Ventricular trabeculae and pectinate muscles are morphological hallmarks of chamber myocardium differentiation. It is not clear, however, to which extent they are homologous regarding the mechanisms of their formation. Pectinate muscles start to form between stages 23-24 in the chick, and around ED11 in the mouse. The ratio of pectinate muscles to free wall in the chick embryo increased steadily between stages 24 and 38 from 6 and 12 up to 62 and 69% of the left and right atrial myocardium, resepctively. Complete mechanical unloading in whole heart culture inhibited their formation, which was not rescued by injection of silicon oil droplet. Left atrial ligation at stage 21 prevented their formation in the ligated portion of the atrium, caused slowing of the impulse propagation, and induced ectopic pacemaking activity. There was a concomitant decrease in expression of hemodynamically-dependent gene KLF2. Unlike ventricular trabeculae, which show a considerably slower proliferative activity than the compact myocardium, the proliferative structure of the atria was more homogeneous with actually higher cell division rate in the pectinate muscles. The atrial wall growth involved thickening of the pectinate muscles with the free wall remaining relatively thin. In conclusion, while there is some structural and functional homology between the ventricular trabeculae and atrial pectinate muscles, there are also significant differences between the atria and ventricles in the terms of myocardial morphogenesis.

Genetics plays a pivotal risk factor for atrial fibrillation (AF), the most prevalent cardiac rhythm disorder. A non-coding region on chromosome 4q25, upstream of the gene encoding the transcription factor PITX2C, is strongly associated with AF. Recent data has suggested that PITX2C regulates cardiomyocyte metabolism and the antioxidant response to stress. However, how the absence of PITX2C leads to metabolic changes and the specific downstream metabolic alterations remain unclear. Using a pitx2c-deficient zebrafish model that displays cardiac phenotypes with significant similarities to the pathologies seen in AF patients, we investigated mRNA levels of selected genes encoding metabolic regulators. Our analysis revealed a significant down-regulation of gapdh, associated with glycolysis, and acsl1b, implicated in lipid metabolism in atrial tissue. Proteomics analyses revealed differential expression of proteins involved in metabolic processes. Additionally, we observed an increase in adipose tissue formation in pitx2c-/- hearts shown by Oil Red O staining, suggesting lipid accumulation. Notably, we established the electron paramagnetic resonance assay using 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) spin probe to measure reactive oxygen species (ROS) levels in zebrafish hearts. Our results reveal elevated ROS levels in pitx2c+/- and pitx2c-/- in adult hearts. We are currently generating transgenic lines to modulate Pitx2c levels and activate endogenous antioxidant genes sod1 and sod2 to test if rescuing Pitx2c expression or expressing antioxidant enzymes ameliorates the metabolic phenotypes and cardiac arrhythmia. We are also assessing the antioxidant defense system function by quantifying GSH/GSSG content and the activity of Sod and Catalase enzymes in pitx2c-/- adult hearts.

During postnatal development, Cx43 undergoes dramatic changes in distribution pattern on the cardiomyocyte surface. Relocation into intercalated disk contribute to acceleratation of conduction velocities (CV) in longitudinal (CVL) as well as transversal direction (CVT). However, little is known about changes in the total and membrane-bound (junctional fraction; JF) levels of Cx43 during postnatal development. The aim of this study was to analyze the quantity and distribution of Cx43 along with electrophysiological parameters at different postnatal developmental stages. Rats hearts were collected on the 1st, 4th, 6th, 11th, 20th, 30th, 40th, 60th, and 90th postnatal days (PD). At these stages, we investigated the amount of Cx43 in the JF and non-junctional fraction (NJF) of the cardiomyocytes together with the total amount of Cx43 using Western blot. Using optical mapping, we analyzed electrophysiological parameters of the left ventricle, including conduction velocity (CV), action potential duration (expressed as APD30 and APD80), and activation pattern. Our results showed that the total amount of Cx43 in the cardiac homogenate increased from the 1st to the 4th PD to 135% and then gradually decreased until the end of the observed period. The amount of Cx43 in JF did not significantly differ over neonatal period. In contrast, the amount of Cx43 in NJF increased until the 20th PD (235% vs. 1st PD), The most pronouced increase in CV was observed between the 1st and 6th PD, by 86% for CVL and by 26% for CVT. Within analyzed time frame, we observed decrease in APD30 and APD80 duration. The activation pattern did not significantly differ during analyzed period. Our data indicate that during neonatal development, the amount of membrane-bound Cx43 is not significantly affected by changes in the total level of Cx43, nor by variations in the NJF level of Cx43. Additionally, the first few postnatal days appear to be highly important for Cx43 dynamics in the rat myocardium. Moreover, revealed changes in NJF indicate potential intacellular shifts in Cx43 localization around 20th PD. Supported by Czech Health Research Council grant no. NU21J-02-00039.

Cardiovascular disease affects men and women differently in terms of prevalence, treatment, and survival rates. However, the underlying mechanisms for these differences are not yet fully understood. To define the molecular mechanisms that lead to cardiac sex differences, our lab performed RNA-seq and quantitative proteomics. This screen identified differentially expressed transcripts and proteins in the adult mouse heart. We uncovered 94 proteins that are differentially expressed between males and females. The majority of the sex-differentially expressed proteins appear to be regulated at the post- transcriptional level. We hypothesized that differential miRNA expression could account for proteomic sex differences. To address the potential post-transcriptional mechanisms that lead to male-female differences in cardiac protein expression, we undertook a screen to identify sex-specific miRNAs. Our screen uncovered 11 miRNAs that show sex- differential expression. Notably, a group of three miRNAs on the X-chromosome are the only miRNA associated with a sex chromosome. These miRNAs are more prominently expressed in females and are restricted in their tissue expression. We have also discovered the same cluster of miRNAs on the X-chromosome of humans. We have demonstrated that a miRNA from the X-chromosome linked cluster, miR-871, regulates male-enriched SRL mRNA translation. SRL is a protein critical in cardiac calcium handling and repolarization. At baseline, women demonstrate prolonged QTc intervals due to differences in intracellular calcium handling. Therefore, we hypothesized that the sex-differential expression of miR-871 regulated SRL translation, leading to sex differences in cardiac electrophysiology. To test this hypothesis, we created cardiac-conditional SRL null mice, SRLfl/fl. We found that SRLfl/fl male mice phenocopy female physiology at baseline, while female SRLfl/fl mice are unaffected. To determine if sex-differential miR-871 expression regulates differential SRL expression in vivo, we designed a novel AAV9 miRNA reporter construct to overexpress miR-871, specifically in cardiomyocytes. We found miR-871 overexpression phenocopied that of male SRLfl/fl mice displaying a prolonged QTc that mirrors that of females at baseline. These findings suggest that differences in miRNA expression between sexes can impact electrophysiology of the heart. These studies may provide new therapeutic options for more equitable treatment of sex-specific cardiac arrhythmias.

The cardiac trabeculae is an important structure contributing to the formation of a functional ventricular wall which facilitates cardiac contractility. The development of the trabecular structure involves complex morphological processes and signalling between the myocardium and encodardium. Abnormal trabeculae formation in humans can lead to hypertrabeculation and hypoplastic wall formation, resulting in compromised cardiac performance. Despite their importance, the precise mechanism of cardiac trabeculae development is not fully understood. Through an analysis of transcriptome and genome-wide chromatin accessibility in zebrafish cardiomyocytes, a cis-regulatory element was identified as a putative cardiac enhancer which we designate as Chr7_51M. A stable transgenic line carrying EGFP reporter under the control of this enhancer exhibited specific reporter expression in a cluster of cells in the monolayered ventricular myocardium following the proliferation, differentiation and migration steps which characterize the trabeculae development. Light sheet microscopy analyses revealed that the Chr7_51M enhancer-driven EGFP expression domain overlapped that of cardiomyocytes but not the endocardium. Moreover, treatment of zebrafish larvae with the Erbb2 inhibitor, which has been shown to inhibit trabeculae development, significantly decreased this EGFP-expressing domain, further supporting their identity as cardiac trabeculae. The nuclear-encoded mitochondrial gene, got2b, were identified as a putative target of this enhancer based on its spatiotemporal expression pattern which matches that of the enhancer-driven reporter. Analysis of transcription factor binding sites within the Chr7_51M enhancer region further revealed potential upstream regulators of trabeculae development, which includes the canonical Wnt signalling. Notably, a genomic region in human homologous to part of the zebrafish Chr7_51M region, containing similar transcription factor binding sites, was identified, suggesting conserved regulatory mechanisms. I will present our ongoing functional analyses of this novel enhancer in the context of cardiac trabeculae development, shedding light on the intricate regulatory networks governing this essential process.

One of the processes which increases myocardial mass is that of trabeculation – formation of myocardial protrusions into the lumen of the heart. In zebrafish, trabeculation occurs when cardiomyocytes from the single layer “compact” myocardium undergo partial delamination to seed the trabecular layer. The earliest known mechanism in this process is mechanical tension, which is increased in select cardiomyocytes driving delamination from ~60 hours post fertilisation (hpf). We have generated a transgenic line (TgBAC(cbfa2t3:gal4;uas:gfp)) which shows expression in individual cardiomyocytes as early as 40 hpf – ~20 hours prior to delamination. Using this line, we observe mosaic eGFP expression in the single-layer compact myocardium and, by 6 days post fertilisation (dpf), all trabecular cardiomyocytes express eGFP, leaving eGFP-negative cardiomyocytes in the compact wall. Lineage tracing through photoconversion and Brainbow labelling show the majority (>90%) of reporter-expressing cardiomyocytes starting in the wall end up in the trabecular layer, suggesting Cbfa2t3 is a marker of trabecular fate. To confirm this, we generated an endogenous knock-in to the cbfa2t3 locus and observe reporter expression restricted to trabecular cardiomyocytes. Chemical treatment and genetic manipulations show that reporter expression at 48 hpf (i.e. prior to trabecular delamination) is independent of Nrg/ErbB signalling, a key driver in trabecular growth. Other factors known to regulate trabeculation, including haemodynamic forces, endocardial signalling and mechanical tension, either mildly reduce or don’t impact the number of Tg(cbfa2t3:gal4;uas:gfp)-positive cardiomyocytes at 48 hpf, suggesting trabecular fate is regulated by mechanisms distinct from trabecular delamination and growth. Finally, to test whether Cbfa2t3 itself is involved in trabeculae development, we have conducted loss- and gain-of-function experiments. We observe a reduction in the number of trabeculae in cbfa2t3 mutants compared with wildtype siblings. Reciprocally, mosaic overexpression of Cbfa2t3-mScarlet in cardiomyocytes promote trabeculation in cells expressing the fusion protein. Based on these data, we propose that fate specification is a new and previously unappreciated step in trabecular development. This identity is genetically hardwired early in cardiac looping and is temporally and genetically separable from existing known mechanisms that regulate trabeculation. Finally, we show that Cbfa2t3 is both a marker of trabecular identity and functionally required for trabeculation.

DCHS1, an atypical cadherin, was the first gene identified as causal to mitral valve prolapse (MVP). While haploinsufficiency partly explained the valve disease, complete knockout of the gene led to a reported high incidence of neonatal lethality. In order to initially identify mechanisms contributing to this lethality, we performed in vivo analyses of DCHS1 expression. By engineering an HA-tagged Dchs1 locus, we were able to successfully demonstrate DCHS1 protein localization to the cell membranes of embryonic and neonatal non-myocyte populations, including endothelial and epicardial cells, as well as fibroblasts. Notably, non-membrane tethered, intracellular DCHS1 protein was detected in a subset of endothelial cells, suggesting potential cleavage of the intracellular domain. This cleavage event was further supported by Western analyses, revealing bands corresponding to both the full-length DCHS1 protein and a smaller 50kDa band. The presence of this smaller molecular weight band was possibly dependent on cell density during late gestation. Single-cell RNA sequencing (scRNAseq) corroborated non-myocyte expression, with a majority of transcripts identified in fibroblast and endothelial cell populations. To investigate Dchs1's role during development, we generated global Dchs1 deletion mice, of which 100% died during neonatal stages with evidence of impaired cardiomyocyte differentiation. Western analyses, immunohistochemistry, and scRNAseq datasets showed a statistically significant increase in proliferation across all cardiac lineages, including myocytes. These findings demonstrate a critical role for DCHS1 in regulating myocyte and non-myocyte cell cycle in a cellular non-autonomous manner. Moreover, these data also suggest that cell density and the contribution of epicardial-derived fibroblasts in the heart likely exert a critical influence on myocyte cell-cycle exit through a DCHS1 mechanism.

The heart develops from gastrulating mesodermal precursors through precisely orchestrated patterns of gene expression. The BRG1/BRM associated factor (BAF) chromatin remodeling complexes are multi-subunit protein assemblies containing a mutually exclusive ATPase BRG1 or BRM and utilize ATP hydrolysis energy to alter nucleosome position and regulate gene expression. BAF complexes are implicated in many different developmental processes, including differentiation of mesoderm precursors to cardiomyocytes. In mesoderm cells, individual Brg1 or Brm loss is tolerated. However, their combined deletion (DKO) severely reduces mesoderm differentiation and abolishes linear heart tube formation, with unknown molecular mechanisms. Here, we investigated the loss of function of Brg1 and Brm in ES cell derived mouse mesoderm cells as well as mouse embryos at late gastrulating mesoderm and linear heart tube stages of development, using simultaneous gene expression and chromatin accessibility measurements in single nuclei. Individual loss of Brg1 or Brm minimally affected gene expression in differentiated mesoderm cells. However, dual BRG1/BRM loss of function using chemical mediated inactivation or PROTAC mediated degradation resulted in formation of cells expressing neuroectodermal genes. In mouse embryos, loss of Brg1, Brm or DKO did not grossly affect cardiogenesis at embryonic day 8 (E8). However, in E8.5 linear heart tubes, BRG1 have specific functions in the formation and differentiation of left ventricle, right ventricle, and outflow tract precursors. In contrast, both BRG1 and BRM appear to be required for the formation anterior second heart field and inflow tract precursors, while DKO caused formation of neural and cardiac/neural hybrid cell types. Further, we observed increased FGF, NCAM and BMP signaling in cells lacking either individual or both the ATPases. Chromatin accessibility in differentiated mesoderm cells as well as in mouse embryos are concordant with observed gene expression. In both mesoderm cells as well as embryos, combined BRG1/BRM loss of function caused increased chromatin accessibility at loci containing CTCF and AHCTF1 motifs. Together, these results suggest that BAF ATPases modulate chromatin accessibility landscape at loci enriched for lineage specific transcription factor motifs, as well as signaling networks, to regulate mesoderm cell fate during cardiogenesis.

