpression is sufficient to induce cardiomyocyte cell cycle responses. Continued overexpression of Runx1 for >1 month continued to promote cardiomyocyte cell cycle activity resulting in substantial hyperpolyploidization (≥8N DNA content). This persistent cell cycle activation was accompanied by ventricular dilation and adverse remodeling. Together, these data demonstrate a role for Runx1 in cardiomyocyte cell cycle, which results in increased diploid or hyperpolyploid CMs depending on developmental state.

Stimulation of cardiac lymphangiogenesis has been shown to improve cardiac function and reduce the progression of heart failure after myocardial infarction (MI). Therefore, understanding the cellular and molecular mechanisms involved in neo-lymphangiogenesis in the infarcted heart would be valuable for improving the effectiveness of treatment. However, how enhanced lymphangiogenesis interacts with various cardiac cell types in the infarcted microenvironment is unclear. Here, we authenticated a series of conserved cardiac lymphatics genes, including T-box transcription factor Tbx1 and chemoattractant Ccl21, specifically activated in the infarcted area. Tbx1 overexpression improved cardiac function in myocardial infarction model. The analysis of single-nucleus RNA-seq showed that lymphatic Tbx1 overexpression protected the mouse heart from ischemic injury and cardiac rupture by promoting cardiomyocytes more resistant to hypoxia stress, inducing extracellular matrix deposition, and enhancing inflammation resolution. Furthermore, our cellular interaction analysis revealed that Arg1+ macrophages communicated with myofibroblasts in the infarcted area via interaction with Cd44. The clinical relevance of these findings was indicated by the observed enrichment of rare TBX1 variants in MI patients with early heart failure. Together, our findings indicated that the Tbx1-mediated lymphatic enhancement might be a potential target for improving post-MI outcomes.

The 1-day old neonatal rat heart contains two subpopulations of ventricular cardiomyocytes (NNVMs) that re-enter the cell cycle distinguished by the absence or de novo expression of the intermediate filament protein nestin. It remains presently unknown whether postnatal development compromises the cell re-entry of each subpopulation in vitro and/or in vivo after cardiac damage. The present study tested the hypothesis that two distinct NNVM subpopulations that re-enter the cell cycle persist in the 7-day old neonatal rat heart. The co-treatment of isolated 7-day old NNVMs with the PKC activator phorbol 12,13-dibutyrate (100 nM) and p38 alpha/beta MAPK inhibitor SB203580 (10 uM) for 3-days induced cell cycle re-entry depicted by 5-bromo-2′-deoxyuridine (BrdU) incorporation and nuclear appearance of the G2-M marker phosphorylated serine10 residue of histone 3 (PHH3). Cell cycle re-entry was identified in two NNVM subpopulations characterized by the absence or de novo nestin expression. Nestin(+)-NNVMs were the predominant subpopulation that incorporated BrdU (77%) and expressed nuclear PHH3 (62%). In the 7-day old neonatal rat heart, cell cycle re-entry was identified in a subpopulation of nestin(-)-NNVMs whereas nestin(+)-NNVMs were absent. Three days following apex resection, the damaged heart was characterized by a cardiac-derived upregulation of a panel of inflammatory cytokine mRNAs (Reg3beta, IL-6, CCL2, CCL22). Apex resection led to the appearance of nestin(+)-NNVMs selectively bordering the damaged region and re-entered the cell cycle depicted by BrdU incorporation and nuclear PPH3 staining. By contrast, cell cycle re-entry of the nestin(-)-NNVM subpopulation was attenuated in the apex-resected neonatal heart. Thus, the 7-day old neonatal rat heart contains two distinct NNVM subpopulations that re-enter the cell cycle following in vitro p38 alpha/beta MAPK inhibition. By contrast, apex resection of the 7-day old neonatal rat heart led to the appearance and selective cell cycle re-entry of nestin(+)-NNVMs in the absence of p38 alpha/beta MAPK inhibition.