Embryonic senescence has emerged as a new embryonic developmental paradigm (1) (2). Dysregulation of this biological process has been observed in congenital pathologies, including hypoplastic left heart syndrome and Cornelia de Lange syndrome (3) (4). Here, we report in mouse embryos that trabeculae cardiomyocytes exit cell cycle and turn into senescence at the onset of compaction. Using Beta galactosidase activity assay and p21 expression, we observed a few senescent cells at E8.5 and E10.5. p21+ cells increased at E13.5 and peaked at E16.5. To modulate this cell process, we used Palbociclib, a CDK4/6 inhibitor that exacerbates senescence and Galunisertib, a TGFβ-RI inhibitor to prevent senescence. Ventricular trabeculation, monitored by HREM combined with fractal analysis revealed hyper-trabeculation and thin ventricular wall or mainly misshapen trabeculae. Altogether this suggests that cell senescence is a major regulator of ventricular compaction. Furthermore, we performed single cell RNA-sequencing of FACS-sorted high tomato+ trabeculae myocytes from both E13.5 and E16.5 hearts collected from SmaCreERT2/Rosa26tdtomato embryos. Gene clustering, profiling and trajectory inference of p21+ cells uncovered the role of specific signaling and metabolic pathways as inducers or regulators of myocyte senescence. CellChatDB, an algorithm to predict cell-cell communication highlighted a dialog between trabeculae and compact zone myocytes through SEMA-PLX pathways as found in human fetal heart (5). Semaphorin signaling is involved in cell migration which likely plays an essential role in compaction (6). We also identified a cluster of macrophages interacting with cardiomyocytes. Immunofluorescence using macrophages markers showed that they are localized in the trabeculae, close to senescent cells. Expression of chemokines by senescent cells point to the presence of a senescence associated secretome phenotype (SASP) that may recruit macrophages for clearing out part of the trabeculae. 1. Muñoz-Espín D et al. Cell. 21 nov 2013;155(5):110418. 2. Storer M et al. Cell. 21 nov 2013;155(5):111930. 3. Gaber N et al. The American Journal of Pathology. 1 sept 2013;183(3):72034. 4. Hachoud C et al. bioRxiv - https://www.biorxiv.org/content/10.1101/2022.07.26.501526v2 5. Farah EN, et al. Nature. 13 mars 2024;111. 6. Epstein JA, Aghajanian H, Singh MK. Cell Metabolism. 3 févr 2015;21(2):16373.

During embryogenesis, the earliest hematopoietic progenitors originate within the yolk sac in mammalian embryos. In recent years alternative hematopoietic sites, such as the endocardium, have been identified. However, the definitive evidence to demonstrate the hematopoietic potential of the endocardium is still missing. Here we used zebrafish embryonic model to test the emergence of hematopoietic progenitors from the endocardium. Expression of an early myeloid marker pu.1/spi1b and pan-leukocytic marker lcp1 was detected within the endocardium and partially overlapped with the endocardial marker nfatc1 expression. Time-lapse imaging and lineage tracing experiments using the Cre/loxP system and Kaede photoconversion demonstrated the emergence of migratory myeloid cells from the endocardium in live zebrafish embryos. Intriguingly, inhibition of Etv2 / Etsrp or Scl / Tal1, two known master regulators of hematopoietic and endothelial development, did not affect the emergence of endocardial-derived myeloid cells. Furthermore, the endocardium contributed to regeneration of myeloid cells in Etv2-inhibited embryos. In contrast, inhibition of Hh signaling resulted in a significantly reduced number of endocardial-derived myeloid cells. We performed single-cell RNA-seq analysis to define the transcriptional profile of endocardial-derived myeloid cells. These data followed by experimental validation suggest that the endocardium is the major source of neutrophilic granulocytes. In summary, our results identify the endocardium as a novel source of neutrophils in zebrafish, characterize their transcriptional profile and begin to dissect molecular mechanisms involved in their emergence. These findings will promote our understanding of alternative mechanisms involved in hematopoiesis, which are likely to be conserved between zebrafish and mammalian embryos.

Fetal heart development is a highly coordinated process directed by complex gene regulatory networks (GRN). Imbalances in these networks can disrupt normal cardiac cell type specification and proliferation, leading to congenital heart defects. These networks include hundreds of transcription factors (TF), many of which are required for normal heart formation, but the extent of their roles and interactions in cardiomyocyte differentiation is not fully understood. In this study, we aim to identify gene regulatory subnetwork drivers of cardiomyocyte specification towards an atrial or ventricular type. We use publicly available single cell mouse RNA and ATAC sequencing data, along with a GRN inference software called CellOracle, to generate seven networks, each with >500 genes and 4,000 directed interactions. The networks represent the enhancer-driven gene regulatory environment for seven distinct cell groups spanning from cardiac progenitor to atrial and ventricular cardiomyocytes at embryonic day 16.5. We use these networks to perform systematic in silico knockdown studies to identify key modulators of cardiac progenitor specification towards atrial or ventricular cardiomyocytes. We identified sets of transcription factors that differentially upregulate atrial (20 TFs) or ventricular (12 TFs) gene programs. By focusing on these 32 TFs, we extract subnetworks with opposing regulation by the atrial and ventricular TF modulators. This novel approach provides a set of testable predictions, both at the TF and subnetwork level, of how cardiac progenitor cells are specified towards atrial or ventricular cardiomyocytes. We plan to validate these predictions through in vitro and in vivo studies.

The neural crest (NC) is a highly migratory embryonic stem cell population unique to vertebrates. It forms at the dorsal side of the neural tube, subsequently migrating throughout the embryo and differentiating into a diverse array of cell types, including a subpopulation of cardiomyocytes (NC-Cms). By driving expression of nitroreductase (NTR) in zebrafish only in NC-Cms, we are able to precisely ablate these cells by treatment with metronidazole (MTZ). Ablation during early heart development causes mild trabeculation defects in embryos as well as adult-onset hypertrophic cardiomyopathy. NC-Cm function was also examined during adult heart regeneration following injury. At 7 days post injury (dpi), NC-Cm ablated hearts contain far fewer proliferating cells compared to controls. Additionally, control hearts express NC marker Sox10 in many proliferating cells, while NC-Cm ablated hearts have reduced or absent Sox10 expression. Control hearts fully regenerate by 30 dpi, while NC-Cm ablated hearts retain scars at the injury site. NC-Cms are enriched for Notch ligand jag2b during heart development, and this embryonic expression appears to be upregulated following adult heart injury. In jag2b null mutants, NC-Cms are able to migrate to the heart and differentiate into cardiomyocytes. However, these mutants phenocopy the adult-onset hypertrophic cardiomyopathy and scarring following injury seen in NC-Cm ablated hearts. Despite the apparent similarity between NC-Cms and Mesoderm-derived cardiomyocytes (Md-Cms), it appears the differentiated NC-Cm population maintains some degree of elasticity well into adulthood, perhaps by maintaining a distinct epigenetic memory. To address this, we are currently exploring the open chromatin structure of these cells versus Md-Cms via ATAC-seq. Additionally, we are utilizing slice culture and lineage tracing to examine the proliferation and migration dynamics of NC-Cms following heart injury ex vivo. These data will further elucidate the role of NC-Cms and how they maintain their NC-Cm identity during adult life.

Cardiomyocyte (CM) polyploidy is prevalent across mammals, which is associated with CM maturation and negatively correlates with the regenerative capacity of the heart. While the kinetics of ventricular CM (vCM) polyploidization has been well-documented in different species, the chamber-specific difference in CM polyploidy and the underlying molecular mechanisms remained poorly understood. To address these questions, we first characterized CM ploidy in the atrium and the ventricle of the mouse heart. In contrast to vCMs that rapidly become polyploid shortly after birth, the majority of atrial CMs (aCMs) remained mononuclear diploid from postnatal day 1 (P1) until adulthood. Intriguingly, similar ploidy difference between a- and vCMs were also observed in pigs. To investigate the molecular mechanisms, we performed transcriptomic analysis and found that Slit signaling components (Slit2, Slit3, Gpc1), key regulators of axon guidance and angiogenesis, are significantly enriched in atria compared with ventricles in P7 mice. Expression of Slit2, 3 is downregulated postnatally in fibroblasts and endothelial cells in the ventricle, and are enriched in mononuclear vCMs of P7 and adult mice, suggestive of a positive role in CM cytokinesis. Functionally, knockdown of Slit2, 3 (ligands) and their CM-enriched receptor Gpc1 significantly promoted primary postnatal vCM polyploidization. Mechanistically, transcriptomic analyzes revealed that many genes related to DNA replication and mitotic cell cycle are significantly downregulated in vCMs upon Slit2, 3 knockdown, including a novel candidate Cep55 that encodes a centrosomal protein critical for successful cell division in neural progenitors. Consistently, knockdown of Cep55 significantly promoted primary postnatal vCM binucleation. Altogether, our findings suggest that (1) Slit signaling is required for CM cytokinesis (hence inhibits polyploidization) via regulating Cep55 and that (2) differential Slit expression and/or Slit signaling activity may underlie, at least in part, the difference in CM ploidy between atria and ventricles.

Adult hearts cannot be repaired after injury, making cardiovascular disease a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising model for studying cardiac development, disease modeling, and regenerative therapy. However, their immature and fetal-like characteristics restrict their use in adult disease modeling and regenerative medicine. Therefore, it is crucial to identify strategies to improve cardiomyocyte maturation. To overcome this hurdle, various strategies have been employed to enhance the maturation of hPSC-CMs. However, molecular mechanisms underlying the maturation remain elusive. Our previous studies demonstrated that RNA-binding protein RBM24 mediates alternative splicing of core myofibrillogenesis genes during sarcomere assembly in a stage-specific manner. Moreover, we now reveal that RBM24 deficiency affects cardiomyocyte maturation, including the failure of myosin isoform switching, metabolism, immature calcium handling, and reduced mitochondria contents. Our mechanistic study further reveals that RBM24 regulates multiple types of non-coding RNAs, including long non-coding RNAs (LncRNAs), micro RNAs (miRNAs), and circular RNAs (CircRNA). Intriguingly, we identify a long non-coding RNA as a critical factor in modulating sarcomere myosin switching—a hallmark of cardiomyocyte maturation. In conclusion, our research reveals that RBM24-mediated cardiomyocyte maturation, involving critical processes like myosin switching, metabolism, and calcium handling, relies on long non-coding RNAs. These findings illuminate potential therapeutic targets for enhancing hPSC-CM maturation and advancing efforts in myocardial regeneration.

Accurate understanding of preoperative anatomical structure and prediction of postoperative cardiac function are crucial for successful surgery of complicated congenital heart disease (CHD). The University of Tokyo has developed a multi-scale and multi-physics heart simulator, "UT-Heart", which faithfully reproduces the heart functions of an adult patient in silico, based on molecular and cellular levels. To apply this cutting-edge technology to surgical decision-making for pediatric CHD, a novel computer simulation system called "ped UT-Heart" has been developed exclusively for CHD, in which a patient-specific finite element model of the heart simulates hemodynamics, wall and valve motion, and electrophysiology to compare different types of surgical procedures before real surgery (digital twin technology). Images of the virtual procedure are also generated under the surgeon's supervision, contributing to post-operative simulation analysis. To validate the efficacy and safety of the “ped UT-Heart“ simulation system, we conducted a single-arm prospective clinical trial of 12 complicated CHDs (jRCTs052210139). The usefulness of the system was confirmed by a 5-point Likert scale evaluation by the primary surgeons. There were no adverse events associated with the simulation system. "ped UT-Heart" would be an ideal and promising technology to support surgery for complicated CHD. ("ped UT-Heart" has been developed in collaboration with Japan Medical Device Corp., PIA Co.Ltd, UT-Heart Inc., and crossMedical Inc.)

Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear-encoded mitochondrial tRNA synthetase that is responsible for charging tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutation of the Aars2 gene is linked to combined oxidative phosphorylation defect type 8 (COXPD8), leading to human infantile cardiomyopathy. However, how Aars2 regulates heart development, cardiac function, and the underlying molecular mechanism remains unknown. Here, we found that an RNA-binding protein, Pcbp1, controls the exon-16 inclusion of Aars2, which is critical for its expression and function. We generated a cTNT-Cre mediated cardiomyocyte-specific Aars2 deletion mutant mice (Aars2-cKOCtnt) that lacks exon-16 and found that they display non-compaction cardiomyopathy at embryonic day E16.5 and are perinatal lethal. To circumvent the perinatal lethality, we use Myh6-Cre to delete Aars2 in later cardiac development (Aars2-cKOMyh6). Aars2-cKOMyh6 displayed severe contractile defects starting at postnatal day (P) 28, leading to dilated cardiomyopathy and early lethality at P55, with a consistent and significant protein reduction that constitutes the oxidative phosphorylation (OXPHOS) complexes. To understand the translational changes caused by the Aars2 loss-of-function, we performed quantitative proteomics on Aars2-cKOMyh6 and control hearts before the onset of the dilated cardiomyopathy at P21. We compared these hearts' transcriptome and proteome to decipher the regulatory contribution from transcription and translation. We found that the mitochondrial-encoded OXPHOS genes are down-regulated only in protein but not in mRNA. Interestingly, 93.3% of the 328 genes differentially regulated by both protein and mRNA in Aars2-cKOMyh6 are regulated in concordance: the genes down-regulated are related to cardiac muscle contraction, and the genes up-regulated are related to mitochondrial and cytosolic translation. Therefore, our study identified Aars2 as a critical regulator of non-compaction and cardiac function, providing important molecular insights into congenital heart defects.

12 out of 17 TBX genes have been associated with human diseases affecting multiple organ systems, most commonly cardiovascular and limb skeletal defects, including TBX4-opathies (TBX4), Holt-Oram Syndrome (TBX5), and 17q22q23.2 microdeletion syndrome (TBX4-TBX2). One of the major challenges of investigating multi-organ disease pathology is the spectrum of phenotypic severity among patients despite shared genetic causes. The overlapping, at times redundant roles of TBX factors during mammalian heart, limb, and lung formation proposes the hypothesis that a cumulative dosage effect across multiple TBX genes could drive patient-specific phenotype variation. In concert, patient-specific genetic variation (i.e. single-nucleotide variants, etc.) provides another layer of influence on the phenotypic severity spectrum between patients. Using zebrafish, we are testing the combinatorial roles of tbx4/5a/5b and tbx4/tbx2b during cardiovascular and fin patterning. Testing potential synergy between tbx4 and tbx5, triple-homozygous tbx4/5a/5b-mutant zebrafish show more severe cardiac phenotypes compared to single and double allele combinations, suggesting a potential cryptic role of tbx4 during heart patterning. Further, distinct from single tbx4 mutants, homozygous mutants for a tbx4/tbx2b double deletion (Δ47 kb) show adult-onset cardiac defects, indicating synergistic effects between tbx4 and tbx2b. We are performing bulk RNA-seq on combinatorial (single, double, triple) tbx4/5a/5b-homozygous mutants to transcriptionally define the compensatory roles between Tbx factors, and to identify affected downstream cardiovascular, cardiopharyngeal, and forelimb patterning genes. Combining our loss-of-function mutants with our novel phiC31 integrase-based transgenesis system (pIGLET), we are in parallel testing patient-specific non-coding and missense SNVs towards understanding their contribution to cardiovascular and limb pathologies. Targeted, reproducible transgene integration now allows quantitative comparisons between enhancer variants using standard reporter assays, and coding variants using inducible overexpression constructs. We apply pIGLET to test putative causal non-coding and missense SNVs associated with human TBX4 and TBX5 in our zebrafish assays. Together, these studies provide new insights into the synergistic versus compensatory roles of distinct TBX factors and establish a platform to dissect the pathology of human multi-organ congenital disease affecting the cardiovascular system and the limbs. This work is supported by NIH/NHLBI K99 5K99HL168148-01, Additional Ventures, CU School of Medicine, Department of Pediatrics. and by the Children’s Hospital Colorado Foundation.

The current drug development paradigm is costly and time-consuming, yet many drug recalls have been reported during post-marketing surveillance due to cardiotoxicity. Therefore, there is an urgent need for a physiologically relevant alternative model in the preclinical stage to increase the prediction of drug safety and efficacy in vitro. Heart-on-a-chip platforms aim to miniaturize and recapitulate cardiac tissue's complex structure and function. Traditionally, microfabricated pillar pairs have been used in these systems, which provide tissue anchorage and determine contractility parameters based on pillar deflection. However, this approach lacks the spatial heterogeneity of local cell- and tissue-scale forces. Here, we established a novel, non-destructive, optical method for continuous micro- and macro-scale contractile force measurements. We developed ultrasoft hydrogel mechanosensors, called edge-labeled micro-spherical stress gauges (eMSGs), that visibly deform under cell- and extracellular matrix (ECM)-generated stress. In addition, the chip was fabricated via soft lithography containing two cell seeding chambers with an array of flexible silicone pillar pairs to support tissue formation and compaction. Each device could be integrated into one well of 12-well plates for high throughput performance. The primary cardiomyocytes (CMs) were further encapsulated in a Fibrin/Geltrex hydrogel mixture (incorporated with eMSGs) and seeded in each chamber of the device. The tissue was gradually compacted and started beating spontaneously. The alignment and elongation of CMs were demonstrated using immunofluorescent staining of cardiac-specific biomarkers, and the functional hallmarks (e.g., calcium transient and tissue-scale beating) were indicated based on pillar deflection. Interestingly, the local cell- and ECM-scale mechanics were investigated based on the shape change of dispersible sensors during 2 weeks in culture. Each sensor's radial and circumferential stresses were further calculated using a MATLAB code. Finally, the platform was validated using two drug candidates (Norepinephrine and Blebbistatin), and their effect on contractility was demonstrated.

Noonan syndrome (NS) is a rare disorder resulting from gain-of-function mutations in the members of the RAS/mitogen-activated protein cascade. Patients with NS caused by RAF1 gain-of-function mutations commonly present cardiovascular defects that are associated with substantial morbidity and mortality shortly after birth, notably hypertrophic cardiomyopathy, arrhythmias, and pulmonary valve stenosis. Before birth, diverse cell types contribute to heart development, while disturbed cellular dynamics often lead to heart defects. We hypothesize that RAF1 mutations cause altered dynamics of embryonic and fetal cardiac cells, which subsequently leads to the pathogenesis of NS heart disease. To investigate this, we are studying cardiac cell dynamics in the embryonic heart of a RAF1 gain-of-function NS mouse model using the Clear Unobstructed Brain Imaging Cocktails (CUBIC) clearing method, followed by two-photon microscopy and three-dimensional reconstructions. By combining heart reconstructions with RNA In Situ Hybridization, we are mapping the expression patterns of essential genes for heart development, and measuring cellular hyperplasia, hypertrophy, and morphology. Characterizing altered embryonic cardiac cell dynamics in this model will allow us to identify the pre-natal pathogenic processes that lead to cardiac complications in NS.

Hypoplastic Left Heart Syndrome is a rare congenital heart defect affecting 3% of babies born with congenital heart disease. As a single ventricular heart defect, HLHS affects structures of the left heart, leading to an underdeveloped left ventricle, ascending aorta, and mitral and aortic valves. Due to disruptions in systemic circulation, HLHS is universally fatal without a series of surgical interventions performed promptly after birth. Despite the severity of the disease, the genes responsible for its etiology are mainly unknown. Ets1 is a highly conserved transcription factor that plays a significant role in cardiovascular development, and previous studies have implicated the loss of Ets1 as a cause of HLHS in humans. Our lab investigated the role of Ets1 in heart development using the Xenopus Laevis model. We found that Ets1 KD in the heart mesoderm led to structural and functional defects reminiscent of HLHS in humans, including a smaller and malformed ventricle. Ejection fraction and cardiac output were significantly decreased in embryos with Ets1 KD, suggesting impairments to cardiac contractility. Through proliferation assays and lineage-traced tadpoles, we found that the morphological defects were not attributable to cardiomyocyte proliferation or specification. However, endocardial and nuclear staining showed severe endocardial cell maturation and alignment disruptions. The endocardial and ventricular defects could be rescued with wild-type tissue transplantation, and bulk RNA sequencing revealed that Ets1 KD downregulated cell adhesion and movement processes. Our results suggested that Ets1 plays a crucial role in endocardial development, which causes secondary effects on ventricular morphology through endocardial-myocardial interactions. Using Fourier Light Field Microscopy, we can further study the morphological and functional effects of various genes in frog ventricular development, allowing us to elucidate the mechanisms underlying human congenital heart defects at a genetic and molecular level.

Marfan syndrome, a genetic connective tissue disorder, is characterized by aortic wall insufficiency in which aortic aneurysm and dissection can occur causing significant morbidity and mortality. This is caused, in part, by deficiency of fibrillin-1 microfibrils, which are necessary for aortic wall structure and function. We recently showed that purified fibrillin-1 bound to ADAMTS6 in a binary interaction assay and an ADAMTS6 cleavage site was subsequently identified in fibrillin-1 via N-terminomics. However, the impact of ADAMTS6 cleavage of fibrillin-1 is unknown during heart development and adult maintenance. Mutant Adamts6 mice die at birth due to a myriad of congenital heart malformations. Therefore, we generated a conditional Adamts6 allele to delineate its role in the postnatal period, specifically in smooth muscle cells in mouse models of Marfan syndrome. Conditional smooth muscle cell deletion of Adamts6 in mouse models of Marfan syndrome results in histologic improvement of aortic wall structure with statistically significant increased staining of fibrillin-1 microfibrils, decreased proteoglycan accumulation, diminished elastin breaks, increased aortic wall thickness, and increased longevity in contrast to mouse models of Marfan syndrome. Together, the data herein suggests that ADAMTS6 regulation of fibrillin-1 microfibril abundance is necessary for aortic wall maintenance. Moreover, it points to ADAMTS6 inhibition as a mediator of fibrillin-1 microfibril quantity in Marfan syndrome and the potential for therapeutic intervention to mitigate aortic aneurysms.

Maternal obesity is a major public health problem leading to a significant increase in the prevalence of severe congenital heart defects (CHDs) in which the arterial pole of the heart, derived from the second heart field (SHF), is a hotspot. The mechanisms underlying the association of maternal obesity with increased risk of CHD are unclear. We have shown that the lipid sensor and metabolic regulator PPARG is involved in outflow tract (OFT) development, suggesting connections between cell metabolism and cellular programs in early embryo. Here, we investigate the metabolic and developmental changes impacting on cardiac morphogenesis in the context of maternal obesity. Transmission electron microscopy (TEM) and MitoTracker studies show the spatio-temporal distribution of the mitochondrial network in the SHF and OFT. Highresolution respirometry quantification demonstrates that the mitochondrial electron transport chain is functional from E9.5 in the ventricles and E10.5 in the OFT with evidence of complex I and complex II ADP-stimulated respiration and ATP synthase coupling. Bodipy uptake in embryo cultures, lipid droplets observation in TEM and Oil Red O staining of sections reveal the presence of lipid vesicles in the SHF and OFT of embryos collected from normal diet (ND) mothers. We show that embryos from obese mothers fed with a high-fat diet (HFD) developed CHDs at higher incidence compared to embryos from ND mothers. RT-qPCR analysis demonstrated increased Pparg as well as Plin4 and Plin5 expression in HFD conditions, suggesting altered lipid metabolism in the SHF and OFT. Progenitor cell proliferation and deployment, as well as mitochondrial network organization and function, are under investigation in embryos from obese mothers. Single-cell RNA sequencing is also underway to identify and classify the metabolic heterogeneity of E9.5 cells from dissected hearts under ND and HFD conditions. By focusing on an unexplored area of early heart development we seek to uncover relevant genes and processes involved in lifethreatening CHD secondary to altered energy metabolism.

Jagged 1 (Jag1) is known to play an important role in cardiac development, where partial deletion of Jag1 is causing severe congenital heart defects. In this study we are comparing embryonic and postnatal hearts with conditional deletion of Jag1 with hearts with single variant in Jag1, which was prepared based on patient data. We analyzed prenatal as well as postnatal hearts of Jag1 floxed, Islet1-cre mouse line and two lines with single variant in Jag1. Morphology of hearts was analyzed on histological sections. Physiological functions were assessed using ultrasound in vivo imaging - Vevo and optical mapping. We found that Jag1, Islet1-cre mouse line exhibits severe heart defects during embryonic development, with variable phenotype ranging from mild abnormalities to Tetralogy of Fallot – double outlet right ventricle, VSD, and valve defects. Surviving postnatal mice present with milder defects, especially valve defects and physiological abnormalities in ventricular activation and contraction. Patients-based mouse model in postnatal stages exhibits valve defects and physiological abnormalities. Our results show that Jag1 is an important player in heart development and its disruption is causing various congenital heart defects. The humanized mouse models help to understand etiology and pathogenesis of congenital heart disease. Supported by Charles University Cooperatio 207029 Cardiovascular Science.

Male and female hearts have well-known differences in anatomy and physiology but the mechanisms that cause these differences are not fully understood. Hormones have been suggested as playing a role, but we have found that non-hormonal networks also contribute to cardiac sex differences. Furthermore, we have shown that sex differences in cardiac protein expression are evident at developmental time points before gonadal development, highlighting a role for sex-chromosome mechanisms. Previous studies suggest that post-transcriptional/post-translational mechanisms regulate these differences, but the mechanisms are unknown. We are investigating if protein turnover contributes to cardiac sex differences. At early cardiac development, it is challenging to define protein stability due to limited total embryonic cardiac protein and the large amount of protein required for standard protein turnover protocols. Therefore, we developed an embryonic whole heart ex-vivo culturing method and used these methodologies to assess protein degradation. This was achieved by dissecting mouse embryonic hearts at E10.5 and culturing the hearts for 2 hours or 24 hours in the presence of DMSO or the proteasome inhibitor MG-132. The resulting samples were assayed for protein stability by quantitative proteomic analysis. Our results demonstrate that we can detect changes to the cardiac proteome in response to MG-132 treatment at both 2-hour and 24-hour time points, indicating the presence of both short-lived and long-lived proteins in the embryonic heart. Cell cycle proteins are upregulated with MG-132 treatment at both time points. Even at a short 2-hour timepoint distinct subsets of proteins are being regulated by MG-132 treatment in male and female hearts and these include proteins that are sex differential at baseline. These findings indicate possible sex differences in cardiac protein turnover between male and female hearts.

The Notch signaling pathway is essential for embryonic development. Mutations in the human Jagged1 (Jag1) gene, which encodes a ligand for the Notch receptor, cause the Alagille syndrome. Symptoms of this inherited disease may include various forms of Tetralogy of Fallot. Here, we generated Jag1flox/flox Islet1Cre/+ mice with conditional Jag1 deletion in the cardiac outflow tract to investigate the impact of Jag1 mutations on cardiac morphology and physiology. Mice with conditional deletion exhibited severe cardiac malformations typical for Tetralogy of Fallot. Islet1 is also expressed in the sinoatrial and atrioventricular node, therefore we used the optical mapping to visualize changes in functioning of the cardiac conduction system. The analysis of E14.5, E16.5 embryos and adult mice showed changes in the activation pattern. In controls, we observed a mature activation from apex to base with two separate activation centres. Mutant embryonic hearts presented with activation originating only from the left ventricle, indicating a perturbed function of the right bundle branch. In mutant adult mice, activation occurred at additional activation centres, distinguishing them from controls where excitation was originating from apex. Vevo ultrasound imaging physiological analysis was performed only on adult heterozygotes, because of the postnatal mortality of the homozygotes. Speckle-based strain analysis revealed vulnerable areas of contractile defect that generated mechanical dyssynchrony pronounced mostly at the anterior wall. In summary, we demonstrated morphological and electrophysiological alterations resulting from conditional deletion of Jag1. Embryonic mice exhibited malformations and irregular activation patterns. Severe malformations were less prevalent in adult mice, primarily due to the survival of heterozygotes only and an increased mortality rate among mice displaying severe congenital defects. Nevertheless, surviving animals exhibited abnormal electrophysiological changes along with physiological alterations resulting in dyssynchronous myocardial contractions observed during strain analysis. Supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104) - Funded by the European Union - Next Generation EU.