Following myocardial infarction (MI) injury, the heart acutely loses functional cardiomyocytes followed by fibrotic extracellular matrix (ECM) deposition. Traditional cell-based treatment strategies, tackling the cellular deficit, have led to limited effects on patient’s cardiac function post-MI. A recent innovative approach focused on restoring tissue homeostasis by supplying a healthy ECM to treat MI, has shown much promise. However due to the variety in study designs, it has been difficult to discern to what extend these approaches truly affect heart tissue following a MI. We performed a systematic review and meta-analysis to investigate the effect of ECM treatments on cardiac function and tissue damage post-MI in pre-clinical studies. Following our systematic search in the SCOPUS and PubMed databases, 71 articles met our inclusion criteria. Our meta-analysis demonstrated that ECM treatment significantly improved LVEF (MD: 10.9% [8.5;13.2%], p < 0.0001), fractional shortening (MD: 5.4%, [3.7;7.2%], p < 0.0001), stroke volume (SMD: 0.7, [0.1;1.3], p = 0.02), and LV wall thickness (SMD: 1.3, [1.0;1.6], p < 0.0001) while reducing infarct size (MD: -12.9%, [-16.5;-9.4%], p < 0.0001). Interestingly, we found no effect of various study characteristics (species, sex, ECM source, ect.) on treatment efficacy. We show that ECM-based treatment strategies have a significant beneficial effect in the pre-clinical setting by improving LVEF, fractional shortening, stroke volume and LV wall thickening, while reducing infarct size, irrespective of species, sex or ECM source. This establishes a foundation for future research directions targeting tissue homeostasis as a means to repair the injured myocardium.

Cardiomyocyte proliferation is a challenging metric to assess. Current methodologies either fall short of demonstrating the generation of new cardiomyocytes or are fraught with technical challenges reducing their applicability. Here, we describe a cell suspension-based methodology that can be universally employed by any lab and to any animal model in a multitude of experimental conditions. We discuss steps of the procedure where edits can be made to tailor the method to a lab’s specific biological question. We highlight additional metrics that can be gathered from the same cell preparation enabling additional relevant analyses to be performed, including general ploidy across cardiomyocytes, total cardiomyocyte numbers, total cycling cardiomyocytes, and cell dimension. Further, we incorporate additional antibody stains to the traditional protocol to disprove potential technical concerns of miscounting. Finally, we employ this methodology with a unique dual-thymidine analog labeling approach to a post-infarction murine model. Our findings from this dual-labeling experiment provide clues as to the timing of CM cell cycle activation and proliferation after injury, continue to point to major differences between genetic backgrounds, including dimensional changes, and demonstrate for the first time that a single CM can divide more than once following MI. A complete, detailed protocol for the procedure and the various analyses that can be performed is available upon request.

The molecular mechanisms underlying differences in regenerative capacity between species have yet to be unraveled. Upon cardiac injury, cardiomyocytes located in the border zone undergo profound transcriptional changes. While in the zebrafish heart this leads to cardiomyocyte cell cycle reentry, in the mammalian heart cardiomyocytes do not enter the cell cycle which preventing regeneration. To better understand why border zone cardiomyocytes reenter the cell cycle in zebrafish and not in mice, we employed spatially-resolved transcriptomics on mouse and zebrafish hearts at critical timepoints post injury. We used the spatially-resolved transcriptomics to identify overlapping and diverging gene expression patterns in the border zone, which revealed cellular processes that are either shared or specific to one species. To identify novel drivers of cardiomyocyte proliferation during heart regeneration we selected candidate genes for functional analysis, revealing an important role for high mobility group (HMG) proteins in chromatin remodeling during zebrafish heart regeneration. In addition, combining single cell RNA-sequencing with sortChIC, revealed that ectopic HMG expression reduced H3K27me3 marks on embryonic genes. Furthermore, ectopic HMG expression in the border zone of injured adult mouse hearts induced similar epigenetic changes, stimulated cardiomyocyte proliferation and improved cardiac function. Together, these results indicate that HMG-mediated chromatin remodeling underlies differences in regenerative capacity between zebrafish and mammalian hearts.