Development requires the coordinated regulation of gene expression across diverse cell populations, including the suppression of alternative fate gene programs. One mechanism of gene regulation involves post-translational modifications of histone proteins. Genes involved in histone methylation establishment and maintenance have been associated with human congenital heart disease (CHD). Kleefstra syndrome can result from heterozygous mutations or deletions of EHMT1, which encodes the histone methyltransferase GLP. Kleefstra syndrome is associated with craniofacial abnormalities and a high penetrance of CHD, including double outlet right ventricle and hypoplastic left heart syndrome. Here we demonstrate that murine ablation of GLP and its paralogue G9a in neural crest cells (using Wnt1-cre and floxed alleles of genes encoding GLP and G9a) results in fully penetrant craniofacial defects such as microcephaly and cleft palate. Preliminary analysis of mutant embryos suggests GLP and G9a may be required in neural crest for proper cardiac morphogenesis. Deletion of GLP and G9a in neural crest is also associated with a reduction in dimethylation of lysine 9 on histone H3 (H3K9me2), a modification our group has previously shown to be associated with gene repression and spatial positioning of chromatin at the nuclear lamina. We hypothesize that GLP and G9a govern H3K9me2 enrichment at regulatory regions of tissue-specific genes involved in coordinating cell fate. For example, our work shows that in embryonic cardiac myocytes muscle-specific gene enhancers are depleted of H3K9me2 compared to enhancers associated with non-myocyte genes. Taken together, these data demonstrate a critical role for GLP and G9a in neural crest development that is associated with dysregulated H3K9me2.

In 22q11.2 deletion syndrome (22q11.2DS), TBX1 is an important causative gene of congenital heart disease (CHD), and yet its haploinsufficiency is not sufficient to explain the variability in clinical presentation and severity. Therefore, 22q11.2DS represents a unique population in which to uncover genetic modifiers of CHD. To identify potential modifiers, our lab previously performed whole genome sequence analysis on 1,182 subjects with 22q11.2DS and discovered rare variants in chromatin regulatory genes occurring in 8.5% of the subjects with CHD. This study aims to investigate the interaction of these genes with Tbx1 in mouse models, starting with Kmt2d, a histone methyltransferase that can cause CHD and/or Kabuki syndrome when mutated in humans. To test this interaction, mouse crosses were generated with a conditional deletion of Kmt2d in the Tbx1-Cre lineage, which was found to cause perinatal lethality in all mutants. A significant subset (~50%) of mutants presented with aortic arch anomalies at embryonic day 15.5 (E15.5), including aberrant right subclavian artery (n=6/12) and interrupted aortic arch type B (n=1/12). These phenotypes are caused by defects of the 4th pharyngeal arch arteries (PAAs), and assessment earlier in developmental time, at E10.5, revealed either absence or hypoplasia of the 4th PAAs in 100% of mouse mutants (n=13). The discrepancy between the incidence of 4th PAA defects and aortic branching defects is suggestive of a recovery mechanism, which has been previously reported as a characteristic in mice with Tbx1 deletion. Further characterization of the mutant phenotype has revealed thymic ectopia and/or hypoplasia in 100% of mutants (n=20), as well as nonlethal but distinctive craniofacial dysmorphism that includes a narrow head and mandibular hypoplasia (p<0.05). These findings support the notion that Tbx1 and Kmt2d could genetically interact in critical developmental processes, including those involved in CHD. Additional analysis is needed to uncover the cause of the completely penetrant perinatal lethality; defects such as cleft palate and persistent truncus arteriosus have already been ruled out, but more investigation into intracardiac abnormalities is of high priority. This will further the understanding of the genetic architecture of heart development and the etiology of CHD in 22q11.2DS, knowledge of which will improve diagnosis and treatment.

During early vertebrae embryogenesis, the heart is the first organ to be formed. This process, conserved across species, is orchestrated by a complex network of regulatory elements and transcription factors (TFs) controlling spatiotemporal gene expression and cell fates in developing tissues. Sequence variants in regulatory elements like enhancers often disrupt gene expression dynamics and can contribute to disease, similar to variants in the respective target gene. Therefore, taking a closer look at these non-coding regions can provide new insights into the regulatory landscape during development as well as in diseases. This study focuses on two accessible conserved non-coding elements (aCNEs) predicted to be involved in early cardiac development due to their proximity to known cardiac TFs and previous analysis of chromatin accessibility in the human and zebrafish genomes, published by the Scott and Wilson labs. Specifically, these aCNEs are candidate enhancers for the cardiac TFs MEIS1 and ZFPM2. The homeobox gene MEIS1 is active in various tissues including the developing heart and its disruption causes severe cardiac defects as previously shown in Meis1-knockout mice. ZFPM2, also known as FOG2, interacts with GATA family members, which are crucial TFs for cardiac and hematopoietic differentiation. Here, we used a transgenic GFP zebrafish reporter assay to validate the cardiac activity of these two candidate enhancer regions, testing both the zebrafish (ZaCNE) and human (HaCNE) orthologs. In stable transgenic lines, we observed robust cardiac enhancer activity of HaCNE_MEIS1 and HaCNE_ZFPM2. To examine their functional conservation alongside sequence conservation, we’re creating stable transgenic lines for the respective orthologues in zebrafish (ZaCNE_meis1 and ZaCNE_zfpm2) to compare the expression pattern of these enhancers between humans and zebrafish. Overall, this study aims to investigate the role of deeply conserved enhancers on spatiotemporal gene expression during early heart development.

Heart failure is characterized by re-expression of fetal cardiac genes, which are activated by pathological stressors. Transcription factors, including Gata4, Tbx5, and Mef2, are highly expressed during cardiogenesis and thought to play a role in this reprogramming process. Here, we utilize transcriptional reprogramming caused by thyroid hormone deficiency in the adult mouse heart as a model to study the cardiac gene reprogramming during heart failure at single-cell resolution. We developed a novel Myh6/Myh7 reporter mouse line and induced hypothyroidism in the reporter mice with propylthiouracil (PTU). This resulted in global cardiac gene reprogramming. We conducted single nuclei multiomic sequencing to analyze gene expression and chromatin accessibility changes during reprogramming. Echocardiograph was used to assess cardiac functions in control and PTU-treated mice. Cardiac tissue samples were collected to reveal histopathological changes and validate candidate genes involved in cardiac gene reprogramming. The results revealed that PTU-treated hearts switched from Myh6 to fetal Myh7 expression and showed significant atrophy, chamber dilatation, and reduced cardiac function with cardiac fibrosis. Analysis of multiome datasets uncovered a set of genes that are differentially expressed and accessible during cardiac gene reprogramming. Upregulated and more accessible genes in PTU-treated cardiomyocytes, including Tbx5, Sox4, Wnt2, Rgs2, Hey2, and Mfn2, are involved in muscle cell differentiation, muscle hypertrophy, and mitochondrion disassembly. Downregulated and less accessible genes, including Kcnd3, Jarid2, Epas1, and Slc25a4, are related to heart contraction, regulation of heart rate, and ion membrane transport. These findings show that hypothyroidism effectively induces histological, functional, and transcriptional changes in the adult heart and reveal new mechanistic insights into cardiac gene reprogramming that alters the expression and accessibility of genes essential for heart development and maturation at the single-cell level.

Heart maturation and remodelling during the foetal and early postnatal period are critical for proper survival and growth of the foetus, yet our knowledge of the molecular processes involved are lacking for many cardiac cell types. To gain a deeper understanding of the transcriptional dynamics of the heart during the perinatal period, we performed single-cell RNA-seq on E14.5, E16.5, E18.5, P0, P4 and P7 mouse hearts to establish a catalogue of 49,769 cells. Gene regulatory network and pathway activity analyses underscored that heart maturation is strongly associated with regulation of cell growth and proliferation via pathways such as TGFβ. We additionally identified a common, cell type-independent signature for imprinted genes over time. Surprisingly, bioinformatics analyses and confirmation with RNAscope confirmed that while lncRNA H19 expression decreased over time in multiple cardiac cell types, it remained stably expressed in endocardial cells between E14.5 and P7. This suggests a differential requirement for H19 in the endocardium, and points towards an endocardium-specific maturation process when compared to other cardiac cell types. We envision this dataset to serve as a resource for better understanding perinatal heart maturation at the transcriptomic level, and to help bridge the gap between early developmental and adult heart stages for single-cell transcriptomics.

Essential patterning processes transform the linear heart tube into a compartmentalized organ with distinct chambers and an atrioventricular canal (AVC) at their junction. The AVC is morphologically, physiologically, and molecularly distinct from the adjacent cardiac chambers, and these distinctions are necessary for proper cardiac function. For example, the heightened deposition of particular ECM components in the AVC contributes to the morphogenesis of the endocardial cushions and facilitates signal transduction for pathways that promote AVC differentiation, such as the Wnt pathway. However, the mechanisms that organize the pattern of ECM localization in the developing heart are not fully understood. In prior studies, we have found that the zebrafish gene smarcc1a, encoding a BAF chromatin remodeling complex subunit homologous to mammalian BAF155, plays a key role in refining the gene expression patterns that distinguish the AVC from the cardiac chambers. Here, we show that smarcc1a function is also required to regulate the cardiac distribution of the ECM component hyaluronic acid (HA). In smarcc1a mutants, HA organization is aberrant: instead of displaying particularly heightened deposition of HA within the AVC, smarcc1a mutant hearts instead exhibit excessive and broad distribution of HA. This is likely a consequence of the strong and early ectopic expression of has2, a key player in HA synthesis, throughout the smarcc1a mutant endocardium. Interestingly, in addition to their broad expression of has2, smarcc1a mutants also display abnormally broad expression of several other endocardial genes, including klf2a, wnt9b, and raldh2. The expanded presence of HA in the smarcc1a mutant heart is also accompanied by ectopic Wnt signaling within the cardiac chambers, extending beyond its normally restricted localization in the AVC. Together, our data support a model in which Smarcc1a-containing chromatin remodeling complexes play an important role in patterning the cardiac ECM by restricting the expression of key endocardial genes.

Heterozygous loss-of-function mutations in LMNA, encoding nuclear lamina protein Lamin A/C, cause severe adult-onset dilated cardiomyopathy. A long-standing hypothesis posits that LMNA insufficiency causes nuclear envelope structural defects that ultimately cause the disease. However, mechanisms linking defective nuclear envelopes to cardiomyopathy remain undefined. To determine specific nuclear envelope defects and their consequences, we deleted Lmna in cardiomyocytes of adult mice (LmnaCKO). Strikingly, a modest (50%) reduction of Lamin A/C caused widespread localized ruptures of the nuclear envelope in cardiomyocytes (En et al. bioRxiv 2023). The nuclear envelope ruptures did not cause immediate cell death, but accompanied a strong inflammatory response in the heart, prior to fatal cardiomyopathy. We hypothesized that DNA leaked from ruptured nuclei might elicit the cGAS-STING cytosolic DNA sensing pathway of innate immunity. Contrary to this hypothesis, we did not observe cGAS-STING activation in LmnaCKO cardiomyocytes. This lack of cGAS-STING activation was likely due to the near absence of cGAS protein in adult cardiomyocytes. To investigate the mechanism underlying cardiac inflammation in LmnaCKO mice, we performed time-course single-nucleus RNA-seq. This analysis nominated cardiac fibroblasts as the central mediator of the inflammatory response, receiving ECM-mediated signaling from LmnaCKO cardiomyocytes and recruiting immune cells to the mutant hearts. Finally, we found evidence suggesting that nuclear envelope repair activity counteracts nuclear envelope ruptures in LmnaCKO mice. We found that the ruptured sites co-localized with the ESCRT-III membrane remodeling complex, previously implicated in nuclear envelope repairs. We further found that overexpression of DNA-binding protein BANF1 was sufficient to promote ESCRT-III recruitment to ruptured sites. We are currently investigating whether facilitated nuclear envelope repair ameliorates cardiomyopathy in LmnaCKO mice. If it does, nuclear envelope repair may be a potential therapeutic strategy for LMNA-related dilated cardiomyopathy.

WNT signaling is a key regulator of embryonic heart muscle differentiation. Taking lessons from the embryo, experimental manipulation of WNT pathway activity is now widely used to guide human Embryonic Stem cells toward functional heart muscle (cardiomyocyte) differentiation. Such experimentally differentiated heart muscle cells serve as indispensable experimental models to study human cardiovascular development and may potentially facilitate future therapeutic strategies. For both applications, we now urgently need to understand how WNT signaling regulates cardiomyocyte differentiation. We carried out single-cell RNA sequencing to dissect relevant transcriptional responses downstream of WNT signaling in the established protocol used to guide human cardiomyocyte differentiation toward functional beating. Even though inhibition of WNT signalling is thought to be indispensable for subsequent functional cardiomyocyte differentiation, our analysis reveals it initially causes a surprisingly subtle overall transcriptional change, compared to stage-matched controls. Detailed analysis of this subtle change suggests WNT inhibition guides cell-cycle exit for differentiation toward a striated cardiomyocyte identity, consistent with the subsequent distinctive beating phenotype. Gene Regulatory Network inference analysis of our transcriptomics data suggests roles for known and for currently less known transcription factors in guiding striated cardiomyocyte differentiation, as opposed to differentiation into alternative but less well-defined cardiovascular lineages dependent on WNT inhibition at this crucial stage of development. Our findings raise new questions about detailed molecular mechanisms of this subtle yet consequential transcriptional change, but also about tissues and molecules in intact embryos regulating endogenous WNT signaling during differentiation of cardiomyocytes.

Morphogenesis is intricately linked to tissue and cell topologies. Advancements in imaging methods now enable a deeper analysis of complex tissue organization beyond flat surfaces. A significant challenge is the automated extraction of quantifications for both cell and tissue morphology, as well as the organization of the subcellular network of cells in highly deformed tissues. We designed HARMLESS (High Resolution Analysis of Microscopic single Layered Epithelia from deep learning based Semantic Segmentations), a method that automatically extract precise spatial organization details of given adherent, golgi and cytoskeletal networks in strongly deformed epithelia. Applied to zebrafish cardiac tissues, we show that endocardial cells proceed through a unique mode of morphogenesis during cardiac looping. We anticipate HARMLESS’ broader applicability in the evolving fields of morphogenesis and tissue biomechanics.

Cardiac septal defects are the most common presentation of congenital heart disease. Ventricular septal defects (VSDs) are one of the most prevalent in live births, yet our understanding of the etiology and progression of these defects is hampered by a lack of basic insight into the mechanisms underlying interventricular septum (IVS) formation. We hereby describe the morphogenesis of the human IVS using a range of imaging approaches. We combined High-Resolution Episcopic Microscopy (HREM), lightsheet and confocal imaging to quantify the growth of the IVS in the developmental window spanning Carnegie Stage 14(CS14) to CS20 (5-8pcw, post conception weeks). We developed volumetric analyses together with fluorescent labeling to quantify growth and cell numbers. We extended our analysis by assessing proliferation and visualizing cell shape changes during the extension of the IVS. We are also studying cell contributions to the IVS by retrospective lineage tracing, using somatic mutations as lineage tracers. To complement these approaches, we are developing an ex-vivo 4-chambered slice culture system to image cell dynamics in the growing IVS. Our results present the first systematic study of human IVS morphogenesis, furthering our understanding of human cardiac development and the origin of VSDs.

Failure in the elongation of the cardiac outflow tract results in congenital heart disease due to ventricular septum defects and misalignment of the great vessels. The cardiac outflow tract lengthens by the accretion of progenitors derived from the second heart field (SHF). SHF cells in the splanchnic mesoderm are exquisitely regionalized and organized into an epithelial-like layer forming the dorsal pericardial wall (DPW). Tissue tension, cell polarity, and proliferation within the DPW are important for the addition of SHF-derived cells to the heart and elongation of the cardiac outflow tract. However, our understanding of genes regulating these processes is not complete. Using conditional mutagenesis in the mouse, we show that fibronectin (Fn1) synthesized by the SHF is a central regulator of epithelial architecture in the DPW. The expression of Fn1 by the SHF modulates the interactions of SHF cells with Tenascin C (TnC), an anti-adhesive protein. In the absence of mesodermal Fn1, SHF-TnC interactions prevail, leading to cell rounding, loss of cell orientation and polarity, and decreased nuclear localization of YAP. Furthermore, we show that in the SHF, Fn1 functions in an SHF-cell-autonomous manner to coordinate multiple cellular behaviors in the anterior DPW necessary for elongating the cardiac outflow tract.

Conotruncal anomalies are found in 30% of patients with congenital heart defects (CHDs) and are associated with high disease severity, emphasizing the dire need to investigate underlying etiologies. Studies have shown that myriad conotruncal CHDs arise from errors in establishing the initial dimensions of the outflow tract (OFT) with genetic perturbations and flow-related disturbances compounding early anomalies. Furthermore, the emerging focus on extracellular matrix (ECM) in cardiac development and disease points to this non-cellular protein network as a key player in sculpting the conotruncus. Yet, we have limited appreciation of the molecular, cellular, and biophysical mechanisms regulated by individual ECM proteins during OFT development. It is critical to elucidate how individual matrix components direct OFT growth, titrate elasticity, and alter mechanosensitive pathways to develop novel therapies. Fibulin (Fbln) proteins are ECM glycoproteins that are tightly connected with fibronectin and elastic fibers. To study critical functions of ECM in OFT growth, we focused on fibulin 5 (fbln5) which precisely demarcates the OFT. Given reports of functional redundancy in the Fbln gene family, we generated a triple mutant, fbln1-/-; fbln2-/-; fbln5-/- (fblnTM), and discovered a statically significant reduction in OFT expansion. Our studies implicate that fbln2, expressed in the arterial pole, is required for accumulation of endothelial cells (ECs) to induce OFT growth through Smad3-dependent TGF-β cues. Fbln5 stimulates SMC differentiation and elastin assembly to establish proper OFT caliber. Tissue stiffness of this auxiliary chamber is elevated; this decreased elasticity contributes to altered flow profiles as indicated by diminished klf2a and notch1b, two targets of Piezo1, a mechanosensitive channel operating in ECs and SMCs. Together, our data suggest that Fibulin proteins regulate, in a temporally coordinated manner, EC accumulation via TGF-β signaling, SMC differentiation, elastic fiber deposition, and shear stress responsiveness to propagate mechanobiological feedback to promote OFT expansion. Insights gained from these studies will shed light on the biomechanical mechanisms responsible for elastic deformation at the arterial pole with implications for diseases involving stenosis of aortic and pulmonary valves. Moreover, probing the cell type-specific functions of Fbln proteins will help identify novel therapeutic targets for tissue engineering of surgical OFT conduits.

The pericardium is a mesothelial sac that encapsulates the heart to support its development and cardiac homeostasis over time. Previous work in zebrafish and mouse have described markers to study aspects of pericardial development; however, the developmental trajectory of the pericardium and its integration into embryonic heart development remains undocumented. We characterize pericardium formation as a specific process during mesothelium formation in coordination with the emerging heart tube. Using zebrafish, we recently mapped mesothelial progenitors to the Hand2-expressing lateral plate mesoderm (LPM) using single-cell RNA-sequencing and live imaging, and further linked Hand2 function to pericardium formation. Single cell-based sub-clustering of hand2-expressing LPM cells reveals distinct subpopulations of mesothelial and cardiac precursors, including a putative pericardial cluster that is distinct from cardiomyocyte precursors and other mesothelia. The highest-expressed genes within this sub-cluster display expression patterns unique to mesothelium emergence adjacent to the heart field, defining a gene expression signature for distinct mesothelial progenitor fields along the body axis. Our live-imaging and cell-tracking documents pericardial precursors residing at the anterior edge of the heart field that migrate then fuse at the midline to encapsulate the embryonic heart and form a single pericardial cavity. By implementing machine-learning, we uncover distinct cellular properties between cardiac and pericardial progenitors during cardiogenesis, further defining their individual identities. Mechanistically, using chemical genetics and reporter imaging, we link canonical Wnt signaling to the control of cell number and size during pericardium formation. Taken together, our data extend our understanding of mesothelial and cardiac development by integrating pericardium formation as a distinctive process during initial cardiogenesis. This work is supported by NIH F31HL167580.

Spatial coordinates provide essential information during human morphogenesis, by reflecting cellular niche-related environmental cues and topographically distinct precursor populations. Accordingly, cardiac structures arise through intricate interplays between various cell states in a topographically defined manner during cardiogenesis. To address spatial dynamics of early heart formation, we generated a comprehensive cardiac cell atlas spanning the embryonic and early fetal periods, by combining unbiased single-cell and spatial transcriptomics approaches with targeted imaging-based transcriptomics validation. By analysing almost 80,000 single cells and 70,000 spatially barcoded tissue regions from hearts collected between postconceptional weeks 5.5 and 14, we identified 11 coarse- and 72 fine-grained cell types and states, and spatially linked them to distinct structural and functional components of the developing human heart. Our results offer novel insight into the early specification of the pacemaker-conduction system and the autonomic cardiac innervation and provide the first spatial account of chromaffin cells in the fetal human heart. We describe the detailed cellular structure of the developing cardiac valves and atrial septum and identify valve interstitial cells and endothelial cells on the ventricularis side as main cellular targets of genetic alterations associated with non-syndromic bicuspid aortic disease. By exploring their position-related molecular diversity, we also define spatially distinct cell states in the hitherto elusive cardiac mesenchymal cell and fibroblast population. Furthermore, we utilize the rich spatial dataset to finely resolve the cellular composition of prominent developmental cardiac niches, allowing for the targeted analysis of their molecular interactions. In summary, our study delineates the cellular and molecular landscape of early human cardiogenesis, offering links between the architecture of the developing heart to genetic causes of heart disease.

The transcription factor MEF2C plays a critical role in the development of the linear heart tube. MEF2C-null mice exhibit embryonic-lethal defects, including severely hypoplastic outflow tract structures and an expanded inflow tract. This phenotype suggests that MEF2C has distinct regulatory functions in developing the outflow and inflow segments of the heart tube, and is critical for driving normal morphogenic behaviors of cardiac progenitors. Nevertheless, the mechanisms through which MEF2C uniquely regulates cardiac progenitors in the outflow and inflow tracts remain largely undefined. To address this, we performed combined single-nucleus RNA- and ATAC-sequencing on wild type and MEF2C-null embryos at E7.5 (cardiac crescent), E8.5 (linear heart tube), and E9 (looped heart tube). Initial analyses have revealed aberrant cardiomyocyte differentiation in MEF2C-null embryos, with reduced expression of numerous cardiomyocyte genes, including Ttn, Tnnt2, and Myl2. These embryos also exhibit a broadly “posteriorized” cardiac gene signature, with increased expression of Gata4, Tbx20, and Wnt2 throughout the heart tube. In the ATAC-seq data, we find distinct profiles of differential chromatin accessibility in the outflow and inflow segments of the heart tube, with hundreds of differentially accessible regions (DARs) of chromatin exhibiting both increased and decreased accessibility with loss of MEF2C. In ongoing analyses, we are intersecting these DARs with publicly-available ChIP-seq datasets to identify specific MEF2C-responsive regulatory elements that drive anterior vs. posterior gene expression in the heart tube. In addition to these genomic analyses, we are also conducting live embryo imaging of wild type and MEF2C-null embryos with fluorescent markers that demarcate the first and second heart field lineages. In our preliminary datasets, we observe distinct, lineage-specific changes in the movements of cardiac progenitors between wild type and MEF2C-null embryos. We are in the process of utilizing cell segmentation and tracking approaches to precisely quantify these differences and determine precisely how loss of MEF2C affects the cell migration behaviors that are necessary for normal heart tube morphogenesis. Together, these studies will provide fundamental insights into how the mammalian heart takes shape during embryonic development.

Introduction: This case navigates the intricate diagnostic terrain between endocardial fibroelastosis (EFE) and left ventricular non-compaction cardiomyopathy (LVNC), both of which present uniquely yet can overlap, as demonstrated in a sudden infant death investigation. Case Report: A 1-year-old boy's clinical presentation posed a diagnostic challenge, initially pointing towards EFE. Subsequent findings of pronounced ventricular trabeculation and a MYBPC3 gene mutation, however, led to a diagnosis of LVNC.The autopsy unveiled pronounced myocardial features suggesting EFE, but genetic findings favoring LVNC directed the final diagnosis, highlighting the critical role of molecular analysis.This case underscores the complexity of diagnosing cardiomyopathies with similar manifestations and the indispensable role of genetic insights in distinguishing between EFE and LVNC. Conclusion: Achieving an accurate cardiomyopathy diagnosis demands an integrated approach, combining clinical insights, detailed histopathology, and genetic analysis. This case vividly illustrates how genetic testing is key in resolving diagnostic ambiguities between cardiomyopathies like EFE and LVNC, thereby guiding appropriate patient management.

Congenital heart disease (CHD) is the most common birth defect, occurring in around 1% of all live births. Genetic causes have been identified for ~34% of CHD probands and suspected in up to 90%. Increasing sample sizes in genomic studies will significantly enhance our ability to uncover novel CHD genes. The Pediatrics Cardiac Genomics Consortium (PCGC) has recruited 17,064 CHD probands for study, with exome sequencing (ES) completed for 12,200 probands including 5,707 parent/proband trios. In this study, our analysis of ES of 5,707 trios more than doubles the previous PCGC study in 2017, which discovered seven CHD genes at exome-wide significance. We utilized a calibrated mutation model to statistically assess the mutational burden of rare loss-of-function and damaging missense de novo variants (DNVs). Our analysis identified twenty genes significant at a false discovery rate (FDR) of 0.05, including four novel genes that have not been previously associated with CHD in humans: SETD5, KDM5B, EDNRA, and SLIT3. Notably, these four novel genes reside within the upper quartile of gene expression in E14.5 mouse embryo hearts. Functionally, these genes are implicated in transcriptional regulation during development, either through chromatin modification or signaling pathways. Among these four genes, SETD5, EDNRA, and SLIT3 have demonstrated murine knockout evidence that recapitulated relevant CHD phenotypes. In conclusion, the substantial increase in trio sample size has led to the discovery of four novel CHD genes, providing valuable mechanistic insights into CHD pathogenesis.

The Elongating Multi-Lineage Organized Cardiac (EMLOC) stem cell gastruloid model marks a pivotal innovation in synthetic embryology, as a state-of-the-art recapitulation of the complex process of human heart development in vitro. EMLOC gastruloids represent a leap in multi-system cardiac developmental biology, bridging the gap between traditional in vitro cell, tissue and organoid models and the complex reality of human embryonic heart development as a multisystem integration. This model successfully integrates the pathways of cardiogenesis and neurogenesis, as evidenced by immunofluorescence of spatial patterning and single cell RNAseq analysis. Distinct integrated lineages included in EMLOCs are cardiomyocytes, epicardial cells, cardiac fibroblasts, vascular endothelium, peripheral glia/Schwann cells, trunk neuroectoderm/spinal cord progenitors, neural populations including autonomic, sensory, and motor neurons. Key signaling regulators include BMP, SHH, and WNT that further delineate the intricate developmental processes and offering insights into the spatiotemporal complex interplay of factors driving integrated cardiogenesis and neurogenesis. EMLOCs provide a unique platform for investigating the complex interactions among tissues and structures critical to human cardiogenic development and its associated adult diseases. Here we present a detailed analysis of the EMLOC model, focusing on bioinformatic investigation of pathways relevant to prominent cardiovascular diseases such as arrhythmogenic cardiomyopathy and restrictive cardiomyopathy. Additionally, we are enhancing the model's capabilities by implementing a medium-high throughput analysis system, establishing protocols for EMLOC shipment, and conducting electrophysiological assessments using microelectrode arrays and ion channel analysis through patch clamp techniques, in collaboration with New York based Cytocybernetics Inc. These advancements aim to facilitate the efficient transition from experimental research to clinical therapy applications, enhancing the translatability of our findings and tools for advancing personalized cardiac medicine.

Engineered cardiac tissue comprising human induced pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise to regenerate damaged hearts post cardiovascular diseases. However, the immaturity of hPSC-CMs after differentiation has greatly impeded their applications. Mechanical stimulation of engineered cardiac tissue was shown to improve cardiomyocyte maturation by emulating the mechanical loadings cardiomyocyte experienced during cardiac cycle, namely resistance to contraction (Afterload) and cyclic stretching (Preload). Nevertheless, many existing platforms for cyclic stretching pose risk of inducing human cardiac pathogy. This is due to their use of rigid tissue anchorage that enforces isometric contraction with an infinitely high afterload and prohibits duty cycle. In this study, we developed a novel bioreactor system to more accurately recapitulate the in vivo loading condition within the cardiac cycle, allowing cyclic stretching with active contractile work production and duty cycling incorporated in between each stretch. Our focus was to investigate the functionalities, maturation, and pathological progression of embryonic chicken engineered cardiac tissue subjected to three distinct mechanical stimulation regimens, including (i) static control, (ii) afterload no duty cycle (afterload NoDC), and (iii) afterload duty cycle (afterload DC). We showed that afterload DC upheld the increases of tissue contractile force and frequency relative to the day 0 baseline over 6 days of stimulation peroid. In contrast, both static control and afterload NoDC resulted in decreased contractile force, despite an increase of contractile frequency by afterload NoDC comparable to that by afterloadDC. Aligning with these results, tissues exposed to afterload DC demonstrated superior sarcomeric alignment with physiological sarcomeric length and expressed highest number of TNNT1 transcripts and connexin 43+ area. Moreover, afterload NoDC upregulated transcriptional expressions of pathological hypertrophy and fibrosis markers, including TGFβ2, COL5A2, and POSTN relative to both static control and afterload DC. Collectively, afterload DC significantly promoted the functionality of engineered cardiac tissues by enhancing cardiomyocyte maturation and suppresseing cardiac pathology. This study highlighted the remarkable potential of biomimetic loadings in facilitating tissue maturation.