The cardiovascular system responds to hemodynamic forces generated by blood flow during development and in pathological conditions. Globally, cardiovascular diseases are the leading cause of mortality, and blood flow defects strongly contribute to their pathogenicity. Notably, the development and maintenance of the cardiac valves are under the control of blood flow. Here, we find that the whole endocardium is also mechanosensitive prior to cardiac valve development, and that loss of cardiac contractions/blood flow leads to endocardium-to-venous transdifferentiation. Using single-cell transcriptomic analysis and live imaging of endothelial cells, we show that perturbations in cardiac contractions/blood flow induce the loss of endocardial markers as well as the ectopic expression of venous markers in endocardial cells. Cardiac microsurgery experiments reveal that blood flow, but not cardiac contractions, is essential to repress venous fate in endocardial cells. Mechanistically, we show that cardiac valve mechanosensors are not responsible for endocardial mechanosensation. Furthermore, using loss- and gain-of-function strategies, we identify Notch signaling as the main regulator of venous fate repression in the endocardium. Together, these data suggest that blood flow regulates cardiac valve development by repressing venous fate in the endocardium, increasing our understanding of the consequences of cardiac insufficiency.

Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and can sustain long-term self-renewal. The ability to derive patient-specific HSPCs in culture has immense therapeutic potential to overcome the shortage of compatible donors for HSC transplantations. However, differentiation protocols largely fail to produce long-lived HSCs from human pluripotent stem cells. Understanding the complex genetic networks and signaling pathways required to generate HSCs will facilitate clinical applications in patients. The hemogenic endothelium (HE) is a specialized niche of endothelial cells (ECs) within the ventral portion of the dorsal aorta that gives rise to HSPCs during the definitive wave of hematopoiesis in the zebrafish embryo. Our data reveal that cysteine rich intestinal protein 2 (crip2), a LIM domain-binding protein expressed specifically in the dorsal aorta, has a previously unrecognized function in establishing the proper EC environment for HSPC specification. To investigate the requirement of crip2 in HSPC development, we employed CRISPR mutagenesis to generate loss-of-function alleles in crip2 and crip3, a gene family member with expression in cardiovascular tissues, to avoid potential genetic compensation. crip2-/-;crip3-/- (cripDM) embryos exhibit decreased emergence of HSPCs by 26 hours post fertilization (hpf). Loss of HSPCs in the cripDM results in decreased erythroid, myeloid, and lymphoid lineages during embryogenesis. To decipher the mechanisms underlying the cripDM phenotype, we performed single cell RNA sequencing of sorted, Kdrl:mCherry+ cells at 30 hpf. Our analysis reveals upregulation of genes essential for vascular development and a failure to repress Notch signals in cripDM hemogenic endothelium during the vital transition of HE specification to HSPC emergence. Moreover, inhibition of Notch activation partially rescues the diminished HSPCs in cripDM embryos by facilitating HSPC generation. Taken together, we anticipate that our insights will inform potential therapeutic interventions for improvements of human HSC production in vitro.

Cardiac valve malformations constitute some of the most common human congenital heart defects (CHDs). Perturbations of retinoic acid (RA) signaling have been associated with multiple types of CHDs, including valve defects. Although the earliest requirement of RA signaling is to restrict cardiomyocyte specification, if early RA signaling is also required for valve development remains unclear. Here, we investigated a requirement of early RA signaling on the development of the atrioventricular valve (AVV), the equivalent of the mitral valve in mammals. Inhibiton of RA production with DEAB beginning at 3 hpf and zebrafish aldh1a2 mutants both indicated that RA-deficient embryos lack AVVs by 72 hour post-fertilization (hpf), despite having enlarged hearts. Although examination of AVC cardiomyocyte markers, including bmp4 and tbx2b, showed that the atrioventricular canal (AVC) was expanded in RA deficient embryos, endocardial valve markers, including klf2a and notch1b, were lost in RA deficient embryos, suggesting that the loss of early RA signaling affects a factor that signals from the AVC myocardium to the overlying endocardium to promote AVV induction. Canonical Wnt signaling within AVC cardiomyocytes has been shown to promote AVV development, which we confirmed through inhibition of Wnt signaling with XAV939 treatment from 32-36 hp. Furthermore, we found that RA-deficient zebrafish embryos lack expression of wnt2bb and the 7XTCF-Siam:GFP Wnt reporter within AVC cardiomyocytes by 36 hpf , implicating canonical Wnt signaling as a candidate downstream effector of RA signaling within the AVVs. Altogether, our results suggest that early RA signaling is necessary to establish a population of AVC cardiomyocytes that turn on Wnt signaling and promote AVV development.