Organoid biology has emerged as a promising discipline for investigating the processes of development and disease in a dish. In particular, developmental organoids have tremendous potential to understand early aspects of cellular specification and determination. However, it remains unexplored whether they retain self-organization properties during early development. Here, we address this fundamental question by dissociating and reaggregating embryonic stem cell-based spheroids at different developmental stages. We found that dissociated cells randomly form aggregates at any stages; intriguingly, those dissociated at the gastrulation-like stage were capable of resuming early developmental processes, whereas the ability to reorganize diminished in cells dissociated at later stages. Based on this, we resorted to a gastrulation model, which faithfully recapitulates early germ layer development. Real-time tracking showed that dissociated T/Brachyury-positive cells initially exhibited a 'salt-and-pepper' pattern post-aggregation, eventually coalescing into a cluster and migrating to the posterior end. In-depth analysis, including single-cell-RNA sequencing and in situ hybridization chain reaction, confirmed that lineage compositions, spatial polarity, and expression levels of marker genes in germ layer cells remained comparable. The re-organized gastruloids subsequently induced first and second heart field cells, suggesting a restored developmental program. Our study introduces the concept of self-reorganization, which can shed light on understanding cell autonomy and cell organization during development, disease, and regeneration.

Pharmaceutical drug discovery, toxicity assessments, and cardiovascular disease or regeneration studies are increasingly using 3D, iPSC-derived cardiac organoids as a relevant, reproducible model for evaluating cardiac system function, particularly metrics of contractility (such as “heartbeat” or contractile synchronicity). However, throughput constraints imposed by use of current live imaging tools and analysis software for cardiac organoids can lead to discarded results and massively increased data collection time to reach statistical power. Here we describe a method for high-throughput cardiac organoid imaging and analysis of contraction rates using Multi-Camera Array Microscope (MCAM™) imaging technology. This novel technology substantially increases data collection speeds by simultaneously imaging up to 24 wells and automatically performing functional analysis for each, ultimately reducing both user subjectivity in analysis and total data analysis time. Non-invasive and high-throughput methods for assessing cardiac organoid contractility as a measure of organoid development or treatment effects are quite limited. Although microelectrode arrays (MEAs) can be used to analyze voltage signals across multiple organoids, they can be tedious and expensive (due to the need to physically transfer samples into costly consumables) and in some cases preclude other imaging based endpoints. A major roadblock to running high-throughput experiments with rate of contractility endpoints in cardiac organoids is that almost all currently available imaging systems with sufficient resolution intrinsically cannot rapidly acquire relatively lengthy (30 second to 1 minute long) videos of every unique organoid to assess the rate of contraction without introducing substantial time biases. Using current, single-objective imaging methods, acquiring 30 second videos per well of a 96 well plate for heartbeat or contractility assessments results in a time differential of at least 48 minutes between the acquisition at well A1 vs. at well H12. These intrinsic time delays can have detrimental effects on the health of the organoids as a result of time outside of the incubator and the time differentials are particularly problematic in acute drug or toxin exposure assays. Using the MCAM™, we are able to synchronously acquire 30 seconds of video data at a rate of 20 frames-per-second (fps) for 24 individual wells of a 96 well-plate. In this paradigm, the MCAM™ can rapidly collect 30 seconds of video data for every single well of a 96 well-plate in approximately 2 minutes. This imaging platform allows extension of the video acquisition times wherein the total acquisition time for the entire well-plate will be approximately 4x the time of each video recording. After multiplexed video acquisition was completed, we developed a custom analysis pipeline to plot the change in pixels in each organoid, resulting in a sinusoidal waveform indicating contraction and relaxation of each organoid. This measure of pixel-change over time was then passed through a simple fourier transformation to extract the frequency peak providing the “heartbeat”, or more accurately, the frequency of contraction on a per-organoid basis in beats per minute. This simplified, multiplexed approach to high-throughput data acquisition and analysis greatly reduces experimental time, providing video data across an entire 96 well plate in as little as 2 minutes and analysis outputs in approximately 15 to 20 minutes per plate.

Zebrafish can regenerate their hearts while close-related teleost medaka cannot, providing a unique platform to study heart regeneration by reciprocal analyses. To compare the transcriptomic responses across species, we adopted a novel bioinformatic approach and constructed the regeneration-associated Time-Ordered Gene Coexpression Network (TO-GCN). Based on the transcriptional factors and their co-express genes in the regeneration-associated TO-GCN, we performed Reactome Pathway Analysis and identified regeneration-associated pathways associated with metabolic activation/switch, cell proliferation, and immune response transition from innate to adaptive immunity. Since cardiomyocyte (CM) regeneration rely on the dedifferentiation of pre-existing CMs and reactivation of cardiac progenitor genes (hand2, gata4, nkx2.5, and tbx20) prior to cell cycle re-entry in both zebrafish and mouse, we focused on and characterized novel upstream regulators of these progenitor genes in the regeneration-associated TO-GCN. To shortlist and validate the activation of these candidate genes in CMs, we performed in situ hybridization (ISH) and found that nfic, sox3, srebf1, and tal1 re-activated following cardiac injury in the dedifferentiated CMs colocalized with embCMHC staining and in FACS-sorted CMs by qRT-PCR. Functionally, we generated nfic and sox3 knockout zebrafish and found decreased CM dedifferentiation and proliferation, as well as unresolved scar in mutants compared to WT siblings after cardiac injury. Last but not least, shRNA knockdown of Nfic and Srebf1 in CMs isolated from P1 neonatal mice impaired cell proliferation similar to Yap1 knockdown, a well-known CM mitogen. Altogether, these results revealed key processes of heart regeneration and further identified evolutionarily conserved regulators essential for CM dedifferentiation and proliferation. This work and conference attendance are funded by the Integrated Research Grant (NHRI-EX113-11237SI) from the National Health Research Institute (NHRI).

Background: Coronary heart disease, leading to myocardial infarction (MI), is the largest cause of death globally. Following MI, heightened inflammation and deposition of fibrotic materials leads to pathological remodelling, cardiac dysfunction and ultimately heart failure. The only current cure is transplantation, therefore improved therapeutic approaches are essential for promoting any regenerative capacity, requiring both tissue restoration and environment conditioning through modulating immune and fibrotic responses. We have previously shown that VEGFC-C156S treatment stimulates lymphatic vessel growth in the infarcted heart and elicits immune cell trafficking correlating with improved outcome. This is dependent upon lymphatic endothelial receptor-1 (LYVE-1) which is required for trafficking. Here, we investigated specific immune cell populations that are preferentially cleared following VEGFC-C156S-induced lymphangiogenesis alongside those retained in a LYVE-1 loss-of-function background. Methods: Wild-type mice treated with PBS or VEGFC-C156S or Lyve1-/- mice underwent permanent coronary ligation. Single cell RNA sequencing was conducted on live CD45+ cells isolated from hearts and mediastinal lymph nodes 7 days post-injury. Results and Conclusion: Improved cardiac outcome post-MI following VEGFC-C156S treatment coincides paradoxically with clearance of what have been described traditionally as pro-reparative macrophages from the infarcted heart. Conversely, hearts of Lyve1-/- mice are enriched with recruited immune cell populations promoting responsive inflammatory and fibrotic signalling, consistent with augmented cardiac dysfunction previously observed. We are currently functionally interrogating cleared versus retained immune cell sub-populations involved in these differential responses. Together, these data highlight the potential therapeutic value of optimising the immune environment to promote effective regeneration and improve cardiac function following MI.

Heart regeneration relies on the reconstruction of gene regulatory networks (GRNs) in response to cardiac injury, which is orchestrated by tissue regeneration enhancer elements (TREEs). Identifying groups of TREEs exhibiting similar features will provide a base for elucidating GRNs that control heart regeneration. We previously dissected a cardiac regeneration enhancer in zebrafish to determine the regulatory mechanisms governing heart regeneration. The cardiac leptin b regeneration enhancer (cLEN) exhibits injury-inducible activity near the wound in the heart, which is conferred by multiple injury-activated regulatory elements distributed throughout the enhancer. Our analysis also found that cardiac regeneration enhancers are actively repressed in the absence of injury, demonstrating dual regulatory mechanisms of cardiac TREEs. Our extensive transgenic assays identified a short 22-bp DNA sequence containing a key repressive element responsible for maintaining the inactivation of cLEN in uninjured hearts. To uncover a group of TREEs similar to cLEN present in the genomes of zebrafish, mice, and humans, we devised a strategy to identify cLEN-like enhancer candidates by analyzing sequence similarity, evolutionary conservation, and epigenomic and transcriptomic profiles. For selected hits, we performed transgenic assays in zebrafish to determine which candidates are functional TREEs. Our transgenic assays identified multiple enhancers in the zebrafish and mammalian genomes that exhibit injury-inducible activation in hearts. Identifying additional regeneration enhancers across species will expand our understanding of the regulatory mechanisms underlying heart regeneration and lead to the identification of potential targets for improving heart repair.

Myocardial infarction (MI) induces cardiac muscle death and its subsequent replacement by a non-contractile fibrotic scar, which leads to heart failure. Current treatments restore blood flow and assist with cardiac workload. However, none are regenerative and clinical trials using stem-cell-based approaches have been disappointing. We have previously shown endogenous growth of the cardiac lymphatic system following MI and further stimulation with the lymphatic endothelial specific mutated VEGF-C isoform, VEGFC-C156S, resolves the immune response and improves cardiac function. Whilst this study demonstrates the feasibility of this approach, the short half-life and broad target profile of VEGFC-C156S makes it sub-optimal for clinical use. Thus, new lymphangiogenic compounds are needed to fill this therapeutic gap. We established a sprouting assay using spheroids derived human lymphatic endothelial cells to mimic lymphangiogenesis in vitro and performed a phenotypic screen using focused compound libraries of epigenetic regulator, kinase inhibitors and stem cell modulators (TOCRIS). We selected, clustered and quantified hit molecules using automated imaging and post-hoc analyses. Several GSK3β inhibitors were identified as potent pro-lymphangiogenic regulators acting in a Wnt-independent manner. Among them, the most potent was Kenpaullone which activated the same canonical pro-lymphangiogenic ERK pathway as VEGF-C. However, bulk RNA-seq revealed heterogeneity in the transcriptome response between Kenpaullone and VEGF-C treated spheroids. We have developed Kenpaullone derivatives to identify drug-protein interactions and functional downstream targets and shed light on the mechanisms underpinning its pro-lymphangiogenic effect. Finally, preliminary experiments in a mouse MI model suggest a lymphangiogenic effect of Kenpaullone and accelerated immune cell clearance.

Cardiovascular diseases are the leading cause of mortality worldwide. A major challenge to cardiac health is the limited regenerative capacity of cardiomyocytes (CMs) in the adult heart post-injury. Previous research in our laboratory demonstrated the potential of inducing cell division and cardiac regeneration through the introduction of cyclin-dependent kinase 1 (CDK1), CDK4, Cyclin B, and Cyclin D into CMs. However, these initial investigations relied on a viral-based approach for CDK/cyclin expression, prompting us to explore alternative delivery methods due to concerns regarding safety and practicality. In our present study, we employed Lipid Nanoparticles (LNPs) as a non-viral vector for CDK/cyclin expression in CMs. Our findings demonstrated successful LNP-mediated transduction, with a notable expression of protein derived from mRNA encoding GFP and CDK/cyclins observed in vitro in cultured human iPSC-derived CMs. Additionally, we assessed CM proliferation by analyzing key proliferation markers such as Ki67 and pH3. The data unveiled a significant increase in Ki67 and pH3-positive cells in the presence of the CDKs and cyclins, suggesting that LNP-mediated transfection effectively stimulates CM proliferation. Moreover, time-lapse imaging of cultured iPSC-derived CMs demonstrated cell division upon induction of CDK/cyclin in vitro. Ongoing investigations aim to validate and assess the in vivo regenerative potential of adult CM division induced by LNP-mediated CDK/cyclin transduction.

Developmental cardiac tissue is proliferative capable of robust regenerative response and prefers glycolysis for energy generation after myocardial injury. However, this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Strategies that induce glycolysis promote cardiac repair in the adult heart. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM proliferation. UCP2 is active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression increases neonatal and adult CM cell cycle parallel to increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. To test UCP2 affect on CM proliferation during postnatal development and adult heart after injury, we generated αMHC-UCPKO and αMHC-MCM-UCPKO mice. αMHC-UCPKO showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-MCM-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). Using SILEC-SF, we showed UCP2 induction increases nuclear acetyl-CoA levels in CMs dependent upon ATP-citrate lyase. In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.

The metabolic switch from glycolysis to fatty acid oxidation during maturation of postnatal cardiomyocytes contributes to the loss of regenerative potential in the mammalian heart. However, the regulatory mechanisms underlying this metabolic switch remain poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) is a key component of cardiac development via regulation of cellular metabolism and protein synthesis, yet the importance of mTORC1 regulation during cardiac maturation and neonatal regeneration is undefined. Here, we utilize the neonatal myocardial infarction model, mTORC1 pharmacological inhibition, immunoblotting, immunostaining, quantitative proteomics, and targetted metabolomics to define the role of mTORC1 in cardiac maturation and regeneration. Our results demonstrate that the activity of mTORC1 is dynamically regulated between the regenerating and the non-regenerating hearts. Acute pharmacological inhibition of mTORC1 decreases cardiomyocyte proliferation and cytokinesis, leading to inhibition of neonatal heart regeneration after myocardial infarction. Our quantitative proteomic analysis demonstrates that the acute pharmacological inhibition of mTORC1 during development or after neonatal MI shifts the cardiac proteome of the mTORC1 inhibited heart from glycolysis towards fatty acid oxidation. The proteomic results were supported by targetted metabolomics, where mTORC1 inhibited hearts accumulated more long chain acyl carnitines compared to non-inhibited controls. These results indicate that neonatal mTORC1 inhibition accelerates the postnatal metabolic switch, promoting metabolic maturation, thus impeding cardiomyocyte proliferation and heart regeneration. Taken together, our results define an important role for mTORC1 in regulating postnatal cardiac metabolism during maturation and injury recovery, and may represent a novel therapeutic target to modulate cardiac metabolism and improve patient outcomes after cardiac injury.

The inability of the human heart to regenerate after myocardial infarction has been partially attributed to a dependence on oxidative phosphorylation (OXPHOS) to meet its energy needs, which is considered detrimental to cardiomyocyte proliferation due to the production of reactive oxygen species (ROS). In contrast, adult zebrafish, that are thought to primarily rely on glycolysis, maintain the capacity to regenerate through cardiomyocyte proliferation. Here, by comparing the cardiac regenerative capacity of seven different wild-type zebrafish strains (AB, NA, SAT, TL, TU, WIK, KCL), we find that not all zebrafish regenerate equally. Exploiting these intraspecies differences, using bulk and single-cell (sc) RNAseq analyses, we have ideantified that upregulation of OXPHOS in border zone cardiomyocytes is associated with a better regenerative outcome at 90 days post-cryoinjury. This upregulation of OXPHOS is linked to glycolysis via activation of the malate-aspartate shuttle (MAS) and inhibition of both OXPHOS and the MAS impaired the long-term regenerative ability of the fish, however, without affecting cardiomyocyte proliferation. We found this is possible as cardiomyocyte proliferation and OXPHOS activation are sequentially activated in time, avoiding the presence of ROS during DNA replication. The upregulation of OXPHOS is required for expression of the embryonic sarcomere gene programme in the border zone, driving cardiomyocyte redifferentiation. We validated these findings in Astyanax mexicanus where we observed that, in contrast to the surface fish, the non-regenerative cavefish fail to upregulate OXPHOS following cryoinjury with reduced embryonic sarcomeric expression and consequently regenerative failure. Overall, our results demonstrate that OXPHOS drives the activation of a dynamic sarcomere program allowing for the redifferentiation of cardiomyocytes after the cessation of cardiomyocyte proliferation. This promotes the completion of cardiac wound healing and determines the long-term regenerative outcome.

Macrophages conduct critical roles in heart repair, but the cardiac tissue niche required to nurture and anchor them is poorly studied. Here, we investigated macrophage niche components in the regenerating heart. First, we analyzed cell-cell interactions through scRNA-seq datasets derived from damaged zebrafish and neonatal murine hearts. We identified a strong interaction between fibroblast/epicardial (Fb/Epi) cells and macrophages, in which outgoing signals were predominantly sent by Fb/Epi cells and incoming signals were mainly received by macrophages. Then, we visualized the association of macrophages with Fb/Epi cells and the blockage of the macrophage response after depleting Fb/Epi cells in the regenerating zebrafish heart. Moreover, we found that Fb/Epi cells with pentraxin 3 long a (ptx3a) expression displayed immune regulatory functions and ptx3a+ cell-associated macrophages exhibited reparative characteristics. Experimentally depleting ptx3a+ cells resulted in lower reparative macrophage numbers and enhanced scar formation in the wound. Further, we identified expression of multiple signaling pathways in ptx3a+ cells, including CSF pathway components, and determined that pharmacological inhibition of the csf1a pathway or csf1a knockout blocked the reparative macrophage response. Moreover, we found that genetic overexpression of csf1a enhanced the reparative macrophage response with or without heart injury, and reduced collagen in the regenerating area. Altogether, our studies illuminate a new cardiac Fb/Epi niche which mediates a beneficial macrophage response after heart injury, a process facilitated by upregulating the csf1a gene.

Cardiovascular diseases are a global health concern. Obstruction of the heart vessels (coronaries) leads to myocardial infarction (MI) and causes persistent damage to cardiac muscle which can be fatal. Unlike humans (and other adult mammals), adult zebrafish can effectively regenerate their heart following injury. After cardiac injury, rapid revascularization of the injury by coronaries is essential for efficient regeneration. Coronary endothelial cells (cECs) secrete angiocrine factors capable of activating regenerative programs. To identify pro-regenerative angiocrines, we performed bulk RNA sequencing on regenerating cECs sorted from zebrafish ventricles and found sulf1 to be upregulated. SULF1 is an extracellular sulfatase which removes specific 6-O-sulfate groups from heparan sulfate chains, regulating their interactions with signaling mediators. Sulf1 mRNA levels are upregulated in MI models in mice and increased sulfatase activity has been reported in MI patients. Being this a conserved response, we set out to identify Sulf1 downstream processes activated in a regenerative organism. To this end, we generated sulf1 mutant zebrafish using the CRISPR/Cas9 technology. Phenotypic analyses on these mutants show that abrogation of Sulf1 impairs key processes during regeneration including proliferation, dedifferentiation, and scar resolution. The lack of efficient treatments post-MI shows the urgent need to better understand the biology behind cardiac regeneration. Overall, these findings might motivate therapeutic approaches by using newly identified angiocrine factors.

In contrast to humans and mice, adult zebrafish can regenerate their hearts after injury. A prompt and tightly orchestrated immune response in the acute phase after injury is indispensable for complete regeneration. If delayed, many of the hallmark processes of zebrafish heart regeneration are impaired. However, the diversity and dynamics of immune cell states in the context of heart injury are not well understood. Here, we investigated the complex composition and transitions of immune cell states in the injured zebrafish heart. Specifically, we established single-cell RNA metabolic labeling (scSLAM-seq) in the adult zebrafish heart to directly measure the response of different cell types to injury. This approach allowed us to identify a subtype of macrophages as the sentinel cells of the zebrafish heart, which respond immediatly after injury by upregulating the inflammatory signalling pathways of Toll-like receptors, NOD-like receptors and C-type lectin receptors. Large-scale scRNA-seq of injured adult zebrafish hearts revealed a diverse and dynamic spectrum of transcriptional cell states across time, with substantially higher complexity than what the M1/M2 macrophage polarization model would suggest. In ongoing work, we spatially resolve the injury responsive cell state with high resolution spatial transcriptomics (open-ST), and we perform macrophage-specific perturbations of the macrophage sentinel cell state to functionally validate its impact on downstream hallmarks in zebrafish heart regeneration.

Upon heart injury in zebrafish, nearby cardiomyocytes dedifferentiate, infiltrate the scar, and proliferate to restore the injured myocardium. Despite having the necessary genes and signaling pathways for injury response, the cardiomyocytes of the Japanese medaka, a distantly related teleost fish, remain dormant after injury and are unable to recover injured myocardium. We used single-cell transcriptional profiling and molecular imaging to unveil the cellular and morphological differences between medaka and zebrafish hearts. We found that adult medaka lack primordial cardiomyocytes. By contrast, zebrafish primordial cardiomyocytes are found in a single cell sheet between the trabecular and cortical myocardium, and express genes involved in early heart development. Primordial cardiomyocytes share marker genes with injury-responsive cardiomyocytes in regenerative neonatal mice and adult zebrafish. Together, we hypothesize that the primordial layer is an evolutionarily conserved developmental cardiomyocyte subpopulation that is retained in adult hearts capable of regeneration. We are developing transgenic approaches to label, trace, and ablate the primordial cardiomyocytes to test their role in heart development and regeneration. Collectively, our work may offer new perspectives on the evolutionary links between heart development, morphology, and regenerative capacity among vertebrates.

Revascularization of ischemic myocardium following cardiac damage is an important step in cardiac regeneration. We have previously shown that pre-existing coronary arteries undergo major remodeling events following myocardial infarction (MI) in adult mouse hearts. The mechanism of arteriogenesis has not been well described during cardiac regeneration. Here we investigated a follow up of coronary artery remodeling and collateral growth during cardiac regeneration. Neonatal MI was induced by ligature of the left descending artery (LAD) in postnatal day (P) 1 or P7 pups from the Cx40-GFP mouse line in which GFP is expressed in coronary arterial endothelial cells. The arterial tree was reconstructed in 3D from images of cleared hearts collected at 1, 2, 4, 7 and 14 days after infarction. Our data show a rapid remodeling of the left coronary arterial tree induced by neonatal MI and the formation of numerous collateral arteries, which are transient in regenerating hearts after MI at P1 and persistent in non-regenerating hearts after MI at P7. This difference is accompanied by restoration of a perfused or a non-perfused LAD following MI at P1 or P7 respectively. Interestingly, we found that perfusion precedes collateral growth and lineage tracing analysis demonstrates that the restoration of the LAD occurs by remodeling of arterial cells. These results demonstrate that the restoration of the LAD artery during cardiac regeneration occurs by pruning with the rapid formation of collaterals supporting perfusion of the disconnected lower LAD which subsequently disappear on restoration of a unique LAD. These results highlight a rapid phase of arterial remodeling that plays an important role in vascular repair during cardiac regeneration.

After myocardial infarction (MI), adult mammalian hearts fail to regenerate, and the cardiac microenvironment is irreversibly disrupted. Inactivation of the Hippo signaling pathway in cardiomyocytes (CMs) induces heart renewal, and rebuilds the post-MI microenvironment. We used single-cell RNA-sequencing combined with spatial transcriptomics to examine cellular relationships within the microenvironment of two murine cardiac renewal models: adult hearts expressing a constitutively active YAP (YAP5SA), and neonatal hearts subject to MI. We found in both models a conserved, renewal competent CM cell state with high YAP activity (CM2). CM2 colocalized with cardiac fibroblasts (CFs) expressing complement pathway component 3 (C3), and macrophages (MPs) expressing complement C3a receptor (C3ar1) to form a pro-renewal cellular triad. C3 and C3ar1 loss-of-function suppressed CM proliferation in both neonatal injured hearts and adult YAP5SA hearts, and indicated that C3a signaling between CFs and MPs was required to assemble the CM2, C3+ CF, and C3ar1+ MP cellular triad. Our results demonstrate that CM-YAP drives the coalescence of a pro-renewal niche, which requires complement pathway signaling, during in vivo cardiac renewal.

Heart valves are complex structures whose developmental formation requires coordination between multiple cell types, including valve endothelial cells (VECs) and valve interstitial cells (VICs). Recent studies have identified heterogeneity within these populations – for example, arterial- and ventricular-facing VECs of the aortic valve are molecularly distinct. The signaling mechanisms that govern transcriptional identity leading to the heterogeneity of valve cell subtypes are not fully understood. Here, we report that during embryonic valvulogenesis, VECs specifically express Mfap5, encoding a secreted elastin microfibril component. An 800 bp cis-regulatory element ~4 kB 5’ of Mfap5, termed Mfap5 -4K enh, is sufficient to guide VEC-specific gene expression in E13 transgenic mouse embryos. Our initial in vitro findings indicate that Mfap5 -4K enh governs gene expression through the coordinated actions of the transcription factors glucocorticoid receptor (GR), NFATC1, and CREB1. In the adult aortic valve, Mfap5 is expressed in ventricular-facing VECs, but is notably absent from aortic-facing VECs. The mechanism governing this intra-EC partitioning is currently unknown. By characterizing the regulatory machinery that govern Mfap5 expression, we will gain insight into the transcriptional modules through which VECs interpret extracellular stimuli during development and valve disease, and generate tools to explore or alter VEC identity.

Aortic valve development requires coordinating cellular proliferation, differentiation, and communication, as well as deposition and reorganization of the extracellular matrix (ECM). We previously showed that loss of the matrix metalloproteinase ADAMTS19 causes progressive non-syndromic aortic valve disease in humans and mice. At 6-9 months, Adamts19-/- mice show significant aortic valve stenosis and dysfunction; however, we observed increased expression of the shear-responsive gene Klf2 in valvular endothelial cells (VECs) as early as P21. Based on this and other preliminary data, we hypothesized that the onset of valve disease in Adamts19 mutants is preceded by pre-symptomatic molecular and cellular changes occurring between E14.5 and P21. We therefore aimed to identify the earliest stages of morphological defects and the cellular and molecular changes that lead to progressive valve dysfunction at adult stages. H&E staining showed that the earliest morphological changes occurred at P1 when mutants had substantial thickening of the valve hinges and cusps compared to WT. Pentachrome staining revealed increased proteoglycan deposition in mutants, concurrent with upregulation of Klf2 in VECs at P1, suggesting that shear stress increased in parallel with the development of morphological defects. Immunostaining indicated increased expression of KI-67 and SOX9 in VICs from P1 to P7. Ex vivo P1 aortic valve explant experiments showed reduced migration and invasion capacity of Adamts19-/- cells into the 3D collagen gel, consistent with a loss of ECM remodeling capacity. Additionally, cells showed increased KI-67 and α-SMA expression, consistent with our in vivo observations. To investigate how the loss of ADAMTS19 leads to changes in valve ECM, we aimed to identify potential ADAMTS19 targets that are differentially cleaved between WT and mutants. Previous scRNAseq on WT vs mutant hearts and a Y2H screen followed by validation via co-immunoprecipitation identified Stat3 as a potential candidate. RNAscope confirmed strong co-localization between Adamts19 and Stat3 in P1 and P7 valves, but not atria, further supporting spatially specific regulation of valve development by ADAMTS19 via Stat3 signaling. We are currently investigating how ADAMTS19 regulates intracellular Stat3 function, in addition to further understanding the extracellular role of ADAMTS19 as an ECM remodeler. Taken together, our studies support a model where the loss of ADAMTS19 leads to pre-symptomatic valve defects at early postnatal stages via altered cellular and molecular signaling and reduced ECM remodelling, which then progresses to severe aortic valve disease in adults.

Endocardial cushions in the atrioventricular (AV) canal undergo maturation and remodeling into a ventricular membranous septum and AV valves. Because both BMP2 and Notch 2 are expressed in the AV endocardial cushions, we have tested our hypothesis that BMP-Notch interaction is critical for AV valvulogenesis. In the present study, we used in vitro culture assays and in vivo genetically engineered mouse models. We found that BMP2 induces Notch pathway effector Hey1 and that a BMP signaling intermediate, Smad1 interacts with Notch2 intracellular domain (ICD) in the nuclei of the AV endocardial cushion mesenchymal cells. For our in vivo studies, we generated double mutant, caAlk3; RBPJflox/+; Nfatc1Cre mice by crossing to genetically assess the effect of combining up-regulation of BMP signaling and down regulation of Notch signaling. We found that the double mutant mice exhibited degenerative phenotypes and aberrant deposition of extracellular matrix (ECM) components in the mitral valves after birth. On the contrary, single mutant mice did not exhibit significant valvular defects. Transcriptomic analysis further revealed that genes associated with leukocyte/macrophage infiltration were significantly affected in the anterior leaflet of mitral valves of the double mutant mice. We further performed RT-quantitative PCRs to verify these changes in gene expression and found an increase in critical genes for leukocyte/macrophage infiltration in the anterior leaflet of mitral valves in a time dependent manner. Our present studies indicate that coordinated expression and interaction of BMP and Notch signaling in endocardial cushion mesenchymal cells play critical roles in AV valvulogenesis and regulation of valve diseases progression. Our double mutant mouse would potentially serve as a useful model system to study the mechanisms of AV mitral valve disease progression.