Heart valves are comprised of various cell types which adapt to continuous biomechanical stress and as necessary coordinate extracellular matrix (ECM) homeostasis. While heart valve formation begins during embryogenesis, the valve structures continue to grow and mature throughout postnatal development. The cellular profiles of heart valves during this postnatal maturation stage have been investigated however, the cell types and their transcriptional profiles present at birth which provide the foundation for maturation have not been characterized. Single nuclear gene expression profiles of the entire aortic valve structure from postnatal day one (PND1) mice were generated, and validated by immunofluorescence staining and lineage tracing mouse models were used to follow maturation. PND1 aortic valves consist largely of valve endothelial (VEC), interstitial (VIC), and tissue resident myeloid cells. At birth, there are 4 VEC subpopulations. A unique PND1 VEC subpopulation was identified along with 3 subpopulations with similar transcriptional profiles to VECs published at PND7 and PND30. PND1 VIC subpopulations are transcriptionally unique from PND7 or PND30 and consist of three subpopulations that based on differentially expressed genes we have termed as primitive, remodeling, and bioactive. Primitive VIC are associated with differentiation, proliferation and motility/migration. Fate mapping studies support the notion that primitive VIC occupy various spatial regions throughout maturation and gene markers decreases during maturation. Remodeling VIC are involved in ECM processes and bioactive VIC are highly metabolically active. Bioactive VIC gene markers increase activity from PND1 to PND14. In summary, the molecular profiles of interstitial subpopulations are highly dynamic throughout maturation while endothelial subpopulations are primarily established by birth.

Severe heart valve disease presents a significant clinical burden, yet effective treatment options remain limited to surgical repair or replacement. Normal heart valve function is largely achieved by the coordination of a highly organized extracellular matrix (ECM) structure that provides all the necessary biomechanical properties to withstand the hemodynamic burden of the cardiac cycle. Previous studies have linked ECM disorganization to biomechanical failure, in addition to damage/dysfunction of the valve endothelial cells (VECs) that line the valve cusps and serve as a barrier to limit infiltration of circulating risk factors, and a signaling source for disease-preventing factors. It has been suggested that VEC damage/dysfunction initiates and promotes ECM disorganization in valve disease, and therefore targeting VEC repair may be a beneficial therapy. To address this, our lab has adapted a mouse surgical model to induce mechanical injury to aortic valve (AoV) VECs. We previously showed that following injury, young, but not aged mice have the potential for self-repair through an acute increase in proliferation of VECs and neighboring valve interstitial cells (VICs). This proliferative response is associated with an upregulation of Tgfß1 in remaining VECs, and upregulation of secreted glycoprotein, collagen triple helix repeat containing 1 (Cthrc1) in underlying VICs. The goal of this present study is to further investigate the mechanisms of endothelial-Tgfß1, and interstitial-Cthrc1 function in VEC repair. To address this, wire injury will be performed in mice following endothelial-specific deletion of Tgfß1, in addition to cthrc1-/- mice, and the reparative response (cell proliferation, endothelium function, ECM organization) will be examined. Successful completion of this work will provide insights into molecular and cellular targets for VEC repair in preventing and attenuating valve disease.

Cardiovascular diseases such as mitral valve disease (MVD) and coronary artery disease (CAD) have traditionally been viewed as conditions acquired with age. However, there is increasing evidence supporting developmental origins for these common conditions. Understanding the genetic and cellular mechanisms governing heart development is essential for earlier diagnosis and development of more effective treatments for heart diseases with developmental origin. Our lab recently reported a role for the transcription factor SOX9 in the invasion of epicardial-derived cells that is essential for maintaining atrioventricular (AV) valve homeostasis. RNA-sequencing experiments from this study identified Cd109 as a novel gene associated with valve and coronary system development (Harvey et al., 2023, JMCC). CD109 is a cell-surface coreceptor which modulates signaling downstream of Transforming Growth Factor Beta and Epidermal Growth Factor receptors. It has never before been studied in the context of cardiovascular development or disease. CD109 is expressed by mesenchymal cells of endocardial origin in the developing AV valves, a cell population that is significantly expanded in our mouse models of myxomatous mitral valve degneration caused by impaired epicardial-derived cell contribution to the AV valves during development. It is, to our knowledge, the only gene identified to date which preferentially marks endocardial-derived cells but not epicardial-derived cells in the AV valve mesenchyme. CD109 is also expressed by endothelial cells of the coronary vasculature at all stages of development. Our studies also identify an correlation between epicardial-derived cell invasion and CD109+ vasculature within the ventricular myocardium. Interestingly, recent GWAS studies have identified rare intronic variants in CD109 that are positively associated with CAD, myocardial infarction, and chest pain. Additionally, loss of function variants in CD109 have been identified in at least 6 patients with severe congenital heart defects, highlighting its relevance in cardiovascular development and disease. Here, we are the first to describe a comprehensive expression profile of CD109 during cardiovascular development, and hypothesize roles for CD109 in valve and vessel development.

The heart is the first organ to form and its development requires the coordination of multiple processes for it to become functional. Cardiac vessels, termed coronaries nourish the ventricular wall by providing blood and nutrients. Alterations in coronary development and patterning affect the formation and function of the heart. How the coronary vessels grow and associate with the cardiac muscle to regulate the formation of the heart remains largely unknown. To address this question, we investigate how the coronaries develop and interact with cardiomyocytes in zebrafish. Using high-resolution imaging and genetic models, we find that developing coronaries vigorously populate the ventricular surface from juvenile stages and define four phases of coronary development. To further examine molecular determinants governing coronary vessel formation, we use zebrafish mutants lacking Vegfa signaling, a key regulator of vascular biology. We find that these mutants display alterations in key steps of coronary formation. Furthermore, using transgenic lines to label coronary endothelial cells and growing cardiac muscle, we analyze coronary-cardiomyocyte interactions throughout development. Identifying regulators of this crosstalk might motivate new therapeutic strategies for congenital heart diseases where the balance between coronary and cardiomyocyte development is disrupted.

Background: The embryonic heart develops as a two layered tubular structure consisting of an outer layer of cardiomyocytes and inner layer of endocardium. The endocardium acts as a vital source of multiple cell lineages in the developing heart, including a substantial proportion of the endothelium which is essential for the development of the coronary blood vessels. Despite the evidence of endocardial plasticity during development, there is virtually no insight into the transcriptional mechanisms controlling the specification of the endocardium to form coronary vascular endothelial cells. Materials and methods: Human induced pluripotent stem cells (hiPSC) were differentiated towards an endocardial identity using a novel directed differentiation protocol. This defined protocol begins with canonical Wnt activation to promote commitment to a cardiogenic mesoderm fate, prior to inducing endocardial specification using media containing VEGF-A, FGF2, BMP-10, and XAV-939 (a Wnt pathway inhibitor). Results and conclusion: Induction of mesodermal identity revealed an increase in mesoderm marker MESP1 by day 2 of differentiation. Following mesoderm specification, the addition of BMP-10 and XAV-939 alongside VEGF-A and FGF2 was found to strongly induce expression of endocardial genes such as NPR3, GATA4, and NKX2-5 in day 6 CD31+ cells. Flow cytometric analysis revealed ~60% of day 6 cells to be CD31+/NPR3+ with cells having a characteristic cobblestone morphology. Further culture of hiPSC-derived endocardial cells revealed their capability to upregulate coronary endothelial cell markers whilst simultaneously downregulating endocardial markers, thus recapitulating the developmental mechanism and validating the application of this model to identify novel regulators of endocardial to coronary endothelial cell transition.