Abnormal Aortic valves and anomalies of the ascending aorta (aortopathies) are comorbidities that can originate from maladaptive developmental remodeling of the cardiac outflow tract (OFT) but the factors involved are not well understood. In this study we utilized mice deficient in the extracellular matrix (ECM) protease ADAMTS5 that develop abnormal cardiac valves and aortopathies with full penetrance; anomalies are evident by embryonic day 14.5 (E14.5), the onset of ECM elastic lamelle assembly in the aorta and fetal valve maturation. Cardiac OFTs were dissected from wildtype (WT) (n=8) and Adamts5-/- (n=8) E14.5 hearts to generate protein lysates for liquid chromatography-tandem mass spectrometry (LC-MS/MS). Label free proteomic analysis (DirectDIA+, Spectronaut) revealed 232 proteins that were differentially expressed in the E14.5 Adamts5-/- OFT compared to WT OFTs, (P<0.05, Student’t-test). Gene ontology enrichment analysis using ToppGene and STRING identified immune function, clotting and wound healing as biological processes significanatly enriched among proteins differentially expressed in the Adamts5-/- E14.5 OFTs (B&H FDR <5.53E-7). There were 39 proteins that were differentially expressed with a Log2 > 0.5 enrichment, with a Student’s t-test P< 0.05; Vcan, an ECM substrate of ADAMTS5 was one of the LC-MS/MS proteins we have previously shown is increased in Adamts5-/- malformed cardiac valves and aortopathies thereby validating the LC-MS/MS approach. Novel protein targets SerpinF1 (Adj. P value <0.024; Log2 FC 0.56) and Pentraxin3 (Ptx3; Adj. P value <0.002; Log2 FC 1.9) also were enriched in the Adamts5-/- OFTs and were validated by IHC and Western blots. In situ analysis detected Ptx3 and SerpinF1 mRNA in cells that comprise malformed regions in the Adamts5-/- OFT. STRING analysis suggests Ptx3 and SerpinF1 interconnect the proteoglycan-ECM and immune interactive nodes indicating upregulation of proteins involved in innate immunity may exacerbate abnormal development of cardiac valves and aortopathies. The altered protein profiles discovered in the Adamts5-/- OFTs may lead to novel drug targets for treatment of cardiac valve and vessel diseases.

Blood flow-induced mechanical forces such as wall shear stress (WSS) could regulate long-term adaptation and remodeling of aortic valve structures. This may result in aortic valve stenosis or regurgitation, or both, which eventually leads to heart failure. However, the association between cells molecular processes, biomechanical changes and WSS is largely unknown. Epidermal growth factor receptor (EGFR) signaling contributes to aortic valve development in mice. We used Sm22α-Cre ;Egfrflox/flox mice to specifically delete Egfr in valvular interstitial cells (VICs) and demonstrate that, as in Egfr null mice, the aortic valve leaflets are thicker. Our analysis showed increased proliferation of VICs, and downregulation of genes associated with valve maturation (Egr1, Nos3, Tgf-β1) and extracellular matrix production (Versican, Col1a2, and Col3a1). In vitro experiments using overexpression, shRNA, and luciferase assays revealed that Egr1, known as flow-sensitive transcription factors, induces the expression of Nos3 and Tgf-β. To further examine how WSS controls Egr1 expression in valve cells, we used a unique fluid activation device that applies physiologically relevant pulsatile WSS to the surface of cells. We showed that the WSS induced the phosphorylation of ERK (MAP kinase pathway) leading to an increase in Egr1 expression. When the cells are pretreated with EGFR inhibitor, the increase in Egr1 expression induced by the WSS is significantly reduced. Interestingly, the exposure of cells to EGF induced the phosphorylation of ERK but not an increase in Egr1 expression, indicating the importance of other receptor in the WSS response. The Piezo channel is a mecano-sensitive receptor implicated in various cardiac physiological and pathologic response. The inhibition of Piezo impaired the augmentation of Egr1 expression induced by WSS. A treatment with YODA, Piezo activator, also induced the phosphorylation of ERK and an increase in Egr1 expression similar to the WSS. Altogether our data highlight the crucial role of Egfr in aortic valve development and the response to mechanical stress. Further study is required to gain a deeper understanding of the association between this pathway and the Piezo channel. However, such exploration could offer valuable insights into addressing valve pathology associated with abnormal WSS and valve aging.

Bicuspid aortic valve (BAV) disease is the most prevalent congenital heart defect, characterized by the formation of two, rather than three cusps. This structural malformation is associated with regionalized calcific nodule formation in ~50% of patients and the development of calcific aortic stenosis (CAS). Currently the most effective treatment for severe CAS is valve replacement; therefore, there is a critical need to develop alternative treatment and preventative therapies. However, advancements have been hindered as the mechanisms of CAS pathobiology in BAV patients are poorly understood. Our preliminary data using human data, mouse models, computational modeling and spatial transcriptomics suggest that the BAV anatomy leads to regionalized increases in wall shear stress that promote calcification via ATP signaling. We show that in two BAV mouse models (Type 0 and Type 1), the ATP purinergic receptor, p2rx4 is upregulated in valve interstitial cells within calcific regions associated with high wall shear stress. Further, treatment of porcine aortic valve interstitial cells (pAVICs) with 100uM of ATP promotes expression of the osteogenic marker, alkaline phosphatase and this is attenuated with co-treatment of 2.5 uM of the p2rx4 antagonist, 5-BDBD. Ongoing studies are currently examining ATP release from porcine aortic valve endothelial cells (pAVECs) exposed to BAV-induced wall shear stress and determining the pro-calcification potential. In addition, the application of 5-BDBD as a therapeutic option for CAS is being tested in animal models of BAV. In summary, completion of this work will for the first time directly link high wall shear stress with calcification in BAV and identify the mechanosensitive mechanisms responsible.

Microvessels supply oxygen and nutrients that support brain function. Consistent blood flow is modulated by vascular support cells, such as pericytes, that promote endothelial integrity and regulate micro-vessels in the brain. Dysfunctional or absent pericytes are associated with micro-hemorrhages and stroke. Pericytes originate from both mesoderm and neural crest and migrate and attach to vessels during brain angiogenesis. Developmentally, the precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. We are interested in identifying the genetic pathways guiding generation and differentiation of pericytes in development, and that also act to replenish pericytes in disease or injury. We identify nkx3.1 as a key driver gene for cells that develop into brain pericytes. We show that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express nkx3.1. Sorting precursor cells expressing nkx3.1, we identify the gene signature of precursors using scRNAseq. We find that nkx3.1, foxf2, and cxcl12 are all required in precursors. Cxcl12b- Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. foxf2 similarly regulates pericyte numbers and loss of this transcription factor leads to pericyte defects throughout the lifespan. Through genetic experiments we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Vascularizing avascular heart tissue has significant implications for treating ischemic heart diseases and fostering regenerative responses to tissue damage. Zebrafish, with its well-studied coronary vessel formation on the ventricle and potent regenerative response to injury, serves as an excellent model for such investigations. Notably, unlike mammals, the zebrafish atrium remains avascular, a characteristic shared with other teleost fish species. In our study, we explored the impact of compromised ventricle function due to injury or malformation on the zebrafish atrium. Following restricted ventricle growth or damage, we observed that the zebrafish atrium expands to more than triple its original volume and increased its cardiomyocyte count by nearly fourfold. We found that while Gata4 is required for atrial cardiomyocyte expansion during development, Gata4 is not required for compensatory atrial expansion during regeneration. Remarkably, this extensive chamber adaptation was accompanied by de novo atrial coronary vascularization. This suggests that the evolutionary loss of atrial coronary vessels in teleosts is reversible with the activation of dormant signaling cues. As such this presents an opportunity to study the signaling pathways necessary for coronary vascularization, which could be targeted therapeutically. To identify potential candidates, we performed RNA sequencing of enlarged atriums. We identified several upregulated secreted factors, including Vegfaa. We observed that Vegfaa is upregulated in the atrium epicardium, and ectopic expression of Vegfaa in the atrium is sufficient to drive atrial expansion. Loss of Vegfaa function inhibited expansion and vascularization, normalized in part the detrimental effects of blocked ventricle expansion and resulted in improved survival. This study underscores the potential of VEGFA-signaling as a therapeutic target, both positively by enhancing vascularization and negatively as a potential pathological factor that contributes to adverse cardiac chamber adaptation and progression of chronic disease.

Introduction: Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cells (VSMCs) phenotype. Cellular communication network factor 2 (CCN2) is a matricellular protein with an established role in maintaining tissue and organ homeostasis. Various studies have demonstrated the importance of CCN2 in regulating cardiovascular diseases, however its role in atherosclerosis remains to be investigated. The aim of the current study is to determine the role of VSMC-derived CCN2 in regulating atherosclerosis progression. Methods: In this study, CCN2 was deleted in VSMCs using Cre-lox technology. To induce atherosclerosis, mice were rendered LDLR deficient via AAV8-PCSK9 injection, followed by administration of a high-fat diet for 20 weeks. Aortic atherosclerotic lesion development was then assessed along with single-cell RNA sequencing to map the molecular effects of CCN2 deficiency on the VSMCs phenotype. Gain and loss of function studies in VSMCs were performed to assess the impact of CCN2 alteration on VSMC phenotype switching. Results: SMC-specific CCN2 knockout (SMC-CCN2-KO) mice demonstrated exquisite susceptibility to atherosclerosis formation as evidenced by a significant increase of aortic lipid-rich plaques in aortic roots, arch, thoracic and abdominal aorta. Concomitant with this, a profound vascular inflammation was seen in SMC-CCN2-KO mice. Importantly, these phenotypic changes were associated with a dramatic shift in VSMC phenotype towards a proliferating, lipid-accumulating and macrophage-like cell phenotype. Mechanistic studies point to the requisite role of the KLF4 pathway in VSMC transdifferentiation in SMC-CCN2-KO mice, findings corroborated by cell culture experiments in which KLF4-dependent phenotypic changes were observed in CCN2-null human primary aortic VSMCs. Conclusions: Our findings show that CCN2 deficiency in VSMCs promotes atherosclerosis development through affecting the VSMC phenotypic transition into macrophage-like cells by activation of the KLF4 signaling pathway.

Stem cell-based therapeutic vascular regeneration offers broad potential in treatment of cardiovascular diseases. ETV2 is a transcription factor (TF) that acts as a master regulator for the development of endothelial lineages. The mechanisms by which ETV2 drives endothelial cell specification and differentiation have remained elusive. Here, we developed a highly efficient ETV2-directed differentiation protocol that directs mesoderm to form human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) over a two day period, yielding cells with typical endothelial morphology and function and that were over 90% positive for CD31 and CD144. By applying single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses, we characterized the transcriptomic profiles, chromatin landscapes of endothelial cell differentiation mediated by ETV2. We defined the scope of ETV2 pioneering activity and identified its direct downstream target genes. In combination with functional screening followed by candidate validation, we showed that a set of TFs are essential co-regulators that act with ETV2 to remodel chromatin and promote EC differentiation. In addition to facilitating recruitment of transcriptional activators, ETV2 pioneering activity also enabled recruitment of a key transcriptional repressor that promotes suppresses non-EC lineage genes. Trajectory inference identified dynamic transcription factor activity signatures of endothelial commitment and maturation during differentiation. Collectively, our study provides an unparalleled molecular analysis of endothelial specification at single cell resolution and reveals transcriptomic and epigenomic mechanisms that promote EC specification and differentiation.

Non-immune hydrops fetalis (NIHF), a life-threatening disease characterized by pathological fluid overload in a fetus, affects 1 in 1700 pregnancies, less than half of which will survive to birth. Current treatment for NIHF is limited to high-risk intrauterine surgical intervention. Because of these complications, alternative medical strategies with lower morbidities, such as pharmacological therapies, are desperately needed. However, these require a clearer understanding of the biology underlying NIHF pathogenesis. Some suggest these excessive fluid collections are the non-specific end stage manifestation of various disorders affecting either cardiac output, blood vessel function, or lymphatic vessel function. However, a study showing that 1/3rd of NIHF cases present with genetic alterations within genes encoding proteins involved in the RAS-MAPK pathway challenges this assumption. RASopathies, a group of diseases caused by germline mutations in these same genes, often exhibit heart defects that frequently coincide with lymphatic abnormalities and NIHF. Due to these interrelated phenotypes, the primary mechanism driving NIHF in RASopathies remains unknown. Using whole exome sequencing of fetuses with NIHF, we identified missense and truncating frame-shift mutations in the gene LZTR1, a putative regulator of RAS-MAPK signaling. We established mouse and zebrafish Lztr1 animal models harboring knockout alleles and novel missense variants that we identified in patients. Preliminary data suggest both mutants have defects in lymphatic vessel patterning that mirror murine lymphatic-endothelial Kras gain of function. Herein we will determine how LZTR1 regulates RAS-MAPK signaling to increase our understanding of the etiology of NIHF and will determine if MAPK signaling is a therapeutic vulnerability in fetuses with NIHF.

Smooth muscle cells (SMCs) are pivotal in regulating physiological functions such as contraction and structural maintenance of blood vessels. ACTA2-encoded α-smooth muscle actin (aSMA) plays a central role in SMC proliferation, migration, and function. Mutations in ACTA2, notably R179H, lead to Multi-Systemic Smooth Muscle Dysfunction Syndrome (MSMDS), which is characterized by widespread SMC dysfunction and diverse clinical manifestations, including persistent ductus arteriosus, thoracic aortic aneurysm, and dilated pupils. Diagnostic and therapeutic challenges underscore the need for more effective treatments to rescue SMC function. We hypothesize that organ-specific SMCs are differentially affected by unique ACTA2 mutations, which then leads to the distinct pathologies arising from these mutations. Our project aims to elucidate the role of ACTA2 in SMC function and to characterize cellular phenotypes in normal versus mutant conditions. Our first objective was to optimize in vitro maturation of different organ-specific primary human SMCs in order to establish a more representative cell state for post-natal disease modeling. For pulmonary artery and pulmonary vein SMCs, we observed increased ACTA2 and MYH11 expression after decreasing FBS, and we observed a synergistic effect of FBS and TGFβ1 in one of the cell lines. Future experiments will focus on further investigating the effects of these new culture conditions on SMC function, followed by testing the effects of unique ACTA2 mutations in matured SMCs. Our systematic approach to optimize SMC culture conditions will provide optimal conditions to investigate organ specific MSMDS defects. This will help to better understand the role of ACTA2 and its mutation dependent MSMDS phenotypes, thereby facilitating the development of improved treatments.