Multisystemic smooth muscle dysfunction syndrome (MSMDS) is a rare genetic disorder characterized by malfunctioning smooth muscle cells (SMCs) throughout the body, which can lead to a range of clinical symptoms. MSMDS is associated with mutations that affect unique residues in the gene Actin alpha 2 (ACTA2). Non-synonymous mutations at position R179 are the root cause of the syndrome's most severe form, which includes dysfunction in gastrointestinal (GI), vascular, and cerebrovascular organs. Mutations at R39 result in GI symptoms and may also involve some vascular problems, while mutations at R258 only cause vascular symptoms. Currently, no information is available to explain how different mutations in the ACTA2 gene cause unique organ-specific defects. We hypothesize that the R179, R258, and R39 mutations cause distinct MSMDS phenotypes via organ-specific differential protein-protein interactions. We will investigate these interactions in wild-type versus mutant primary human SMCs via TurboID and downstream mass spectrometry. We will validate differential ACTA2 interactions and their effects with co-immunoprecipitation, immunofluorescence, and functional assays. By carefully comparing how the normal versus mutant ACTA2 protein interacts with other proteins, we may better understand how these unique mutations lead to SMC dysfunction and, subsequently, distinct MSMDS phenotypes.

Significant phenotypic differences exist between the vascular endothelium of different organs, including cell-cell junctions, paracellular fluid transport, shape, and mural cell coverage. These organ-specific morphological features ultimately manifest as different functional capacities, as demonstrated by the dramatic differences in capillary permeability between the leaky vessels of the liver compared to the almost impermeable vasculature of the brain. While these morphological and functional differences have been long appreciated, the epigenetic and transcriptional mechanisms governing endothelial organ specialization remain unclear. To address this problem, we profiled accessible chromatin, as well as gene expression, in six different organs, across three distinct time points, of murine development. After identifying both common, and organ-specific DNA motif usage and transcriptional signatures, we focused our studies on the endothelium of the central nervous system. Critically, we show that these unique regulatory regions and gene expression signatures are evolutionarily conserved in humans. By comparing these bulk epigenetic and transcriptional data with a comprehensive single cell transcriptomic atlas of murine cerebrovascular development, we identified novel gene regulatory networks active in either the angiogenic endothelium of the developing brain or confined to the homeostatic, mature endothelium of the adult brain. Finally, in vitro studies show that a cocktail of the brain-enriched TFs alter the endothelial barrier and upregulate a BBB-like gene signature, while liver-specific TFs have the opposite effect. Collectively, this work provides a valuable resource for identifying the transcriptional regulators that may control organ-specific endothelial specialization and provides novel insight into the gene regulatory networks governing the maturation and maintenance of the cerebrovasculature.

Congenital heart disease (CHD) is one of the most commonly prenatally diagnosed congenital anomalies. Maternal hypertensive disorders are known to be associated with increased rates of CHD. The umbilical cord and placental vasculature functions as an extracorporeal extension of the fetal vascular system. However, we do not well understand changes to the umbilical cord vasculature in pregnancies with fetal CHD. Therefore, we sought to investigate placental and cord alterations as they related to various forms of CHD while also considering maternal hypertensive disorders. In this retrospective study, we reviewed placental pathology according to the Amsterdam Criteria in singleton pregnancies with or without fetal CHD. Prenatally diagnosed CHD was confirmed by postnatal imaging and subcategorized into the following: D-transposition of the great arteries (TGA), obstructive left CHD, obstructive right CHD, Mixing and Other. Categorical variables were compared by Chi squared and Fisher’s exact tests. Continuous variables were compared by t-test, Wilcoxon rank-sum and Kruskal-Wallis rank sum tests as appropriate for sample size and normality. Significance threshold was set at p<0.05. 122 control placentas and 227 fetal CHD placentas met the inclusion criteria. Fetal CHD placentas showed higher rates of cord anomalies (22% vs. 0.8%, p = <0.001) and featured higher rates of abnormal coiling (5.3% vs. 0%, p = 0.010) as well as abnormal insertion (11% vs. 0.8%, p = 0.001), and abnormal vasculature (6.6% vs 0%, p = 0.004) of the umbilical cord. Comparisons across the types of CHD demonstrated no difference across the types of CHD. However, left-obstructive cardiac lesions showed higher rates of chorangiosis (9.4% compared to 0%-1.5%, p = 0.044) than the other categories. Within a large group of fetal CHD placentas, we were able to demonstrate that umbilical abnormalities were present. Rates of abnormalities did not differ significantly by CHD type, indicating that it is a potentially common and consistent risk to fetal development and infant health. Further research is warranted to explore the association between anatomical umbilical cord alterations and CHD.